Sample records for n-formyl peptide receptors
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Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A; Bothner, Brian; Jesaitis, Algirdas J
2013-09-20
Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.
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Mitochondrial N-formyl peptides induce cardiovascular collapse and sepsis-like syndrome
McCarthy, Cameron G.; Szasz, Theodora; Goulopoulou, Styliani; Webb, R. Clinton
2015-01-01
Fifty percent of trauma patients who present sepsis-like syndrome do not have bacterial infections. This condition is known as systemic inflammatory response syndrome (SIRS). A unifying factor of SIRS and sepsis is cardiovascular collapse. Trauma and severe blood loss cause the release of endogenous molecules known as damage-associated molecular patterns. Mitochondrial N-formyl peptides (F-MIT) are damage-associated molecular patterns that share similarities with bacterial N-formylated peptides and are potent immune system activators. The goal of this study was to investigate whether F-MIT trigger SIRS, including hypotension and vascular collapse via formyl peptide receptor (FPR) activation. We evaluated cardiovascular parameters in Wistar rats treated with FPR or histamine receptor antagonists and inhibitors of the nitric oxide pathway before and after F-MIT infusion. F-MIT, but not nonformylated peptides or mitochondrial DNA, induced severe hypotension via FPR activation and nitric oxide and histamine release. Moreover, F-MIT infusion induced hyperthermia, blood clotting, and increased vascular permeability. To evaluate the role of leukocytes in F-MIT-induced hypotension, neutrophil, basophil, or mast cells were depleted. Depletion of basophils, but not neutrophils or mast cells, abolished F-MIT-induced hypotension. Rats that underwent hemorrhagic shock increased plasma levels of mitochondrial formylated proteins associated with lung damage and antagonism of FPR ameliorated hemorrhagic shock-induced lung injury. Finally, F-MIT induced vasodilatation in isolated resistance arteries via FPR activation; however, F-MIT impaired endothelium-dependent relaxation in the presence of blood. These data suggest that F-MIT may be the link among trauma, SIRS, and cardiovascular collapse. PMID:25637548
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Bommakanti, R K; Bokoch, G M; Tolley, J O; Schreiber, R E; Siemsen, D W; Klotz, K N; Jesaitis, A J
1992-04-15
Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.
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Neutrophil formyl-peptide receptors. Relationship to peptide-induced responses and emphysema.
Stockley, R A; Grant, R A; Llewellyn-Jones, C G; Hill, S L; Burnett, D
1994-02-01
A reproducible assay was established to assess the number of formyl-peptide receptors expressed on the surface of human polymorphonuclear leukocytes (PMN). Using this assay the number of receptors was shown to demonstrate wide within- and between-subject variability. However, the receptor numbers were related to the chemotactic response (r = 0.572) and degranulation response (r = 0.512) to the peptide formyl-methionyl-leucyl-phenylalanine. Subsequent studies showed increased receptor numbers on PMN from patients with emphysema (median, 459 x 10(3)/cell; range, 207 to 1,080) as compared with age-matched control subjects (median, 288; range, 168 to 519; p < 0.02), which may explain the increased chemotactic response of the PMN to formyl peptides. This difference was not observed in patients with bronchiectasis, suggesting that the increased receptor number is a feature of emphysema. Furthermore, the increase was largely a feature of smokers with emphysema (median, 463; range, 362 to 1,080), whereas age-matched smokers without emphysema had lower numbers of receptors (p < 0.001; median, 332; range, 243 to 411). This observation suggests a mechanism that may explain the susceptibility of some smokers to the development of emphysema.
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Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C
1994-04-15
Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.
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Nakata, Makoto; Otsubo, Kouji; Kikuchi, Tomoko; Itou, Takuya; Sakai, Takeo
2010-02-01
This study describes a chemotaxis assay of ferret polymorphonuclear cells (PMNs). The optimal conditions for this chemotaxis assay were investigated for three chemoattractants: zymosan activated serum (ZAS), recombinant human interleukin-8 (rhIL-8) and N-formyl-Met-Leu- Phe (fMLF). In this study, ferret polymorphonuclear cells (PMNs) reacted to ZAS and rhIL-8, but not fMLF. The optimal concentration of ZAS and rhIL-8 were 5% and 100 ng/ml, respectively. The optimal incubation time of each reagent was 60 min. Due to the lack of response shown from fMLF, the existence of formyl peptide receptors (FPR) on ferret PMNs was investigated by evaluating FPR binding using flow cytometry. The receptor was not detected, implying that ferret neutrophils may lack FPR. This study confirms the fundamental experimental conditions for ferret PMNs chemotaxis and elucidates new findings concerning FPR in ferret neutrophils. Copyright 2009 Elsevier Ltd. All rights reserved.
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Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors
2005-01-01
activated phagocytes. Therefore, we examined the chemotactic migration of platelets qualita- tively by videomicroscopy . Platelets in medium were al- lowed...significantly decreased M. Czapiga et al. /Experimental Hematology 33 (2005) 73–84 79Figure 3. Videomicroscopy of human platelets in response to formyl...selected platelets during videomicroscopy from the time of the addition of fMLF (104 M in 1 µL) or PBS. Movement between markers represents 10 frames
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Gilbert, T L; Bennett, T A; Maestas, D C; Cimino, D F; Prossnitz, E R
2001-03-27
After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.
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Stephenson, Karin A; Banerjee, Sangeeta Ray; Sogbein, Oyebola O; Levadala, Murali K; McFarlane, Nicole; Boreham, Douglas R; Maresca, Kevin P; Babich, John W; Zubieta, Jon; Valliant, John F
2005-01-01
A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-alpha-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the K(d) values were found to be in the low micromolar range (0.5-3.0 microM). Compound 5j was subsequently labeled with (99m)Tc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.
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Bennett, T A; Maestas, D C; Prossnitz, E R
2000-08-11
Following activation by ligand, the N-formyl peptide receptor (FPR) undergoes processing events initiated by phosphorylation that lead to receptor desensitization and internalization. Our previous results have shown that FPR internalization can occur in the absence of receptor desensitization, suggesting that FPR desensitization and internalization are controlled by distinct mechanisms. More recently, we have provided evidence that internalization of the FPR occurs via a mechanism that is independent of the actions of arrestin, dynamin, and clathrin. In the present report, we demonstrate that stimulation of the FPR with agonist leads to a significant translocation of arrestin-2 from the cytosol to the membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A mutant FPR, which does not undergo activation or phosphorylation in response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required for FPR internalization and demonstrate for the first time that a common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.
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N-formyl peptide receptors internalize but do not recycle in the absence of arrestins.
Vines, Charlotte M; Revankar, Chetana M; Maestas, Diane C; LaRusch, Leah L; Cimino, Daniel F; Kohout, Trudy A; Lefkowitz, Robert J; Prossnitz, Eric R
2003-10-24
Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonstrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the post-endocytic trafficking of GPCRs.
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Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik
2015-01-01
In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484] PMID:26129676
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Fay, S.P.; Domalewski, M.D.; Houghton, T.G.
1994-02-01
Environmentally sensitive molecules have many potential cellular applications. The authors have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphthofluorescein), because in previous studies protonation has been used to explain the quenching when the fluorescinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonatedmore » and unprotonated forms can be examined by multiparameter flow cytometry. The authors found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pK[sub a] of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed. 22 refs., 4 figs.« less
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Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R
2003-06-24
G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.
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Perretti, Mauro; Getting, Stephen J.; Solito, Egle; Murphy, Philip M.; Gao, Ji-Liang
2001-01-01
An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides. PMID:11395373
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Regulation of N-formyl peptide-mediated degranulation by receptor phosphorylation.
Vines, Charlotte M; Xue, Mei; Maestas, Diane C; Cimino, Daniel F; Prossnitz, Eric R
2002-12-15
One of the major functions of the N-formyl peptide receptor (FPR) is to mediate leukocyte degranulation. Phosphorylation of the C-terminal domain of the FPR is required for receptor internalization and desensitization. Although arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of novel signaling cascades for a number of G protein-coupled receptors, their roles in FPR regulation and signaling remain unclear. CXCR1-mediated degranulation of RBL-2H3 cells is promoted by arrestin binding. To determine whether receptor phosphorylation or arrestin binding is required to promote FPR-mediated degranulation, we used RBL-2H3 cells stably transfected with either the wild-type FPR or a mutant form, DeltaST, which is incapable of undergoing ligand-stimulated phosphorylation. We observed that stimulation of wild-type FPR resulted in very low levels of degranulation compared with that mediated by cross-linking of the Fc(epsilon)RI receptor. Stimulation of the DeltaST mutant, however, resulted in levels of degranulation comparable to those of the Fc(epsilon)RI receptor, demonstrating that neither receptor phosphorylation nor arrestin binding was necessary to initiate FPR-mediated degranulation. Degranulation initiated by the DeltaST mutant was proportional to the level of active cell surface receptor, suggesting that either receptor internalization or desensitization may be responsible for terminating degranulation of the wild-type FPR. To distinguish between these possibilities, we used a partially phosphorylation-deficient mutant of the FPR that can undergo internalization, but not desensitization. Degranulation by this mutant FPR was indistinguishable from that of the DeltaST mutant, indicating that FPR phosphorylation or binding of arrestin but not internalization terminates the degranulation response.
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Potter, Ross M; Maestas, Diane C; Cimino, Daniel F; Prossnitz, Eric R
2006-05-01
Adaptation, defined as the diminution of receptor signaling in the presence of continued or repeated stimulation, is critical to cellular function. G protein-coupled receptors (GPCRs) undergo multiple adaptive processes, including desensitization and internalization, through phosphorylation of cytoplasmic serine and threonine residues. However, the relative importance of individual and combined serine and threonine residues to these processes is not well understood. We examined this mechanism in the context of the N-formyl peptide receptor (FPR), a well-characterized member of the chemoattractant/chemokine family of GPCRs critical to neutrophil function. To evaluate the contributions of individual and combinatorial serine and threonine residues to internalization, desensitization, and arrestin2 binding, 30 mutant forms of the FPR, expressed in the human promyelocytic U937 cell line, were characterized. We found that residues Ser(328), Ser(332), and Ser(338) are individually critical, and indeed sufficient, for internalization, desensitization, and arrestin2 binding, but that the presence of neighboring threonine residues can inhibit these processes. Additionally, we observed no absolute correlation between arrestin binding and either internalization or desensitization, suggesting the existence of arrestin-independent mechanisms for these processes. Our results suggest C-terminal serine and threonine residues of the FPR represent a combinatorial code, capable of both positively and negatively regulating signaling and trafficking. This study is among the first detailed analyses of a complex regulatory site in a GPCR, and provides insight into GPCR regulatory mechanisms.
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Boer, J C; Domanska, U M; Timmer-Bosscha, H; Boer, I G J; de Haas, C J C; Joseph, J V; Kruyt, F A E; de Vries, E G E; den Dunnen, W F A; van Strijp, J A G; Walenkamp, A M E
2013-01-01
Background: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. Methods and results: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I–IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). Conclusion: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients. PMID:23322202
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Designer cells programming quorum-sensing interference with microbes.
Sedlmayer, Ferdinand; Hell, Dennis; Müller, Marius; Ausländer, David; Fussenegger, Martin
2018-05-08
Quorum sensing is a promising target for next-generation anti-infectives designed to address evolving bacterial drug resistance. The autoinducer-2 (AI-2) is a key quorum-sensing signal molecule which regulates bacterial group behaviors and is recognized by many Gram-negative and Gram-positive bacteria. Here we report a synthetic mammalian cell-based microbial-control device that detects microbial chemotactic formyl peptides through a formyl peptide sensor (FPS) and responds by releasing AI-2. The microbial-control device was designed by rewiring an artificial receptor-based signaling cascade to a modular biosynthetic AI-2 production platform. Mammalian cells equipped with the microbial-control gene circuit detect formyl peptides secreted from various microbes with high sensitivity and respond with robust AI-2 production, resulting in control of quorum sensing-related behavior of pathogenic Vibrio harveyi and attenuation of biofilm formation by the human pathogen Candida albicans. The ability to manipulate mixed microbial populations through fine-tuning of AI-2 levels may provide opportunities for future anti-infective strategies.
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Yoshikawa, Masaaki
2015-10-01
We have found various bioactive peptides derived from animal and plant proteins, which interact with receptors for endogenous bioactive peptides such as opioids, neurotensin, complements C3a and C5a, oxytocin, and formyl peptides etc. Among them, rubiscolin, a δ opioid peptide derived from plant RuBisCO, showed memory-consolidating, anxiolytic-like, and food intake-modulating effects. Soymorphin, a μ opioid peptide derived from β-conglycinin showed anxiolytic-like, anorexigenic, hypoglycemic, and hypotriglyceridemic effects. β-Lactotensin derived from β-lactoglobulin, the first natural ligand for the NTS2 receptor, showed memory-consolidating, anxiolytic-like, and hypocholesterolemic effects. Weak agonist peptides for the complements C3a and C5a receptors were released from many proteins and exerted various central effects. Peptides showing anxiolytic-like antihypertensive and anti-alopecia effects via different types of receptors such as OT, FPR and AT2 were also obtained. Based on these study, new functions and post-receptor mechanisms of receptor commom to endogenous and exogenous bioactive peptides have been clarified. Copyright © 2015 Elsevier Inc. All rights reserved.
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Antagonism of Human Formyl Peptide Receptor 1 with Natural Compounds and their Synthetic Derivatives
Schepetkin, Igor A.; Khlebnikov, Andrei I.; Kirpotina, Liliya N.; Quinn, Mark T.
2015-01-01
Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small-molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca2+ mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes. PMID:26382576
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Multiparameter flow cytometry of a pH sensitive ligand bound to receptors and inside cells
DOE Office of Scientific and Technical Information (OSTI.GOV)
Fay, S.P.; Habbersett, R.; Posner, R.G.
1993-01-01
Because fluoresceinated ligands of the neutrophil formyl peptide receptor can be protonated either upon binding to the receptor on the cell surface or in acidified intracellular compartments, the authors synthesized a ligand conjugated to the pH sensitive fluorescent probe SNAFL (CHO-Met-Leu-Phe-Phe-Lys-SNAFL). In the three laser flow cytometer at LANL, protonated dye is excited at 488 nm and emits at 530 nm; unprotonated dye is excited at 568 nm and emits at 650 nm. Detection at the isobestic and isoemissive points at 528 and 600 nm is used to keep track of variations in ligand concentration from sample to sample. Themore » SNAFL-ligand bound to HL-60 cells (which overexpress the formyl peptide receptor) was compared to the free ligand in solution over a pH range from 6.5 to 9.0. The results suggest that the ligand bound to cell surface receptors was protonated in the binding pocket, possibly by virtue of its proximity to His 90, based on sequence data. When the cells were raised from 4[degrees] to 37[degrees], they also observed a time-dependent acidification of the ligand, indicative of ligand-receptor processing beginning 3-4 minutes after internalization.« less
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Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A4
Brigatte, Patrícia; Faiad, Odair Jorge; Ferreira Nocelli, Roberta Cornélio; Landgraf, Richardt G.; Palma, Mario Sergio; Cury, Yara; Curi, Rui; Sampaio, Sandra Coccuzzo
2016-01-01
We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs. PMID:27190493
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Qin, Cheng Xue; May, Lauren T.; Li, Renming; Cao, Nga; Rosli, Sarah; Deo, Minh; Alexander, Amy E.; Horlock, Duncan; Bourke, Jane E.; Yang, Yuan H.; Stewart, Alastair G.; Kaye, David M.; Du, Xiao-Jun; Sexton, Patrick M.; Christopoulos, Arthur; Gao, Xiao-Ming; Ritchie, Rebecca H.
2017-01-01
Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI. PMID:28169296
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Pei, L; Zhang, J; Zhao, F; Su, T; Wei, H; Tian, J; Li, M; Shi, J
2011-12-01
Annexin 1 (ANXA1) has analgesic effects in inflammatory pain. We aimed to investigate the anti-nociceptive role of ANXA1, at the dorsal root ganglion (DRG) level, through an interaction with formyl-peptide-receptor-like 1 (FPR2/ALX). Inflammatory pain was evoked by injecting complete Freund's adjuvant (CFA, 50 μl) into the hindpaw of male Sprague-Dawley rats. The distribution of ANXA1 and FPR2/ALX in L4/5 DRGs was evaluated by immunofluorescence. The expression of ANXA1 was measured by western blot. The involvement of FPR2/ALX in the anti-nociception of ANXA1 was investigated by thermal (irradiant heat) and mechanical (von Frey filament) pain tests with intrathecal (i.t.) ANXA1-derived peptide (Anxa1(2-26)), FPR2/ALX agonist 5(S)-6(R)-7-trihydroxyheptanoic-acid-methyl-ester (BML-111), and antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc1). ANXA1 and FPR2/ALX localized in the satellite glial cells and neurones in L4/5 DRGs. CFA treatment (n=20) increased ANXA1 expression in L4/5 DRGs within 7 days (P<0.01). I.T. Anxa1(2-26) (20 and 100 µg µl(-1)) and BML-111 (10 and 100 nmol) reduced CFA-induced thermal and mechanical nociception within 48 h (n=40) (P<0.05). However, i.t. Boc1 10 µg intensified inflammatory pain (P<0.05) and reversed the anti-nociceptive effect of Anxa1(2-26) (n=25) (P<0.05). Moreover, ANXA1 expression increased in L4/5 DRGs after i.t. Anxa1(2-26) (20 µg µl(-1)) (P<0.05) and BML-111 (10 nmol) (P<0.01) but decreased after i.t. Boc1 (10 and 100 µg) alone (P<0.01) or Boc1 (10 µg) co-injection with Anxa1(2-26) (20 µg µl(-1)) (P<0.05). Endogenous ANXA1 expression at the DRG level is involved in CFA-induced inflammatory pain, and i.t. ANXA1 20 µg µl(-1) produces its anti-nociceptive effect through FPR2/ALX.
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Yang, Jingyu; Wang, Qing; Zhao, Ruijun; Sun, Baoshan; Wang, Lihui; Hou, Yue; Li, Xiaoqin; Wu, Chunfu
2013-04-01
Formyl peptide receptor 1 (FPR1) plays an important role in the rapid progression of glioblastoma and has been considered as a molecular target for the treatment. Previously, we have shown that oligomer proanthocyanidins (F2, degree of polymerization 2-15), isolated from grape seeds, inhibited FPR1-mediated chemotaxis of U-87 glioblastoma cells. In the present study, we investigated the capacity of F2 to interact with FPR1. The cross attenuation of chemotaxis revealed that F2 shared FPR1 with formyl-methionyl-leucyl-phenylalanine (fMLF), which is a prototype agonist of FPR1. F2 was chemotactic for U-87 cells, and the chemotactic response was abolished when FPR1 gene was silenced or FPR1 was competitively occupied. We further show that F2 specifically blocked the binding of fluorescent agonist to FPR1. Interestingly, F2 exhibited the characteristic of a partial agonist for FPR1, as shown by its capacity to activate FPR1-mediated PI3K-PKC-MAPK pathways. Meanwhile, F2 also attenuated fMLF-triggered MAPK activation, suggesting that F2 could antagonize the effect of an agonist. Furthermore, F2 abolished the invasion of U-87 cells induced by fMLF. Thus, we have identified F2 as a novel, partial agonist for FPR1, which may be useful for glioblastoma therapy. Copyright © 2013 Elsevier B.V. All rights reserved.
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Antagonism of Human Formyl Peptide Receptor 1 (FPR1) by Chromones and Related Isoflavones
Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Cheng, Ni; Ye, Richard D.; Quinn, Mark T.
2014-01-01
Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. Because FPRs play an important role in the regulation of inflammatory reactions implicated in disease pathogenesis, FPR antagonists may represent novel therapeutics for modulating innate immunity. Previously, 4H-chromones were reported to be potent and competitive FPR1 antagonists. In the present studies, 96 additional chromone analogs, including related synthetic and natural isoflavones were evaluated for FPR1 antagonist activity. We identified a number of novel competitive FPR1 antagonists that inhibited fMLF-induced intracellular Ca2+ mobilization in FPR1-HL60 cells and effectively competed with WKYMVm-FITC for binding to FPR1 in FPR1-HL60 and FPR1-RBL cells. Compound 10 (6-hexyl-2-methyl-3-(1-methyl-1H-benzimidazol-2-yl)-4-oxo-4H-chromen-7-yl acetate) was found to be the most potent FPR1-specific antagonist, with binding affinity Ki~100 nM. These chromones inhibited Ca2+ flux and chemotaxis in human neutrophils with nanomolar-micromolar IC50 values. In addition, the most potent novel FPR1 antagonists inhibited fMLF-induced phosphorylation of extracellular signal-regulated kinases (ERK1/2) in FPR1-RBL cells. These antagonists were specific for FPR1 and did not inhibit WKYMVM/WKYMVm-induced intracellular Ca2+ mobilization in FPR2-HL60 cells, FPR3-HL60 cells, RBL cells transfected with murine Fpr1, or interleukin 8-induced Ca2+ flux in human neutrophils and RBL cells transfected with CXC chemokine receptor 1 (CXCR1). Moreover, pharmacophore modeling showed that the active chromones had a significantly higher degree of similarity with the pharmacophore template as compared to inactive analogs. Thus, the chromone/isoflavone scaffold represents a relevant backbone for development of novel FPR1 antagonists. PMID:25450672
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Richter, J; Andersson, R; Edvinsson, L; Gullberg, U
1992-01-01
The effect of the neuropeptides substance P, neurokinin A and alpha-calcitonin gene-related peptide (CGRP) on human neutrophil granulocytes was investigated. Substance P induced secondary granule secretion at a concentration of 100 microM. CGRP induced a significant secretory response at 10 microM and thus appeared to be about 10 times more potent than substance P. Calcitonin and a fragment of CGRP, CGRP(8-37), had no effect on neutrophil degranulation. The chemotactic peptide antagonist BOC-MLP (100 microM) inhibited lactoferrin secretion mediated both by CGRP and chemotactic peptide FMLP almost completely, while secretion in response to tumour necrosis factor (TNF) was unaffected. Results from receptor binding studies showed that CGRP and N-formyl-methionyl-leucyl-phenylalanine (FMLP) do not compete for binding. This indicates that CGRP does not exert its effects by binding to the chemotactic peptide receptor. CGRP induced a rapid increase in the cytosolic-free calcium concentration and this increase was not, unlike that induced by FMLP, abolished by preincubation of the cells with pertussis toxin (1000 ng/ml). Therefore CGRP signal transduction in neutrophils appears to involve rapid changes in the cytosolic-free calcium concentration but not a pertussis toxin-sensitive G-protein. In summary, this is the first report to show that CGRP can directly activate neutrophil granulocytes, and this probably occurs via a cell surface receptor which is distinct from that of FMLP although both the CGRP and FMLP-mediated effects can be blocked by BOC-MLP. Images Figure 3 PMID:1282494
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Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor
Thompson, Dawn; McArthur, Simon; Hislop, James N.; Flower, Roderick J.; Perretti, Mauro
2014-01-01
Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes β-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation. PMID:25326384
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Modulation of Neutrophil Apoptosis by Antimicrobial Peptides
Nagaoka, Isao; Suzuki, Kaori; Niyonsaba, François; Tamura, Hiroshi; Hirata, Michimasa
2012-01-01
Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Human antimicrobial peptides, α-defensins (human neutrophil peptides, HNPs), human β-defensins (hBDs), and cathelicidin (LL-37) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria, but also function as immunomodulatory molecules by inducing cytokine and chemokine production, and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, we provided the evidence that HNP-1, hBD-3, and LL-37 cannot only destroy bacteria but also potently modulate (suppress) neutrophil apoptosis, accompanied with the phosphorylation of ERK-1/-2, the downregulation of tBid (an proapoptotic protein) and upregulation of Bcl-xL (an antiapoptotic protein), and the inhibition of mitochondrial membrane potential change and caspase 3 activity, possibly via the actions on the distinct receptors, the P2Y6 nucleotide receptor, the chemokine receptor CCR6, and the low-affinity formyl-peptide receptor FPRL1/the nucleotide receptor P2X7, respectively. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion. PMID:23724322
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The GIT–PIX complexes regulate the chemotactic response of rat basophilic leukaemia cells
Gavina, Manuela; Za, Lorena; Molteni, Raffaella; Pardi, Ruggero; Curtis, Ivan de
2009-01-01
Background information. Cell motility entails the reorganization of the cytoskeleton and membrane trafficking for effective protrusion. The GIT–PIX protein complexes are involved in the regulation of cell motility and adhesion and in the endocytic traffic of members of the family of G-protein-coupled receptors. We have investigated the function of the endogenous GIT complexes in the regulation of cell motility stimulated by fMLP (formyl-Met-Leu-Phe) peptide, in a rat basophilic leukaemia RBL-2H3 cell line stably expressing an HA (haemagglutinin)-tagged receptor for the fMLP peptide. Results. Our analysis shows that RBL cells stably transfected with the chemoattractant receptor expressed both GIT1–PIX and GIT2–PIX endogenous complexes. We have used silencing of the different members of the complex by small interfering RNAs to study the effects on a number of events linked to agonist-induced cell migration. We found that cell adhesion was not affected by depletion of any of the proteins of the GIT complex, whereas agonist-enhanced cell spreading was inhibited. Analysis of agonist-stimulated haptotactic cell migration indicated a specific positive effect of GIT1 depletion on trans-well migration. The internalization of the formyl-peptide receptor was also inhibited by depletion of GIT1 and GIT2. The effects of the GIT complexes on trafficking of the receptors was confirmed by an antibody-enhanced agonist-induced internalization assay, showing that depletion of PIX, GIT1 or GIT2 protein caused decreased perinuclear accumulation of internalized receptors. Conclusions. Our results show that endogenous GIT complexes are involved in the regulation of chemoattractant-induced cell motility and receptor trafficking, and support previous findings indicating an important function of the GIT complexes in the regulation of different G-protein-coupled receptors. Our results also indicate that endogenous GIT1 and GIT2 regulate distinct subsets of agonist-induced responses and suggest a possible functional link between the control of receptor trafficking and the regulation of cell motility by GIT proteins. PMID:19912111
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Effect of hypothermia on splenic leukocyte modulation and survival duration in severely septic rats.
Willis, Rhett N; Charles, Eric J; Guidry, Christopher A; Chordia, Mahendra D; Davies, Stephen W; Yang, Zequan; Sawyer, Robert G
2017-07-01
Therapeutic hypothermia (HT) in severe septic shock is associated with prolonged survival. We hypothesized that moderate HT would prolong survival and modulate the inflammatory response in rats with septic shock by exerting its therapeutic effect on splenic leukocytes. Severe septic shock was created in rats by cecal ligation and incision (CLI). One hour after CLI or laparotomy, rats were randomized to sham, normothermia (NT), or 4 h of HT followed by 2 h of rewarming. HT (31 ± 1°C) was induced using a cooling blanket and monitored via a rectal temperature probe. Survival duration was 2.78 ± 1.0 h in NT rats and 8.33 ± 0.32 h in HT rats (n = 8/group, P < 0.0001). In separate groups, 3 h after CLI, the spleen weight was significantly smaller in NT rats (769 ± 100 mg) than in HT rats (947 ± 157 mg, P = 0.04). Fluorescent immunostaining of formyl peptide receptors on leukocytes in spleen tissue showed considerably higher formyl peptide receptor expression in HT rats than in NT rats. Significantly elevated proinflammatory cytokines and myeloperoxidase enzyme in plasma were found in NT rats compared with HT rats. Anti-inflammatory cytokine, interleukin-10, was significantly higher in HT rats. Both proinflammatory cytokines and plasma myeloperoxidase were significantly reduced in splenectomized NT rats. Moderate hypothermic therapy significantly prolongs the survival duration of rats with severe septic shock. HT dampens the inflammatory response during septic shock by modulating the spleen to an anti-inflammatory mode and preventing the spleen from releasing activated splenic leukocytes into the blood. Copyright © 2017 Elsevier Inc. All rights reserved.
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Antiviral activity of formyl peptide receptor 2 antagonists against influenza viruses.
Courtin, Noémie; Fotso, Aurélien Fotso; Fautrad, Pierre; Mas, Floriane; Alessi, Marie-Christine; Riteau, Béatrice
2017-07-01
Influenza viruses are one of the most important respiratory pathogens worldwide, causing both epidemic and pandemic infections. The aim of the study was to evaluate the effect of FPR2 antagonists PBP10 and BOC2 on influenza virus replication. We determined that these molecules exhibit antiviral effects against influenza A (H1N1, H3N2, H6N2) and B viruses. FPR2 antagonists used in combination with oseltamivir showed additive antiviral effects. Mechanistically, the antiviral effect of PBP10 and BOC2 is mediated through early inhibition of virus-induced ERK activation. Finally, our preclinical studies showed that FPR2 antagonists protected mice from lethal infections induced by influenza, both in a prophylactic and therapeutic manner. Thus, FPR2 antagonists might be explored for novel treatments against influenza. Copyright © 2017 Elsevier B.V. All rights reserved.
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LL-37 modulates human neutrophil responses to influenza A virus
Tripathi, Shweta; Verma, Anamika; Kim, Eun-Jeong; White, Mitchell R.; Hartshorn, Kevan L.
2014-01-01
Recent studies have shown that the human cathelicidin, LL-37, has antiviral activity against IAV in vitro and in vivo. Neutrophils are important cellular components of the initial innate response to IAV infection. In addition to its direct antimicrobial activities, LL-37 has important immunomodulatory effects. In this study, we explore how LL-37 affects interactions of IAV with human neutrophils. LL-37 did not alter neutrophil uptake of IAV but significantly increased neutrophil H2O2 responses to the virus. IAV stimulated production of NETs in vitro, and this response was increased by preincubating the virus with LL-37. NADPH-oxidase blockade did not reduce IAV-induced NET formation or the increased NET response stimulated by LL-37 + IAV. The increased respiratory burst and NET responses were, however, inhibited by preincubating cells with a formyl peptide receptor blocker, indicating that LL-37 engages these receptors when complexed with IAV. Responses to IAV alone were not inhibited by formyl peptide receptor blockade. It has been reported that LL-37 reduces proinflammatory cytokine responses during IAV infection in vivo. We now show that IAV alone potentiated release of IL-8 from neutrophils, and preincubation with LL-37 reduced IAV-stimulated IL-8 release. These results confirm that LL-37 modulates human neutrophil responses to IAV in a distinctive manner and could have important bearing on the protective effects of LL-37 during IAV infection in vivo. PMID:25082153
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Bednar, Filip; Song, Changcheng; Bardi, Giuseppe; Cornwell, William; Rogers, Thomas J.
2014-01-01
The cross-regulation of G protein-coupled receptors (GPCRs) plays an important role in the immune response. Studies from several laboratories have suggested that a hierarchy of sensitivities to cross-desensitization exists for the chemoattractant GPCRs. We carried out experiments to study the capacity of the formyl peptide receptor-1 (FPR1) to desensitize chemokine receptors CCR1 and CCR2. Our results show that activation of FPR1 resulted in the desensitization and partial internalization of CCR1, but not CCR2, in both primary human monocytes and HEK293 cells co-expressing CCR1, CCR2, and FPR1 (HR1R2F cells). The desensitization of CCR1 by FPR1 stimulation was not due to the simple depletion of the Ca2+ stores, but was dependent on activation of PKC. Furthermore, we found that the cross-desensitization of CCR1 by FPR1 was associated with CCR1 phosphorylation, and moderate reduction of CCR1 cell surface expression. In contrast, CCR2 was not phosphorylated or internalized following FPR1 activation. Additional studies showed that optimal cross-talk between FPR1 and CCR1 were dependent on the functional activity of PKCβ. These results provide a mechanistic basis for the capacity of certain GPCR ligands to exert rapid and selective cross-inactivation of other chemoattractant receptors, and suggest that FPR1 is able to exert “traffic control” in the migration of inflammatory cells by rapidly inhibiting the cell responses to potentially “low priority” chemoattractants such as CCR1 agonists without inhibiting the response to “higher priority” CCR2 chemoattractants. PMID:24778447
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Peptide Deformylase Inhibitors as Potent Antimycobacterial Agents▿ †
Teo, Jeanette W. P.; Thayalan, Pamela; Beer, David; Yap, Amelia S. L.; Nanjundappa, Mahesh; Ngew, Xinyi; Duraiswamy, Jeyaraj; Liung, Sarah; Dartois, Veronique; Schreiber, Mark; Hasan, Samiul ; Cynamon, Michael; Ryder, Neil S.; Yang, Xia; Weidmann, Beat; Bracken, Kathryn ; Dick, Thomas; Mukherjee, Kakoli
2006-01-01
Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC50) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC90 values of <1 μM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of ≤5 × 10−7 in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents. PMID:16966397
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Tardif, M; Mery, L; Brouchon, L; Boulay, F
1993-04-15
Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.
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Proinflammatory Activity of a Cecropin-Like Antibacterial Peptide from Helicobacter pylori
Bylund, Johan; Christophe, Thierry; Boulay, Francois; Nyström, Thomas; Karlsson, Anna; Dahlgren, Claes
2001-01-01
Helicobacter pylori, the bacterial pathogen associated with gastritis and peptic ulcers, is highly successful in establishing infection in the human gastric mucosa, a process typically associated with massive infiltration of inflammatory cells. Colonization of the mucosa is suggested to be facilitated by H. pylori-produced cecropin-like peptides with antibacterial properties, giving the microbe a competitive advantage over other bacteria. We show that a cecropin-like antibacterial peptide from H. pylori, Hp(2-20), not only has a potent bactericidal effect but also induces proinflammatory activities in human neutrophils, e.g., upregulation of integrins (Mac-1), induction of chemotaxis, and activation of the oxygen radical producing NADPH-oxidase. Furthermore, we show that these effects are mediated through binding of Hp(2-20) to the promiscuous, G-protein-linked lipoxin A4 receptor–formyl peptide-like receptor 1. PMID:11353614
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Bidasee, Keshore R; Nallani, Karuna; Yu, Yongqi; Cocklin, Ross R; Zhang, Yinong; Wang, Mu; Dincer, U Deniz; Besch, Henry R
2003-07-01
Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M+H(+)) >or=500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M+H(+) peaks corresponding to theoretical RyR2 peptides with single N(epsilon)-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetes-induced RyR2 dysfunction.
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Stimler-Gerard, N P
1987-01-01
The responsiveness of isolated guinea pig lung parenchymal strips to substance P was enhanced by at least 100-fold in the presence of the endopeptidase inhibitors phosphoramidon (1 microM) or thiorphan (1 microM), but not with the converting enzyme inhibitor, captopril, or an inhibitor of serum carboxypeptidase N (both 1 microM). Responses of guinea pig tracheal rings to substance P were also markedly potentiated by phosphoramidon. The increase in tissue responsiveness by these inhibitors was relatively specific for substance P among several other spasmogenic peptides, including formyl-methionyl-leucyl-phenylalanine and the complement peptides C3a and C5a. The enhanced responses appear to result from a decrease in the rate of substance P degradation in the presence of neutral endopeptidase inhibitors. Specific binding of substance P to its receptor on bronchial membranes was increased by three- to fourfold in the presence of phosphoramidon. These data demonstrate an enhanced potential for substance P to contract lung tissues when degradation by a neutral endopeptidase-like enzyme is blocked. PMID:2438306
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The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation
Volmering, Stephanie; Block, Helena; Boras, Mark; Lowell, Clifford A.; Zarbock, Alexander
2016-01-01
SUMMARY Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton’s tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation. PMID:26777396
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Salminen, Antero; Ojala, Johanna; Kauppinen, Anu; Kaarniranta, Kai; Suuronen, Tiina
2009-02-01
The inflammatory process has a fundamental role in the pathogenesis of Alzheimer's disease (AD). Recent studies indicate that inflammation is not merely a bystander in neurodegeneration but a powerful pathogenetic force in the disease process. Increased production of amyloid-beta peptide species can activate the innate immunity system via pattern recognition receptors (PRRs) and evoke Alzheimer's pathology. We will focus on the role of innate immunity system of brain in the initiation and the propagation of inflammatory process in AD. We examine here in detail the significance of amyloid-beta oligomers and fibrils as danger-associated molecular patterns (DAMPs) in the activation of a wide array of PRRs in glial cells and neurons, such as Toll-like, NOD-like, formyl peptide, RAGE and scavenger receptors along with complement and pentraxin systems. We also characterize the signaling pathways triggered by different PRRs in evoking inflammatory responses. In addition, we will discuss whether AD pathology could be the outcome of chronic activation of the innate immunity defence in the brain of AD patients.
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Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza
2016-08-23
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.
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Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza
2016-01-01
The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88–92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88–92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream. PMID:27323409
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Tripathi, Shweta; Wang, Guangshun; White, Mitchell; Rynkiewicz, Michael; Seaton, Barbara; Hartshorn, Kevan
2015-01-01
The human cathelicidin LL-37 has been shown to play a role in host defense against influenza A viruses (IAV) through direct antiviral effects and through modulating inflammatory responses to infection. We recently showed that LL-37 increases neutrophil respiratory burst and neutrophil extracellular trap (NET) responses to IAV through engaging formyl peptide receptor 2 (FPR-2). In this paper we show that a fragment of LL-37, GI-20, which is composed of the central helical segment of the peptide, has similar effects as LL-37 on neutrophil activation. In addition to increasing respiratory burst and NET responses of the cells to IAV through an FPR-2 dependent mechanism, it reduces neutrophil IL-8 production to IAV (also like LL-37). The N-terminal fragment, LL-23, did not have similar effects. Both GI-20 and LL-37 increase neutrophil intracellular calcium levels and their ability to increase neutrophil activation responses was calcium dependent and partially inhibited by pertussis toxin. These studies show that the central helix of LL-37 retains the ability of LL-37 to modulate neutrophil responses through FPR-2. Based on our findings we developed a homology model of FPR-2 and performed docking experiments of LL-37 and GI-20 with the receptor. PMID:26308522
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Gouwy, Mieke; De Buck, Mieke; Abouelasrar Salama, Sara; Vandooren, Jennifer; Knoops, Sofie; Pörtner, Noëmie; Vanbrabant, Lotte; Berghmans, Nele; Opdenakker, Ghislain; Proost, Paul; Van Damme, Jo; Struyf, Sofie
2018-01-01
Serum amyloid A1 (SAA1) is a prototypic acute phase protein, induced to extremely high levels by physical insults, including inflammation and infection. Human SAA and its NH2-terminal part have been studied extensively in the context of amyloidosis. By contrast, little is known about COOH-terminal fragments of SAA. Intact SAA1 chemoattracts leukocytes via the G protein-coupled receptor formyl peptide receptor like 1/formyl peptide receptor 2 (FPR2). In addition to direct leukocyte activation, SAA1 induces chemokine production by signaling through toll-like receptor 2. We recently discovered that these induced chemokines synergize with intact SAA1 to chemoattract leukocytes in vitro and in vivo. Gelatinase B or matrix metalloproteinase-9 (MMP-9) is also induced by SAA1 during infection and inflammation and processes many substrates in the immune system. We demonstrate here that MMP-9 rapidly cleaves SAA1 at a known consensus sequence that is also present in gelatins. Processing of SAA1 by MMP-9 at an accessible loop between two alpha helices yielded predominantly three COOH-terminal fragments: SAA1(52–104), SAA1(57–104), and SAA1(58–104), with a relative molecular mass of 5,884.4, 5,327.3, and 5,256.3, respectively. To investigate the effect of proteolytic processing on the biological activity of SAA1, we chemically synthesized the COOH-terminal SAA fragments SAA1(52–104) and SAA1(58–104) and the complementary NH2-terminal peptide SAA1(1–51). In contrast to intact SAA1, the synthesized SAA1 peptides did not induce interleukin-8/CXCL8 in monocytes or fibroblasts. Moreover, these fragments possessed no direct chemotactic activity for neutrophils, as observed for intact SAA1. However, comparable to intact SAA1, SAA1(58–104) cooperated with CXCL8 in neutrophil activation and migration, whereas SAA1(1–51) lacked this potentiating activity. This cooperative interaction between the COOH-terminal SAA1 fragment and CXCL8 in neutrophil chemotaxis was mediated by FPR2. Hence, proteolytic cleavage of SAA1 by MMP-9 fine tunes the inflammatory capacity of this acute phase protein in that only the synergistic interactions with chemokines remain to prolong the duration of inflammation.
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Surve, Chinmay R; Lehmann, David; Smrcka, Alan V
2014-06-20
Our laboratory has identified a number of small molecules that bind to G protein βγ subunits (Gβγ) by competing for peptide binding to the Gβγ "hot spot." M119/Gallein were identified as inhibitors of Gβγ subunit signaling. Here we examine the activity of another molecule identified in this screen, 12155, which we show that in contrast to M119/Gallein had no effect on Gβγ-mediated phospholipase C or phosphoinositide 3-kinase (PI3K) γ activation in vitro. Also in direct contrast to M119/Gallein, 12155 caused receptor-independent Ca(2+) release, and activated other downstream targets of Gβγ including extracellular signal regulated kinase (ERK), protein kinase B (Akt) in HL60 cells differentiated to neutrophils. We show that 12155 releases Gβγ in vitro from Gαi1β1γ2 heterotrimers by causing its dissociation from GαGDP without inducing nucleotide exchange in the Gα subunit. We used this novel probe to examine the hypothesis that Gβγ release is sufficient to direct chemotaxis of neutrophils in the absence of receptor or G protein α subunit activation. 12155 directed chemotaxis of HL60 cells and primary neutrophils in a transwell migration assay with responses similar to those seen for the natural chemotactic peptide n-formyl-Met-Leu-Phe. These data indicate that release of free Gβγ is sufficient to drive directional chemotaxis in a G protein-coupled receptor signaling-independent manner. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Wood, Matthew P; Cole, Amy L; Eade, Colleen R; Chen, Li-Mei; Chai, Karl X; Cole, Alexander M
2014-01-01
Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1. PMID:24617769
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Crystal structure of an EfPDF complex with Met-Ala-Ser based on crystallographic packing.
Nam, Ki Hyun; Kim, Kook-Han; Kim, Eunice Eun Kyeong; Hwang, Kwang Yeon
2009-04-17
PDF (peptide deformylase) plays a critical role in the production of mature proteins by removing the N-formyl polypeptide of nascent proteins in the prokaryote cell system. This protein is essential for bacterial growth, making it an attractive target for the design of new antibiotics. Accordingly, PDF has been evaluated as a drug target; however, architectural mechanism studies of PDF have not yet fully elucidated its molecular function. We recently reported the crystal structure of PDF produced by Enterococcus faecium [K.H. Nam, J.I. Ham, A. Priyadarshi, E.E. Kim, N. Chung, K.Y. Hwang, "Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure", Proteins 74 (2009) 261-265]. Here, we present the crystal structure of the EfPDF complex with MAS (Met-Ser-Ala), thereby not only delineating the architectural mechanism for the recognition of mimic-peptides by N-terminal cleaved expression peptide, but also suggesting possible targets for rational design of antibacterial drugs. In addition to their implications for drug design, these structural studies will facilitate elucidation of the architectural mechanism responsible for the peptide recognition of PDF.
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A colorimetric micro method for the determination of formyl groups
Lakshmi, S. Usha; Ramachandran, L. K.
1969-01-01
The characteristic purple colour formed by N-formyl-N′-2,4-dinitrophenyl-hydrazine in the presence of piperidine and acetone was made the basis of a new quantitative method for the determination of formyl groups. Samples containing N-formyl groups (up to 0·4μmole) are hydrazinolysed at 97–98° for 1hr. and are dinitrophenylated after the removal of excess of hydrazine. Interference from 2,4-dinitrophenylhydrazine is eliminated by subjecting the dinitrophenylated samples to chromatography on an alumina column. Interference arising from the formation of N-acetyl-N′-2,4-dinitrophenylhydrazine, when determining formyl groups in samples containing acetyl, can be avoided by a paper-chromatographic separation before analysis. A standard procedure is described. The method gives satisfactory results when applied to N-formyl-amino acids. Gramicidin, when analysed by this method, was found to contain 0·89 mole of formyl group/mole for a molecular weight of 1880. The method indicated the absence of formyl groups from lysozyme, a protein known not to contain such groups. Generally, the analytical values obtained by the method are within 100±4% of theory. PMID:5774469
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Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A
2017-07-05
The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yasui, K.; Yamazaki, M.; Miyagawa, Y.
Childhood chronic neutropenia with decreased numbers of chemotactic factor receptors as well as defective chemotaxis was first demonstrated in an 8-month-old girl. Chemotactic factor receptors on neutrophils were assayed using tritiated N-formyl-methionyl-leucyl-phenylalanine (/sup 3/H-FMLP). The patient's neutrophils had decreased numbers of the receptors: numbers of the receptors were 20,000 (less than 3 SD) as compared with those of control cells of 52,000 +/- 6000 (mean +/- SD) (n = 10). The neutropenia disappeared spontaneously by 28 months of age parallel with the improvement of chemotaxis and increase in numbers of chemotactic factor receptors. These results demonstrate a transient decrease ofmore » neutrophil chemotactic factor receptors as one of the pathophysiological bases of a transient defect of neutrophil chemotaxis in this disorder.« less
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N-formylation of amines via the aerobic oxidation of methanol over supported gold nanoparticles.
Ishida, Tamao; Haruta, Masatake
2009-01-01
Dress code: formyl. Gold nanoparticles supported on NiO catalyze the one-pot N-formylation of amines with methanol and molecular oxygen to produce formamide at a selectivity of 90 %. This process generates methyl formate in situ, followed by reaction with amines.
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Young, Susan M; Curry, Mark S; Ransom, John T; Ballesteros, Juan A; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S
2004-03-01
HyperCyt, an automated sample handling system for flow cytometry that uses air bubbles to separate samples sequentially introduced from multiwell plates by an autosampler. In a previously documented HyperCyt configuration, air bubble separated compounds in one sample line and a continuous stream of cells in another are mixed in-line for serial flow cytometric cell response analysis. To expand capabilities for high-throughput bioactive compound screening, the authors investigated using this system configuration in combination with automated cell sorting. Peptide ligands were sampled from a 96-well plate, mixed in-line with fluo-4-loaded, formyl peptide receptor-transfected U937 cells, and screened at a rate of 3 peptide reactions per minute with approximately 10,000 cells analyzed per reaction. Cell Ca(2+) responses were detected to as little as 10(-11) M peptide with no detectable carryover between samples at up to 10(-7) M peptide. After expansion in culture, cells sort-purified from the 10% highest responders exhibited enhanced sensitivity and more sustained responses to peptide. Thus, a highly responsive cell subset was isolated under high-throughput mixing and sorting conditions in which response detection capability spanned a 1000-fold range of peptide concentration. With single-cell readout systems for protein expression libraries, this technology offers the promise of screening millions of discrete compound interactions per day.
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Bozinovski, Steven; Uddin, Mohib; Vlahos, Ross; Thompson, Michelle; McQualter, Jonathan L; Merritt, Anne-Sophie; Wark, Peter A B; Hutchinson, Anastasia; Irving, Louis B; Levy, Bruce D; Anderson, Gary P
2012-01-17
Chronic obstructive pulmonary disease (COPD) will soon be the third most common cause of death globally. Despite smoking cessation, neutrophilic mucosal inflammation persistently damages the airways and fails to protect from recurrent infections. This maladaptive and excess inflammation is also refractory to glucocorticosteroids (GC). Here, we identify serum amyloid A (SAA) as a candidate mediator of GC refractory inflammation in COPD. Extrahepatic SAA was detected locally in COPD bronchoalveolar lavage fluid, which correlated with IL-8 and neutrophil elastase, consistent with neutrophil recruitment and activation. Immunohistochemistry detected SAA was in close proximity to airway epithelium, and in vitro SAA triggered release of IL-8 and other proinflammatory mediators by airway epithelial cells in an ALX/FPR2 (formyl peptide receptor 2) receptor-dependent manner. Lipoxin A(4) (LXA(4)) can also interact with ALX/FPR2 receptors and lead to allosteric inhibition of SAA-initiated epithelial responses (pA(2) 13 nM). During acute exacerbation, peripheral blood SAA levels increased dramatically and were disproportionately increased relative to LXA(4). Human lung macrophages (CD68(+)) colocalized with SAA and GCs markedly increased SAA in vitro (THP-1, pEC(50) 43 nM). To determine its direct actions, SAA was administered into murine lung, leading to induction of CXC chemokine ligand 1/2 and a neutrophilic response that was inhibited by 15-epi-LXA(4) but not dexamethasone. Taken together, these findings identify SAA as a therapeutic target for inhibition and implicate SAA as a mediator of GC-resistant lung inflammation that can overwhelm organ protective signaling by lipoxins at ALX/FPR2 receptors.
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Design of chimeric peptide ligands to galanin receptors and substance P receptors.
Langel, U; Land, T; Bartfai, T
1992-06-01
Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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Of pheromones and kairomones: what receptors mediate innate emotional responses?
Fortes-Marco, Lluis; Lanuza, Enrique; Martinez-Garcia, Fernando
2013-09-01
Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation. Copyright © 2013 Wiley Periodicals, Inc.
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Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis.
Cai, Yanfei; Chandrangsu, Pete; Gaballa, Ahmed; Helmann, John D
2017-02-01
Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes.
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Yin, Yanting; Zhou, X Edward; Hou, Li; Zhao, Li-Hua; Liu, Bo; Wang, Gaihong; Jiang, Yi; Melcher, Karsten; Xu, H Eric
2016-01-01
The glucagon-like peptide-1 receptor is a class B G protein coupled receptor (GPCR) that plays key roles in glucose metabolism and is a major therapeutic target for diabetes. The classic two-domain model for class B GPCR activation proposes that the apo-state receptor is auto-inhibited by its extracellular domain, which physically interacts with the transmembrane domain. The binding of the C-terminus of the peptide hormone to the extracellular domain allows the N-terminus of the hormone to insert into the transmembrane domain to induce receptor activation. In contrast to this model, here we demonstrate that glucagon-like peptide-1 receptor can be activated by N-terminally truncated glucagon-like peptide-1 or exendin-4 when fused to the receptor, raising the question regarding the role of N-terminal residues of peptide hormone in glucagon-like peptide-1 receptor activation. Mutations of cysteine 347 to lysine or arginine in intracellular loop 3 transform the receptor into a G protein-biased receptor and allow it to be activated by a nonspecific five-residue linker that is completely devoid of exendin-4 or glucagon-like peptide-1 sequence but still requires the presence of an intact extracellular domain. Moreover, the extracellular domain can activate the receptor in trans in the presence of an intact peptide hormone, and specific mutations in three extracellular loops abolished this extracellular domain trans-activation. Together, our data reveal a dominant role of the extracellular domain in glucagon-like peptide-1 receptor activation and support an intrinsic agonist model of the extracellular domain, in which peptide binding switches the receptor from the auto-inhibited state to the auto-activated state by releasing the intrinsic agonist activity of the extracellular domain. PMID:27917297
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Charve, Joséphine; Manganiello, Sonia; Glabasnia, Arne
2018-02-28
Corn sauce, an ingredient obtained from the fermentation of enzymatically hydrolyzed corn starch and used in culinary applications to provide savory taste, was investigated in this study. The links between its sensory properties and taste compounds were assessed using a combination of analytical and sensory approaches. The analyses revealed that glutamic acid, sodium chloride, and acetic acid were the most abundant compounds, but they could not explain entirely the savory taste. The addition of other compounds, found at subthreshold concentrations (alanine, glutamyl peptides, and one Amadori compound), contributed partly to close the sensory gap between the re-engineered sample and the original product. Further chemical breakdown, by a sensory-guided fractionation approach, led to the isolation of two fractions with taste-modulating effects. Analyses by mass spectrometry and nuclear magnetic resonance showed that the fractions contained glutamyl peptides, pyroglutamic acid, glutamic acid, valine, N-formyl-glutamic acid, and N-acetyl-glutamine.
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Chain, Benjamin M; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward
2008-10-23
The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes.
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Chain, Benjamin M.; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward
2008-01-01
The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes. PMID:18765264
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Cao, Jun
2015-06-28
In the present work, the combined electronic structure calculations and dynamics simulations have been performed to explore photocleavages of 2-formyl-2H-azirine and isoxazole in the gas phase and the subsequent rearrangement reactions. The carbonyl n → π(*) transition induces a cleavage of the C-N single bond of 2-formyl-2H-azirine to yield β-formylvinylnitrene in open-shell singlet state. However, the n → π(*) excitation of the imine chromophore results in a cleavage of the C-C single bond, producing a nitrile ylide intermediate through an internal conversion to the ground state. β-formylvinylnitrene and nitrile ylide with the carbonyl group are easily transformed into 2-formyl-2H-azirine and oxazole, respectively. The N-O bond cleavages on both S1((1)ππ(*)) and S2((1)nNπ(*)) of isoxazole are ultrafast processes, and they give products of 2-formyl-2H-azirine, 3-formylketenimine, HCN + CHCHO, and HCO + CHCHN. Both 2H-azirines and ketenimines were suggested to be formed from the triplet vinylnitrenes by intersystem crossing in the previous studies. However, our calculations show that the singlet β-formylvinylnitrene is responsible for the formation of 2-formyl-2H-azirine and 3-formylketenimine, and the singlet vinylnitrenes can play a key role in the photoinduced reactions of both 2H-azirines and isoxazoles.
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Virtual Screening Approach of Bacterial Peptide Deformylase Inhibitors Results in New Antibiotics.
Merzoug, Amina; Chikhi, Abdelouahab; Bensegueni, Abderrahmane; Boucherit, Hanane; Okay, Sezer
2018-03-01
The increasing resistance of bacteria to antibacterial therapy poses an enormous health problem, it renders the development of new antibacterial agents with novel mechanism of action an urgent need. Peptide deformylase, a metalloenzyme which catalytically removes N-formyl group from N-terminal methionine of newly synthesized polypeptides, is an important target in antibacterial drug discovery. In this study, we report the structure-based virtual screening of ZINC database in order to discover potential hits as bacterial peptide deformylase enzyme inhibitors with more affinity as compared to GSK1322322, previously known inhibitor. After virtual screening, fifteen compounds of the top hits predicted were purchased and evaluated in vitro for their antibacterial activities against one Gram positive (Staphylococcus aureus) and three Gram negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella. pneumoniae) bacteria in different concentrations by disc diffusion method. Out of these, three compounds, ZINC00039650, ZINC03872971 and ZINC00126407, exhibited significant zone of inhibition. The results obtained were confirmed using the dilution method. Thus, these proposed compounds may aid the development of more efficient antibacterial agents. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P; Veldkamp, Christopher T
2016-01-01
In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis. © 2016 Elsevier Inc. All rights reserved.
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Seibert, Christoph; Sanfiz, Anthony; Sakmar, Thomas P.; Veldkamp, Christopher T.
2016-01-01
In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by post-translational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8 and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the liability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods from sulfopeptide analysis. PMID:26921955
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Cao, Jun, E-mail: caojunbnu@mail.bnu.edu.cn
2015-06-28
In the present work, the combined electronic structure calculations and dynamics simulations have been performed to explore photocleavages of 2-formyl-2H-azirine and isoxazole in the gas phase and the subsequent rearrangement reactions. The carbonyl n → π{sup *} transition induces a cleavage of the C—N single bond of 2-formyl-2H-azirine to yield β-formylvinylnitrene in open-shell singlet state. However, the n → π{sup *} excitation of the imine chromophore results in a cleavage of the C—C single bond, producing a nitrile ylide intermediate through an internal conversion to the ground state. β-formylvinylnitrene and nitrile ylide with the carbonyl group are easily transformed intomore » 2-formyl-2H-azirine and oxazole, respectively. The N—O bond cleavages on both S{sub 1}({sup 1}ππ{sup *}) and S{sub 2}({sup 1}n{sub N}π{sup *}) of isoxazole are ultrafast processes, and they give products of 2-formyl-2H-azirine, 3-formylketenimine, HCN + CHCHO, and HCO + CHCHN. Both 2H-azirines and ketenimines were suggested to be formed from the triplet vinylnitrenes by intersystem crossing in the previous studies. However, our calculations show that the singlet β-formylvinylnitrene is responsible for the formation of 2-formyl-2H-azirine and 3-formylketenimine, and the singlet vinylnitrenes can play a key role in the photoinduced reactions of both 2H-azirines and isoxazoles.« less
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Hoaglund Hyzer, Cherokee S; Williamson, Michele L; Jansen, Patrick J; Kopach, Michael E; Scherer, R Brian; Baertschi, Steven W
2017-05-01
Edivoxetine (LY2216684 HCl), although a chemically stable drug substance, has shown the tendency to degrade in the presence of carbohydrates that are commonly used tablet excipients, especially at high excipient:drug ratios. The major degradation product has been identified as N-formyl edivoxetine. Experimental evidence including solution and solid-state investigations, is consistent with the N-formylation degradation pathway resulting from a direct reaction of edivoxetine with (1) formic acid (generated from decomposition of microcrystalline cellulose or residual glucose) and (2) the reducing sugar ends (aldehydic carbons) of either residual glucose or the microcrystalline cellulose polymer. Results of labeling experiments indicate that the primary source of the formyl group is the C1 position from reducing sugars. Presence of water or moisture accelerates this degradation pathway. Investigations in solid and solution states support that the glucose Amadori Rearrangement Product does not appear to be a direct intermediate leading to N-formyl degradation of edivoxetine, and oxygen does not appear to play a significant role. Solution-phase studies, developed to rapidly assess propensity of amines toward Maillard reactivity and formylation, were extended to show comparative behavior with example systems. The cyclic amine systems, such as edivoxetine, showed the highest propensity toward these side reactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Cao, Jun
2015-06-01
In the present work, the combined electronic structure calculations and dynamics simulations have been performed to explore photocleavages of 2-formyl-2H-azirine and isoxazole in the gas phase and the subsequent rearrangement reactions. The carbonyl n → π* transition induces a cleavage of the C—N single bond of 2-formyl-2H-azirine to yield β-formylvinylnitrene in open-shell singlet state. However, the n → π* excitation of the imine chromophore results in a cleavage of the C—C single bond, producing a nitrile ylide intermediate through an internal conversion to the ground state. β-formylvinylnitrene and nitrile ylide with the carbonyl group are easily transformed into 2-formyl-2H-azirine and oxazole, respectively. The N—O bond cleavages on both S1(1ππ*) and S2(1nNπ*) of isoxazole are ultrafast processes, and they give products of 2-formyl-2H-azirine, 3-formylketenimine, HCN + CHCHO, and HCO + CHCHN. Both 2H-azirines and ketenimines were suggested to be formed from the triplet vinylnitrenes by intersystem crossing in the previous studies. However, our calculations show that the singlet β-formylvinylnitrene is responsible for the formation of 2-formyl-2H-azirine and 3-formylketenimine, and the singlet vinylnitrenes can play a key role in the photoinduced reactions of both 2H-azirines and isoxazoles.
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Sulfonyl fluoride-based prosthetic compounds as potential 18F labelling agents.
Inkster, James A H; Liu, Kate; Ait-Mohand, Samia; Schaffer, Paul; Guérin, Brigitte; Ruth, Thomas J; Storr, Tim
2012-08-27
Nucleophilic incorporation of [(18)F]F(-) under aqueous conditions holds several advantages in radiopharmaceutical development, especially with the advent of complex biological pharmacophores. Sulfonyl fluorides can be prepared in water at room temperature, yet they have not been assayed as a potential means to (18)F-labelled biomarkers for PET chemistry. We developed a general route to prepare bifunctional 4-formyl-, 3-formyl-, 4-maleimido- and 4-oxylalkynl-arylsulfonyl [(18)F]fluorides from their sulfonyl chloride analogues in 1:1 mixtures of acetonitrile, THF, or tBuOH and Cs[(18)F]F/Cs(2)CO(3(aq.)) in a reaction time of 15 min at room temperature. With the exception of 4-N-maleimide-benzenesulfonyl fluoride (3), pyridine could be used to simplify radiotracer purification by selectively degrading the precursor without significantly affecting observed yields. The addition of pyridine at the start of [(18)F]fluorination (1:1:0.8 tBuOH/Cs(2)CO(3(aq.))/pyridine) did not negatively affect yields of 3-formyl-2,4,6-trimethylbenzenesulfonyl [(18)F]fluoride (2) and dramatically improved the yields of 4-(prop-2-ynyloxy)benzenesulfonyl [(18)F]fluoride (4). The N-arylsulfonyl-4-dimethylaminopyridinium derivative of 4 (14) can be prepared and incorporates (18)F efficiently in solutions of 100 % aqueous Cs(2)CO(3) (10 mg mL(-1)). As proof-of-principle, [(18)F]2 was synthesised in a preparative fashion [88(±8) % decay corrected (n=6) from start-of-synthesis] and used to radioactively label an oxyamino-modified bombesin(6-14) analogue [35(±6) % decay corrected (n=4) from start-of-synthesis]. Total preparation time was 105-109 min from start-of-synthesis. Although the (18)F-peptide exhibited evidence of proteolytic defluorination and modification, our study is the first step in developing an aqueous, room temperature (18)F labelling strategy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Dihydrogenphosphate recognition: Assistance from the acidic OH moiety of the anion
NASA Astrophysics Data System (ADS)
Das, Rituraj; Pathak, Nibedan; Choudhury, Samarjit; Borah, Suchibrata; Mahanta, Sanjeev Pran
2017-11-01
The binding affinity of the acidic hydrogen i.e. OH moiety of dihydrogenphosphate was investigated with receptors having competent H-bond donor and H-bond acceptor component. Three derivatives of 2, 3-dipyrrol-2‧-ylquinoxaline substituted with H-bond acceptor moiety at pyrrole α- positions were synthesized and their dihydrogenphosphate affinity was studied. All the three receptors shows general affinity towards fluoride, acetate and cyanide ions in DMSO solution. Interestingly, formyl substitution at both the pyrrole α-positions of 2, 3-dipyrrol-2‧-ylquinoxaline leads to binding of H2PO4-. 1H-NMR study rules out the involvement of the H-bond donor unit of the receptor in the biding event and reveals that the binding occurs predominantly via the Osbnd H⋯O interaction between the acidic OH moiety of the anion and the Cdbnd O of the formyl group of the receptor.
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Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.
Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J
1993-06-15
alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM).
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Structure-activity analysis of synthetic alpha-thrombin-receptor-activating peptides.
Van Obberghen-Schilling, E; Rasmussen, U B; Vouret-Craviari, V; Lentes, K U; Pavirani, A; Pouysségur, J
1993-01-01
alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM). PMID:7686363
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Lack of formylated methionyl-tRNA has pleiotropic effects on Bacillus subtilis
Cai, Yanfei; Chandrangsu, Pete; Gaballa, Ahmed; Helmann, John D
2017-01-01
Bacteria initiate translation using a modified amino acid, N-formylmethionine (fMet), adapted specifically for this function. Most proteins are processed co-translationally by peptide deformylase (PDF) to remove this modification. Although PDF activity is essential in WT cells and is the target of the antibiotic actinonin, bypass mutations in the fmt gene that eliminate the formylation of Met-tRNAMet render PDF dispensable. The extent to which the emergence of fmt bypass mutations might compromise the therapeutic utility of actinonin is determined, in part, by the effects of these bypass mutations on fitness. Here, we characterize the phenotypic consequences of an fmt null mutation in the model organism Bacillus subtilis. An fmt null mutant is defective for several post-exponential phase adaptive programmes including antibiotic resistance, biofilm formation, swarming and swimming motility and sporulation. In addition, a survey of well-characterized stress responses reveals an increased sensitivity to metal ion excess and oxidative stress. These diverse phenotypes presumably reflect altered synthesis or stability of key proteins involved in these processes. PMID:27983482
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Koole, Cassandra; Reynolds, Christopher A.; Mobarec, Juan C.; Hick, Caroline; Sexton, Patrick M.; Sakmar, Thomas P.
2017-01-01
The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state. PMID:28283573
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Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides
Bond, Cherie E.; Zimmermann, Martina; Greenfield, Susan A.
2009-01-01
Background The alpha-7 nicotinic acetylcholine receptor (α7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the α7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the α7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of α7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance The results reported here demonstrate a hitherto unknown relationship between the α7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration. PMID:19287501
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Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin
2016-03-10
Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.
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Láng, András; Csizmadia, Imre G; Perczel, András
2005-02-15
The conformational space of the most biologically significant backbone folds of a suitable methionine peptide model was explored by density functional computational method. Using a medium [6-31G(d)] and a larger basis set [6-311++G(2d,2p)], the systematic exploration of low-energy backbone structures restricted for the "L-region" in the Ramachandran map of N-formyl-L-methioninamide results in conformers corresponding to the building units of an extended backbone structure (betaL), an inverse gamma-turn (gammaL), or a right-handed helical structure (alphaL). However, no poly-proline II type (epsilonL) fold was found, indicating that this conformer has no intrinsic stability, and highlighting the effect of molecular environment in stabilizing this backbone structure. This is in agreement with the abundance of the epsilonL-type backbone conformation of methionine found in proteins. Stability properties (DeltaE) and distinct backbone-side-chain interactions support the idea that specific intramolecular contacts are operative in the selection of the lowest energy conformers. Apart from the number of different folds, all stable conformers are within a 10 kcal x mol(-1) energy range, indicating the highly flexible behavior of methionine. This conformational feature can be important in supporting catalytic processes, facilitating protein folding and dimerization via metal ion binding. In both of the biological examples discussed (HIV-1 reverse transcriptase and PcoC copper-resistant protein), the conformational properties of Met residues were found to be of key importance. Spatial proximity to other types of residues or the same type of residue seems to be crucial for the structural integrity of a protein, whether Met is buried or exposed.
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Role of spatial inhomogenity in GPCR dimerisation predicted by receptor association-diffusion models
NASA Astrophysics Data System (ADS)
Deshpande, Sneha A.; Pawar, Aiswarya B.; Dighe, Anish; Athale, Chaitanya A.; Sengupta, Durba
2017-06-01
G protein-coupled receptor (GPCR) association is an emerging paradigm with far reaching implications in the regulation of signalling pathways and therapeutic interventions. Recent super resolution microscopy studies have revealed that receptor dimer steady state exhibits sub-second dynamics. In particular the GPCRs, muscarinic acetylcholine receptor M1 (M1MR) and formyl peptide receptor (FPR), have been demonstrated to exhibit a fast association/dissociation kinetics, independent of ligand binding. In this work, we have developed a spatial kinetic Monte Carlo model to investigate receptor homo-dimerisation at a single receptor resolution. Experimentally measured association/dissociation kinetic parameters and diffusion coefficients were used as inputs to the model. To test the effect of membrane spatial heterogeneity on the simulated steady state, simulations were compared to experimental statistics of dimerisation. In the simplest case the receptors are assumed to be diffusing in a spatially homogeneous environment, while spatial heterogeneity is modelled to result from crowding, membrane micro-domains and cytoskeletal compartmentalisation or ‘corrals’. We show that a simple association-diffusion model is sufficient to reproduce M1MR association statistics, but fails to reproduce FPR statistics despite comparable kinetic constants. A parameter sensitivity analysis is required to reproduce the association statistics of FPR. The model reveals the complex interplay between cytoskeletal components and their influence on receptor association kinetics within the features of the membrane landscape. These results constitute an important step towards understanding the factors modulating GPCR organisation.
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Cotham, Victoria C; Shaw, Jared B; Brodbelt, Jennifer S
2015-09-15
Fast online chemical derivatization of peptides with an aromatic label for enhanced 193 nm ultraviolet photodissociation (UVPD) is demonstrated using a dual electrospray reactor implemented on the front-end of a linear ion trap (LIT) mass spectrometer. The reactor facilitates the intersection of protonated peptides with a second population of chromogenic 4-formyl-1,3-benzenedisulfonic acid (FBDSA) anions to promote real-time formation of ion/ion complexes at atmospheric pressure. Subsequent collisional activation of the ion/ion intermediate results in Schiff base formation generated via reaction between a primary amine in the peptide cation and the aldehyde moiety of the FBDSA anion. Utilizing 193 nm UVPD as the subsequent activation step in the MS(3) workflow results in acquisition of greater primary sequence information relative to conventional collision induced dissociation (CID). Furthermore, Schiff-base-modified peptides exhibit on average a 20% increase in UVPD efficiency compared to their unmodified counterparts. Due to the efficiency of covalent labeling achieved with the dual spray reactor, we demonstrate that this strategy can be integrated into a high-throughput LC-MS(n) workflow for rapid derivatization of peptide mixtures.
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West, Graham M.; Willard, Francis S.; Sloop, Kyle W.; Showalter, Aaron D.; Pascal, Bruce D.; Griffin, Patrick R.
2014-01-01
Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands. PMID:25180755
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Locke, Landon W; Kothandaraman, Shankaran; Tweedle, Michael; Chaney, Sarah; Wozniak, Daniel J; Schlesinger, Larry S
2018-01-01
Granulomas are the histopathologic hallmark of tuberculosis (TB), both in latency and active disease. Diagnostic and therapeutic strategies that specifically target granulomas have not been developed. Our objective is to develop a probe for imaging relevant immune cell populations infiltrating the granuloma. We report the binding specificity of Cyanine 3 (Cy3)-labeled cFLFLFK-PEG 12 to human leukocytes and cellular constituents within a human in vitro granuloma model. We also report use of the probe in in vivo studies using a mouse model of lung granulomatous inflammation. We found that the probe preferentially binds human neutrophils and macrophages in human granuloma structures. Inhibition studies showed that peptide binding to human neutrophils is mediated by the receptor formyl peptide receptor 1 (FPR1). Imaging the distribution of intravenously administered cFLFLFK-PEG 12 -Cy3 in the mouse model revealed probe accumulation within granulomatous inflammatory responses in the lung. Further characterization revealed that the probe preferentially associated with neutrophils and cells of the monocyte/macrophage lineage. As there is no current clinical diagnostic imaging tool that specifically targets granulomas, the use of this probe in the context of latent and active TB may provide a unique advantage over current clinical imaging probes. We anticipate that utilizing a FPR1-targeted radiopharmaceutical analog of cFLFLFK in preclinical imaging studies may greatly contribute to our understanding of granuloma influx patterns and the biological roles and consequences of FPR1-expressing cells in contributing to disease pathogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Kuribayashi, Kageaki; Nakamura, Kiminori; Tanaka, Maki; Sato, Tsutomu; Kato, Junji; Sasaki, Katsunori; Takimoto, Rishu; Kogawa, Katsuhisa; Terui, Takeshi; Takayama, Tetsuji; Onuma, Takayuki; Matsunaga, Takuya; Niitsu, Yoshiro
2007-03-26
Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) zeta was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCzeta in SASH1 cells, myristoylated PKCzeta peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.
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Kuribayashi, Kageaki; Nakamura, Kiminori; Tanaka, Maki; Sato, Tsutomu; Kato, Junji; Sasaki, Katsunori; Takimoto, Rishu; Kogawa, Katsuhisa; Terui, Takeshi; Takayama, Tetsuji; Onuma, Takayuki; Matsunaga, Takuya; Niitsu, Yoshiro
2007-01-01
Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) ζ was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCζ in SASH1 cells, myristoylated PKCζ peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway. PMID:17389234
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Control of cellular influx in lung and its role in pulmonary toxicology.
Lynn, W S
1984-01-01
The pulmonary influx of cytotoxic inflammatory cells, normally, in response to external toxins, is now thought to be etiologic in many of the disease syndromes of man, such as bronchitis and emphysema. Many types of effector inflammatory cells are involved, e.g., eosinophils, neutrophils, T-lymphocytes, monocytes. The diseases are characterized either by tissue destruction or by tissue hyperplasia. Agents which initiate the influx and cytotoxic secretions by these cells are legion and in general are not cell-specific. They include agents, such as phorbol esters, formyl peptides-complement fragments, elastin fragments, fatty acids (leukotrienes) as well as many uncharacterized excretions of inflammatory cells themselves, which react with specific receptors on the inflammatory cells, and secreted proteins such as fibronectin. Other agents, such as linoleic acid, digitonin and hydroxy fatty acids which are not bound by specific receptors also activate motility of inflammatory cells. The precise role of the above multiple cytotoxins in specific cellular fluxes in most pulmonary disease remains undefined. Similarly, the mechanism of cytotoxicity used by specific invading cells in specific pulmonary syndromes remains unclear. In general, macrophages are thought to destroy using specific proteases, neutrophils use oxidant radicals and proteases and eosinophils use basic surface active peptides. T-cells kill by unknown mechanisms. However, in specific clinical syndromes, it is usually not clear which cell is the cytotoxic culprit, nor is the mechanism of destruction usually known. PMID:6376103
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Durek, Thomas; Cromm, Philipp M; White, Andrew M; Schroeder, Christina I; Kaas, Quentin; Weidmann, Joachim; Ahmad Fuaad, Abdullah; Cheneval, Olivier; Harvey, Peta J; Daly, Norelle L; Zhou, Yang; Dellsén, Anita; Österlund, Torben; Larsson, Niklas; Knerr, Laurent; Bauer, Udo; Kessler, Horst; Cai, Minying; Hruby, Victor J; Plowright, Alleyn T; Craik, David J
2018-04-26
Ultrastable cyclic peptide frameworks offer great potential for drug design due to their improved bioavailability compared to their linear analogues. Using the sunflower trypsin inhibitor-1 (SFTI-1) peptide scaffold in combination with systematic N-methylation of the grafted pharmacophore led to the identification of novel subtype selective melanocortin receptor (MCR) agonists. Multiple bicyclic peptides were synthesized and tested toward their activity at MC1R and MC3-5R. Double N-methylated compound 18 showed a p K i of 8.73 ± 0.08 ( K i = 1.92 ± 0.34 nM) and a pEC 50 of 9.13 ± 0.04 (EC 50 = 0.75 ± 0.08 nM) at the human MC1R and was over 100 times more selective for MC1R. Nuclear magnetic resonance structural analysis of 18 emphasized the role of peptide bond N-methylation in shaping the conformation of the grafted pharmacophore. More broadly, this study highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery.
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Li, Xuechen; Danishefsky, Samuel J.
2008-01-01
Thermolysis of isonitriles with carboxylic acids provides, in one step, N-formyl imides (see, for example 8 + 19 → 21). The resultant N-formyl group can be converted to N-H, NCH2OH or NCH3. This chemistry allows for a new route for synthesizing β-N (asparagine) linked glycosyl amino acids. PMID:18370392
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Gustafsson, Erika; Haas, Pieter-Jan; Walse, Björn; Hijnen, Marcel; Furebring, Christina; Ohlin, Mats; van Strijp, Jos AG; van Kessel, Kok PM
2009-01-01
Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG. PMID:19284584
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New peptide deformylase inhibitors design, synthesis and pharmacokinetic assessment.
Lv, Fengping; Chen, Chen; Tang, Yang; Wei, Jianhai; Zhu, Tong; Hu, Wenhao
2016-08-01
The docking approach for the screening of designed small molecule ligands, led to the identification of a critical arginine residue in peptide deformylase for spiro cyclopropyl PDF inhibitor's extra hydrophobic binding, providing us a useful tool for searching more efficient PDF inhibitors to fight for horrifying antibiotics resistance. Further synthetic modification was undertaken to optimize the potency of amide compounds. To lower metabolic susceptibility and in turn reduce unwanted metabolic toxicity that was observed clinically, while retaining desired antibacterial activity, the use of azoles as amide bioisosteres had also been investigated. After the completion of chemical synthesis, all the compounds were evaluated through in vitro antibacterial activity assay, some of which were further subject to in vivo rat pharmacokinetic assessment. Those findings in this letter showed that spiro cyclopropyl proline N-formyl hydroxylamines, and especially the bioisosteric azoles, can represent a promising class of PDF inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Berg, Alexander K; Manokaran, Sumathra; Eiler, Daniel; Kooren, Joel; Mallik, Sanku; Srivastava, D K
2008-01-01
Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.
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Mauzy, C; Wu, L H; Egloff, A M; Mirzadegan, T; Chung, F Z
1992-01-01
In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.
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Identification of olfactory receptor genes in the Japanese grenadier anchovy Coilia nasus.
Zhu, Guoli; Wang, Liangjiang; Tang, Wenqiao; Wang, Xiaomei; Wang, Cong
2017-01-01
Olfaction is essential for fish to detect odorant elements in the environment and plays a critical role in navigating, locating food and detecting predators. Olfactory function is produced by the olfactory transduction pathway and is activated by olfactory receptors (ORs) through the binding of odorant elements. Recently, four types of olfactory receptors have been identified in vertebrate olfactory epithelium, including main odorant receptors (MORs), vomeronasal type receptors (VRs), trace-amine associated receptors (TAARs) and formyl peptide receptors (FPRs). It has been hypothesized that migratory fish, which have the ability to perform spawning migration, use olfactory cues to return to natal rivers. Therefore, obtaining OR genes from migratory fish will provide a resource for the study of molecular mechanisms that underlie fish spawning migration behaviors. Previous studies of OR genes have mainly focused on genomic data, however little information has been gained at the transcript level. In this study, we identified the OR genes of an economically important commercial fish Coilia nasus through searching for olfactory epithelium transcriptomes. A total of 142 candidate MOR, 52 V2R/OlfC, 32 TAAR and two FPR putative genes were identified. In addition, through genomic analysis we identified several MOR genes containing introns, which is unusual for vertebrate MOR genes. The transcriptome-scale mining strategy proved to be fruitful in identifying large sets of OR genes from species whose genome information is unavailable. Our findings lay the foundation for further research into the possible molecular mechanisms underlying the spawning migration behavior in C. nasus .
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Characterization and distribution of natriuretic peptide receptors in the rat uterus.
Dos Reis, A M; Fujio, N; Dam, T V; Mukaddam-Daher, S; Jankowski, M; Tremblay, J; Gutkowska, J
1995-10-01
Atrial natriuretic peptide (ANP) receptors were characterized in rat uterus. The binding of [125I]ANP to uterine membranes was completely competed for by increasing concentrations of unlabeled ANP (Kd = 0.39 nM) and brain natriuretic peptide (Kd = 1.24 nM) and partially by C-type natriuretic peptide (CNP; Kd = 80.4 nM), but not by C-ANF. Also, [125I]Tyr-CNP bound to uterine membranes was completely competed by unlabeled CNP (Kd = 1.12 nM). Cross-linking of [125I]ANP to uterine membranes revealed the presence of one band of 130 kilodaltons, corresponding to the guanylyl cyclase (GC-A and/or GC-B) subtypes of natriuretic peptide receptors. The presence of messenger RNA coding for genes of both GC-A and GC-B receptors was shown by quantitative reverse transcriptase polymerase chain reaction. Furthermore, ANP and, to a lesser degree, CNP stimulated the production of cGMP in rat uterus. Autoradiographic studies localized the highest binding of [125I]ANP in the endometrium, whereas [125I]Tyr-CNP binding was distributed in the endometrium as well as in the myometrium. These results demonstrate that rat uterine ANP receptors are of the guanylyl cyclase-coupled subtypes. The uterus is a target of natriuretic peptides where ANP induces its biological effects through the production of cGMP.
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Liu, Shuangxi; Gao, Yan; Yu, Xiaoli; Zhao, Baoming; Liu, Lu; Zhao, Yin; Luo, Zhenzhao; Shi, Jing
2016-01-01
Annexin-1 (ANXA1) has shown neuroprotective effects and microglia play significant roles during central nervous system injury, yet the underlying mechanisms remain unclear. This study sought to determine whether ANXA1 regulates microglial response to oxygen–glucose deprivation/reperfusion (OGD/R) treatment and to clarify the downstream molecular mechanism. In rat hippocampal slices, OGD/R treatment enhanced the ANXA1 expression in neuron, the formyl peptide receptor (FPRs) expression in microglia, and the microglial activation in the CA1 region (cornu ammonis 1). These effects were reversed by the FPRs antagonist Boc1. The cell membrane currents amplitude of BV-2 microglia (the microglial like cell-line) was increased when treated with Ac2-26, the N-terminal peptide of ANXA1. Ac2-26 treatment enhanced BV-2 microglial migration whereas Boc1 treatment inhibited the migration. In BV-2 microglia, both the expression of the CK2 target phosphorylated α-E-catenin and the binding of casein kinase II (CK2) with α-E-catenin were elevated by Ac2-26, these effects were counteracted by the CK2 inhibitor TBB and small interfering (si) RNA directed against transcripts of CK2 and FPRs. Moreover, both TBB and siRNA-mediated inhibition of CK2 blocked Ac2-26-mediated BV-2 microglia migration. Our findings indicate that ANXA1 promotes microglial activation and migration during OGD/R via FPRs, and CK2 target α-E-catenin phosphorylation is involved in this process. PMID:27782092
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Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru
2003-01-01
This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.
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Millimeter-wave spectroscopy of syn formyl azide (HC(O)N3) in seven vibrational states
NASA Astrophysics Data System (ADS)
Walters, Nicholas A.; Amberger, Brent K.; Esselman, Brian J.; Woods, R. Claude; McMahon, Robert J.
2017-01-01
Millimeter-wave spectra for formyl azide (HC(O)N3) were obtained from 240 to 360 GHz at ambient temperature. For the ground state of syn formyl azide, over 1500 independent rotational transitions were measured and least-squares fit to a complete S-reduced 8th order centrifugal distortion/rigid rotor Hamiltonian. The decomposition of formyl azide was monitored over a period of several hours, the half-life (t½ = 30 min) was determined, and its decomposition products were investigated. Transitions from five vibrational satellites of syn formyl azide (ν9, ν12, 2ν9, ν9 + ν12, and ν11) were observed, measured, and least-squares fit to complete or nearly complete octic centrifugally-distorted, single-state S-reduced models. A less complete single-state fit of 3ν9 (509.3 cm-1) was obtained from an unperturbed subset of its assignable transitions. This state is apparently coupled to the fundamental ν8 (489.4 cm-1) and the overtone 2ν12 (503.6 cm-1), but the coupling remains unanalyzed. Anharmonic CCSD(T)/ANO1 estimates of the vibrational frequencies of syn formyl azide were in close agreement with previously published experimental and computational values. Experimentally determined vibration-rotation interaction (αi) values were in excellent agreement with coupled-cluster predicted αi values for the fundamentals ν9, ν12, and ν11.
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Targeting the atypical chemokine receptor ACKR3/CXCR7 for the treatment of cancer and other diseases
NASA Astrophysics Data System (ADS)
Vestal, Richard D., Jr.
One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's N-terminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, showed no effect on the viability or proliferation upon exposure to MCF-7 and U87-MG cells. Membrane binding, colocalization, and cellular uptake were confirmed by whole-cell ELISA and confocal microscopy. The recovered peptides did not activate the receptor as confirmed by a Beta-Arrestin recruitment assay. The data shows that the peptide sequences recovered from the phage display protocol are viable candidates for targeting cancer cells and delivering material to them.
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Watkins, H A; Walker, C S; Ly, K N; Bailey, R J; Barwell, J; Poyner, D R; Hay, D L
2014-01-01
Background and Purpose Receptor activity-modifying proteins (RAMPs) define the pharmacology of the calcitonin receptor-like receptor (CLR). The interactions of the different RAMPs with this class B GPCR yield high-affinity calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors. However, the mechanism for this is unclear. Experimental Approach Guided by receptor models, we mutated residues in the N-terminal helix of CLR, RAMP2 and RAMP3 hypothesized to be involved in peptide interactions. These were assayed for cAMP production with AM, AM2 and CGRP together with their cell surface expression. Binding studies were also conducted for selected mutants. Key Results An important domain for peptide interactions on CLR from I32 to I52 was defined. Although I41 was universally important for binding and receptor function, the role of other residues depended on both ligand and RAMP. Peptide binding to CLR/RAMP3 involved a more restricted range of residues than that to CLR/RAMP1 or CLR/RAMP2. E101 of RAMP2 had a major role in AM interactions, and F111/W84 of RAMP2/3 was important with each peptide. Conclusions and Implications RAMP-dependent effects of CLR mutations suggest that the different RAMPs control accessibility of peptides to binding residues situated on the CLR N-terminus. RAMP3 appears to alter the role of specific residues at the CLR-RAMP interface compared with RAMP1 and RAMP2. PMID:24199627
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Wootten, Denise; Reynolds, Christopher A; Koole, Cassandra; Smith, Kevin J; Mobarec, Juan C; Simms, John; Quon, Tezz; Coudrat, Thomas; Furness, Sebastian G B; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M
2016-03-01
The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiologic significance of this is yet to be elucidated. Previous work has implicated R2.60(190), N3.43(240), Q7.49(394), and H6.52(363) as key residues involved in peptide-mediated biased agonism, with R2.60(190), N3.43(240), and Q7.49(394) predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53(364)A, N3.43(240)Q, Q7.49(394)N, and N3.43(240)Q/Q7.49(394)N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium, or extracellular signal-regulated kinase (ERK) signaling. A polar interaction between E6.53(364) and R2.60(190) was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49(394), but not R2.60(190)/E6.53(364) was critical for calcium mobilization for all three peptides. Mutation of N3.43(240) and Q7.49(394) had differential effects on individual peptides, providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
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Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.
Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha
2010-06-01
The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.
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The family B1 GPCR: structural aspects and interaction with accessory proteins.
Couvineau, Alain; Laburthe, Marc
2012-01-01
G protein coupled receptors (GPCRs) play a crucial role in physiology and pathophysiology in humans. Beside the large family A (rhodopsin-like receptors) and family C GPCR (metabotropic glutamate receptors), the small family B1 GPCR (secretin-like receptors) includes important receptors such as vasoactive intestinal peptide receptors (VPAC), pituitary adenylyl cyclase activating peptide receptor (PAC1R), secretin receptor (SECR), growth hormone releasing factor receptor (GRFR), glucagon receptor (GCGR), glucagon like-peptide 1 and 2 receptors (GLPR), gastric inhibitory peptide receptor (GIPR), parathyroid hormone receptors (PTHR), calcitonin receptors (CTR) and corticotropin-releasing factor receptors (CRFR). They represent very promising targets for the development of drugs having therapeutical impact on many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of family B1 receptors plays a pivotal role in natural ligand recognition. Structural analysis of some family B1 GPCR N-teds revealed the existence of a Sushi domain fold consisting of two antiparallel β sheets stabilized by three disulfide bonds and a salt bridge. The family B1 GPCRs promote cellular responses through a signaling pathway including predominantly the Gsadenylyl cyclase-cAMP pathway activation. Family B1 GPCRs also interact with a few accessory proteins which play a role in cell signaling, receptor expression and/or pharmacological profiles of receptors. These accessory proteins may represent new targets for the design of new drugs. Here, we review the current knowledge regarding: i) the structure of family B1 GPCR binding domain for natural ligands and ii) the interaction of family B1 GPCRs with accessory proteins.
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Vestal, R D; LaJeunesse, D R; Taylor, E W
2016-01-01
One of the greatest challenges in fighting cancer is cell targeting and biomarker selection. The Atypical Chemokine Receptor ACKR3/CXCR7 is expressed on many cancer cell types, including breast cancer and glioblastoma, and binds the endogenous ligands SDF1/CXCL12 and ITAC/CXCL11. A 20 amino acid region of the ACKR3/CXCR7 N-terminus was synthesized and targeted with the NEB PhD-7 Phage Display Peptide Library. Twenty-nine phages were isolated and heptapeptide inserts sequenced; of these, 23 sequences were unique. A 3D molecular model was created for the ACKR3/CXCR7 N-terminus by mutating the corresponding region of the crystal structure of CXCR4 with bound SDF1/CXCL12. A ClustalW alignment was performed on each peptide sequence using the entire SDF1/CXCL12 sequence as the template. The 23-peptide sequences showed similarity to three distinct regions of the SDF1/CXCL12 molecule. A 3D molecular model was made for each of the phage peptide inserts to visually identify potential areas of steric interference of peptides that simulated CXCL12 regions not in contact with the receptor's Nterminus. An ELISA analysis of the relative binding affinity between the peptides identified 9 peptides with statistically significant results. The candidate pool of 9 peptides was further reduced to 3 peptides based on their affinity for the targeted N-terminus region peptide versus no target peptide present or a scrambled negative control peptide. The results clearly show the Phage Display protocol can be used to target a synthesized region of the ACKR3/CXCR7 N-terminus. The 3 peptides chosen, P20, P3, and P9, will be the basis for further targeting studies.
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Cobb, K A; Novotny, M V
1992-01-01
The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.
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Drug forecast - the peptide deformylase inhibitors as antibacterial agents.
Guay, David R P
2007-08-01
The relatively rapid development of microbial resistance after the entry of every new antimicrobial into the marketplace necessitates a constant supply of new agents to maintain effective pharmacotherapy. Despite extensive efforts to identify novel lead compounds from molecular targets, only the peptide deformylase inhibitors (PDIs) have shown any real promise, with some advancing to phase I human trials. Bacterial peptide deformylase, which catalyzes the removal of the N-formyl group from N-terminal methionine following translation, is essential for bacterial protein synthesis, growth, and survival. The majority of PDIs are pseudopeptide hydroxamic acids and two of these (IV BB-83698 and oral NVP LBM-415) entered phase I human trials. However, agents to the present have suffered from major potential liabilities. Their in vitro activity has been limited to gram-positive aerobes and some anaerobes and has been quite modest against the majority of such species (MIC(90) values ranging from 1-8 mg/L). They have exerted bacteriostatic, not bacteriocidal, activity, thus reducing their potential usefulness in the management of serious infections in the immunocompromised. The relative ease with which microorganisms have been able to develop resistance and the multiple available mechanisms of resistance (mutations in fmt, defB, folD genes; AcrAB/TolC efflux pump; overexpression of peptide deformylase) are worrisome. These could portend a short timespan of efficacy after marketing. Despite these current liabilities, further pursuit of more potent and broader spectrum PDIs which are less susceptible to bacterial mechanisms of resistance is still warranted.
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Sachleben, Joseph R.; McElroy, Craig A.; Gollnick, Paul; Foster, Mark P.
2010-01-01
Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT12, composed of a tetramer of trimers, AT3. Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT3), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT3 to form inactive AT12 is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pKa for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors “active” trimeric AT and protonation favors “inactive” dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH. PMID:20713740
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Townsend, Steven D.; Wu, Xiangyang; Danishefsky, Samuel J.
2012-01-01
α,β-Unsaturated imides, formylated at the nitrogen atom, comprise a new and valuable family of dienophiles for servicing Diels-Alder reactions. These systems are assembled through extension of recently discovered isonitrile chemistry to the domain of α,β-unsaturated acids. Cycloadditions are facilitated by Et2AlCl, presumably via chelation between the two carbonyl groups of the N-formyl amide. Applications of the isonitrile/Diels-Alder logic to the IMDA reaction, as well as methodologies to modify the N-formyl amide of the resultant cycloaddition product, are described. It is expected that this easily executed chemistry will provide a significant enhancement for application of Diels-Alder reactions to many synthetic targets. PMID:22708980
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D'Agostino, Bruno; Marrocco, Giuseppina; De Nardo, Marilisa; Calò, Girolamo; Guerrini, Remo; Gallelli, Luca; Advenier, Charles; Rossi, Francesco
2005-01-01
Nociceptin/orphanin FQ (N/OFQ) is the endogenous peptide ligand for a specific G-protein coupled receptor, the N/OFQ peptide receptor (NOP). The N/OFQ-NOP receptor system has been reported to play an important role in pain, anxiety and appetite regulation. In airways, N/OFQ was found to inhibit the release of tachykinins and the bronchoconstriction and cough provoked by capsaicin. Here we evaluated the effects of NOP receptor activation in bronchoconstriction and airway microvascular leakage induced by intraesophageal (i.oe.) hydrochloric acid (HCl) instillation in rabbits. We also tested the effects of NOP receptor activation in SP-induced plasma extravasation and bronchoconstriction. In anesthetized New Zealand rabbits bronchopulmonary function (total lung resistance (RL) and dynamic compliance (Cdyn)) and airway microvascular leakage (extravasation of Evans blue dye) were evaluated. Infusion of i.oe. HCl (1 N) led to a significant increase in bronchoconstriction and plasma extravasation in the main bronchi and trachea of rabbits pretreated with propranolol, atropine and phosphoramidon. Bronchoconstriction and airway microvascular leakage were inhibited by N/OFQ (3–30 μg kg−1 i.v.) in a dose-dependent manner. The NOP receptor agonist [Arg14,Lys15]N/OFQ mimicked the inhibitory effect of N/OFQ, being 10-fold more potent, UFP-101, a peptide selective NOP receptor antagonist, blocked the inhibitory effects of both agonists. Under the same experimental conditions, N/OFQ and [Arg14,Lys15]N/OFQ did not counteract the bronchoconstriction and airway microvascular leakage induced by substance P. These results suggest that bronchoconstriction and airway plasma extravasation induced by i.oe. HCl instillation are inhibited by activation of prejunctional NOP receptors. PMID:15685213
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[The effect of substance P on functional proteins in human neutrophil].
Yang, Lin; Fa, Xiang-guang
2002-02-01
To explore the effect of substance P (SP) on the functional proteins on plasma membrane of neutrophil (Np). The response of Np to SP was examined by measuring the level of respiratory burst, the activities of ACP and ALP, the fluoroscopy intensity of CR3, CD45 and FM-LP. It was found that SP could increase respiratory burst of Np, decrease the activity of acid phosphatase (ACP), but had no effect on alkaline phosphatase (ALP). SP could also promote the amount of CD45, complement receptor type 3 (CR3) and N-Formyl-Met-Leu-Phe (FMLP) receptors. The results showed that the effects of SP on functional proteins in human Np membrane were universality and diversity. It implied that SP could affect various inflammation responses in Np.
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Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.
Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer
2008-04-25
The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.
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Prévost, M; Vertongen, P; Waelbroeck, M
2012-10-01
Glucagon plays an essential role in the glycemia maintenance during fasting, but also aggravates hyperglycemia in diabetic patients. A series of analogues of glucagon were synthesized replacing each amino acid of the C-terminal region (residues 15-29) with alanine. The residues affecting the binding to the glucagon receptor are found to be located on one face of the glucagon helix. Several 3-dimensional models of the N-terminal domain of the glucagon receptor in complex with its ligand peptide were built and used to analyze the peptide-receptor interface in terms of the nature of the peptide residues and the interactions they form with the receptor. The models suggest that glucagon keeps its native helical structure upon binding, and that a large part of the interface formed with the receptor is hydrophobic. We find that in the C-terminal region, F22, V23, M27, and D15 are the most important residues for peptide binding. They bury a large portion of their solvent accessible surface area and make numerous interactions with the receptor mainly of the hydrophobic type. © Georg Thieme Verlag KG Stuttgart · New York.
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Planagumà, A; Domenech, T; Jover, I; Ramos, I; Sentellas, S; Malhotra, R; Miralpeix, M
2013-08-01
Neutrophil recruitment and survival are important control points in the development and resolution of inflammatory processes. 15-epi-lipoxin (LX)A interaction with formyl peptide receptor 2 (FPR2)/ALX receptor is suggested to enhance anti-inflammatory neutrophil functions and mediate resolution of airway inflammation. However, it has been reported that 15-epi-LXA₄ analogues can also bind to cysteinyl leukotriene receptor 1 (CysLT1) and that the CysLT1 antagonist MK-571 binds to FPR2/ALX, so cross-reactivity between FPR2/ALX and CysLT1 ligands cannot be discarded. It is not well established whether the resolution properties reported for 15-epi-LXA4 are mediated through FPR2/ALX, or if other receptors such as CysLT1 may also be involved. Evaluation of specific FPR2/ALX ligands and CysLT1 antagonists in functional biochemical and cellular assays were performed to establish a role for both receptors in 15-epi-LXA₄-mediated signalling and function. In our study, a FPR2/ALX synthetic peptide (WKYMVm) and a small molecule FPR2/ALX agonist (compound 43) induced FPR2/ALX-mediated signalling, enhancing guanosine triphosphate-gamma (GTPγ) binding and decreasing cyclic adenosine monophosphate (cAMP) levels, whereas 15-epi-LXA₄ was inactive. Furthermore, 15-epi-LXA4 showed neither binding affinity nor signalling towards CysLT1. In neutrophils, 15-epi-LXA₄ showed a moderate reduction of interleukin (IL)-8-mediated neutrophil chemotaxis but no effect on neutrophil survival was observed. In addition, CysLT1 antagonists were inactive in FPR2/ALX signalling or neutrophil assays. In conclusion, 15-epi-LXA₄ is not a functional agonist or an antagonist of FPR2/ALX or CysLT1, shows no effect on IL-8-induced neutrophil survival and produces only moderate inhibition in IL-8-mediated neutrophil migration. Our data do not support an anti-inflammatory role of 15-epi-LXA₄- FPR2/ALX interaction in IL-8-induced neutrophil inflammation. © 2013 British Society for Immunology.
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Targeted Type 1 phototherapeutic agents using azido-peptide bioconjugates
NASA Astrophysics Data System (ADS)
Rajagopalan, Raghavan; Achilefu, Samuel I.; Jimenez, Hermo N.; Webb, Elizabeth G.; Schmidt, Michelle A.; Bugaj, Joseph E.; Dorshow, Richard B.
2001-07-01
Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.
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Natriuretic peptide system in the rat submaxillary gland.
Jankowski, M; Petrone, C; Tremblay, J; Gutkowska, J
1996-04-09
Natriuretic peptides and their receptors were characterized in rat submaxillary glands (SGs). Reverse phase-high performance liquid chromatography (HPLC) of rat SGs extracts revealed the presence of the 28-amino-acid (AA) circulating peptide ANP (Ser99-Tyr126) and the 126-AA prohormone (Asn1-Tyr126). The presence of ANP prohormone indicated that SGs are a site of ANP synthesis. Indeed, ANP mRNAs were demonstrated. ANP mRNA was 10 times lower than in the lung and only about 7 times lower than in the hypothalamus. ANP content in SG was determined as 30 +/- 8 ng/mg of protein (n = 7). In addition the presence of another member of the natriuretic peptide family, C-type natriuretic peptide (CNP), was found in SG. The CNP level of 293 +/- 38 pg/mg protein was significantly higher than in the lungs (44 +/- 6 pg/mg protein, P < 0.001, n = 5), but about 15 times lower than in hypothalamus (4.5 +/- 0.6 ng/mg protein, P < 0.001, n = 6). Both guanylyl cyclase and clearance receptors were expressed in SG. The presence of natriuretic peptide transcripts and their receptors suggests a role in rat SG functions.
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Revealing the membrane-bound structure of neurokinin A using neutron diffraction
NASA Astrophysics Data System (ADS)
Darkes, Malcolm J. M.; Hauss, Thomas; Dante, Silvia; Bradshaw, Jeremy P.
2000-03-01
Neurokinin A (or substance K) belongs to the tachykinin family, a group of small amphipathic peptides that bind to specific membrane-embedded, G-protein coupled receptors. The agonist/receptor complex is quaternary in nature because the receptor binding sites are thought to be located within the lipid bilayer and because the role of water cannot be ignored. The cell membrane acts as a solvent to accumulate peptide and an inducer of peptide secondary structure. The three-dimensional shape that the peptide assumes when associated to the cell membrane will be an important parameter with regards to the receptor selectivity and affinity. Neutron diffraction measurements were carried out in order to define the location of the N-terminus of the peptide in synthetic phospholipid multi-bilayer stacks.
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O-linked oligosaccharides on insulin receptor
DOE Office of Scientific and Technical Information (OSTI.GOV)
Collier, E.; Gorden, P.
1991-02-01
The insulin receptor, an integral membrane glycoprotein, is synthesized as a single-chain precursor that is cleaved to produce two mature subunits, both of which contain N-linked oligosaccharide chains and covalently linked fatty acids. We report that the beta-subunit also contains O-linked oligosaccharides. The proreceptor, alpha-subunit, and beta-subunit were labeled with (3H)mannose and (3H)galactose in the presence or absence of an inhibitor of O-linked glycosylation. Tryptic peptides from each component were separated by reverse-phase high-performance liquid chromatography. N- and O-linked oligosaccharide chains were identified on these peptides by specific enzymatic digestions. The proreceptor and alpha-subunit contained only N-linked oligosaccharides, whereas themore » beta-subunit contained both N- and O-linked oligosaccharides. The O-linked oligosaccharide chains were attached to a single tryptic fraction of the beta-subunit, which also contained N-linked chains. This fraction was further localized to the NH2-terminal tryptic peptide of the beta-subunit by specific immunoprecipitation with an anti-peptide antibody with specificity for this region. Binding of insulin and autophosphorylation of the beta-subunit were not dependent on O-linked glycosylation, because cells grown in the presence of the inhibitor exhibited a normal dose response to insulin. Therefore, the insulin receptor contains O-linked oligosaccharides on the NH2-terminal tryptic peptide of the beta-subunit, and these O-linked oligosaccharides are not necessary to the binding or autophosphorylation function of the receptor.« less
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Nabi, A H M Nurun; Biswas, Kazal Boron; Nakagawa, Tsutomu; Ichihara, Atsuhiro; Inagami, Tadashi; Suzuki, Fumiaki
2009-07-01
This study investigated a role of decoy peptide region (R10PIFLKRMPSI19P) in prorenin prosegment for prorenin binding to the (pro)renin receptor using the surface plasmon resonance technique. Three kinds of anti-receptor antibodies labeled as anti-107/121, anti-221/235 and anti-His tag antibody were prepared. The respective antigens D107SVANSIHSLFSEET121 (close to the N-terminal side of receptor), E221IGKRYGEDSEQFRD235 (N-terminal side of the transmembrane part of receptor) and 10xHis sequence (C-terminus) were designed based on the sequence of the receptor. These antibodies were immobilized on the CM5 sensor chip by amine coupling and allowed to bind to the receptor. Human prorenin, renin and the decoy bound to the receptor associated with antibodies. Their association (ka) and dissociation (kd) rate constants were measured and the dissociation constants (KD) were determined using Langmuir 1:1 kinetic binding model. The KD for interaction of prorenin and receptor associated to anti-107/121, anti-221/235 and anti-His tag antibodies were 2.9, 1.2 and 7.8 nM, respectively and for renin they were 9.3, 4.4 and 7.1 nM. The decoy bound to the respective immobilized receptor-antibody complexes at KD's of 6.2, 3.5 and 15.2 nM. Prorenin, renin and decoy had lower KD at the nanomolar ranges compared to those of L1PPTD4P in the prorenin prosegment and A248KKRLFDYVV257 in the C-domain of mature renin. The decoy reduced the binding of not only prorenin but also renin to (P)RR. These data are direct evidence that prorenin, renin and the peptides bind to (P)RR and the decoy reduces prorenin binding, supporting our hypothesis that decoy peptide region has a crucial role in prorenin binding.
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Signal Diversity of Receptor for Advanced Glycation End Products.
Sakaguchi, Masakiyo; Kinoshita, Rie; Putranto, Endy Widya; Ruma, I Made Winarsa; Sumardika, I Wayan; Youyi, Chen; Tomonobu, Naoko; Yamamoto, Ken-Ichi; Murata, Hitoshi
2017-12-01
The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.
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Bombesin-like peptide receptors in human bronchial epithelial cells.
Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A
1996-01-01
Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.
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Park, Ki Hun; Choi, Jeong-Heui; Abd El-Aty, A M; Cho, Soon-Kil; Park, Jong-Hyouk; Kim, Bo Mi; Yang, Angel; Na, Tae Woong; Rahman, Musfiqur; Im, Geon-Jae; Shim, Jae-Han
2012-10-15
In this study, a simultaneous method was developed for the determination of spinetoram (XDE-175-J and XDE-175-L) and its demethyl metabolites (N-demethyl-175-J and N-demethyl-175-L) and formyl metabolites (N-formyl-175-J and N-formyl-175-L) in the minor crops; amaranth and parsley. The method uses quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction. Afterwards, the analytes were quantified and confirmed via liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM). Calibration curves were linear over the calibration ranges for all the analytes tested with r(2)>0.993. Limits of detection and quantitation were 0.01 and 0.03 mg/kg for all the tested analytes in amaranth and parsley, respectively. Recovery values, at spiking levels 0.05 and 0.25 mg/kg, ranged from 71.0% to 115.2% with relative standard deviations <15%, except for N-formyl-175-J in both amaranth and parsley. This method was applied to field-incurred samples and was shown to provide an adequate sensitivity and performance for the simultaneous determination of spinetoram and metabolites. To the best of our knowledge, this is the first time spinetoram and its metabolites were quantified using LC-MS/MS in minor crops. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Orwig, Kevin S; Lassetter, McKensie R; Hadden, M Kyle; Dix, Thomas A
2009-04-09
Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.
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Shin, Y; Moni, R W; Lueders, J E; Daly, J W
1994-04-01
1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A1-adenosine receptors. 2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTP gamma S to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins. 3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin. N-Ethylmaleimide inhibits the response to both peptides. 4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTP gamma S-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects. 5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered by N-ethylmaleimide. 6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.
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Nagai, Chiaki; Mabashi-Asazuma, Hideaki; Nagasawa, Hiromichi; Nagata, Shinji
2014-01-01
Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC50 values of 1.1–2.6 × 10−8 m, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori. PMID:25278025
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Haskell-Luevano, C; Holder, J R; Monck, E K; Bauzo, R M
2001-06-21
The central melanocortin receptors, melanocortin-4 (MC4R) and melanocortin-3 (MC3R), are involved in the regulation of satiety and energy homeostasis. The MC4R in particular has become a pharmaceutical industry drug target due to its direct involvement in the regulation of food intake and its potential therapeutic application for the treatment of obesity-related diseases. The melanocortin receptors are stimulated by the native ligand, alpha-melanocyte stimulating hormone (alpha-MSH). The potent and enzymatically stable analogue NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH(2)) is a lead peptide for the identification of melanocortin amino acids important for receptor molecular recognition and stimulation. We have synthesized nine peptide fragments of NDP-MSH, deleting N- and C-terminal amino acids to determine the "minimally active" sequence of NDP-MSH. Additionally, five peptides were synthesized to study stereochemical inversion at the Phe 7 and Trp 9 positions in attempts to increase tetra- and tripeptide potencies. These peptide analogues were pharmacologically characterized at the mouse melanocortin MC1, MC3, MC4, and MC5 receptors. This study has identified the Ac-His-DPhe-Arg-Trp-NH(2) tetrapeptide as possessing 10 nM agonist activity at the brain MC4R. The tripeptide Ac-DPhe-Arg-Trp-NH(2) possessed micromolar agonist activities at the MC1R, MC4R, and MC5R but only slight stimulatory activity was observed at the MC3R (at up to 100 microM concentration). This study has also examined to importance of both N- and C-terminal NDP-MSH amino acids at the different melanocortin receptors, providing information for drug design and identification of putative ligand-receptor interactions.
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Cardona, C; Bleehen, N M; Reeve, J G
1992-01-01
The ligand-binding properties of the gastrin-releasing peptide (GRP) receptor and the cellular processing of GRP have been studied in the small-cell lung cancer (SCLC) cell line COR-L42. Scatchard analysis of GRP receptor expression indicated a single class of high-affinity receptors (Kd 1.5 nM) and approx. 6700 receptors/cell. GRP bound to its receptor with a Ki of 2.4 nM. The bombesin-related peptides neuromedin B (NMB) and phyllolitorin also bound to GRP receptors with Ki values of 22.7 and 59.1 nM respectively. Binding of 125I-GRP to COR-L42 cells increased rapidly at 37 degrees, achieved a maximum at 10 min and declined rapidly thereafter. At 4 degrees C, maximum binding was achieved at 30 min and the subsequent decline in cell-associated radioactivity was slower than that seen at 37 degrees C. Acid/salt extraction, to separate surface-bound ligand from internalized GRP, indicated that after receptor binding 125I-GRP was rapidly internalized. To determine the pathway of 125I-GRP degradation, binding studies were carried out with the lysosomotropic agent chloroquine (5 mM), and with phosphoramidon (10 microM), an inhibitor of the membrane-bound enzyme (EC 3.4.24.11). Both agents markedly inhibited the degradation of GRP, indicating that this process involves a lysosomal pathway and a phosphoramidon-sensitive pathway, possibly involving the EC 3.4.24.11 enzyme. GRP receptor down-regulation was observed following a 10 min exposure to 100 nM-GRP. With longer pretreatment times the number of binding sites recovered to 80% of control values. Treatment with 5 mM-chloroquine plus GRP or cycloheximide (10 micrograms/ml) plus GRP demonstrated that the majority of GRP receptors are recycled. NMB and phyllolitorin pretreatment did not influence the subsequent binding of 125I-GRP, suggesting that these peptides do not down-regulate GRP receptors. PMID:1310003
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Thompson, Aaron A.; Liu, Wei; Chun, Eugene
Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, {delta}, {kappa} and {mu} ({delta}-OR, {kappa}-OR and {mu}-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes ({approx}60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptidemore » N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors {kappa} (ref. 5) and {mu} (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.« less
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USDA-ARS?s Scientific Manuscript database
Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by po...
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Absence of C-type natriuretic peptide receptors in hamster glomeruli.
Luk, J K; Wong, E F; Wong, N L
1994-01-01
The distribution of atrial natriuretic peptide receptor B (ANPR-B) varies between tissues and species. The aim of this study is to determine whether ANPR-B is present in the hamster glomeruli. In vitro C-type natriuretic peptide (CNP)- and atrial natriuretic factor (ANF)-stimulated cGMP accumulation studies were performed in hamster glomeruli. Elevated cGMP accumulations were observed upon ANF addition. No cGMP response was seen with CNP. Competitive receptor-binding experiments were performed with 125I-CNP and 125I-ANF against their respective cold peptides in hamster glomeruli. Although no CNP binding was detected, positive ANF binding was found and two types of ANF receptor were demonstrated. The affinity (Kdl) and maximum binding capacity (Bmaxl) of the high-affinity ANF receptor were 0.014 +/- 0.001 nM and 60.4 +/- 10.2 fmol/mg protein, respectively. Those of the low-affinity receptor (Kd2 and Bmax2) were 45.7 +/- 6.2 nM and 28.3 +/- 6.3 pmol/mg protein, respectively. Similarly, saturation binding experiments also failed to show any CNP receptor binding in hamster glomeruli. This finding suggests that ANPR-B is not present in hamster glomeruli and CNP is not a direct physiological regulator of hamster renal function.
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Delineation of the peptide binding site of the human galanin receptor.
Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T
1996-01-01
Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199
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1994-01-01
Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose- dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N- formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria. PMID:8270862
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Activation of formylmethanofuran synthesis in cell extracts of Methanobacterium thermoautotrophicum.
Bobik, T A; Wolfe, R S
1989-01-01
In cell extracts of Methanobacterium thermoautotrophicum, formylmethanofuran (formyl-MFR) synthesis (an essential CO2 fixation reaction that is an early step in CO2 reduction to methane) is subject to a complex activation that involves a heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine O3-phosphate (CoM-S-S-HTP). In this paper we report that titanium(III) citrate, a low-potential reducing agent, stimulated CO2 reduction to methane and activated formyl-MFR synthesis in cell extracts. Titanium(III) citrate functioned as the sole source of electrons for formyl-MFR synthesis and enabled this reaction to occur independently of CoM-S-S-HTP. In addition, CoM-S-S-HTP was found to activate an unknown electron carrier that reduced metronidazole. The activation of formyl-MFR synthesis by CoM-S-S-HTP may involve the activation of a low-potential electron carrier. PMID:2921239
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New features of the delta opioid receptor: conformational properties of deltorphin I analogues.
Balboni, G; Marastoni, M; Picone, D; Salvadori, S; Tancredi, T; Temussi, P A; Tomatis, R
1990-06-15
Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.
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Structural Basis for Hormone Recognition by the Human CRFR2[alpha] G Protein-coupled Receptor
DOE Office of Scientific and Technical Information (OSTI.GOV)
Pal, Kuntal; Swaminathan, Kunchithapadam; Xu, H. Eric
2012-05-09
The mammalian corticotropin releasing factor (CRF)/urocortin (Ucn) peptide hormones include four structurally similar peptides, CRF, Ucn1, Ucn2, and Ucn3, that regulate stress responses, metabolism, and cardiovascular function by activating either of two related class B G protein-coupled receptors, CRFR1 and CRFR2. CRF and Ucn1 activate both receptors, whereas Ucn2 and Ucn3 are CRFR2-selective. The molecular basis for selectivity is unclear. Here, we show that the purified N-terminal extracellular domains (ECDs) of human CRFR1 and the CRFR2{alpha} isoform are sufficient to discriminate the peptides, and we present three crystal structures of the CRFR2{alpha} ECD bound to each of the Ucn peptides.more » The CRFR2{alpha} ECD forms the same fold observed for the CRFR1 and mouse CRFR2{beta} ECDs but contains a unique N-terminal {alpha}-helix formed by its pseudo signal peptide. The CRFR2{alpha} ECD peptide-binding site architecture is similar to that of CRFR1, and binding of the {alpha}-helical Ucn peptides closely resembles CRF binding to CRFR1. Comparing the electrostatic surface potentials of the ECDs suggests a charge compatibility mechanism for ligand discrimination involving a single amino acid difference in the receptors (CRFR1 Glu104/CRFR2{alpha} Pro-100) at a site proximate to peptide residue 35 (Arg in CRF/Ucn1, Ala in Ucn2/3). CRFR1 Glu-104 acts as a selectivity filter preventing Ucn2/3 binding because the nonpolar Ala-35 is incompatible with the negatively charged Glu-104. The structures explain the mechanisms of ligand recognition and discrimination and provide a molecular template for the rational design of therapeutic agents selectively targeting these receptors.« less
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Cyclic mu-opioid receptor ligands containing multiple N-methylated amino acid residues.
Adamska-Bartłomiejczyk, Anna; Janecka, Anna; Szabó, Márton Richárd; Cerlesi, Maria Camilla; Calo, Girolamo; Kluczyk, Alicja; Tömböly, Csaba; Borics, Attila
2017-04-15
In this study we report the in vitro activities of four cyclic opioid peptides with various sequence length/macrocycle size and N-methylamino acid residue content. N-Methylated amino acids were incorporated and cyclization was employed to enhance conformational rigidity to various extent. The effect of such modifications on ligand structure and binding properties were studied. The pentapeptide containing one endocyclic and one exocyclic N-methylated amino acid displayed the highest affinity to the mu-opioid receptor. This peptide was also shown to be a full agonist, while the other analogs failed to activate the mu opioid receptor. Results of molecular docking studies provided rationale for the explanation of binding properties on a structural basis. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Peptide and non-peptide opioid-induced hyperthermia in rabbits
NASA Technical Reports Server (NTRS)
Kandasamy, S. B.; Williams, B. A.
1983-01-01
The intracerebroventricular administration of prototype nonpeptide opioid receptor (mu, kappa, and sigma) agonists, morphine, ketocyclazocine, and N-allyl-normetazocine was found to induce hyperthermia in rabbits. The similar administration of peptide opioids like beta-endorphin (BE), methionine-enkephalin (ME), and its synthetic analogue D-ala2-methionine-enkephalinamide (DAME) was also found to cause hyperthermia. Results indicate that only the liver-like transport system is important to the ventricular inactivation of BE and DAME. Prostaglandins and norepinephrine were determined not to be involved in peptide and nonpeptide opioid-induced hyperthermia. In addition, cAMP was not required since a phosphodiesterase inhibitor, theophylline, did not accentuate the hyperthermia due to peptide and nonpeptide opioids. Naloxone-sensitive receptors were found to be involved in the induction of hyperthermia by morphine, BE, ME, and DAME since naloxone attenuated them. However, the hyperthermic response to ketocyclazocine and N-allyl-normetazocine was not antagonized by naloxone.
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Pescatore, Robyn; Marrone, Gina F; Sedberry, Seth; Vinton, Daniel; Finkelstein, Netanel; Katlowitz, Yitzchak E; Pasternak, Gavril W; Wilson, Krista R; Majumdar, Susruta
2015-06-17
Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-d-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [d-Ala(2)]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the μ- and κ-opioid receptors. It is a full agonist in [(35)S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which (125)I is incorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe(3). The peptides were characterized for binding affinity at the μ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology.
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Synthesis and Pharmacology of Halogenated δ-Opioid-Selective [D-Ala2]Deltorphin II Peptide Analogues
Pescatore, Robyn; Marrone, Gina F.; Sedberry, Seth; Vinton, Daniel; Finkelstein, Netanel; Katlowitz, Yitzchak E.; Pasternak, Gavril W.; Wilson, Krista R.; Majumdar, Susruta
2015-01-01
Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-D-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [D-Ala2]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the μ- and κ-opioid receptors. It is a full agonist in [35S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which 125I isincorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe3. The peptides were characterized for binding affinity at the μ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology. PMID:25844930
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Bajracharya, Prati; Lu, Hsiao-Ling; Pietrantonio, Patricia V.
2014-01-01
Neuropeptides and their receptors play vital roles in controlling the physiology and behavior of animals. Short neuropeptide F (sNPF) signaling regulates several physiological processes in insects such as feeding, locomotion, circadian rhythm and reproduction, among others. Previously, the red imported fire ant (Solenopsis invicta) sNPF receptor (S. invicta sNPFR), a G protein-coupled receptor, was immunolocalized in queen and worker brain and queen ovaries. Differential distribution patterns of S. invicta sNPFR protein in fire ant worker brain were associated both with worker subcastes and with presence or absence of brood in the colony. However, the cognate ligand for this sNPFR has not been characterized and attempts to deorphanize the receptor with sNPF peptides from other insect species which ended in the canonical sequence LRLRFamide, failed. Receptor deorphanization is an important step to understand the neuropeptide receptor downstream signaling cascade. We cloned the full length cDNA of the putative S. invicta sNPF prepropeptide and identified the putative “sNPF” ligand within its sequence. The peptide ends with an amidated Tyr residue whereas in other insect species sNPFs have an amidated Phe or Trp residue at the C-terminus. We stably expressed the HA-tagged S. invicta sNPFR in CHO-K1 cells. Two S. invicta sNPFs differing at their N-terminus were synthesized that equally activated the sNPFR, SLRSALAAGHLRYa (EC50 = 3.2 nM) and SALAAGHLRYa (EC50 = 8.6 nM). Both peptides decreased the intracellular cAMP concentration, indicating signaling through the Gαi-subunit. The receptor was not activated by sNPF peptides from other insect species, honey bee long NPF (NPY) or mammalian PYY. Further, a synthesized peptide otherwise identical to the fire ant sequence but in which the C-terminal amidated amino acid residue ‘Y’ was switched to ‘F’, failed to activate the sNPFR. This discovery will now allow us to investigate the function of sNPY and its cognate receptor in fire ant biology. PMID:25310341
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Naydenova, Emilia; Todorov, Petar; Zamfirova, Rositza
2015-01-01
The heptadecapeptide nociceptin, also called orphanin FQ (N/OFQ), is the endogenous agonist of the N/OFQ peptide receptor (NOP receptor) and is involved in several central nervous system pathways, such as nociception, reward, tolerance, and feeding. The discovery of small molecule ligands for NOP is being actively pursued for several therapeutic applications. This review presents overview of the several recently reported NOP ligands (agonists and antagonists), with an emphasis of the structural features that may be important for modulating the intrinsic activity of these ligands. In addition, a brief account on the characterization of newly synthesized ligands of NOP receptor with aminophosphonate moiety and β-tryptophan analogues will be presented. © 2015 Elsevier Inc. All rights reserved.
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Vulfius, Catherine A; Spirova, Ekaterina N; Serebryakova, Marina V; Shelukhina, Irina V; Kudryavtsev, Denis S; Kryukova, Elena V; Starkov, Vladislav G; Kopylova, Nina V; Zhmak, Maxim N; Ivanov, Igor A; Kudryashova, Ksenia S; Andreeva, Tatyana V; Tsetlin, Victor I; Utkin, Yuri N
2016-10-01
Phospholipase A 2 (named bitanarin) possessing capability to block nicotinic acetylcholine receptors (nAChRs) was isolated earlier (Vulfius et al., 2011) from puff adder Bitis arietans venom. Further studies indicated that low molecular weight fractions of puff adder venom inhibit nAChRs as well. In this paper, we report on isolation from this venom and characterization of three novel peptides called baptides 1, 2 and 3 that reversibly block nAChRs. To isolate the peptides, the venom of B. arietans was fractionated by gel-filtration and reversed phase chromatography. The amino acid sequences of peptides were established by de novo sequencing using MALDI mass spectrometry. Baptide 1 comprised 7, baptides 2 and 3-10 amino acid residues, the latter being acetylated at the N-terminus. This is the first indication for the presence of such post-translational modification in snake venom proteins. None of the peptides contain cysteine residues. For biological activity studies the peptides were prepared by solid phase peptide synthesis. Baptide 3 and 2 blocked acetylcholine-elicited currents in isolated Lymnaea stagnalis neurons with IC 50 of about 50 μM and 250 μM, respectively. In addition baptide 2 blocked acetylcholine-induced currents in muscle nAChR heterologously expressed in Xenopus oocytes with IC 50 of about 3 μM. The peptides did not compete with radioactive α-bungarotoxin for binding to Torpedo and α7 nAChRs at concentration up to 200 μM that suggests non-competitive mode of inhibition. Calcium imaging studies on α7 and muscle nAChRs heterologously expressed in mouse neuroblastoma Neuro2a cells showed that on α7 receptor baptide 2 inhibited acetylcholine-induced increasing intracellular calcium concentration with IC 50 of 20.6 ± 3.93 μM. On both α7 and muscle nAChRs the suppression of maximal response to acetylcholine by about 50% was observed at baptide 2 concentration of 25 μM, the value being close to IC 50 on α7 nAChR. These data are in accord with non-competitive inhibition as follows from α-bungarotoxin binding experiments. The described peptides are the shortest peptides without disulfide bridges isolated from animal venom and capable to inhibit nAChR by non-competitive way. Copyright © 2016. Published by Elsevier Ltd.
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Reinartz, Michael T; Kälble, Solveig; Littmann, Timo; Ozawa, Takeaki; Dove, Stefan; Kaever, Volkhard; Wainer, Irving W; Seifert, Roland
2015-01-01
Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the β2-adrenoceptor (β2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using β2AR-Gsα and β2AR-Giα fusion proteins, (R,S')- and (S,S')-isomers of 4'-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via β-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of β2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased β2AR agonists.
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Wilczynski, Andrzej; Wilson, Krista R; Scott, Joseph W; Edison, Arthur S; Haskell-Luevano, Carrie
2005-04-21
The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for (1)H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[beta-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) and Tyr-c[beta-Asp-His-DNal(1')-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a gamma-turn secondary structure involving the DPhe(4) in the central position of the Tyr-c[beta-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology.
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Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori
2016-08-09
The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.
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Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd
2008-06-01
Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met(24). The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin.
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Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A; Katritch, Vsevolod; Coin, Irene
2017-01-01
The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors. DOI: http://dx.doi.org/10.7554/eLife.27711.001 PMID:28771403
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NASA Technical Reports Server (NTRS)
Kandasamy, S. B.; Williams, B. A.
1983-01-01
The intracerebroventricular administration of prototype nonpeptide opioid receptor (mu, kappa, and sigma) agonists, morphine, ketocyclazocine, and N-allyl normetazocine and an agonist at both kappa and sigma receptors, pentazocine, was found to induce hyperthermia in guinea pigs. The similar administration of peptide opioids like beta endorphin, methionine endkephalin, leucine endkephaline, and several of their synthetic analogues was also found to cause hyperthermia. Only the liver-like transport system of the three anion transport systems (iodide, hippurate, and liver-like) present in the choroid plexus was determined to be important to the central inactivation of beta-endorphin and two synthetic analogues. Prostaglandins and norepinephrine (NE) as well as cAMP were not involved in peptide and nonpeptide opioid-induced hyperthermia. Naloxone-sensitive receptors were found to be involved in the induction of hyperthermia by morphine and beta-endorphin, while hyperthermic responses to ketocyclazocine, N-allyl normetazocine, pentazocine, Met-enkephalin, Leu-enkephalin, and two of the synthetic analogues were not antagonized by nalozone. The lack of antagonism of naloxone on pyrogen, arachidonic acid, PGE2, dibutyryl cAMP, and NE-induced hyperthermia shows that endogenous opioid peptides are not likely to be central mediators of the hyperthermia induced by these agents.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Manning, B.W.; Cerniglia, C.E.; Federle, T.W.
The nitropolycyclic aromatic hydrocarbon 1-nitropyrene (1-NP) is an environmental pollutant, a potent bacterial and mammalian mutagen, and a carcinogen. The metabolism of 1-NP by the human intestinal microbiota was studied using a semicontinuous culture system that simulates the colonic lumen. (/sup 3/H)-1-Nitropyrene was metabolized by the intestinal microbiota to 1-aminopyrene (1-AP) and N-formyl-1-aminopyrene (FAP) as determined by high-performance liquid chromatography (HPLC) and mass spectrometry. Twenty-four hours after the addition of (/sup 3/H)-1-NP, the formylated compound and 1-AP accounted for 20 and 80% of the total metabolism respectively. This percentage increased to 66% for FAP after 24 h following 10 dmore » of chronic exposure to unlabeled 1-NP, suggesting metabolic adaptation to 1-NP by the microbiota. Both 1-AP and FAP have been shown to be nonmutagenic towards Salmonella typhimurium TA98, which indicates that the intestinal microflora may potentially detoxify 1-NP.« less
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Yang, Yoosoo; Kong, Byoungjae; Jung, Younghoon; Park, Joon-Bum; Oh, Jung-Mi; Hwang, Jaesung; Cho, Jae Youl; Kweon, Dae-Hyuk
2018-01-01
Vesicle-associated V-soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans -SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.
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Xu, Bo; Vasile, Silvana; Østergaard, Søren; Paulsson, Johan F; Pruner, Jasna; Åqvist, Johan; Wulff, Birgitte S; Gutiérrez-de-Terán, Hugo; Larhammar, Dan
2018-04-01
Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y 2 receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment ( 32 TRQRY 36 -amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y 2 receptor. The amidated C-terminus would be stabilized by polar interactions with Gln288 6.55 and Tyr219 5.39 , while Gln130 3.32 contributes to interactions with Q 34 in the peptide and T 32 is close to the tip of TM7 in the receptor. This leaves the core, α -helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y 2 system and can be used as a basis for optimization of Y 2 receptor agonists. Copyright © 2018 by The Author(s).
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Miller, Philip S; Barwell, James; Poyner, David R; Wigglesworth, Mark J; Garland, Stephen L; Donnelly, Dan
2010-01-01
The receptor for calcitonin gene-related peptide (CGRP) has been the target for the development of novel small molecule antagonists for the treatment of migraine. Two such antagonists, BIBN4096BS and MK-0974, have shown great promise in clinical trials and hence a deeper understanding of the mechanism of their interaction with the receptor is now required. The structure of the CGRP receptor is unusual since it is comprised of a hetero-oligomeric complex between the calcitonin receptor-like receptor (CRL) and an accessory protein (RAMP1). Both the CLR and RAMP1 components have extracellular domains which interact with each other and together form part of the peptide-binding site. It seems likely that the antagonist binding site will also be located on the extracellular domains and indeed Trp-74 of RAMP1 has been shown to form part of the binding site for BIBN4096BS. However, despite a chimeric study demonstrating the role of the N-terminal domain of CLR in antagonist binding, no specific residues have been identified. Here we carry out a mutagenic screen of the extreme N-terminal domain of CLR (residues 23-63) and identify a mutant, Met-42-Ala, which displays 48-fold lower affinity for BIBN4096BS and almost 900-fold lower affinity for MK-0974. In addition, we confirm that the Trp-74-Lys mutation at human RAMP1 reduces BIBN4096BS affinity by over 300-fold and show for the first time a similar effect for MK-0974 affinity. The data suggest that the non-peptide antagonists occupy a binding site close to the interface of the N-terminal domains of CLR and RAMP1. Copyright 2009 Elsevier Inc. All rights reserved.
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Reduction of Blood Pressure by AT1 Receptor Decoy Peptides.
Re, Richard N; Chen, Ben; Alam, Jawed; Cook, Julia L
2013-01-01
We previously identified the binding of the chaperone protein gamma-aminobutyric acid receptor-associated protein (GABARAP) to a sequence on the carboxy-terminus of the angiotensin II AT1 receptor (AT1R) and showed that this binding enhances AT1R trafficking to the cell surface as well as angiotensin signaling. In this study, we treated sodium-depleted mice with decoy peptides consisting either of a fusion of the cell-penetrating peptide penetratin and the GABARAP/AT1R binding sequence or penetratin fused to a mutated AT1R sequence. We used telemetry to measure blood pressure. Systolic and diastolic pressure fell during the 24 hours following decoy peptide injection but not after control peptide injection. Active cell-penetrating decoy peptide decreased 24-hour average systolic blood pressure from 129.8 ± 4.7 mmHg to 125.0 ± 6.0 mmHg (mean ± standard deviation). Diastolic blood pressure fell from 99.0 ± 7.1 mmHg to 95.0 ± 9.2 mmHg (n=5). Administration of the control peptide raised systolic blood pressure from 128.7 ± 1.3 mmHg to 131.7 ± 2.9 mmHg and diastolic pressure from 93.9 ± 4.5 mmHg to 95.9 ± 4.2 mmHg (n=5). The decreases in both systolic and diastolic blood pressure after active peptide administration were statistically significant compared to control peptide administration (P<0.05, two-tailed Wilcoxon rank-sum test). These results indicate the physiological and potentially therapeutic relevance of inhibitors of GABARAP/AT1R binding.
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Singh, Anamika; Wilczynski, Andrzej; Holder, Jerry R.; Witek, Rachel M.; Dirain, Marvin L.; Xiang, Zhimin; Edison, Arthur S.; Haskell-Luevano, Carrie
2011-01-01
Using a solid-phase synthetic approach, a bioactive reverse turn heterocyclic was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R-MC5R) are G-protein coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nM potency. Biophysical structural analysis [2D 1H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families as compared to the reference peptide and result in a novel template for further structure-function studies. PMID:21306168
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Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G
1989-07-01
Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin.
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Erspamer, V; Melchiorri, P; Falconieri-Erspamer, G; Negri, L; Corsi, R; Severini, C; Barra, D; Simmaco, M; Kreil, G
1989-01-01
Deltorphins are endogenous linear heptapeptides, isolated from skin extracts of frogs belonging to the genus Phyllomedusa, that have a higher affinity and selectivity for delta opioid binding sites than any other natural compound known. Two deltorphins with the sequence Tyr-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH2 have been isolated from skin extracts of Phyllomedusa bicolor. The alanine in position 2 is in the D configuration. These peptides, [D-Ala2]deltorphins I and II, show an even higher affinity for delta receptors than the previously characterized deltorphin, which contains D-methionine as the second amino acid. These peptides show some similarity to another constituent of Phyllomedusa skin, dermorphin, which is highly selective for mu-opioid receptors. These peptides all have the N-terminal sequence Tyr-D-Xaa-Phe, where D-Xaa is either D-alanine or D-methionine. While this structure seems to be capable of activating both mu and delta opioid receptors, differences in the C-terminal regions of these peptides are probably responsible for the observed high receptor selectivity of dermorphin and deltorphin. PMID:2544892
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Jain, Rinku; Hao, Bing; Liu, Ren-Peng; Chan, Michael K
2005-04-06
E. coli peptide deformylase (PDF) catalyzes the deformylation of nascent polypeptides generated during protein synthesis. While PDF was originally thought to be a zinc enzyme, subsequent studies revealed that the active site metal is iron. In an attempt to understand this unusual metal preference, high-resolution structures of Fe-, Co-, and Zn-PDF were determined in complex with its deformylation product, formate. In all three structures, the formate ion binds the metal and forms hydrogen-bonding interactions with the backbone nitrogen of Leu91, the amide side chain of Gln50, and the carboxylate side chain of Glu133. One key difference, however, is how the formate binds the metal. In Fe-PDF and Co-PDF, formate binds in a bidentate fashion, while in Zn-PDF, it binds in a monodentate fashion. Importantly, these structural results provide the first clues into the origins of PDF's metal-dependent activity differences. On the basis of these structures, we propose that the basis for the higher activity of Fe-PDF stems from the better ability of iron to bind and activate the tetrahedral transition state required for cleavage of the N-terminal formyl group.
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Stalder, Anna K; Lott, Dominik; Strasser, Daniel S; Cruz, Hans G; Krause, Andreas; Groenen, Peter M A; Dingemanse, Jasper
2017-03-01
The main objectives of these two phase I studies were to investigate safety and tolerability as well as the pharmacokinetic/pharmacodynamic profile of the novel potent and selective formyl peptide receptor type 2 (FPR2)/Lipoxin A 4 receptor (ALX) agonist ACT-389949. A challenge model was used to assess the drug's anti-inflammatory potential, with the aim of selecting a dosing regimen for future patient studies. Two double-blind, randomized phase I studies investigated the safety, tolerability, pharmacokinetics and pharmacodynamics of ACT-389949 at different doses and dosing regimens. Drug exposure was correlated with target engagement markers such as receptor internalization and cytokine measurements. The effect of FPR2/ALX agonism on neutrophil migration was studied in a lipopolysaccharide (LPS) inhalation model. ACT-389949 was well tolerated. Maximum concentrations were reached around 2 h after dosing, with a mean terminal half-life of 29.3 h [95% confidence interval (CI) 25.5, 33.7]. After multiple-dose administration, exposure increased by 111% (95% CI 89, 136), indicating drug accumulation. Administration of ACT-389949 resulted in a dose-dependent, long-lasting internalization of FPR2/ALX into leukocytes. Pro- and anti-inflammatory cytokines were dose-dependently but transiently upregulated only after the first dose. No pharmacological effect on neutrophil count was observed in the LPS challenge test performed at steady state. FPR2/ALX agonism with ACT-389949 was shown to be safe and well tolerated in healthy subjects. Receptor internalization and downstream mediators pointed towards a desensitization of the system, which may explain the lack of effect on neutrophil recruitment in the LPS challenge model. © 2016 The British Pharmacological Society.
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NASA Astrophysics Data System (ADS)
Lee, ImShik; Marchant, Roger E.
2001-10-01
The peptide sequence arginine-glycine-aspartate (RGD) found in fibrinogen, von Willebrand factor, fibronectin, and vitronectin, plays a critical role in platelet adhesion and thrombus formation, when bound to the platelet α IIbβ 3 integrin receptor. Using atomic force microscopy (AFM), we have measured the debonding interaction between an RGD peptide-modified AFM probe tip and a human platelet surface from pN to nN levels of force. The peptide sequence, GSSSGRGDSPA, which contains the biologically active RGDSP sequence with a hydrophilic spacer sequence (GSSSG), was covalently coupled to AFM probe tips. Direct measurements on the debonding force for the RGD ligand - α IIbβ 3 platelet receptor system were carried out in Tyrode buffer at room temperature. Our results show three distinct distributions of debonding forces at a loading rate of 12 nN/s, from which we estimate the debonding force for the single ligand-receptor to be ˜93 pN. The results also show evidence for considerable extension in the flexible sample surface during the debonding process, and a linear correlation between the debonding force and the logarithm of the rate of loading. From our analysis, the zero kinetic off-rate Koff(0), the single molecular binding energy Eb, and the transition state xB, assuming rigid binding, were extracted from the data, and estimated to be 22.6 s -1, -2.64×10 -20 J and 0.1 nm, respectively.
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Kleczkowska, Patrycja; Bojnik, Engin; Leśniak, Anna; Kosson, Piotr; Van den Eynde, Isabelle; Ballet, Steven; Benyhe, Sandor; Tourwé, Dirk; Lipkowski, Andrzej W
2013-01-01
Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity. The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.
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Engineered Peptides for Applications in Cancer-Targeted Drug Delivery and Tumor Detection.
Soudy, R; Byeon, N; Raghuwanshi, Y; Ahmed, S; Lavasanifar, A; Kaur, K
2017-01-01
Cancer-targeting peptides as ligands for targeted delivery of anticancer drugs or drug carriers have the potential to significantly enhance the selectivity and the therapeutic benefit of current chemotherapeutic agents. Identification of tumor-specific biomarkers like integrins, aminopeptidase N, and epidermal growth factor receptor as well as the popularity of phage display techniques along with synthetic combinatorial methods used for peptide design and structure optimization have fueled the advancement and application of peptide ligands for targeted drug delivery and tumor detection in cancer treatment, detection and guided therapy. Although considerable preclinical data have shown remarkable success in the use of tumor targeting peptides, peptides generally suffer from poor pharmacokinetics, enzymatic instability, and weak receptor affinity, and they need further structural modification before successful translation to clinics is possible. The current review gives an overview of the different engineering strategies that have been developed for peptide structure optimization to confer selectivity and stability. We also provide an update on the methods used for peptide ligand identification, and peptide- receptor interactions. Additionally, some applications for the use of peptides in targeted delivery of chemotherapeutics and diagnostics over the past 5 years are summarized. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Glucagon Like Peptide-1 Receptor Expression in the Human Thyroid Gland
Gier, Belinda; Butler, Peter C.; Lai, Chi K.; Kirakossian, David; DeNicola, Matthew M.
2012-01-01
Background: Glucagon like peptide-1 (GLP-1) mimetic therapy induces medullary thyroid neoplasia in rodents. We sought to establish whether C cells in human medullary thyroid carcinoma, C cell hyperplasia, and normal human thyroid express the GLP-1 receptor. Methods: Thyroid tissue samples with medullary thyroid carcinoma (n = 12), C cell hyperplasia (n = 9), papillary thyroid carcinoma (n = 17), and normal human thyroid (n = 15) were evaluated by immunofluorescence for expression of calcitonin and GLP-1 receptors. Results: Coincident immunoreactivity for calcitonin and GLP-1 receptor was consistently observed in both medullary thyroid carcinoma and C cell hyperplasia. GLP-1 receptor immunoreactivity was also detected in 18% of papillary thyroid carcinoma (three of 17 cases). Within normal human thyroid tissue, GLP-1 receptor immunoreactivity was found in five of 15 of the examined cases in about 35% of the total C cells assessed. Conclusions: In humans, neoplastic and hyperplastic lesions of thyroid C cells express the GLP-1 receptor. GLP-1 receptor expression is detected in 18% papillary thyroid carcinomas and in C cells in 33% of control thyroid lobes. The consequence of long-term pharmacologically increased GLP-1 signaling on these GLP-1 receptor-expressing cells in the thyroid gland in humans remains unknown, but appropriately powered prospective studies to exclude an increase in medullary or papillary carcinomas of the thyroid are warranted. PMID:22031513
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Giuliani, S; Patacchini, R; Barbanti, G; Turini, D; Rovero, P; Quartara, L; Giachetti, A; Maggi, C A
1993-11-01
The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lentz, T.L.
1991-11-12
Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similarmore » segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.« less
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Xiao, Li; Ding, Mengmeng; Zhang, Yi; Chordia, Mahendra; Pan, Dongfeng; Shimer, Adam; Shen, Francis; Glover, David; Jin, Li; Li, Xudong
2017-10-01
Inflammation plays a key role in the progression of intervertebral disk (IVD) herniation and associated low back pain. However, real-time spatial diagnosis of inflammation associated with acute disk herniation has not been investigated. We sought to detect local neutrophil and macrophage infiltration near disk herniation via the formyl peptide receptor 1 (FPR1)-mediated molecular imaging in a disk puncture mouse model to elucidate pathophysiological process of disk herniation. Disk herniation was induced in mouse with an established needle puncture procedure. Degenerative change of disk and infiltration of neutrophils and macrophages were detected with Safranin-O, hematoxylin and eosin (H&E), and immunohistochemical staining after injury. FPR1-specific imaging probes cFLFLF-PEG-Cy7 and [ 99m Tc]HYNIC-PEG-cFLFLF were administered systemically to sham and disk injury mice. Leukocyte infiltration was tracked by in vivo near-infrared fluorescence (NIRF) and single-photon emission tomography (SPECT) imaging. The peptide-receptor binding specificity was further investigated with FPR1 -/- mice via ex vivo NIRF scan and in vitro binding assays. Safranin-O staining exhibited disorganized disk structure and loss of proteoglycan after puncture. Massive inflammatory cells were observed in the anterior region of punctured annulus in the injury group. The majority of neutrophils were detected at 1 through 3 days, while infiltration of macrophages appeared the most at 7 days after injury. NIRF and SPECT images revealed preferential accumulation of cFLFLF probes in herniation site in wild-type mice but not in FPR1 -/- mice. Binding of the cFLFLF peptide to FPR1 was also observed in RAW 267.4 cells and macrophages isolated from wild-type mice, whereas much less signal was observed in macrophages from FPR1 -/- mice. The presence of macrophage infiltration was also detected in human-herniated disk samples by immunohistochemistry. For the first time, leukocyte infiltration around acute disk herniation site was detected directly and non-invasively in a timely fashion using FPR1-targeted molecular imaging modalities. Such functional imaging of disk herniation via infiltrated leukocytes would advance the understanding of etiology and facilitate drug delivery and treatment monitoring of disk herniation.
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Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N
2000-02-18
Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione¿, a PI3-kinase inhibitor, attenuated only calcitonin gene-related peptide-induced ERK and not P38 MAPK activation. Thus, these data suggest that activation of ERK by calcitonin gene-related peptide involves a H89-sensitive protein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene-related peptide involves only the H89 sensitive pathway and is independent of PI3 kinase. This also suggests that although both ERK and P38 can be activated by protein kinase A, the distal signaling components to protein kinase A in the activation of these two kinases (ERK and P38) are different.
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Zielińska, Joanna; Stadnik, Jacek; Bierczyńska-Krzysik, Anna; Stadnik, Dorota
2018-05-16
Isolation and identification of unknown impurities of recombinant insulin lispro (produced at IBA) formed during accelerated stability testing of pharmaceutical solutions. For comparative purposes also commercially available formulations of recombinant human insulin (Humulin S®; Lilly), recombinant insulin lispro (Humalog®; Lilly), recombinant insulin aspart (NovoRapid® Penfill®; Novo Nordisk), recombinant insulin detemir (Levemir®; Novo Nordisk) and recombinant insulin glargine (Lantus®; Sanofi-Aventis) were analyzed. The impurities of insulin analogs were isolated by RP-HPLC and identified with peptide mass fingerprinting using MALDI-TOF/TOF mass spectrometry. The identified derivatives were N-terminally truncated insulin analog impurities of decreased molecular mass of 119, 147 and 377 Da related to the original protein. The modifications resulting in a mass decrease were detected at the N-terminus of B chains of insulin lispro, insulin aspart, human insulin, insulin glargine, insulin detemir in all tested formulations. To our knowledge it is the first time that these impurities are reported. The following derivatives formed by truncation of the B chain in insulin analogs were identified in pharmaceutical formulations: desPhe B1 -N-formyl-Val B2 derivative, desPhe B1 derivative, pyroGlu B4 derivative.
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Time averaging of NMR chemical shifts in the MLF peptide in the solid state.
De Gortari, Itzam; Portella, Guillem; Salvatella, Xavier; Bajaj, Vikram S; van der Wel, Patrick C A; Yates, Jonathan R; Segall, Matthew D; Pickard, Chris J; Payne, Mike C; Vendruscolo, Michele
2010-05-05
Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density functional theory (DFT). We measured the chemical shifts of the N-formyl-L-methionyl-L-leucyl-L-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the (13)C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which accounts for the periodic nature of the crystal structure, obtaining an overall accuracy of 4.2 ppm. In order to understand the origin of the difference between experimental and calculated chemical shifts, we carried out first-principles molecular dynamics simulations to characterize the molecular motion of the MLF peptide on the picosecond time scale. We found that (13)C chemical shifts experience very rapid fluctuations of more than 20 ppm that are averaged out over less than 200 fs. Taking account of these fluctuations in the calculation of the chemical shifts resulted in an accuracy of 3.3 ppm. To investigate the effects of averaging over longer time scales we sampled the rotameric states populated by the MLF peptides in the solid state by performing a total of 5 micros classical molecular dynamics simulations. By averaging the chemical shifts over these rotameric states, we increased the accuracy of the chemical shift calculations to 3.0 ppm, with less than 1 ppm error in 10 out of 22 cases. These results suggests that better DFT-based predictions of chemical shifts of peptides and proteins will be achieved by developing improved computational strategies capable of taking into account the averaging process up to the millisecond time scale on which the chemical shift measurements report.
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The Hallucinogen N,N-Dimethyltryptamine (DMT) Is an Endogenous Sigma-1 Receptor Regulator
Fontanilla, Dominique; Johannessen, Molly; Hajipour, Abdol R.; Cozzi, Nicholas V.; Jackson, Meyer B.; Ruoho, Arnold E.
2010-01-01
The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor. PMID:19213917
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The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator.
Fontanilla, Dominique; Johannessen, Molly; Hajipour, Abdol R; Cozzi, Nicholas V; Jackson, Meyer B; Ruoho, Arnold E
2009-02-13
The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.
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Ye, Weihua; Spånning, Erika; Unnerståle, Sofia; Gotthold, David; Glaser, Elzbieta; Mäler, Lena
2012-10-01
Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide. © 2012 The Authors Journal compilation © 2012 FEBS.
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Ali, Ahmed M; Taylor, Scott D
2010-04-01
Tyrosine sulfation is a post translational modification that occurs on integral membrane and secreted proteins, and is required for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmoc-based solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group (FmocTyr(SO(3)DCV)OH, 1). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here we demonstrate that sTyr peptides containing Met or Trp residues can be prepared using our sulfate-protecting group strategy by preparing peptides corresponding to residues 1-20 of chemokine receptor CXCR6 and 8-42 of chemokine receptor DARC. Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp, by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for removing the DCV group and superiour to our original conditions using H(2), ammonium formate, Pd/C. The presence of Met was found not to interfere with the removal of the DCV group. The use of pseudoproline dipeptides and N-backbone protection with the 2,4-dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation during the synthesis of these peptides. We also report an alternative and more cost effective synthesis of amino acid 1. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.
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Synthesis and evaluation of novel multimeric neurotensin(8-13) analogs.
Hultsch, Christina; Pawelke, Beate; Bergmann, Ralf; Wuest, Frank
2006-09-01
Neurotensin(8-13) is a hexapeptide with subnanomolar affinity to the neurotensin receptor 1 which is expressed with high incidence in several human tumor entities. Thus, radiolabeled neurotensin(8-13) might be used for tumor targeting. However, its application is limited by insufficient metabolic stability. The present study aims at improving metabolic stability by the synthesis of multimeric neurotensin(8-13) derivatives rather than commonly employed chemical modifications of the peptide itself. Thus, different dimeric and tetrameric peptides carrying C- or N-terminal attached neurotensin(8-13) moieties have been synthesized and their binding affinity toward the neurotensin receptor has been determined. The results demonstrate that branched compounds containing neurotensin(8-13) attached via its C-terminus only show low receptor affinities, whilst derivatives with neurotensin(8-13) attached via the N-terminus show IC50 values in the nanomolar range. Moreover, within the multimeric neurotensin(8-13) derivatives with neurotensin(8-13) attached via the N-terminus an increasing number of branching units lead to higher binding affinities toward the neurotensin receptor.
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Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels
2006-01-01
The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566
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Cai, Minying; Marelli, Udaya Kiran; Mertz, Blake; Beck, Johannes G; Opperer, Florian; Rechenmacher, Florian; Kessler, Horst; Hruby, Victor J
2017-08-15
Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His 6 -d-Nal(2') 7 -NMe-Arg 8 -Trp 9 -Lys]-NH 2 (15) and Ac-Nle-c[Asp-His 6 -d-Nal(2') 7 -NMe-Arg 8 -NMe-Trp 9 -NMe-Lys]-NH 2 (17). It is known that the pharmacophore (His 6 -DNal 7 -Arg 8 -Trp 9 ) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal 7 -Arg 8 . The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp 9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg 8 and Trp 9 side chains are involved in a majority of the interactions with the receptor. While Arg 8 forms polar contacts with D154 and D158 of hMC3R, Trp 9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp 9 -hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.
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Zaki, Mohamed A; Nanayakkara, N P Dhammika; Hetta, Mona H; Jacob, Melissa R; Khan, Shabana I; Mohammed, Rabab; Ibrahim, Mohamed A; Samoylenko, Volodymyr; Coleman, Christina; Fronczek, Frank R; Ferreira, Daneel; Muhammad, Ilias
2016-09-23
Two new flavonoids, rac-6-formyl-5,7-dihydroxyflavanone (1) and 2',6'-dihydroxy-4'-methoxy-3'-methylchalcone (2), together with five known derivatives, rac-8-formyl-5,7-dihydroxyflavanone (3), 4',6'-dihydroxy-2'-methoxy-3'-methyldihydrochalcone (4), rac-7-hydroxy-5-methoxy-6-methylflavanone (5), 3'-formyl-2',4',6'-trihydroxy-5'-methyldihydrochalcone (6), and 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7), were isolated from the leaves of Eugenia rigida. The individual (S)- and (R)-enantiomers of 1 and 3, together with the corresponding formylated flavones 8 (6-formyl-5,7-dihydroxyflavone) and 9 (8-formyl-5,7-dihydroxyflavone), as well as 2',4',6'-trihydroxychalcone (10), 3'-formyl-2',4',6'-trihydroxychalcone (11), and the corresponding 3'-formyl-2',4',6'-trihydroxydihydrochalcone (7) and 2',4',6'-trihydroxydihydrochalcone (12), were synthesized. The structures of the isolated and synthetic compounds were established via NMR, HRESIMS, and electronic circular dichroism data. In addition, the structures of 3, 5, and 8 were confirmed by single-crystal X-ray diffraction crystallography. The isolated and synthetic flavonoids were evaluated for their antimicrobial and cytotoxic activities against a panel of microorganisms and solid tumor cell lines.
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Couture, Leah A; Piao, Wenji; Ru, Lisa W; Vogel, Stefanie N; Toshchakov, Vladimir Y
2012-07-13
Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal) is an adapter protein that facilitates recruitment of MyD88 to TLR4 and TLR2 signaling complexes. We previously generated a library of cell-permeating TLR4 TIR-derived decoy peptides fused to the translocating segment of the Drosophila Antennapedia homeodomain and examined each peptide for the ability to inhibit TLR4 signaling (Toshchakov, V. Y., Szmacinski, H., Couture, L. A., Lakowicz, J. R., and Vogel, S. N. (2011) J. Immunol. 186, 4819-4827). We have now expanded this study to test TIRAP decoy peptides. Five TIRAP peptides, TR3 (for TIRAP region 3), TR5, TR6, TR9, and TR11, inhibited LPS-induced cytokine mRNA expression and MAPK activation. Inhibition was confirmed at the protein level; select peptides abolished the LPS-induced cytokine production measured in cell culture 24 h after a single treatment. Two of the TLR4 inhibitory peptides, TR3 and TR6, also inhibited cytokine production induced by a TLR2/TLR1 agonist, S-(2,3-bis(palmitoyloxy)-(2R,2S)-propyl)-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH; however, a higher peptide concentration was required to achieve comparable inhibition of TLR2 versus TLR4 signaling. Two TLR4 inhibitory peptides, TR5 and TR6, were examined for the ability to inhibit TLR4-driven cytokine induction in mice. Pretreatment with either peptide significantly reduced circulating TNF-α and IL-6 in mice following LPS injection. This study has identified novel TLR inhibitory peptides that block cellular signaling at low micromolar concentrations in vitro and in vivo. Comparison of TLR4 inhibition by TLR4 and TIRAP TIR-derived peptides supports the view that structurally diverse regions mediate functional interactions of TIR domains.
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Nagai-Okatani, Chiaki; Nagasawa, Hiromichi
2016-01-01
Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR)-A24 as an ion transport peptide-like (ITPL) receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs), we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1–5). In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling. PMID:27248837
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Dam, T.V.; Takeda, Y.; Krause, J.E.
1990-01-01
The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide (gamma-PPT-(72-92)-NH2), a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeledmore » Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.« less
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Saptal, Vitthal B; Bhanage, Bhalchandra M
2016-08-09
In this report, the activity of N-heterocyclic olefins (NHOs) as a newly emerging class of organocatalyst is investigated for the chemical fixation of carbon dioxide through reactions with aziridines to form oxazolidinones and the N-formylation of amines with polymethylhydrosiloxane (PMHS) or 9-borabicyclo[3.3.1]nonane (9-BBN) as the reducing agent under mild conditions. The exocyclic carbon atoms of NHOs are highly nucleophilic owing to the electron-donating ability of the two nitrogen atoms. This high nucleophilicity of the NHOs activates CO2 molecules to form zwitterionic NHO-carboxylate (NHO-CO2 ) adducts, which are active in formylation reactions as well as the carboxylation of aziridines to oxazolidinones. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Hardeland, Rüdiger; Pandi-Perumal, S R; Cardinali, Daniel P
2006-03-01
Melatonin, originally discovered as a hormone of the pineal gland, is produced by bacteria, protozoa, plants, fungi, invertebrates, and various extrapineal sites of vertebrates, including gut, skin, Harderian gland, and leukocytes. Biosynthetic pathways seem to be identical. Actions are pleiotropic, mediated by membrane and nuclear receptors, other binding sites or chemical interactions. Melatonin regulates the sleep/wake cycle, other circadian and seasonal rhythms, and acts as an immunostimulator and cytoprotective agent. Circulating melatonin is mostly 6-hydroxylated by hepatic P450 monooxygenases and excreted as 6-sulfatoxymelatonin. Pyrrole-ring cleavage is of higher importance in other tissues, especially the brain. The product, N1-acetyl-N2-formyl-5-methoxykynuramine, is formed by enzymatic, pseudoenzymatic, photocatalytic, and numerous free-radical reactions. Additional metabolites result from hydroxylation and nitrosation. The secondary metabolite, N1-acetyl-5-methoxykynuramine, supports mitochondrial function and downregulates cyclooxygenase 2. Antioxidative protection, safeguarding of mitochondrial electron flux, and in particular, neuroprotection, have been demonstrated in many experimental systems. Findings are encouraging to use melatonin as a sleep promoter and in preventing progression of neurodegenerative diseases.
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Monastyrnaya, Margarita; Peigneur, Steve; Zelepuga, Elena; Sintsova, Oksana; Gladkikh, Irina; Leychenko, Elena; Isaeva, Marina; Tytgat, Jan; Kozlovskaya, Emma
2016-01-01
Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1–APHC3 from H. crispa, and clusters with the peptides from so named “analgesic cluster” of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10−7 and 7.0 × 10−7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21–TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides. PMID:27983679
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Monastyrnaya, Margarita; Peigneur, Steve; Zelepuga, Elena; Sintsova, Oksana; Gladkikh, Irina; Leychenko, Elena; Isaeva, Marina; Tytgat, Jan; Kozlovskaya, Emma
2016-12-15
Sea anemone venoms comprise multifarious peptides modulating biological targets such as ion channels or receptors. The sequence of a new Kunitz-type peptide, HCRG21, belonging to the Heteractis crispa RG (HCRG) peptide subfamily was deduced on the basis of the gene sequence obtained from the Heteractis crispa cDNA. HCRG21 shares high structural homology with Kunitz-type peptides APHC1-APHC3 from H. crispa , and clusters with the peptides from so named "analgesic cluster" of the HCGS peptide subfamily but forms a separate branch on the NJ-phylogenetic tree. Three unique point substitutions at the N-terminus of the molecule, Arg1, Gly2, and Ser5, distinguish HCRG21 from other peptides of this cluster. The trypsin inhibitory activity of recombinant HCRG21 (rHCRG21) was comparable with the activity of peptides from the same cluster. Inhibition constants for trypsin and α-chymotrypsin were 1.0 × 10 -7 and 7.0 × 10 -7 M, respectively. Electrophysiological experiments revealed that rHCRG21 inhibits 95% of the capsaicin-induced current through transient receptor potential family member vanilloid 1 (TRPV1) and has a half-maximal inhibitory concentration of 6.9 ± 0.4 μM. Moreover, rHCRG21 is the first full peptide TRPV1 inhibitor, although displaying lower affinity for its receptor in comparison with other known ligands. Macromolecular docking and full atom Molecular Dynamics (MD) simulations of the rHCRG21-TRPV1 complex allow hypothesizing the existence of two feasible, intra- and extracellular, molecular mechanisms of blocking. These data provide valuable insights in the structural and functional relationships and pharmacological potential of bifunctional Kunitz-type peptides.
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Zhao, Hongqiong; Matsuda, Seinosuke; Thanthan, Sint; Yannaing, Swe; Kuwayama, Hideto
2012-10-01
This study was designed to determine the effects of bombesin-like peptides (BLPs) on the secretion of growth hormone (GH) and to characterize the receptor subtypes mediating these effects in cattle. Four experiments were conducted: (1) six steers were randomly assigned to receive intravenous (IV) bolus injections of 0, 0.2, 1.0, 12.5 and 50.0 μg/kg neuromedin C (NMC); (2) seven pre-weaned calves were IV injected with 1.0 μg/kg NMC; (3) six steers were IV injected with 2.5μg/kg bovine gastrin-releasing peptide (GRP), 1.0 μg/kg NMC combined with 20.0 μg/kg [d-Lys(3)]-GHRP-6 (an antagonist for the GH secretagogue receptor type 1a [GHS-R1a]), 1.0 μg/kg NMC combined with 20.0 μg/kg N-acetyl-GRP(20-26)-OCH(2)CH(3) (N-GRP-EE, an antagonist for the GRP receptor), 20.0 μg/kg N-GRP-EE alone, 1.0 μg/kg neuromedin B (NMB); and (4) four rats were IV injected 1.0 μg/kg NMC. A serial blood sample was collected before and after injection. Plasma GH levels dose-dependently increased at 5 min after NMC injection and the minimal effective dose was 1.0 μg/kg. Plasma GH level was elevated by GRP, but not by NMB. The NMC-induced elevation of GH was completely blocked by N-GRP-EE. The administration of NMC elevated GH level in pre-weaned calves but not in rats. Ghrelin level was unaffected by any treatments; and [d-Lys(3)]-GHRP-6 did not block the NMC-induced elevation of GH. The results indicate BLP-induced elevation of GH levels is mediated by the GRP receptor but not through a ghrelin/GHS-R1a pathway in cattle. Copyright © 2012 Elsevier Inc. All rights reserved.
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Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo)
Bird, Mark F.; Malfacini, Davide; Vezzi, Vanessa; Molinari, Paola; Micheli, Laura; Mannelli, Lorenzo Di Cesare; Ghelardini, Carla; Guerrini, Remo; Calò, Girolamo; Lambert, David G.
2016-01-01
Introduction Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. Methods We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. Results DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. Conclusion Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties. PMID:27272042
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Processing of carcinoembryonic antigen by Kupffer cells: recognition of a penta-peptide sequence.
Gangopadhyay, A; Thomas, P
1996-10-01
Carcinoembryonic antigen (CEA) binds to an 80-kDa cell surface receptor on Kupffer cells via the peptide sequence PELPK (residues 108-112) located at the hinge region between the N and Al immunoglobulin-like domains. This study is aimed at analyzing the specificity of the peptide binding, determining biodistribution of 80-kDa receptor, and processing of CEA by this receptor. We synthesized a number of bovine serum albumin (BSA) derivatives carrying PELPK and related sequences. A series of peptides (YPELPK, YPDLPK, YPDLPR, and YPELGK) were conjugated to bovine serum albumin using N-hydroxysuccinimidyl-4-azidobenzoate. When 125I peptide conjugates, CEA, and BSA were injected intravenously into rats CEA and the PELPK-albumin conjugate were cleared rapidly. The other peptide conjugates and BSA cleared at a much slower rate. Activity of 125I-labeled CEA and PELPK-albumin conjugate per gram of tissue was highest for the liver and spleen. Clearance of 125I-CEA was inhibited by the presence of higher concentrations of the PELPK-albumin conjugate. With isolated rat Kupffer cells, only CEA and the PELPK-albumin conjugate were bound and internalized in vitro and CEA binding was inhibited by higher concentrations of the PELPK-albumin conjugate. Similarly, binding of the PELPK-albumin conjugate was inhibited by the presence of unlabeled CEA. Use of a heterobifunctional cross linking agent demonstrated reaction of the PELPK-albumin with an 80-kDa protein on the Kupffer cell surface by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This semisynthetic ligand (PELPK-albumin) allows us to examine the function of the 80-kDa receptor without interference due to other properties of CEA including its ability to bind lectins and to cause homotypic aggregation of cells. The consequences of CEA binding to the 80-kDa receptor may have implications in the development of hepatic metastasis from colorectal cancer.
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Haynes, John M; Hill, Stephen J; Selbie, Lisa A
1997-01-01
The effects of peptide YY (PYY), neuropeptide Y (NPY) and structurally related peptides upon field stimulation-induced and phenylephrine-mediated contractile responses in the cauda epididymis of the guinea-pig were investigated.Preparations of cauda epididymis responded to field stimulation with contractions which were completely attenuated by both the neurotoxin, tetrodotoxin (500 nM), and also by the α-adrenoceptor antagonist, phentolamine (3 μM). PYY (n=7) and the truncated peptide analogue PYY(3–36) (n=5) inhibited field stimulation-induced contractions (pIC50+s.e.mean: 8.9±0.2 and 9.4±0.2, respectively). Pancreatic polypeptide (PP, up to 1 μM, n=6), NPY (up to 100 nM, n=6) and the NPY analogues [Leu31,Pro34]NPY (n=6) and NPY (13–36) (both up to 1 μM, n=5) had no significant effect.The NPY Y1 receptor antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N[(4-hydroxyphenyl)-methyl]-argininamide) at 750 nM (n=6) and 7.5 μM (n=6) did not affect the PYY-mediated inhibition of field stimulation-induced contractions (pIC50 8.9±0.3 and 9.0±0.3, respectively). In the presence of BIBP3226 (7.5 μM), NPY (n=6) inhibited field stimulation-induced contractions (pIC50 8.0±0.2).NPY, PYY and PYY(3–36) inhibited [3H]-noradrenaline release from preparations of epididymis (pIC50 values 7.9±0.7, 9.6±0.8 and 10.0±0.9, respectively, all n=6). The agonists PP and [Leu31,Pro34]PYY (both up to 100 nM) were without significant effect (both n=6).In preparations of cauda epididymis, stimulated with threshold concentrations of the α1-adrenoceptor agonist, phenylephrine (1 μM), both NPY (n=6) and PYY (n=7) elicited concentration-dependent increases in contractile force (with pEC50 values of 8.9±0.2 and 8.6±0.1, respectively). The effects of both NPY (n=6) and PYY (n=6) were antagonized by preincubation with BIBP3226 (75 nM; apparent pKB±s.e. values 8.3±1.0 and 8.2±0.6, respectively). The peptide analogues NPY(13–36) (n=5), PYY (3–36) (n=7) and [Leu31,Pro34]NPY (n=5) did not significantly augment responses to threshold concentrations of phenylephrine.These results are consistent with the proposal that distinct NPY receptors mediate the (prejunctional) inhibition of field stimulation-induced contractions and the (postjunctional) potentiation of responses to phenylephrine in the cauda epididymis of the guinea-pig. The rank order of agonist potency (NPY⩾PYY≫NPY(13–36), [Leu31,Pro34]NPY and PYY(3–36) and the high potency of BIBP3226 indicate that the postjunctional receptor may be Y1-like. The rank orders of agonist potency in inhibiting field stimulation-induced contractile responses and [3H]-noradrenaline release (PYY(3–36)⩾PYY> NPY≫PP, NPY(13–36), [Leu31,Pro34]NPY and PYY(3–36)⩾PYY>NPY≫;PP,[Leu31,Pro34]PYY, respectively) are consistent with the action of these peptides at a PYY-preferring receptor subtype, which may be distinct from the presently characterized NPY receptor subtypes. PMID:9421306
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Mining proteomic data to expose protein modifications in Methanosarcina mazei strain Gö1
Leon, Deborah R.; Ytterberg, A. Jimmy; Boontheung, Pinmanee; ...
2015-03-05
Proteomic tools identify constituents of complex mixtures, often delivering long lists of identified proteins. The high-throughput methods excel at matching tandem mass spectrometry data to spectra predicted from sequence databases. Unassigned mass spectra are ignored, but could, in principle, provide valuable information on unanticipated modifications and improve protein annotations while consuming limited quantities of material. Strategies to “mine” information from these discards are presented, along with discussion of features that, when present, provide strong support for modifications. In this study we mined LC-MS/MS datasets of proteolytically-digested concanavalin A pull down fractions from Methanosarcina mazei Gö1 cell lysates. Analyses identified 154more » proteins. Many of the observed proteins displayed post-translationally modified forms, including O-formylated and methyl-esterified segments that appear biologically relevant (i.e., not artifacts of sample handling). Interesting cleavages and modifications (e.g., S-cyanylation and trimethylation) were observed near catalytic sites of methanogenesis enzymes. Of 31 Methanosarcina protein N-termini recovered by concanavalin A binding or from a previous study, only M. mazei S-layer protein MM1976 and its M. acetivorans C2A orthologue, MA0829, underwent signal peptide excision. Experimental results contrast with predictions from algorithms SignalP 3.0 and Exprot, which were found to over-predict the presence of signal peptides. Proteins MM0002, MM0716, MM1364, and MM1976 were found to be glycosylated, and employing chromatography tailored specifically for glycopeptides will likely reveal more. This study supplements limited, existing experimental datasets of mature archaeal N-termini, including presence or absence of signal peptides, translation initiation sites, and other processing. Methanosarcina surface and membrane proteins are richly modified.« less
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li, Lin; Pian, Yaya; Chen, Shaolong; Hao, Huaijie; Zheng, Yuling; Zhu, Li; Xu, Bin; Liu, Keke; Li, Min; Jiang, Hua; Jiang, Yongqiang
2016-01-01
Vascular leakage frequently occurs in patients with severe Staphylococcus aureus infection. However, the mechanism underlying S. aureus infection-induced vascular leakage remains unclear. Here, we identified the S. aureus virulence factor phenol-soluble modulin (PSM)α4 from the culture supernatant of strain USA300 as a stimulator of heparin-binding protein (HBP) release from polymorphonuclear neutrophils (PMNs) and demonstrated that PSMα4-induced HBP release from PMNs leads to vascular leakage. PSMα4 appeared less cytolytic than PSMα1–3 and was insensitive to lipoproteins; it significantly increased myeloperoxidase and elastase release from PMNs and cell surface CD63 expression in PMNs. PSMα4-induced HBP release required formyl peptide receptor 2 (FPR2) and phosphoinositide 3-kinase (PI3K) and depended on Ca2+ influx and cytoskeleton rearrangement. Thus, PSMα4 may stimulate HBP release by activating FPR2 and PI3K to initiate PMN degranulation. PSMα4-induced HBP release from PMNs increased endothelial cell monolayer permeability in vitro and induced vascular leakage in mice. This novel function of PSMα4 may contribute to the pathogenesis of S. aureus and may be a potential therapeutic target. PMID:27383625
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Riand, J; Baron, D; Nicolas, P; Benajiba, A; Teng, Y; Naim, M
1999-12-01
The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.
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NASA Astrophysics Data System (ADS)
Ogura, Kenji; Okamura, Hideyasu
2013-10-01
Growth factor receptor-bound protein 2 (Grb2) is a small adapter protein composed of a single SH2 domain flanked by two SH3 domains. The N-terminal SH3 (nSH3) domain of Grb2 binds a proline-rich region present in the guanine nucleotide releasing factor, son of sevenless (Sos). Using NMR relaxation dispersion and chemical shift analysis methods, we investigated the conformational change of the Sos-derived proline-rich peptide during the transition between the free and Grb2 nSH3-bound states. The chemical shift analysis revealed that the peptide does not present a fully random conformation but has a relatively rigid structure. The relaxation dispersion analysis detected conformational exchange of several residues of the peptide upon binding to Grb2 nSH3.
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Brown, Haley A; Vinogradov, Evgeny; Gilbert, Michel; Holden, Hazel M
2018-05-15
Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined. This article is protected by copyright. All rights reserved. © 2018 The Protein Society.
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Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.
Philips, M R; Pillinger, M H; Staud, R; Volker, C; Rosenfeld, M G; Weissmann, G; Stock, J B
1993-02-12
In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.
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NASA Astrophysics Data System (ADS)
Yu-Wai-Man, Cynthia; Tagalakis, Aristides D.; Manunta, Maria D.; Hart, Stephen L.; Khaw, Peng T.
2016-02-01
There is increasing evidence that the Myocardin-related transcription factor/Serum response factor (MRTF/SRF) pathway plays a key role in fibroblast activation and that knocking down MRTF can lead to reduced scarring and fibrosis. Here, we have developed a receptor-targeted liposome-peptide-siRNA nanoparticle as a non-viral delivery system for MRTF-B siRNA in conjunctival fibrosis. Using 50 nM siRNA, the MRTF-B gene was efficiently silenced by 76% and 72% with LYR and LER nanoparticles, respectively. The silencing efficiency was low when non-targeting peptides or siRNA alone or liposome-siRNA alone were used. LYR and LER nanoparticles also showed higher silencing efficiency than PEGylated LYR-P and LER-P nanoparticles. The nanoparticles were not cytotoxic using different liposomes, targeting peptides, and 50 nM siRNA. Three-dimensional fibroblast-populated collagen matrices were also used as a functional assay to measure contraction in vitro, and showed that MRTF-B LYR nanoparticles completely blocked matrix contraction after a single transfection treatment. In conclusion, this is the first study to develop and show that receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient and safe non-viral siRNA delivery system that could be used to prevent fibrosis after glaucoma filtration surgery and other contractile scarring conditions in the eye.
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Sekiguchi, Toshio; Kuwasako, Kenji; Ogasawara, Michio; Takahashi, Hiroki; Matsubara, Shin; Osugi, Tomohiro; Muramatsu, Ikunobu; Sasayama, Yuichi; Suzuki, Nobuo; Satake, Honoo
2016-01-29
The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.
Yoshino, K; Suzuki, N
1992-06-15
We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.
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Rosenberg, R L; Isaacson, J S; Tsien, R W
1989-01-01
These experiments provide a starting point for biochemical characterization of Ca channels from neuronal membranes, using omega-CgTX as a specific marker. The purification of the omega-CgTX receptors is far from complete. Each of the purification steps described results in only a two- to fivefold enrichment of the receptor proteins, and is accompanied by a loss of receptor concentration and stability, so the maximal specific activity achieved by a combination of these steps falls several orders of magnitude short of that of a large, homogeneous, active protein. Nevertheless, these studies have yielded important information about the omega-CgTX receptor. The Stokes' radius, determined from gel exclusion chromatography, is approximately 87 A, and the sedimentation coefficient, determined from sucrose gradient sedimentation, is approximately 19 S. These values are similar to those found for the DHP receptors solubilized in digitonin. We have also found that at least some of the omega-CgTX receptors have complex carbohydrate moieties that are recognized by WGA, together with evidence of heterogeneity of receptor glycosylation. Additionally, we have been able to use the solubilized, partially purified receptors in cross-linking experiments to tentatively identify the molecular weights of the omega-CgTX targets from rat brain. A large peptide of approximately 300 kDa, similar to that identified in photoaffinity studies, is very clearly labeled by the chemical incorporation of [125I]omega-CgTX into partially purified receptor preparations, but some ambiguity remains because of the faint labeling of peptides in the 120-170-kDa range. The approximately 300-kDa peptide is much larger than any single peptide component of DHP receptors from skeletal muscle, and it may be related to a molecular combination of the 170-kDa and 135-kDa subunits of the DHP receptor. Because [125I]omega-CgTX presumably labels both N- and L-type neuronal Ca channels, both channel types will probably be found in the purified preparations. Thus, at some time, it will be necessary to separate DHP-sensitive L-type channels from preparations of L- and N-type channels identified by omega-CgTX binding.
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Knoll-Köhler, Elisabeth; Stiebel, Juliane
2002-08-01
Amine hydrofluorides are widely used to prevent caries. As an acidulated gel, they were also studied for their applicability to reduce pathogenic bacteria in periodontal pockets. We assessed the toxicity of this pharmaceutical amine hydrofluoride preparation on human polymorphonuclear leukocytes in vitro by measuring Trypan blue exclusion and the generation of superoxide anions (O2) by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) after a 3-min contact with gel. Depending on the experimental conditions, gel dilutions up to 1.3 x 10(4) resulted in an increase in Trypan blue-colored cells and liberation of beta-glucuronidase. Dilutions between 3 x 10(4) and 1 x 10(5) augmented the fMLP-mediated O2- generation, which could be prevented by Ca2+ chelation with BAPTA-AM (1,2'-bis (o-aminophenoxyethane-N.N.N'.N'-tetraacetic acid tetra (acetoxymethyl) ester) and ethyleneglycoltetraacetic acid (EGTA) or inhibition of protein kinase C (PKC) with staurosporine and bisindolylmaleimide I. respectively. Compared with data published on the minimal inhibitory concentration for periodontal pathogenic bacteria, the cytotoxicity of amine hydrofluorides on eukaryotic cells is much greater and thus of consequence for their clinical use.
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Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.
2010-01-01
Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024
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Shamshirian, Danial; Erfani, Mostafa; Beiki, Davood; Fallahi, Babak; Shafiei, Mohammad
2015-10-01
Melanocortin-1 (MC1) receptor is an attractive melanoma-specific target which has been used for melanoma imaging and therapy. In this work, a new lactam bridge α-MSH analog was labeled with (99m)Tc via HYNIC and EDDA/tricine as coligands including gamma aminobutyric acid (GABA) as a three carbon chain spacer between HYNIC and the N-terminus of the cyclic peptide. Also, stability in human serum, receptor bound internalization, in vivo tumor uptake, and tissue biodistribution were thoroughly investigated. HYNIC-GABA-Nle-CycMSHhept was synthesized using a standard Fmoc strategy. Labeling was performed at 95 °C and analysis involved instant thin layer chromatography and high performance liquid chromatography methods. The receptor bound internalization rate was studied in MC1 receptor expressing B16/F10 cells. Biodistribution of radiopeptide was studied in nude mice bearing B16/F10 tumor. Labeling yield of >98 % (n = 3) was obtained corresponding to a specific activity of 81 MBq/nmol. Peptide conjugate showed efficient stability in the presence of human serum. The radioligand showed specific internalization into B16/F10 cells (12.45 ± 1.1 % at 4 h). In biodistribution studies, a receptor-specific uptake was observed in MC1 receptor-positive organs so that after 2 h the uptake in mouse tumor was 5.10 ± 0.08 % ID/g, while low accumulation in the kidney uptake was observed (4.58 ± 0.68 % ID/g at 2 h after injection). The obtained results show that the presented new designed labeled peptide conjugate may be a suitable candidate for diagnosis of malignant tumors.
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Murray, J S; Fois, S D S; Schountz, T; Ford, S R; Tawde, M D; Brown, J C; Siahaan, T J
2002-03-01
Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.
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α7nAchR/NMDAR coupling affects NMDAR function and object recognition.
Li, Shupeng; Nai, Qiang; Lipina, Tatiana V; Roder, John C; Liu, Fang
2013-12-20
The α7 nicotinic acetylcholine receptor (nAchR) and NMDA glutamate receptor (NMDAR) are both ligand-gated ion channels permeable to Ca2+ and Na+. Previous studies have demonstrated functional modulation of NMDARs by nAchRs, although the molecular mechanism remains largely unknown. We have previously reported that α7nAchR forms a protein complex with the NMDAR through a protein-protein interaction. We also developed an interfering peptide that is able to disrupt the α7nAchR-NMDAR complex and blocks cue-induced reinstatement of nicotine-seeking in rat models of relapse. In the present study, we investigated whether the α7nAchR-NMDAR interaction is responsible for the functional modulation of NMDAR by α7nAchR using both electrophysiological and behavioral tests. We have found that activation of α7nAchR upregulates NMDAR-mediated whole cell currents and LTP of mEPSC in cultured hippocampal neurons, which can be abolished by the interfering peptide that disrupts the α7nAchR-NMDAR interaction. Moreover, administration of the interfering peptide in mice impairs novel object recognition but not Morris water maze performance. Our results suggest that α7nAchR/NMDAR coupling may selectively affect some aspects of learning and memory.
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Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain*
Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi
2012-01-01
The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions. PMID:22654118
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Zhu, Cui-Feng; Li, Guan-Zhi; Peng, Hong-Bin; Zhang, Fan; Chen, Yun; Li, Yong
2010-04-01
To explore Effects of marine collagen peptides (MCPs) on markers of metablic nuclear receptors, i.e peroxisome proliferator-activated receptor (PPARs), liver X receptor (LXRs) and farnesoid X receptor (FXRs) in type 2 diabetic patients with/without hypertension. METHOD Study population consisted of 200 type 2 diabetic patients with/without hypertension and 50 healthy subjects, all of whom were randomly assigned to MCPs-treated diabetics (n = 50), placebo-treated diabetics (n = 50), MCPs-treated diabetics with hypertension (n=50), placebo-treated diabetics with hypertension (n = 50), and healthy controls (n = 50). MCPs or placebo (water-soluble starch) were given daily before breakfast and bedtime over three months. Levels of free fatty acid, cytochrome P450, leptin, resistin, adiponectin, bradykinin, NO, and Prostacyclin were determined before intervention, and 1.5 months, and 3 months after intervention. Hypoglycemia and the endpoint events during the study were recorded and compared among the study groups. At the end of the study period, MCPs-treated patients showed marked improvement compared with patients receiving placebo. The protection exerted by MCPs seemed more profound in diabetics than in diabetics with hypertension. In particular, after MCPs intervention, levels of free fatty acid, hs-CRP, resistin, Prostacyclin decreased significantly in diabetics and tended to decrease in diabetic and hypertensive patients whereas levels of cytochrome P450, leptin, NO tended to decrease in diabetics with/without hypertension. Meanwhile, levels of adiponectin and bradykinin rose markedly in diabetics following MCPs administration. MCPs could offer protection against diabetes and hypertension by affecting levels of molecules involved in diabetic and hypertensive pathogenesis. Regulation on metabolic nuclear receptors by MCPs may be the possible underlying mechanism for its observed effects in the study. Further study into its action may shed light on development of new drugs based on bioactive peptides from marine sources.
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Novel Markers to Delineate Murine M1 and M2 Macrophages
Jablonski, Kyle A.; Amici, Stephanie A.; Webb, Lindsay M.; Ruiz-Rosado, Juan de Dios; Popovich, Phillip G.; Partida-Sanchez, Santiago; Guerau-de-Arellano, Mireia
2015-01-01
Classically (M1) and alternatively activated (M2) macrophages exhibit distinct phenotypes and functions. It has been difficult to dissect macrophage phenotypes in vivo, where a spectrum of macrophage phenotypes exists, and also in vitro, where low or non-selective M2 marker protein expression is observed. To provide a foundation for the complexity of in vivo macrophage phenotypes, we performed a comprehensive analysis of the transcriptional signature of murine M0, M1 and M2 macrophages and identified genes common or exclusive to either subset. We validated by real-time PCR an M1-exclusive pattern of expression for CD38, G-protein coupled receptor 18 (Gpr18) and Formyl peptide receptor 2 (Fpr2) whereas Early growth response protein 2 (Egr2) and c-Myc were M2-exclusive. We further confirmed these data by flow cytometry and show that M1 and M2 macrophages can be distinguished by their relative expression of CD38 and Egr2. Egr2 labeled more M2 macrophages (~70%) than the canonical M2 macrophage marker Arginase-1, which labels 24% of M2 macrophages. Conversely, CD38 labeled most (71%) in vitro M1 macrophages. In vivo, a similar CD38+ population greatly increased after LPS exposure. Overall, this work defines exclusive and common M1 and M2 signatures and provides novel and improved tools to distinguish M1 and M2 murine macrophages. PMID:26699615
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Lipoxin A4 activates ALX/FPR2 receptor to regulate conjunctival goblet cell secretion.
Hodges, R R; Li, D; Shatos, M A; Bair, J A; Lippestad, M; Serhan, C N; Dartt, D A
2017-01-01
Conjunctival goblet cells play a major role in maintaining the mucus layer of the tear film under physiological conditions as well as in inflammatory diseases like dry eye and allergic conjunctivitis. Resolution of inflammation is mediated by proresolution agonists such as lipoxin A 4 (LXA 4 ) that can also function under physiological conditions. The purpose of this study was to determine the actions of LXA 4 on cultured rat conjunctival goblet cell mucin secretion, intracellular [Ca 2+ ] ([Ca 2+ ] i ), and identify signaling pathways activated by LXA 4 . ALX/FPR2 (formyl peptide receptor2) was localized to goblet cells in rat conjunctiva and in cultured goblet cells. LXA 4 significantly increased mucin secretion, [Ca 2+ ] i , and extracellular regulated kinase 1/2 (ERK 1/2) activation. These functions were inhibited by ALX/FPR2 inhibitors. Stable analogs of LXA 4 increased [Ca 2+ ] i to the same extent as LXA 4 . Sequential addition of either LXA 4 or resolvin D1 followed by the second compound decreased [Ca 2+ ] i of the second compound compared with its initial response. LXA 4 activated phospholipases C, D, and A 2 and downstream molecules protein kinase C, ERK 1/2, and Ca 2+ /calmodulin-dependent kinase to increase mucin secretion and [Ca 2+ ] i . We conclude that conjunctival goblet cells respond to LXA 4 to maintain the homeostasis of the ocular surface and could be a novel treatment for dry eye diseases.
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Chen, Jing; Zhong, Jian; Yu, Hao; Xu, Xingsen; He, Hongbo; Yan, Zhencheng; Scholze, Alexandra; Liu, Daoyan; Zhu, Zhiming; Tepel, Martin
2012-01-01
Increased transient receptor potential canonical type 3 (TRPC3) channels have been observed in patients with essential hypertension. In the present study we tested the hypothesis that increased monocyte migration is associated with increased TRPC3 expression. Monocyte migration assay was performed in a microchemotaxis chamber using chemoattractants formylated peptide Met-Leu-Phe (fMLP) and tumor necrosis factor-α (TNF-α). Proteins were identified by immunoblotting and quantitative in-cell Western assay. The effects of TRP channel-inhibitor 2–aminoethoxydiphenylborane (2-APB) and small interfering RNA knockdown of TRPC3 were investigated. We observed an increased fMLP-induced migration of monocytes from hypertensive patients compared with normotensive control subjects (246±14% vs 151±10%). The TNF-α-induced migration of monocytes in patients with essential hypertension was also significantly increased compared to normotensive control subjects (221±20% vs 138±18%). In the presence of 2-APB or after siRNA knockdown of TRPC3 the fMLP-induced monocyte migration was significantly blocked. The fMLP-induced changes of cytosolic calcium were significantly increased in monocytes from hypertensive patients compared to normotensive control subjects. The fMLP-induced monocyte migration was significantly reduced in the presence of inhibitors of tyrosine kinase and phosphoinositide 3-kinase. We conclude that increased monocyte migration in patients with essential hypertension is associated with increased TRPC3 channels. PMID:22438881
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Effect of diazepam and clonazepam on the function of isolated rat platelet and neutrophil.
Rajtar, Grazyna; Zółkowska, Dorota; Kleinrok, Zdzisław
2002-04-01
Benzodiazepine binding sites distinct from the GABA-receptor-chloride-complex in the central nervous system have been recognized in many peripheral tissues, but their physiological role remains unexplained. Our study was undertaken to examine the effects of diazepam, clonazepam, and PK 11195, a peripheral benzodiazepine receptor antagonist, on the functional and biochemical responses of platelets and neutrophils stimulated by different physiological agonists. The experiments were conducted on isolated washed rat platelets activated by arachidonic acid (AA), adenosine 5'-diphosphate (ADP), or thrombin and on isolated rat neutrophils activated by a chemotactic peptide, formyl methionyl leucyl phenylalanine (fMLP). The results showed that neither diazepam nor clonazepam nor PK 11195 alone augmented the response of resting platelets or modified neutrophil response, but diazepam and clonazepam in a concentration-dependent manner inhibited thrombin, ADP or AA-stimulated platelet aggregation and the thrombin-induced increase in free intracellular Ca2+. Both drugs also exerted an inhibitory effect on reactive oxygen species (ROS) produced by fMLP-stimulated neutrophils. However, diazepam was about 10 times more effective than clonazepam. PK11195 did not influence platelet and neutrophil function stimulated by agonists, but reversed the inhibitory action of both benzodiazepines on platelet activation and ROS production. The results indicated that in vitro diazepam, and in a much smaller degree clonazepam, may down-regulate platelet activation and release of some proinflammatory mediators by stimulated neutrophils. These effects are probably exerted by a specific benzodiazepine binding sites.
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Gazal, S; Masterson, L R; Barany, G
2005-12-01
C-Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin-bound Weinreb amide precursor. Thus, 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyramido-polyethylene glycol-polystyrene (BAL-PEG-PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin-bound methoxylamine, which was efficiently acylated by different Fmoc-amino acids upon bromo-tris-pyrrolidone-phosphonium hexafluorophosphate (PyBrOP) activation. Solid-phase chain elongation gave backbone amide-linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C-terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68-83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16-53% and initial purities in the range of 30-40%. Under the LiAlH4 cleavage conditions used, those model peptides containing t-Bu-protected aspartate residues underwent partial side chain reduction to the corresponding homoserine-containing peptides. Similar results were obtained when working with high-load aminomethyl-polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.
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Arai, Kazune; Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki
2015-02-15
A group of synthetic substance P (SP) antagonists, such as [Arg(6),D-Trp(7,9),N(Me)Phe(8)]-substance P(6-11) and [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]-substance P, bind to a range of distinct G-protein-coupled receptor (GPCR) family members, including V1a vasopressin receptors, and they competitively inhibit agonist binding. This extended accessibility enabled us to identify a GPCR subset with a partially conserved binding site structure. By combining pharmacological data and amino acid sequence homology matrices, a pharmacological lineage of GPCRs that are sensitive to these two SP antagonists was constructed. We found that sensitivity to the SP antagonists was not limited to the Gq-protein-coupled V1a and V1b receptors; Gs-coupled V2 receptors and oxytocin receptors, which couple with both Gq and Gi, also demonstrated sensitivity. Unexpectedly, a dendrogram based on the amino acid sequences of 222 known GPCRs showed that a group of receptors sensitive to the SP antagonists are located in close proximity to vasopressin/oxytocin receptors. Gonadotropin-releasing peptide receptors, located near the vasopressin receptors in the dendrogram, were also sensitive to the SP analogs, whereas α1B adrenergic receptors, located more distantly from the vasopressin receptors, were not sensitive. Our finding suggests that pharmacological lineage analysis is useful in selecting subsets of candidate receptors that contain a conserved binding site for a ligand with broad-spectrum binding abilities. The knowledge that the binding site of the two broad-spectrum SP analogs partially overlaps with that of distinct peptide agonists is valuable for understanding the specificity/broadness of peptide ligands. Copyright © 2015 Elsevier B.V. All rights reserved.
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Consumption of peptide-included and free tryptophan induced by peroxyl radicals: A kinetic study.
Fuentes, E; López-Alarcón, C
2014-10-01
It is well-known that tryptophan residues are efficiently oxidized by peroxyl radicals, generating kynurenine, and N-formyl kynurenine as well as hydroperoxide derivatives as products. In the present work we studied the kinetic of such reaction employing free and peptide-included tryptophan. Two azocompounds were used to produce peroxyl radicals: AAPH (2,2'-Azobis(2-methylpropionamidine) dihydrochloride) and ABCVA (4,4'-Azobis(4-cyanovaleric acid)), which generate cationic and anionic peroxyl radicals, respectively. Tryptophan consumption was assessed by fluorescence spectroscopy and the reactions were carried out in phosphate buffer (75mM, pH 7.4) at 45°C. Only a slight effect of the peroxyl radical charge was evidenced on the consumption of free tryptophan and the dipeptide Gly-Trp. Employing AAPH as peroxyl radical source, at low free tryptophan concentrations (1-10µM) near 0.3 mol of tryptophan were consumed per each mol of peroxyl radicals introduced into the system. However, at high free tryptophan concentrations (100µM-1mM) such stoichiometry increased in a tryptophan concentration-way. At 1mM three moles of tryptophan were consumed per mol of AAPH-derived peroxyl radicals, evidencing the presence of chain reactions. A similar behavior was observed when di and tri-peptides (Gly-Trp, Trp-Gly, Gly-Trp-Gly, Trp-Ala, Ala-Trp-Ala) were studied. Nonetheless, at low initial concentration (5µM), the initial consumption rate of tryptophan included in the peptides was two times higher than free tryptophan. In contrast, at high concentration (1mM) free and peptide-included tryptophan showed similar initial consumption rates. These results could be explained considering a disproportionation process of tryptophanyl radicals at low free tryptophan concentrations, a process that would be inhibited when tryptophan is included in peptides. Copyright © 2014. Published by Elsevier Inc.
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IL-5 Induces Suspended Eosinophils to Undergo Unique Global Reorganization Associated with Priming
Han, Shih-Tsung
2014-01-01
The experiments described herein define a unique program of polarization of suspended human eosinophils stimulated with IL-5 family cytokines. We found that eosinophil granules and the nucleus move in opposite directions to form, respectively, a granular compartment and the nucleopod, a specialized uropod occupied by the nucleus and covered with adhesion receptors, including P-selectin glycoprotein ligand-1, CD44, and activated αMβ2 integrin. Ligated IL-5 family receptors localize specifically at the tip of the nucleopod in proximity to downstream signaling partners Janus tyrosine kinase 2, signal transducer and activator of transcription-1 and -5, and extracellular signal–regulated kinase. Microscopy and effects of cytochalasin B and nocodazole indicate that remodeling of filamentous actin and reorientation of the microtubule network are required for eosinophil polarization and nucleopod formation. IL-5 induces persistent polarization and extracellular signal–regulated kinase redistribution that are associated with eosinophil priming, a robust response on subsequent stimulation with N-formyl-methionyl-leucyl-phenylalanine. Global reorganization of cytoskeleton, organelles, adhesion receptors, and signaling molecules likely facilitates vascular arrest, extravasation, migration, granule release, and survival of eosinophils entering inflamed tissues from the bloodstream. PMID:24156300
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Goncalves, Gleisy Kelly; de Oliveira, Thiago Henrique Caldeira; de Oliveira Belo, Najara
2017-01-01
Background Heart failure in women increases around the time of menopause when high-fat diets may result in obesity. The heart produces brain natriuretic peptide (BNP), also known as B-type natriuretic peptide. This aims of this study were to assess cardiac hypertrophy and BNP levels in ovariectomized rats fed a high-fat diet. Material/Methods Forty-eight female Wistar rats were divided into four groups: sham-operated rats fed a control diet (SC) (n=12); ovariectomized rats fed a control diet (OC) (n=12); sham-operated rats fed a high-fat diet (SF) (n=12); and ovariectomized rats fed a high-fat diet (OF) (n=12). Body weight and blood pressure were measured weekly for 24 weeks. Rats were then euthanized, and plasma samples and heart tissue were studied for gene expression, hydroxyproline levels, and histological examination. Results A high-fat diet and ovariectomy (group OF) increased the weight body and the systolic blood pressure after three months and five months, respectively. Cardiomyocyte hypertrophy was associated with increased expression of ventricular BNP, decreased natriuretic peptide receptor (NPR)-A and increased levels of hydroxyproline and transforming growth factor (TGF)-β. The plasma levels of BNP and estradiol were inversely correlated; expression of estrogen receptor (ER)β and ERα were reduced. Conclusions The findings of this study showed that, in the ovariectomized rats fed a high-fat diet, the BNP-NPR-A receptor complex was involved in cardiac remodeling. BNP may be a marker of cardiac hypertrophy in this animal model. PMID:29249795
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Mild Hyperthermia Downregulates Receptor-dependent Neutrophil Function
Fröhlich, Dieter; Wittmann, Sigrid; Rothe, Gregor; Sessler, Daniel I.; Vogel, Peter; Taeger, Kai
2005-01-01
Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of un-opsonized bacteria. We evaluated various functions at 33 to 41°C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecules expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature independent. However, upregulation of CD11b with TNF-α was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature dependent. Bacterial uptake was inversely related to temperature, more so with E. coli than S. aureus. Temperature dependence of phagocytosis occurred only with opsonized bacteria. Hypothermia slightly increased N-Formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) receptors on neutrophils: hyperthermia decreased expression, especially with TNF-α. FMLP-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-α. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to non-receptor dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated. PMID:15281545
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Giblin, Michael F.; Wang, Nannan; Hoffman, Timothy J.; Jurisson, Silvia S.; Quinn, Thomas P.
1998-01-01
α-Melanocyte stimulating hormone (α-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind α-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, d-Phe7–α-MSH4-13 analog. Structural analysis of the Re–peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate–metal–thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re–peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re–α-MSH analog. Characterization of the second-generation Re–peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of α-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties. PMID:9788997
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99mTc-D(LPR): A novel retro-inverso peptide for VEGF receptor-1 targeted tumor imaging.
Rezazadeh, Farzaneh; Sadeghzadeh, Nourollah; Abedi, Seyed Mohammad; Abediankenari, Saeid
2018-05-31
The aim of this study was to evaluate the ability of D (LPR), a novel retro-inverso peptidomimetic derivative for imaging colon cancer. Two different D (LPR) analogs were designed and compared based on conjugation of HYNIC at peptide's C or N terminal and then labeled with technetium-99m using tricine/EDDA as an exchange coligands. The radiolabeled conjugates were assessed for in vitro stability in saline and serum. The VEGFR-1 and NRP-1 receptors affinity, in vitro internalization and also dissociation Constance was evaluated. SPCET imaging and biodistribution studies were performed in nude mice bearing HT-29 xenograft tumors. Both peptides labeled with technetium-99m in high radiochemical yield (˃97%). Peptide stability studies indicated a high metabolic stability of the radiopeptides in solution and serum. In vitro blocking studies demonstrated specific binding and internalization of [ 99m Tc]Tc-HYNIC-peptides in cultured HUVEC cells. The K d value for 99m Tc-peptide 1 and 99m Tc-peptide 2 were found to be 56.8 ± 12.9 nM and 71.6 ± 17.9 nM respectively. The tumor to muscle ratio was significant at 0.5 and 1 h after injection (4.5 and 4 for 99m Tc-peptide 1 and 4.9 and 4.4 for 99m Tc-peptide 2 at 0.5 and 1 h p.i. respectively). SPECT imaging studies revealed that both radioconjugates had prominent activity accumulation in VEGFR-1 and NRP-1 expressing HT-29 tumors. This study is the first instance of using a radiolabeled retro-inverso peptide for tumor imaging which is a promising tool to improve the performance of fragile peptide probes in vivo as imaging agents and warrant further investigations in other peptide-target systems. Copyright © 2018 Elsevier Inc. All rights reserved.
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Kudryavtsev, Denis; Shelukhina, Irina; Vulfius, Catherine; Makarieva, Tatyana; Stonik, Valentin; Zhmak, Maxim; Ivanov, Igor; Kasheverov, Igor; Utkin, Yuri; Tsetlin, Victor
2015-05-14
Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.
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Kudryavtsev, Denis; Shelukhina, Irina; Vulfius, Catherine; Makarieva, Tatyana; Stonik, Valentin; Zhmak, Maxim; Ivanov, Igor; Kasheverov, Igor; Utkin, Yuri; Tsetlin, Victor
2015-01-01
Nicotinic acetylcholine receptors (nAChRs) fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt), and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds. PMID:26008231
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Hastrup, H; Schwartz, T W
1996-12-16
The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.
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Izquierdo, Esther; Bednarczyk, Audrey; Schaeffer, Christine; Cai, Yimin; Marchioni, Eric; Van Dorsselaer, Alain; Ennahar, Saïd
2008-01-01
Enterococcus faecium IT62, isolated from ryegrass in Japan, was shown to produce three different bacteriocins, two of which had molecular masses and amino acid sequences that corresponded to those of enterocin L50A and enterocin L50B. These peptides existed, however, as chemically modified forms that were either N formylated or N formylated and oxidized at Met24. The third bacteriocin, named enterocin IT, had a molecular mass of 6,390 Da, was made up of 54 amino acids, and did not correspond to any known bacteriocin. However, enterocin IT was identical to the C-terminal part of the 16-amino-acid-longer bacteriocin 32 (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). For the first time, the antimicrobial activity spectra for enterocins L50A and L50B were determined separately and included a wide range of gram-positive bacteria but also a few gram-negative strains that were weakly sensitive. Slight differences in the activities of enterocins L50A and L50B were observed, as gram-positive bacteria showed an overall higher level of sensitivity to L50A than to L50B, as opposed to gram-negative ones. Conversely, enterocin IT showed a very narrow antimicrobial spectrum that was limited to E. faecium strains, one strain of Bacillus subtilis, and one strain of Lactococcus lactis. This study showed that E. faecium IT62, a grass-borne strain, produces bacteriocins with very different activity features and structures that may be found in strains associated with food or those of clinical origin, which demonstrates that a particular enterocin structure may be widespread and not related to the producer's origin. PMID:18391036
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Improved Synthesis of and Nucleophilic Addition to 2-Formyl-2-Cyclohexenone
Adary, Elan M.; Chang, Chih-wei; Auria, Damian T. D’; Nguyen, Phuc M.; Polewacz, Klaudyna; Reinicke, Justin A.; Seo, Hannah; Berger, Gideon O.
2014-01-01
A preparation of 2-formyl-2-cyclohexenone in nearly quantitative yield and purity of approximately 95% is described. It is scalable and has been extended to the synthesis of the 5- and 7-membered ring homologs with comparable yields. Conditions have also been developed for the successful conjugate addition of dimethylmalonate to 2-formyl-2-cyclohexenone, in good and scalable yield (60%). This result has been extended to 5 other nucleophile classes, and the dimethylmalonate conjugate addition has been demonstrated with 2-formyl-2-cyclopentenone and 2-formyl-2-cycloheptenone. PMID:25593375
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Emanuel, R L; Torday, J S; Mu, Q; Asokananthan, N; Sikorski, K A; Sunday, M E
1999-11-01
Previously, we have shown that bombesin-like peptide (BLP) promotes fetal lung development in rodents and humans but mediates postnatal lung injury in hyperoxic baboons. The present study analyzed the normal ontogeny of BLP and BLP receptors as well as the effects of BLP on cultured normal fetal baboon lungs. Transcripts encoding gastrin-releasing peptide (GRP), a pulmonary BLP, were detectable on gestational day 60 (ED60), peaked on approximately ED90, and then declined before term (ED180). Numbers of BLP-immunopositive neuroendocrine cells peaked from ED80 to ED125 and declined by ED160, preceding GRP-receptor mRNAs detected from ED125 until birth. BLP (0.1-10 nM) stimulated type II cell differentiation in organ cultures as assessed by [(3)H]choline incorporation into surfactant phospholipids, electron microscopy, and increased surfactant protein (SP) A- and/or SP-C-immunopositive cells and SP-A mRNA. BLP also induced neuroendocrine differentiation on ED60. Cell proliferation was induced by GRP, peaking on ED90. Similarly, blocking BLP degradation stimulated lung growth and maturation, which was completely reversed by a BLP-specific antagonist. The dissociation between GRP and GRP-receptor gene expression during ontogeny suggests that novel BLP receptors and/or peptides might be implicated in these responses.
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Liu, Dijie; Overbey, Douglas; Watkinson, Lisa; Giblin, Michael F.
2009-01-01
This study describes the synthesis and preliminary biologic evaluation of an 111Inlabeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla)2-K-K(DOTA)-NH2 was synthesized and conjugated with the DOTA chelating moiety via conventional Solid-Phase Peptide Synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC50 of 240 ± 125 nM for the peptide, compared with IC50’s of 0.44 ± 0.02 and 0.75 ± 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 hr post-injection of 111In-DOTA-peptide, and compared with data obtained using a scrambled control peptide, as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the 111In-labeled peptide from the blood pool, with 0.12 ± 0.06% ID/g remaining in blood at 4 hr pi. Elimination was seen primarily via the renal/urinary route, with 83.9 ± 2.2%ID in the urine at the same timepoint. Tumor uptake at this time was 0.53 ± 0.11%ID/g, resulting in tumor: blood and tumor: muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo. PMID:19354275
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Zhang, Chunfen; McDougall, Jason J
2006-01-01
The peripheral effect of the ‘opioid-like' peptide nociceptin/orphanin FQ (N/OFQ) on joint blood flow was investigated in acutely inflamed rats. Sensory neuropeptide release from capsaicin-sensitive nerves and the involvement of synovial mast cells and leukocytes on these vasomotor responses were also studied. Blood flow measurements of exposed knee joints were performed in urethane-anaesthetised rats (2 mg kg−1 intraperitoneal) using laser Doppler perfusion imaging. Topical administration of N/OFQ (10−13–10−8 mol) to acutely inflamed joints caused a dose-dependent increase in synovial perfusion with an ED50 of 4.0 × 10−10 mol. This vasodilatatory response was blocked by the selective NOP receptor antagonist [Phe1-(CH2-NH)-Gly2]-Nociceptin(1–13)-NH2 (10−9 mol) (P<0.0001). Co-administration of N/OFQ with the neurokinin-1 (NK1) receptor antagonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-Substance P (10−12 mol), the vasoactive intestinal peptide (VIP) receptor antagonist VIP6–28 (10−9 mol) or the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP8–37 (10−9 mol) all blocked the hyperaemic effect of N/OFQ (P<0.0001). Treatment of acutely inflamed knees with capsaicin (8-methyl-N-vanillyl-6-noneamide) to destroy unmyelinated joint afferents also inhibited N/OFQ vasomotor activity. Stabilisation of synovial mast cells with disodium cromoglycate (cromolyn) ameliorated N/OFQ responses, whereas inactivation of circulating leukocytes with the pan-selectin inhibitor fucoidin completely blocked N/OFQ-induced hyperaemia in these joints. These experiments show that in acutely inflamed knee joints, N/OFQ acts on NOP receptors located on synovial mast cells and leukocytes leading to the secondary release of proinflammatory mediators into the joint. These agents subsequently stimulate sensory neuropeptide release from capsaicin-sensitive nerves culminating in vasodilatation and increased articular blood flow. PMID:16783411
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Zishiri, Vincent K; Hunter, Roger; Smith, Peter J; Taylor, Dale; Summers, Robert; Kirk, Kiaran; Martin, Rowena E; Egan, Timothy J
2011-05-01
A series of 12 new dibemethin (N-benzyl-N-methyl-1-phenylmethanamine) derivatives bearing an N-aminomethyl group attached to the one phenyl ring and an H, Cl, OCH3 or N(CH3)2 group on the other have been synthesized. These compounds all showed strong chloroquine chemosensitizing activity, comparable to verapamil, when present at 1 μM in an in vitro culture of the chloroquine-resistant W2 strain of the human malaria parasite, Plasmodium falciparum. Their N-formylated derivatives also exhibited resistance-reversing activity, but only at substantially higher IC10 concentrations. A number of the dibemethin derivatives were shown to inhibit chloroquine transport via the parasite's 'chloroquine resistance transporter' (PfCRT) in a Xenopus laevis oocyte expression system. The reduced resistance-reversing activity of the formylated compounds relative to their free amine counterparts can probably be ascribed to two factors: decreased accumulation of the formylated dibemethins within the parasite's internal digestive vacuole (believed to be the site of action of chloroquine), and a reduced ability to inhibit PfCRT. The resistance-reversing activity of the compounds described here demonstrates that the amino group need not be attached to the two aromatic rings via a three or four carbon chain as has been suggested by previous QSAR studies. These compounds may be useful as potential side chains for attaching to a 4,7-dichloroquinoline group in order to generate new resistance-reversing chloroquine analogues with inherent antimalarial activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.
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Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.
Katragadda, M; Chopra, A; Bennett, M; Alderfer, J L; Yeagle, P L; Albert, A D
2001-07-01
An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.
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Ceraudo, Emilie; Hierso, Régine; Tan, Yossan-Var; Murail, Samuel; Rouyer-Fessard, Christiane; Nicole, Pascal; Robert, Jean-Claude; Jamin, Nadège; Neumann, Jean-Michel; Robberecht, Patrick; Laburthe, Marc; Couvineau, Alain
2012-05-01
Vasoactive intestinal peptide (VIP) plays a major role in pathophysiology. Our previous studies demonstrated that the VIP sequence 6-28 interacts with the N-terminal ectodomain (N-ted) of its receptor, VPAC1. Probes for VIP and receptor antagonist PG97-269 were synthesized with a photolabile residue/Bpa at various positions and used to explore spatial proximity with VPAC1. PG97-269 probes with Bpa at position 0, 6, and 24 behaved as high-affinity receptor antagonists (K(i)=12, 9, and 7 nM, respectively). Photolabeling experiments revealed that the [Bpa(0)]-VIP probe was in physical contact with VPAC1 Q(135), while [Bpa(0)]-PG97-269 was covalently bound to G(62) residue of N-ted, indicating different binding sites. In contrast, photolabeling with [Bpa(6)]- and [Bpa(24)]-PG97-269 showed that the distal domains of PG97-269 interacted with N-ted, as we previously showed for VIP. Substitution with alanine of the K(143), T(144), and T(147) residues located in the first transmembrane domain of VPAC1 induced a loss of receptor affinity (IC(50)=1035, 874, and 2070 nM, respectively), and pharmacological studies using VIP2-28 indicated that these three residues play an important role in VPAC1 interaction with the first histidine residue of VIP. These data demonstrate that VIP and PG97-269 bind to distinct domains of VPAC1.
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Stefano, G B; Zhu, W; Cadet, P; Bilfinger, T V; Mantione, K
2004-03-01
Studies from our laboratory have revealed a novel micro opiate receptor, micro(3), which is expressed in both human vascular tissues and leukocytes. The micro(3) receptor is selective for opiate alkaloids, insensitive to opioid peptides and is coupled to constitutive nitric oxide (cNO) release. We now identify the micro(3) receptor characteristics in mammalian gut tissues. It appears that the various regions of the mouse gut release low levels of NO (0.02 to 4.6 nM ) in a pulsatile manner. We demonstrate that morphine stimulates cNO release (peak level 17 nM) in the mouse stomach, small intestine and large intestine in a naloxone and L-NAME antagonizable manner. Opioid peptides do not exhibit cNO-stimulating capabilities in these tissues. Taken together, we surmise morphine acts as a hormone to limit gut activity via micro(3) coupled to NO release since micro opiate receptors are found in the gut and endogenous morphine is not but is found in blood.
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Strack, Martin; Bedini, Andrea; Yip, King T; Lombardi, Sara; Siegmund, Daniel; Stoll, Raphael; Spampinato, Santi M; Metzler-Nolte, Nils
2016-10-04
Herein, the selective enforcement of one particular receptor-ligand interaction between specific domains of the μ-selective opioid peptide dermorphin and the μ opioid receptor is presented. For this, a blocking group scan is described which exploits the steric demand of a bis(quinolinylmethyl)amine rhenium(I) tricarbonyl complex conjugated to a number of different, strategically chosen positions of dermorphin. The prepared peptide conjugates lead to the discovery of two different binding modes: An expected N-terminal binding mode corresponds to the established view of opioid peptide binding, whereas an unexpected C-terminal binding mode is newly discovered. Surprisingly, both binding modes provide high affinity and agonistic activity at the μ opioid receptor in vitro. Furthermore, the unprecedented C-terminal binding mode shows potent dose-dependent antinociception in vivo. Finally, in silico docking studies support receptor activation by both dermorphin binding modes and suggest a biological relevance for dermorphin itself. Relevant ligand-protein interactions are similar for both binding modes, which is in line with previous protein mutation studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Structure of the glucagon receptor in complex with a glucagon analogue.
Zhang, Haonan; Qiao, Anna; Yang, Linlin; Van Eps, Ned; Frederiksen, Klaus S; Yang, Dehua; Dai, Antao; Cai, Xiaoqing; Zhang, Hui; Yi, Cuiying; Cao, Can; He, Lingli; Yang, Huaiyu; Lau, Jesper; Ernst, Oliver P; Hanson, Michael A; Stevens, Raymond C; Wang, Ming-Wei; Reedtz-Runge, Steffen; Jiang, Hualiang; Zhao, Qiang; Wu, Beili
2018-01-03
Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.
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Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki
2013-10-01
The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.
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What peptides these deltorphins be.
Lazarus, L H; Bryant, S D; Cooper, P S; Salvadori, S
1999-02-01
The deltorphins are a class of highly selective delta-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe, comparable to dermorphin, which is the prototype of a group of mu-selective opioids from the same source. The D-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high delta-receptor affinity, bioactivity and peptide conformation. This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the delta-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the delta-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.
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Yang, Na; Sun, Chaomin
2016-01-01
Vibrio sp. is the most serious pathogen in marine aquaculture, and the development of anti-Vibrio agents is urgently needed. However, it is extreme lack of high-throughput screening (HTS) model for searching anti-Vibrio compounds. Here, we established a protein-based HTS screening model to identify agents targeting peptide deformylase (PDF) of Vibrio anguillarum. To find potential anti-Vibrio compounds, crude extracts derived from marine actinomycetes were applied for screening with this model. Notably, crude extract of strain Streptomyces sp. NHF165 inhibited dramatically both on V. anguillarum PDF (VaPDF) activity and V. anguillarum cell growth. And actinonin was further identified as the functional component. Anti-VaPDF and anti-V. anguillarum activities of actinonin were dose-dependent, and the IC50 values were 6.94 and 2.85 μM, respectively. To understand the resistance of V. anguillarum against actinonin, spontaneous V. anguillarum mutants with resistance against actinonin were isolated. Surprisingly, for the resistant strains, the region between 774 and 852 base pairs was found to be absent in the gene folD which produces 10-formyl-tetrahydrofolate, a donor of N-formyl to Met-tRNAfmet. When compared to the wild type strain, ΔfolD mutant showed eight times of minimum inhibition concentration on actinonin, however, the folD complementary strain could not grow on the medium supplemented with actinonin, which suggested that folD gene mutation was mainly responsible for the actinonin resistance. To our knowledge, this is the first report showing that marine derived Streptomyces sp. could produce actinonin with anti-VaPDF activity and the resistance against actinonin by V. anguillarum is mediated by mutation in folD gene. PMID:27679625
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Roosenburg, S; Laverman, P; Joosten, L; Cooper, M S; Kolenc-Peitl, P K; Foster, J M; Hudson, C; Leyton, J; Burnet, J; Oyen, W J G; Blower, P J; Mather, S J; Boerman, O C; Sosabowski, J K
2014-11-03
Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.
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Accardo, Antonella; Galli, Filippo; Mansi, Rosalba; Del Pozzo, Luigi; Aurilio, Michela; Morisco, Anna; Ringhieri, Paola; Signore, Alberto; Morelli, Giancarlo; Aloj, Luigi
2016-12-01
Overexpression of the gastrin-releasing peptide receptor (GRP-R) has been documented in several human neoplasms such as breast, prostate, and ovarian cancer. There is growing interest in developing radiolabeled peptide-based ligands toward these receptors for the purpose of in vivo imaging and radionuclide therapy of GRP-R-overexpressing tumors. A number of different peptide sequences, isotopes, and labeling methods have been proposed for this purpose. The aim of this work is to perform a direct side-by-side comparison of different GRP-R binding peptides utilizing a single labeling strategy to identify the most suitable peptide sequence. Solid-phase synthesis of eight derivatives (BN1-8) designed based on literature analysis was carried out. Peptides were coupled to the DOTA chelator through a PEG4 spacer at the N-terminus. Derivatives were characterized for serum stability, binding affinity on PC-3 human prostate cancer cells, biodistribution in tumor-bearing mice, and gamma camera imaging at 1, 6, and 24 h after injection. Serum stability was quite variable among the different compounds with half-lives ranging from 16 to 400 min at 37 °C. All compounds tested showed K d values in the nanomolar range with the exception of BN3 that showed no binding. Biodistribution and imaging studies carried out for compounds BN1, BN4, BN7, and BN8 showed targeting of the GRP-R-positive tumors and the pancreas. The BN8 compound (DOTA-PEG-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2) showed high affinity, the longest serum stability, and the highest target-to-background ratios in biodistribution and imaging experiments among the compounds tested. Our results indicate that the NMeGly for Gly substitution and the Sta-Leu substitution at the C-terminus confer high serum stability while maintaining high receptor affinity, resulting in biodistribution properties that outperform those of the other peptides.
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Todorovic, Aleksandar; Holder, Jerry Ryan; Bauzo, Rayna M; Scott, Joseph Walker; Kavanagh, Renny; Abdel-Malek, Zalfa; Haskell-Luevano, Carrie
2005-05-05
The melanocortin system is involved in the regulation of a diverse number of physiologically important pathways including pigmentation, feeding behavior, weight and energy homeostasis, inflammation, and sexual function. All the endogenous melanocortin agonist ligands possess the conserved His-Phe-Arg-Trp tetrapeptide sequence that is postulated to be important for melanocortin receptor molecular recognition and stimulation. Previous studies by our laboratory resulted in the discovery that increasing alkyl chain length at the N-terminal "capping" region of the His-dPhe-Arg-Trp-NH(2) tetrapeptide resulted in a 100-fold increased melanocortin receptor agonist potency. This study was undertaken to systematically evaluate the pharmacological effects of increasing N-capping alkyl chain length of the CH(3)(CH(2))(n)CO-His-dPhe-Arg-Trp-NH(2) (n = 6-16) tetrapeptide template. Twelve analogues were synthesized and pharmacologically characterized at the mouse melanocortin receptors MC1R and MC3R-MC5R and human melanocytes known to express the MC1R. These peptides demonstrated melanocortin receptor selectivity profiles different from those of previously published tetrapeptides. The most notable results of enhanced ligand potency (20- to 200-fold) and receptor selectivity were observed at the MC1R. Tetrapeptides that possessed greater than nine alkyl groups were superior to alpha-MSH in terms of the stimulation of human melanocyte tyrosinase activity. Additionally, the n-pentadecanoyl derivative had a residual effect on tyrosinase activity that existed for at least 4 days after the peptide was removed from the human melanocyte culture medium. These data demonstrate the utility, potency, and residual effect of melanocortin tetrapeptides by adding N-terminal fatty acid moieties.
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Swedberg, Joakim E.; Schroeder, Christina I.; Mitchell, Justin M.; Fairlie, David P.; Edmonds, David J.; Griffith, David A.; Ruggeri, Roger B.; Derksen, David R.; Loria, Paula M.; Price, David A.; Liras, Spiros; Craik, David J.
2016-01-01
Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22–27) directing the binding of Phe22 into a hydrophobic pocket on the GLP-1R. PMID:27226591
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Kumar, Virendra; Guo, Deqi; Marella, Michael; Cassel, Joel A; Dehaven, Robert N; Daubert, Jeffrey D; Mansson, Erik
2008-06-15
A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.
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NASA Astrophysics Data System (ADS)
Zhang, Zhengfeng; Li, Jianping; Chen, Yanke; Xie, Huayong; Yang, Jun
2017-12-01
In this letter, we propose a robust heteronuclear dipolar recoupling method for proteins in magic-angle spinning (MAS) solid-state NMR. This method is as simple, robust and efficient as the well-known TEDOR in the aspect of magnetization transfer between 15N and 13C. Deriving from our recent band-selective dual back-to-back pulses (DBP) (Zhang et al., 2016), this method uses new phase-cycling schemes to realize broadband DBP (Bro-DBP). For broadband 15N-13C magnetization transfer (simultaneous 15N → 13C‧ and 15N → 13Cα), Bro-DBP has almost the same 15N → 13Cα efficiency while offers 30-40% enhancement on 15N → 13C‧ transfer, compared to TEDOR. Besides, Bro-DBP can also be used as a carbonyl (13C‧)-selected method, whose 15N → 13C‧ efficiency is up to 1.7 times that of TEDOR and is also higher than that of band-selective DBP. The performance of Bro-DBP is demonstrated on the N-formyl-[U-13C,15N]-Met-Leu-Phe-OH (fMLF) peptide and the U-13C, 15N labeled β1 immunoglobulin binding domain of protein G (GB1) microcrystalline protein. Since Bro-DBP is as robust, simple and efficient as TEDOR, we believe it is very useful for protein studies in MAS solid-state NMR.
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Design and structure of stapled peptides binding to estrogen receptors.
Phillips, Chris; Roberts, Lee R; Schade, Markus; Bazin, Richard; Bent, Andrew; Davies, Nichola L; Moore, Rob; Pannifer, Andrew D; Pickford, Andrew R; Prior, Stephen H; Read, Christopher M; Scott, Andrew; Brown, David G; Xu, Bin; Irving, Stephen L
2011-06-29
Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.
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Poujade, C; Lavielle, S; Torrens, Y; Beaujouan, J C; Glowinski, J; Marquet, A
1984-09-01
Glycosylated analogues of the C-terminal heptapeptide of substance P either free or blocked on the N-terminal glutamine were synthesized in order to develop a metabolically stable peptide that would have an increased specificity for one type of receptor. Of the analogue described, (N-alpha-Boc-beta-D-Glc-p (1----5) Gln) -Gln-Phe-Phe-Gly-Leu-Met-NH2 is highly resistant to degradation on exposure to rat hypothalamic slices. This glycosylated peptide is about one third as potent as substance P in eliciting contractions of the guinea-pig ileum and is almost devoided of affinity for the 125I-Bolton Hunter-SP specific binding sites on rat brain synaptosomes.
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C-type natriuretic peptide and atrial natriuretic peptide receptors of rat brain.
Brown, J; Zuo, Z
1993-03-01
Natriuretic peptide receptors in rat brain were mapped by in vitro autoradiography using 125I-labeled [Tyr0]CNP-(1-22) to bind atrial natriuretic peptide receptor (ANPR)-B and ANPR-C receptors selectively, and 125I-labeled alpha-ANP to select ANPR-A and ANPR-C receptors. Des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4- 23)-amide (C-ANP) was used for its selectivity for ANPR-C over ANPR-A. Specific binding of 125I-[Tyr0]CNP-(1-22) with a dissociation constant (Kd) approximately 1 nM occurred in olfactory bulb, cerebral cortex, lateral septal nucleus, choroid plexus, and arachnoid mater. This binding was abolished by C-type natriuretic peptide [CNP-(1-22)], alpha-ANP and C-ANP, and conformed to ANPR-C. 125I-alpha-ANP bound to all structures that bound 125I-[Tyr0]CNP-(1-22). This binding was also inhibited by both CNP-(1-22) and C-ANP, confirming the presence of ANPR-C-like binding sites. However, ANPR-C-like binding sites were heterogenous because only some had high affinities for 125I-[Tyr0]CNP-(1-22) and CNP-(1-22). 125I-alpha-ANP also bound sites without affinities for C-ANP or CNP-(1-22). These sites were consistent with ANPR-A. They occurred mainly on the olfactory bulb, the choroid plexus, and the subfornical organ. Guanosine 3',5'-cyclic monophosphate production was strongly stimulated by alpha-ANP but not by CNP-(1-22) in olfactory bulb. Neither ligand stimulated it in cortical tissue. Thus the natriuretic peptide binding sites of rat brain conformed to ANPR-A and to heterogenous ANPR-C-like sites. No ANPR-B were detected.
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2015-01-01
The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, β-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp7 residue, was synthesized using solid phase peptide synthesis and characterized by a β-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D 1H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII β-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands. PMID:25141170
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Cheng, Dengfeng; Yin, Duanzhi; Li, Gucai; Wang, Mingwei; Li, Shiqiang; Zheng, Mingqiang; Cai, Hancheng; Wang, Yongxian
2006-12-01
In an effort to develop a peptide-based radiopharmaceutical for the detection of tumors overexpressed vasoactive intestinal peptide receptors with positron emission tomography, we have prepared a novel [R(8,15,21), L17]-VIP peptide for 18F-labeling. This peptide inhibited 125I-VIP binding to rats lung membranes with high affinity [half-maximal inhibitory concentrations (IC50) of 0.12 nm]. Additionally, [R(8,15,21), L17]-VIP showed higher stability than native vasoactive intestinal peptide in vivo of mice. With N-succinimidyl 4-[18F] fluorobenzoate as labeling prosthetic group, [18F]FB-[R(8,15,21), L17]-VIP was obtained in >99% radiochemical purity within 100 min in decay-for-corrected radiochemical yield of 33.6 +/- 3% (n = 5) and a specific radioactivity 255 GBq/micromol at the end of synthesis. Stability of [18F]FB-[R(8,15,21), L17]-VIP in vitro and in vivo were investigated. Biodistribution of this trace was carried out in mice with induced C26 colorectal tumor. Fast clearance of [18F]FB-[R(8,15,21), L17]-VIP from non-target tissues and specific uptakes by tumors realized higher tumor-to-muscle ratio (3.55) and tumor-to-blood ratio (2.37) 60 min postinjection. Clear difference was observed between the blocking and unblocking experiments in biodistribution and whole body radioautography. [18F]FB-[R(8,15,21), L17]-VIP has demonstrated its potential for diagnosing tumors overexpressed vasoactive intestinal peptide receptors both in vitro and in vivo.
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The Janus Kinase (JAK) FERM and SH2 Domains: Bringing Specificity to JAK-Receptor Interactions.
Ferrao, Ryan; Lupardus, Patrick J
2017-01-01
The Janus kinases (JAKs) are non-receptor tyrosine kinases essential for signaling in response to cytokines and interferons and thereby control many essential functions in growth, development, and immune regulation. JAKs are unique among tyrosine kinases for their constitutive yet non-covalent association with class I and II cytokine receptors, which upon cytokine binding bring together two JAKs to create an active signaling complex. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains, both of which are classical mediators of peptide interactions. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich "Box1" and hydrophobic "Box2," which are present in the intracellular domain of cytokine receptors. While the general sidechain chemistry of Box1 and Box2 peptides is conserved between receptors, they share very weak primary sequence homology, making it impossible to posit why certain JAKs preferentially interact with and signal through specific subsets of cytokine receptors. Here, we review the structure and function of the JAK FERM and SH2 domains in light of several recent studies that reveal their atomic structure and elucidate interaction mechanisms with both the Box1 and Box2 receptor motifs. These crystal structures demonstrate how evolution has repurposed the JAK FERM and SH2 domains into a receptor-binding module that facilitates interactions with multiple receptors possessing diverse primary sequences.
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Permatasari, Galuh W; Utomo, Didik H; Widodo
2016-10-01
A designing peptide as agent for inducing diabetes mellitus type 2 (T2DM) in an animal model is challenging. The computational approach provides a sophisticated tool to design a functional peptide that may block the insulin receptor activity. The peptide that able to inhibit the binding between insulin and insulin receptor is a warrant for inducing T2DM. Therefore, we designed a potential peptide inhibitor of insulin receptor as an agent to generate T2DM animal model by bioinformatics approach. The peptide has been developed based on the structure of insulin receptor binding site of insulin and then modified it to obtain the best properties of half life, hydrophobicity, antigenicity, and stability binding into insulin receptor. The results showed that the modified peptide has characteristics 100h half-life, high-affinity -95.1±20, and high stability 28.17 in complex with the insulin receptor. Moreover, the modified peptide has molecular weight 4420.8g/Mol and has no antigenic regions. Based on the molecular dynamic simulation, the complex of modified peptide-insulin receptor is more stable than the commercial insulin receptor blocker. This study suggested that the modified peptide has the promising performance to block the insulin receptor activity that potentially induce diabetes mellitus type 2 in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Large Extremity Peripheral Nerve Repair
2014-10-01
has also been shown to produce human-beta-3-defensin. These antimicrobial peptides are implicated in the resistance of epithelial surfaces to...gonadotrophin receptors that regulate prostaglandin production and activity. Epithelial cells manufacture multiple vasoactive peptides , growth factors...200734 P-RCT (n Z 102) PT burns Processed Amnion vs topical antimicrobials . Significantly less dressing changes with amnion. Time to healing, length
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The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism.
Wootten, Denise; Reynolds, Christopher A; Smith, Kevin J; Mobarec, Juan C; Koole, Cassandra; Savage, Emilia E; Pabreja, Kavita; Simms, John; Sridhar, Rohan; Furness, Sebastian G B; Liu, Mengjie; Thompson, Philip E; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M
2016-06-16
Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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Guerreiro, Serge; Florence, Clélia; Rousseau, Erwann; Hamadat, Sabah; Hirsch, Etienne C; Michel, Patrick P
2015-01-01
To determine whether orexinergic hypothalamic peptides can influence the survival of brainstem dopamine (DA) neurons, we used a model system of rat midbrain cultures in which DA neurons degenerate spontaneously and progressively as they mature. We established that orexin (OX)-B provides partial but significant protection to spontaneously dying DA neurons, whereas the homologous peptide OXA has only marginal effects. Importantly, DA neurons rescued by OXB accumulated DA efficiently by active transport, suggesting that they were functional. G-protein-coupled OX1 and OX2 receptors were both present on DA neurons, but the protective effect of OXB was attributable solely to OX2 receptors; a selective inhibitor of this receptor subtype, N-ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide (EMPA), suppressed this effect, whereas a selective agonist, [Ala(11), d-Leu(15)]OXB, reproduced it. Survival promotion by OXB required intracellular calcium mobilization via inositol-1,4,5-triphosphate and ryanodine receptors. Nicotine, a well known neuroprotective molecule for DA neurons, improved OXB-mediated rescue through the activation of α-bungarotoxin-sensitive (presumably α7) nicotinic receptors, although nicotine had no effect on its own. Altogether, our data suggest that the loss of hypothalamic orexinergic neurons that occurs in Parkinson's disease might confer an increased vulnerability to midbrain DA neurons in this disorder. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
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Ngoka, L C; Gross, M L
2000-02-01
We previously showed by using mass spectrometry that endothelin A selective receptor antagonists BQ123 and JKC301 form novel coordination compounds with sodium ions. This property may underlie the ability of an ET(A) antagonist to induce net tubular sodium reabsorption in the proximal tubule cells and reverse acute renal failure induced by severe ischemia. We have now defined the metal binding sites on BQ123 and JKC301 by subjecting the metal-containing peptides to multiple stages of collisionally activated decomposition (CAD) in an ion trap mass spectrometer. When submitted to low-energy CAD, the ring opens at the Asp-Pro amide bond. The metal ion, which bonds, inter alia, to the carbonyl oxygen of the proline residue, acts as a fixed charge site, and directs a charge-remote, sequence-specific fragmentation of the ring-opened peptide. Amino acid residues are sequentially cleaved from the C-terminal end, and the terminal aziridinone structure moves one step toward the N-terminus with each C-terminal amino acid residue removed. These observations are the basis of a new method to sequence cyclic peptides. Amino acid residues are observed as sets of three ions, a*(n)PD, b*(n)PD and c*(n)PD where n is the number of amino acid residues in the peptide. Copyright 2000 John Wiley & Sons, Ltd.
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Gergel, J R; McNamara, D J; Dobrusin, E M; Zhu, G; Saltiel, A R; Miller, W T
1994-12-13
Photoaffinity labeling and site-directed mutagenesis have been used to identify amino acid residues of the phospholipase C gamma 1 (PLC gamma 1) N-terminal SH2 domain involved in recognition of the activated epidermal growth factor receptor (EGFR). The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into phosphotyrosine-containing peptides derived from EGFR autophosphorylation sites Tyr992 and Tyr1068. Irradiation of these labels in the presence of SH2 domains showed cross-linking which was time-dependent and specific; labeling was inhibited with non-Bpa-containing peptides from EGFR in molar excess. The phosphotyrosine residue on the peptides was important for SH2 recognition, as dephosphorylated peptides did not cross-link. Radiolabeled peptides were used to identify sites of cross-linking to the N-terminal SH2 of PLC gamma 1. Bpa peptide-SH2 complexes were digested with trypsin, and radioactive fragments were purified by HPLC and analyzed by Edman sequencing. These experiments showed Arg562 and an additional site in the alpha A-beta B region of the SH2 domain, most likely Glu587, to be labeled by the Tyr992-derived peptide. Similar analysis of the reaction with the Tyr1068-derived photoaffinity label identified Leu653 as the cross-linked site. Mutation of the neighboring residues of Glu587 decreased photo-cross-linking, emphasizing the importance of this region of the molecule for recognition. These results are consistent with evidence from the v-Src crystal structure and implicate the loop spanning residues Gln640-Ser654 of PLC gamma 1 in specific recognition of phosphopeptides.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tai, Xi-Shi, E-mail: taixs@wfu.edu.cn; Wang, Xin
2017-03-15
A new Ca(II) coordination polymer, ([CaL(H{sub 2}O){sub 4}] · (H{sub 2}O){sub 4}){sub n} (L = 4-formyl-1,3-benzenedisulfonate-2-furoic acid hydrazide) has been prepared by one-pot synthesis method. And it was characterized by elemental analysis, IR and thermal analysis. The result of X-ray single-crystal diffraction analysis shows that the Ca(II) complex molecules form one-dimensional chain structure by the bridging oxygen atoms. The anti-tumor activity of L ligand and the Ca(II) coordination polymer has also been studied.
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Molino, Yves; David, Marion; Varini, Karine; Jabès, Françoise; Gaudin, Nicolas; Fortoul, Aude; Bakloul, Karima; Masse, Maxime; Bernard, Anne; Drobecq, Lucile; Lécorché, Pascaline; Temsamani, Jamal; Jacquot, Guillaume; Khrestchatisky, Michel
2017-05-01
The blood-brain barrier (BBB) prevents the entry of many drugs into the brain and, thus, is a major obstacle in the treatment of CNS diseases. There is some evidence that the LDL receptor (LDLR) is expressed at the BBB and may participate in the transport of endogenous ligands from blood to brain, a process referred to as receptor-mediated transcytosis. We previously described a family of peptide vectors that were developed to target the LDLR. In the present study, in vitro BBB models that were derived from wild-type and LDLR-knockout animals ( ldlr -/- ) were used to validate the specific LDLR-dependent transcytosis of LDL via a nondegradative route. We next showed that LDLR-targeting peptide vectors, whether in fusion or chemically conjugated to an Ab Fc fragment, promote binding to apical LDLR and transendothelial transfer of the Fc fragment across BBB monolayers via the same route as LDL. Finally, we demonstrated in vivo that LDLR significantly contributes to the brain uptake of vectorized Fc. We thus provide further evidence that LDLR is a relevant receptor for CNS drug delivery via receptor-mediated transcytosis and that the peptide vectors we developed have the potential to transport drugs, including proteins or Ab based, across the BBB.-Molino, Y., David, M., Varini, K., Jabès, F., Gaudin, N., Fortoul, A., Bakloul, K., Masse, M., Bernard, A., Drobecq, L., Lécorché, P., Temsamani, J., Jacquot, G., Khrestchatisky, M. Use of LDL receptor-targeting peptide vectors for in vitro and in vivo cargo transport across the blood-brain barrier. © FASEB.
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Ferro-Flores, Guillermina; Arteaga de Murphy, Consuelo; Rodriguez-Cortés, Jeanette; Pedraza-López, Martha; Ramírez-Iglesias, María Teresa
2006-04-01
Bombesin is a peptide that was initially isolated from frog skin and which belongs to a large group of neuropeptides with many biological functions. The human equivalent is gastrin-releasing peptide (GRP), whose receptors are over-expressed in a variety of malignant tumours. To prepare a HYNIC-[Lys 3]-bombesin analogue that could be easily labelled with 99mTc from lyophilized kit formulations and to evaluate its potential as an imaging agent for GRP receptor-positive tumours. HYNIC was conjugated to the epsilon-amino group of Lys 3 residue at the N-terminal region of bombesin via succinimidyl-N-Boc-HYNIC at pH 9.0. 99mTc labelling was performed by addition of sodium pertechnetate solution and 0.2 M phosphate buffer pH 7.0 to a lyophilized formulation. Stability studies were carried out by reversed phase HPLC and ITLC-SG analyses in serum and cysteine solutions. In-vitro internalization was tested using human prostate cancer PC-3 cells with blocked and non-blocked receptors. Biodistribution and tumour uptake were determined in PC-3 tumour-bearing nude mice. 99mTc-EDDA/HYNIC-[Lys 3]-bombesin was obtained with radiochemical purities >93% and high specific activity ( approximately 0.1 GBq.nmol). Results of in-vitro studies demonstrated a high stability in serum and cysteine solutions, specific cell receptor binding and rapid internalization. Biodistribution data showed a rapid blood clearance, with predominantly renal excretion and specific binding towards GRP receptor-positive tissues such as pancreas and PC-3 tumours. 99mTc-EDDA/HYNIC-[Lys 3]-bombesin obtained from lyophilized kit formulations has promising characteristics for the diagnosis of malignant tumours that over-express the GRP receptor.
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Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie
2003-12-01
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. Alpha-melanocyte stimulating hormone (alpha-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.
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Alteration of lung atrial natriuretic peptide receptors in genetic cardiomyopathy.
Mukaddam-Daher, S; Tremblay, J; Fujio, N; Koch, C; Jankowski, M; Quillen, E W; Gutkowska, J
1996-07-01
These studies were designed to characterize the atrial natriuretic peptide (ANF) receptor subtypes [guanylyl cyclase natriuretic peptide receptors (NPR-A, NPR-B) and NPR-C] in lungs of normal hamsters and to evaluate alterations in receptor kinetics in genetic cardiomyopathy (CMO), a model of human congestive heart failure. Lung membranes were obtained from normal and CMO 200-to 230-day-old hamsters. Cross-linking and competitive binding receptor assays using 125I-labeled human ANF showed that lung membranes exhibit NPR, mainly guanylyl cyclase NPR-A and clearance NPR-C receptors. Stimulation of guanylyl cyclase by ANF and C-type natriuretic peptide (CNP) confirmed the presence of NPR-A and NPR-B. The maximum binding capacity of total ANF binding sites (442 +/- 68 vs. 271 +/- 57 fmol/mg protein, P < 0.05) was reduced, but dissociation constant (0.26 +/- 0.04 vs. 0.41 +/- 0.08 nM) was not altered in CMO animals. Similar reductions were observed in the binding sites for brain natriuretic peptide (BNP; 438 +/- 83 vs. 236 +/- 53 fmol/mg protein) and CNP (321 +/- 80 vs. 165 +/- 56 fmol/mg protein, P < 0.05) which may reflect a decline in NPR-A and NPR-B and/or NPR-C. Acid wash improved binding of 125I-labeled rat ANF to lung membranes of both normal and CMO hamsters, but the tendency towards reduced binding in CMO hamsters did not reach statistical significance, implying that downregulation may not have been due only to prior occupancy of the receptors. Transcripts of NPR-A, NPR-B, and NPR-C receptors in hamster lungs were detected by quantitative polymerase chain reaction. Compared with normal controls, the CMO hamster lung NPR-A mRNA was reduced by 50%, but NPR-B mRNA and NPR-C mRNA were not altered. Moreover, CMO hamster lungs showed less activation of guanylyl cyclase by ANF. These studies demonstrate that lung NPR are downregulated in hamster CMO.
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Fenspiride and membrane transduction signals in rat alveolar macrophages.
Féray, J C; Mohammadi, K; Taouil, K; Brunet, J; Garay, R P; Hannaert, P
1997-07-15
Fenspiride inhibits the calcium signal evoked by the inflammatory peptide formyl-Met-Leu-Phe (fMLP) in peritoneal macrophages, but at concentrations (approximately 1 mM) far above the therapeutic range (approximately 1 microM). Here, in rat alveolar macrophages, high fenspiride concentrations (1 mM) were required to inhibit the calcium signals evoked by the calcium agonist Bay K8644 or by ionomycin. Moreover, fenspiride (1 mM) was a poor inhibitor of the cell membrane depolarization induced by gramicidine D. By contrast, fenspiride blocked Na+-H+ antiport activation by (i) fMLP with an IC50 = 3.1 +/- 1.9 nM and (ii) PMA (phorbol 12-myristate 13-acetate) with an IC50 = 9.2 +/- 3.1 nM. Finally, protein kinase C (PKC) activity of macrophage homogenate was not significantly modified by 10 or 100 microM fenspiride (at 100 microM: 2.57 +/- 1.60 vs. 2.80 +/- 1.71 pmol/10(6) cells/min). In conclusion, fenspiride inhibits fMLP- and PMA-induced pH signals in rat alveolar macrophages, probably by acting distally on the PKC transduction signal. This pH antagonistic action may be relevant for the antiinflammatory mechanism of fenspiride and requires further investigation.
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Drago-Serrano, Maria Elisa; Campos-Rodriguez, Rafael; Carrero, Julio Cesar; de la Garza, Mireya
2018-03-27
Lactoferrin (Lf) is a conserved cationic non-heme glycoprotein that is part of the innate immune defense system of mammals. Lf is present in colostrum, milk and mucosal sites, and it is also produced by polymorphonuclear neutrophils and secreted at infection sites. Lf and Lf N-terminus peptide-derivatives named lactoferricins (Lfcins) are molecules with microbiostatic and microbicidal action in a wide array of pathogens. In addition, they display regulatory properties on components of nonspecific immunity, including toll-like receptors, pro- and anti-inflammatory cytokines, and reactive oxygen species. Mechanisms explaining the ability of Lf and Lfcins to display both up- and down-modulatory properties on cells are not fully understood but result, in part, from their interactions with membrane receptors that elicit biochemical signal pathways, whereas other receptors enable the nuclear translocation of these molecules for the modulation of target genes. The dual role of Lf and Lfcins as antimicrobials and immunomodulators is of biotechnological and pharmaceutical interest. Native Lf and its peptide-derivatives from human and bovine sources, the recombinant versions of the human protein, and their synthetic peptides have potential application as adjunctive agents in therapies to combat infections caused by multi-resistant bacteria and those caused by fungi, protozoa and viruses, as well as in the prevention and reduction of several types of cancer and response to LPS-shock, among other effects. In this review, we summarize the immunomodulatory properties of the unique multifunctional protein Lf and its N-terminus peptides. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.
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Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven
2018-04-01
Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.
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Kimura, Richard H; Cheng, Zhen; Gambhir, Sanjiv Sam; Cochran, Jennifer R
2009-01-01
There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with low nM affinity to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or 64Cu-DOTA to their N-termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. Near-infrared fluorescence and microPET imaging both demonstrated that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 h post injection for two high affinity (IC50 ∼20 nM) 64Cu-DOTA-conjugated knottin peptides was 4.47 ± 1.21 and 4.56 ± 0.64 % injected dose/gram (%ID/g), compared to a low affinity knottin peptide (IC50 ∼0.4 μM; 1.48 ± 0.53 %ID/g) and c(RGDyK) (IC50 ∼1 μM; 2.32 ± 0.55 %ID/g), a low affinity cyclic pentapeptide under clinical development. Furthermore, 64Cu-DOTA-conjugated knottin peptides generated lower levels of non-specific liver uptake (∼2 %ID/g) compared to c(RGDyK) (∼4 %ID/g) 1 h post injection. MicroPET imaging results were confirmed by in vivo biodistribution studies. 64Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers. PMID:19276378
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Developing a novel catalytic approach for imine formation by using self-replicating catalyst
NASA Astrophysics Data System (ADS)
Nasir, Fatin Ilyani; Philp, Douglas; Hasbullah, Siti Aishah; Hassan, Nurul Izzaty
2015-09-01
Synthesis of imine compounds usually results in moderate yield due its reversibility characteristic and prone to hydrolysis. Hence, to increase the formation of imine compound, self-replicating catalyst was introduced. The self-replicating catalyst is the imine product itself. The first imine compound, 4-{[4-(3,5-Dimethyl-phenylcarbamoyl)-benzylidene]-amino}-phenyl)-acetic acid has been synthesized from 4-Amino-N-(3,5-dimethyl-phenyl)-benzamide and (4-formyl-phenyl)-acetic acid. Simultaneously, 4-formylbenzoic acid was reacted with thionyl chloride to produce 4-formylbenzoyl chloride, which was then reacted with 2-amino-4,6-dimethylpyridine in the presence of triethylamine to afford N-(4,6-dimethyl-pyridin-2-yl)-4-formyl-benzamide. N-(4,6-dimethyl-pyridin-2-yl)-4-formyl-benzamide formed then reacted with 4-amino-2-methylbenzoic acid to form the second imine derivative, 4-{[4-(4,6-dimethyl-pyridin-2-ylcarbamoyl)-benzylidene]-amino}-2-methyl-benzoic acid. The concentration time profile for the synthesis of self-replicating imine 1 reveals the classic sigmoidal shape characteristics of an autocatalytic process and the rate of the reaction are higher than that observed in the absence of recognition. In order to demonstrate the nature of self-replicating catalyst, a preformed imine 1 was doped into the reaction mixture of amine 1 and the corresponding aldehyde, 4-formylbenzoic acid. The insertion of substoichiometric amounts (15 mol%) of imine 1 at the start of the reaction has accelerated the rate formation of imine 1.
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Hsu, Cheng-Lung; Liu, Jai-Shin; Wu, Po-Long; Guan, Hong-Hsiang; Chen, Yuh-Ling; Lin, An-Chi; Ting, Huei-Ju; Pang, See-Tong; Yeh, Shauh-Der; Ma, Wen-Lung; Chen, Chung-Jung; Wu, Wen-Guey; Chang, Chawnshang
2014-12-01
Treatment with individual anti-androgens is associated with the development of hot-spot mutations in the androgen receptor (AR). Here, we found that anti-androgens-mt-ARs have similar binary structure to the 5α-dihydrotestosterone-wt-AR. Phage display revealed that these ARs bound to similar peptides, including BUD31, containing an Fxx(F/H/L/W/Y)Y motif cluster with Tyr in the +5 position. Structural analyses of the AR-LBD-BUD31 complex revealed formation of an extra hydrogen bond between the Tyr+5 residue of the peptide and the AR. Functional studies showed that BUD31-related peptides suppressed AR transactivation, interrupted AR N-C interaction, and suppressed AR-mediated cell growth. Combination of peptide screening and X-ray structure analysis may serve as a new strategy for developing anti-ARs that simultaneously suppress both wt and mutated AR function. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Gladysz, J. A.
1980-11-10
Mechanistic studies of anionic formyl complexes were made. The rate of decomposition of the formyl (CO)/sub 4/Mn(COC/sub 6/H/sub 5/)(CHO)/sup -/ anion as a function of counter-ion and trialkylborane present was examined. The deuterated formyl (CO)/sub 4/Mn(COC/sub 6/H/sub 5/)(CDO)/sup -/ was prepared and its decomposition was monitored by H/sup 2/ NMR. The data obtained indicate that the formyl decomposes by a disproportionative pathway not by initial CO loss, as commonly noted for formyl complexes.
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RFamide Peptides: Structure, Function, Mechanisms and Pharmaceutical Potential
Findeisen, Maria; Rathmann, Daniel; Beck-Sickinger, Annette G.
2011-01-01
Different neuropeptides, all containing a common carboxy-terminal RFamide sequence, have been characterized as ligands of the RFamide peptide receptor family. Currently, five subgroups have been characterized with respect to their N-terminal sequence and hence cover a wide pattern of biological functions, like important neuroendocrine, behavioral, sensory and automatic functions. The RFamide peptide receptor family represents a multiligand/multireceptor system, as many ligands are recognized by several GPCR subtypes within one family. Multireceptor systems are often susceptible to cross-reactions, as their numerous ligands are frequently closely related. In this review we focus on recent results in the field of structure-activity studies as well as mutational exploration of crucial positions within this GPCR system. The review summarizes the reported peptide analogs and recently developed small molecule ligands (agonists and antagonists) to highlight the current understanding of the pharmacophoric elements, required for affinity and activity at the receptor family. Furthermore, we address the biological functions of the ligands and give an overview on their involvement in physiological processes. We provide insights in the knowledge for the design of highly selective ligands for single receptor subtypes to minimize cross-talk and to eliminate effects from interactions within the GPCR system. This will support the drug development of members of the RFamide family.
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Blanes-Mira, Clara; Merino, Jaime M; Valera, Elvira; Fernández-Ballester, Gregorio; Gutiérrez, Luis M; Viniegra, Salvador; Pérez-Payá, Enrique; Ferrer-Montiel, Antonio
2004-01-01
Synthetic peptides patterned after the C-terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N-terminal-derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N-terminus of SNAP25, specially the segment that encompasses 22Ala-44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an alpha-helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+-dependent exocytosis from detergent-permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia-induced, glutamate-mediated excitotoxicity with a potency that rivalled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N-terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.
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Corin, Karolina; Baaske, Philipp; Ravel, Deepali B; Song, Junyao; Brown, Emily; Wang, Xiaoqiang; Wienken, Christoph J; Jerabek-Willemsen, Moran; Duhr, Stefan; Luo, Yuan; Braun, Dieter; Zhang, Shuguang
2011-01-01
A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.
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Corin, Karolina; Baaske, Philipp; Ravel, Deepali B.; Song, Junyao; Brown, Emily; Wang, Xiaoqiang; Wienken, Christoph J.; Jerabek-Willemsen, Moran; Duhr, Stefan; Luo, Yuan; Braun, Dieter; Zhang, Shuguang
2011-01-01
A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins. PMID:22132066
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Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.
2011-01-01
The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452
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Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric
2016-07-15
G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Lammerich, Hans-Peter; Busmann, Annette; Kutzleb, Christian; Wendland, Martin; Seiler, Petra; Berger, Claudia; Eickelmann, Peter; Meyer, Markus; Forssmann, Wolf-Georg; Maronde, Erik
2003-01-01
The human orphan G-protein coupled receptor bombesin receptor subtype 3 (hBRS-3) was screened for peptide ligands by a Ca2+ mobilization assay resulting in the purification and identification of two specific ligands, the naturally occurring VV-hemorphin-7 (VV-H-7) and LVV-hemorphin-7 (LVV-H-7), from human placental tissue. These peptides were functionally characterized as full agonists with unique specificity albeit low affinity for hBRS-3 compared to other bombesin receptors. VV-H-7 and LVV-H-7 induced a dose-dependent response in hBRS-3 overexpressing CHO cells, as well as in NCI-N417 cells expressing the hBRS-3 endogenously. The affinity of VV-H-7 was higher in NCI-N417 cells compared to overexpressing CHO cells. In detail, the EC50 values were 45±15 μM for VV-H-7 and 183±60 μM for LVV-H-7 in CHO cells, and 19±6 μM for VV-H-7 and 38±18 μM for LVV-H-7 in NCI-N417 cells. Other hemorphins had no effect. Gastrin-releasing peptide (GRP) and neuromedin B (NMB) showed similar EC50 values of 13–20 μM (GRP) and of 1–2 μM (NMB) on both cell lines. Structure-function analysis revealed that both the N-terminal valine and the C-terminal phenylalanine residues of VV-H-7 are critical for the ligand-receptor interaction. Endogenous hBRS-3 in NCI-N417 activated by VV-H-7 couples to phospholipase C resulting in changes of intracellular calcium, which is initially released from an inositol trisphosphate (IP3)-sensitive store followed by a capacitive calcium entry from extracellular space. VV-H-7-induced hBRS-3 activation led to phosphorylation of p42/p44-MAP kinase in NCI-N417 cells, but did not stimulate cell proliferation. In contrast, phosphorylation of focal adhesion kinase (p125FAK) was not observed. PMID:12721098
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Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke
2007-07-01
Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100< pmol/mouse) than when administered i.t. (ED(50): 1.34-4.51 pmol/mouse). These results suggest the involvement of nociceptin-like agonistic effects of the Ac-RYYRIK pharmacophore in the peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.
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Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells
NASA Technical Reports Server (NTRS)
Whitson, P. A.; Huls, M. H.; Sams, C. F.
1991-01-01
Atrial natriuretic peptide (ANP) binding and ANP-induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid-regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time- and concentration-dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase-linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5-27 (ANP 5-27) to modulate the accumulation of cGMP was found to be ANP greater than BNP much greater than ANP 5-27. Affinity cross-linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60-70 kD receptor, indicating the presence of the nonguanylate cyclase-linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5-27 and suggested that a large proportion of the ANP receptors present in blood-brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood-brain barrier endothelial cells.
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Nagasaki, Y; Kutsuna, T; Iijima, M; Kato, M; Kataoka, K; Kitano, S; Kadoma, Y
1995-01-01
Well-defined poly(ethylene oxide) (PEO) with a formyl group at one end and a hydroxyl group at the other terminus was synthesized by the anionic ring opening polymerization of ethylene oxide (EO) with a new organometallic initiator possessing an acetal moiety, potassium 3,3-diethoxypropyl alkoxide. Hydrolysis of the acetal moiety produced a formyl group-terminated heterobifunctional PEO with a hydroxyl group at the other end.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou
Tracheary Element Differentiation Inhibitory Factor (TDIF) belongs to the family of post-translationally modified CLE (CLAVATA3/embryo surrounding region (ESR)-related) peptide hormones that control root growth and define the delicate balance between stem cell proliferation and differentiation in SAM (shoot apical meristem) or RAM (root apical meristem). In Arabidopsis, Tracheary Element Differentiation Inhibitory Factor Receptor (TDR) and its ligand TDIF signaling pathway is involved in the regulation of procambial cell proliferation and inhibiting its differentiation into xylem cells. Here we present the crystal structures of the extracellular domains (ECD) of TDR alone and in complex with its ligand TDIF resolved at 2.65more » Åand 2.75 Å respectively. These structures provide insights about the ligand perception and specific interactions between the CLE peptides and their cognate receptors. Our in vitro biochemical studies indicate that the interactions between the ligands and the receptors at the C-terminal anchoring site provide conserved binding. While the binding interactions occurring at the N-terminal anchoring site dictate differential binding specificities between different ligands and receptors. Our studies will open different unknown avenues of TDR-TDIF signaling pathways that will enhance our knowledge in this field highlighting the receptor ligand interaction, receptor activation, signaling network, modes of action and will serve as a structure function relationship model between the ligand and the receptor for various similar leucine-rich repeat receptor-like kinases (LRR-RLKs).« less
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Pike, Victor W.; Rash, Karen S.; Chen, Zhaogen; Pedregal, Concepción; Statnick, Michael A.; Kimura, Yasuyuki; Hong, Jinsoo; Zoghbi, Sami S.; Fujita, Masahiro; Toledo, Miguel A.; Diaz, Nuria; Gackenheimer, Susan L.; Tauscher, Johannes T.; Barth, Vanessa N.; Innis, Robert B.
2011-01-01
Positron emission tomography (PET) coupled to an effective radioligand could provide an important tool for understanding possible links between neuropsychiatric disorders and brain NOP (nociceptin/orphanin FQ peptide) receptors. We sought to develop such a PET radioligand. High-affinity NOP ligands were synthesized based on a 3-(2'-fluoro-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)-2(2-halobenzyl)-N-alkylpropanamide scaffold and from experimental screens in rats, with ex vivo LC-MS/MS measures, three ligands were identified for labeling with carbon-11 and evaluation with PET in monkey. Each ligand was labeled by 11C-methylation of an N-desmethyl precursor and studied in monkey under baseline and NOP receptor-preblock conditions. The three radioligands, [11C](S)-10a–c, gave similar results. Baseline scans showed high entry of radioactivity into brain to give a distribution reflecting that expected for NOP receptors. Pre-block experiments showed high early peak levels of brain radioactivity which rapidly declined to a much lower level than seen in baseline scans, thereby indicating a high level of receptor-specific binding in baseline experiments. Overall, [11C](S)-10c showed the most favorable receptor-specific signal and kinetics and is now selected for evaluation in human subjects. PMID:21438532
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Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu
2017-07-01
N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.
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Cardiac distribution of the binding sites for natriuretic peptides in vertebrates.
Cerra, M C
1994-12-01
Natriuretic peptides are hormones that play an important role in the cardiovascular control of mammalian and non-mammalian vertebrates. They have been classified into four groups. Of these, ANP (atrial natriuretic peptide), BNP (brain atriuretic peptides), CNP (C-type natriuretic peptide) are detected in cardiac and non cardiac tissues of all vertebrates; while VNP (ventricular natriuretic peptide) has been isolated only from the fish ventricle. All peptides have shown a high degree of sequence homology. The expression of the three principal types of natriuretic peptide (ANP, BNP and CNP) in cardiac tissues is developmentally and functionally regulated in a highly tissue-specific manner. Three types of natriuretic peptide receptors have been identified in numerous target tissues. Two receptors are transmembrane guanylyl cyclases (ANPR-A and ANPR-B) that mediate biological effects of natriuretic peptides; the third one (ANPR-C) has no guanylyl cyclase and is called "clearance receptor." The presence of natriuretic peptide binding sites in the heart suggests new aspects of paracrine control of cardiac function. A relevant localization of natriuretic peptide receptors was found in those cardiac regions particularly suitable for monitoring blood volume and pressure oscillations such as the inflow tract and the outflow tract. For example, in birds (quail) the highest levels of natriuretic peptide receptors were detected in the inflow tract represented by the vena cava. In both fish and birds, the outflow chamber, the bulbus cordis, had a high number of natriuretic peptide binding sites. In mammals, a remarkable concentration of natriuretic peptide receptors was also observed in the coronary vessels. This zoning of cardiac natriuretic peptide receptors indicates an intracardiac action of the hormones and adds a humoral dimension to the morphofunctional design of the vertebrate heart.
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Ulfig, Agnes; Freudl, Roland
2018-05-11
The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.
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Lu, Jie; Hathaway, Helen J; Royce, Melanie E; Prossnitz, Eric R; Miao, Yubin
2014-02-01
The purpose of this study was to examine whether the introduction of D-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated D-Lys(6)-GnRH peptides. Building upon the construct of DOTA-Ahx-(D-Lys(6)-GnRH1) we previously reported, an aromatic amino acid of D-Phe was inserted either between the DOTA and Ahx or between the Ahx and D-Lys(6) to generate new DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) or DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) peptides. Compared to DOTA-Ahx-(D-Lys(6)-GnRH1) (36.1 nM), the introduction of D-Phe improved the GnRH receptor binding affinities of DOTA-D-Phe-Ahx-(D-Lys(6)-GnRH) (16.3 nM) and DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1), (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using (111)In-DOTA-Ahx-D-Phe-(D-Lys(6)-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future. Copyright © 2014 Elsevier Ltd. All rights reserved.
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New trends in the development of opioid peptide analogues as advanced remedies for pain relief.
Gentilucci, Luca
2004-01-01
The search for new peptides to be used as analgesics in place of morphine has been mainly directed to develop peptide analogues or peptidomimetics having higher biological stability and receptor selectivity. Indeed, most of the alkaloid opioid counterindications are due to the scarce stability and the contemporary activation of different receptor types. However, the development of several extremely stable and selective peptide ligands for the different opioid receptors, and the recent discovery of the micro-receptor selective endomorphins, rendered this search less fundamental. In recent years, other opioid peptide properties have been investigated in the search for new pharmacological tools. The utility of a drug depends on its ability to reach appropriate receptors at the target tissue and to remain metabolically stable in order to produce the desired effect. This review deals with the recent investigations on peptide bioavailability, in particular barrier penetration and resistance against enzymatic degradation; with the development of peptides having activity at different receptors; with chimeric peptides, with propeptides, and with non-conventional peptides, lacking basic pharmacophoric features.
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Stabilization of exosome-targeting peptides via engineered glycosylation.
Hung, Michelle E; Leonard, Joshua N
2015-03-27
Exosomes are secreted extracellular vesicles that mediate intercellular transfer of cellular contents and are attractive vehicles for therapeutic delivery of bimolecular cargo such as nucleic acids, proteins, and even drugs. Efficient exosome-mediated delivery in vivo requires targeting vesicles for uptake by specific recipient cells. Although exosomes have been successfully targeted to several cellular receptors by displaying peptides on the surface of the exosomes, identifying effective exosome-targeting peptides for other receptors has proven challenging. Furthermore, the biophysical rules governing targeting peptide success remain poorly understood. To evaluate one factor potentially limiting exosome delivery, we investigated whether peptides displayed on the exosome surface are degraded during exosome biogenesis, for example by endosomal proteases. Indeed, peptides fused to the N terminus of exosome-associated transmembrane protein Lamp2b were cleaved in samples derived from both cells and exosomes. To suppress peptide loss, we engineered targeting peptide-Lamp2b fusion proteins to include a glycosylation motif at various positions. Introduction of this glycosylation motif both protected the peptide from degradation and led to an increase in overall Lamp2b fusion protein expression in both cells and exosomes. Moreover, glycosylation-stabilized peptides enhanced targeted delivery of exosomes to neuroblastoma cells, demonstrating that such glycosylation does not ablate peptide-target interactions. Thus, we have identified a strategy for achieving robust display of targeting peptides on the surface of exosomes, which should facilitate the evaluation and development of new exosome-based therapeutics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Singh, Anamika; Dirain, Marvin; Witek, Rachel; Rocca, James R.; Edison, Arthur S; Haskell-Luevano, Carrie
2013-01-01
The melanocortin-3 (MC3) and melanocortin-4 (MC4) receptors regulate energy homeostasis, food intake, and associated physiological conditions. The MC4R has been studied extensively. Less is known about specific physiological roles of the MC3R. A major obstacle to this lack of knowledge is attributed to a limited number of identified MC3R selective ligands. We previously reported a spatial scanning approach of a 10-membered thioether-heterocycle ring incorporated into a chimeric peptide template that identified a lead nM MC4R ligand. Based upon those results, 17 compounds were designed and synthesized that focused upon modification in the pharmacophore domain. Notable results include the identification of a 0.13 nM potent 5800-fold mMC3R selective antagonist/slight partial agonist versus a 760 nM mMC4R full agonist (ligand 11). Biophysical experiments (2D 1H NMR and computer assisted molecular modeling) of this ligand resulted in the identification of an inverse γ-turn secondary structure in the ligand pharmacophore domain. PMID:23432160
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Nagai, Junya; Saito, Masaki; Adachi, Yoshinori; Yumoto, Ryoko; Takano, Mikihisa
2006-05-01
Our previous studies showed that coadministration of cytochrome c and a 20-residue basic peptide, N-WASP181-200 (NISHTKEKKKGKAKKKRLTK, pI=10.87) inhibits renal accumulation of gentamicin. In this study, we examined effects of ligands of megalin, an endocytic receptor involved in renal uptake of gentamicin, and basic peptides including N-WASP180-200 and its mutant peptides on gentamicin binding to isolated rat renal brush-border membrane (BBM). Gentamicin binding to BBM was inhibited by megalin ligands, basic peptide fragments of cytochrome c, and N-WASP181-200 in a concentration-dependent manner. Klotz plot analysis showed that N-WASP181-200 inhibited the binding of gentamicin in a competitive manner. By substituting glycines for lysines in N-WASP181-200 at positions 9 and 15, the inhibitory effect on gentamicin binding to BBM was reduced, which may be related to a decrease in the alpha-helix content in the peptide. Gentamicin binding to BBM treated with trypsin, in which megalin completely disappeared, was significantly but not completely decreased compared with the native BBM. In addition, treatment of BBM with trypsin led to a decrease in the inhibitory effect of N-WASP181-200 on gentamicin binding. These observations support that megalin ligands and basic peptides including N-WASP181-200 decrease renal accumulation of gentamicin by inhibiting its binding to BBM of proximal tubule cells, partly interacting with megalin. In addition, the alpha-helix conformation may play an important role in the inhibitory effect of N-WASP181-200 on the binding of gentamicin to BBM.
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Trujillo, Cleber A.; Sathler, Luciana B.; Juliano, Maria A.; Juliano, Luiz; Ulrich, Henning; Ferreira, Sergio T.
2013-01-01
Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD. PMID:23894286
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Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun
2015-06-01
Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.
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Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander
2015-12-01
We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Real Hernandez, Luis M; Gonzalez de Mejia, Elvira
2017-04-01
Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (-7.2 to -7.0kcal/mol) and the cowpea bean dipeptide Lys-Asp (-7.0kcal/mol) had higher binding affinities than ezetimibe (-6.6kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (-7.2kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption. Copyright © 2017 Elsevier Inc. All rights reserved.
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Gupta, Shyam L; Dhiman, Vikas; Jayasekharan, T; Sahoo, N K
2016-06-15
The increased use of silver nanoparticles (AgNPs) for various biological applications, and over-expression of various peptide receptors in different tumors/cancer cells, necessitate the need for dedicated investigations on the intrinsic binding ability of Ag with various biologically important peptides for better understanding of AgNPs-peptide interactions and for the future development of contrasting agents as well as drugs for imaging/biomedical applications. The [M+(Ag)n ](+) complexes are prepared and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Silver complexes of the peptides [M+(Ag)n ](+) , where M = oxytocin, arg(8) -vasopressin, bradykinin, bombesin, somatostatin, and neurotensin, have been investigated for their intrinsic Ag(+) -binding ability. Unusual binding of up to seven Ag(+) with these small peptides is observed. The mass spectra show n = 1-5 for bombesin and somatostatin, n = 1-6 for bradykinin and arg(8) -vasopressin, and n = 1-7 for oxytocin and neurotensin. In addition, oxytocin and arg(8) -vasopressin show the formation of dimers and their complexes [M2 +(Ag)n ](+) with n = 1-8 and n = 1-5, respectively. The possible amino acid residues responsible for Ag(+) binding in each peptide have been identified on the basis of density functional theory (DFT)-calculated binding energy values of Ag(+) towards individual amino acids. Mass spectrometric evidence indicates that the peptides, viz., oxytocin, arg(8) -vasopressin, bradykinin, bombesin, somatostatin, and neurotensin, show greater affinity for Ag(+) . Hence, they may be used as carriers for AgNPs in targeted drug delivery as well as an alternative for iodinated contrasting agents in dual energy X-ray imaging techniques. Radio-labeled Ag with these peptides can also be used in radio-pharmaceuticals for diagnostic and therapeutic applications. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
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Wang, Jia-Hui; Shao, Xiao-Xia; Hu, Meng-Jun; Wei, Dian; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun
2017-05-01
Relaxin family peptide receptor 3 (RXFP3) is an A-class G protein-coupled receptor that is implicated in the regulation of food intake and stress response upon activation by its cognate agonist relaxin-3. To study its interaction with various ligands, we developed a novel bioluminescence resonance energy transfer (BRET)-based binding assay using the brightest NanoLuc as an energy donor and a newly developed cyan-excitable orange fluorescent protein (CyOFP) as an energy acceptor. An engineered CyOFP without intrinsic cysteine residues but with an introduced cysteine at the C-terminus was overexpressed in Escherichia coli and chemically conjugated to the A-chain N-terminus of an easily labeled chimeric R3/I5 peptide via an intermolecular disulfide linkage. After the CyOFP-conjugated R3/I5 bound to a shortened human RXFP3 (removal of 33 N-terminal residues) fused with the NanoLuc reporter at the N-terminus, high BRET signals were detected. Saturation binding and real-time binding assays demonstrated that this BRET pair retained high binding affinity with fast association/dissociation. Using this BRET pair, binding potencies of various ligands with RXFP3 were conveniently measured through competition binding assays. Thus, the novel BRET-based binding assay facilitates interaction studies of RXFP3 with various ligands. The engineered CyOFP without intrinsic cysteine residues may also be applied to other BRET-based binding assays in future studies.
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Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.
Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram
2002-02-01
We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.
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Toward an effective peripheral visceral analgesic: responding to the national opioid crisis.
Camilleri, Michael
2018-06-01
This minireiew summarizes recent new developments in visceral analgesics. This promising field is important, as a new approach to address abdominal pain with peripheral visceral analgesics is considered a key approach to addressing the current opioid crisis. Some of the novel compounds address peripheral pain mechanisms through modulation of opioid receptors via biased ligands, nociceptin/orphanin FQ opioid peptide (NOP) receptor, or dual action on NOP and μ-opioid receptor, buprenorphine and morphiceptin analogs. Other compounds target nonopioid mechanisms, including cannabinoid (CB2), N-methyl-d-aspartate, calcitonin gene-related peptide, estrogen, and adenosine A 2B receptors and transient receptor potential (TRP) channels (TRPV1, TRPV4, and TRPM8). Although current evidence is based predominantly on animal models of visceral pain, early human studies also support the evidence from the basic and animal research. This augurs well for the development of nonaddictive, visceral analgesics for treatment of chronic abdominal pain, an unmet clinical need.
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Myoinhibiting peptides are the ancestral ligands of the promiscuous Drosophila sex peptide receptor
USDA-ARS?s Scientific Manuscript database
Male insects change behaviors of female partners by co-transferring accessory gland proteins (Acps) like sex peptide (SP), with their sperm. The Drosophila sex peptide receptor (SPR) is a G protein-coupled receptor expressed in the female’s nervous system and genital tract. While most Acps show a fa...
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Ma, Xianyue; Cline, Kenneth
2013-03-01
Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.
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Antipas, Georgios S E; Germenis, Anastasios E
2015-06-01
The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC) and a T cell receptor (TCR) is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1) peptide along with three artificially altered variants, all of which were presented by the (Class I) HLA-A201 protein in complexation with the human (CD8(+)) A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist) and peptide pair distribution function (PDF). In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT) datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3 (+)) terminal group while antagonist peptides are not.
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Khan, Mahjabin; Huang, Tao; Lin, Cheng-Yuan; Wu, Jiang; Fan, Bao-Min; Bian, Zhao-Xiang
2017-01-01
Lung cancer, claiming millions of lives annually, has the highest mortality rate worldwide. This advocates the development of novel cancer therapies that are highly toxic for cancer cells but negligibly toxic for healthy cells. One of the effective treatments is targeting overexpressed surface receptors of cancer cells with receptor-specific drugs. The receptors-in-focus in the current review are the G-protein coupled receptors (GPCRs), which are often overexpressed in various types of tumors. The peptide subfamily of GPCRs is the pivot of the current article owing to the high affinity and specificity to and of their cognate peptide ligands, and the proven efficacy of peptide-based therapeutics. The article summarizes various ectopically expressed peptide GPCRs in lung cancer, namely, Cholecystokinin-B/Gastrin receptor, the Bombesin receptor family, Bradykinin B1 and B2 receptors, Arginine vasopressin receptors 1a, 1b and 2, and the Somatostatin receptor type 2. The autocrine growth and pro-proliferative pathways they mediate, and the distinct tumor-inhibitory effects of somatostatin receptors are then discussed. The next section covers how these pathways may be influenced or ‘corrected’ through therapeutics (involving agonists and antagonists) targeting the overexpressed peptide GPCRs. The review proceeds on to Nano-scaled delivery platforms, which enclose chemotherapeutic agents and are decorated with peptide ligands on their external surface, as an effective means of targeting cancer cells. We conclude that targeting these overexpressed peptide GPCRs is potentially evolving as a highly promising form of lung cancer therapy. PMID:29262666
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Burov, Sergey; Moskalenko, Yulia; Dorosh, Marina; Shkarubskaya, Zoya; Panarin, Evgeny
2009-11-01
N-terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N-amidino-amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block-N-amidino-pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N-amidino-proline using RuO(4) did not produce positive results, N-amidino-Glp-Phe-OH was synthesized on Wang polymer by cyclization of alpha-guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N-amidino-Glp-Phe-OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N-amidino-Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach.
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Alexander, Jodi L.; Oliphant, Andrew; Wilcockson, David C.; Audsley, Neil; Down, Rachel E.; Lafont, Rene; Webster, Simon G.
2018-01-01
Neuropeptides play a central role as neurotransmitters, neuromodulators and hormones in orchestrating arthropod physiology. The post-genomic surge in identified neuropeptides and their putative receptors has not been matched by functional characterization of ligand-receptor pairs. Indeed, until very recently no G protein-coupled receptors (GPCRs) had been functionally defined in any crustacean. Here we explore the structurally-related, functionally-diverse gonadotropin-releasing hormone paralogs, corazonin (CRZ) and red-pigment concentrating hormone (RPCH) and their G-protein coupled receptors (GPCRs) in the crab, Carcinus maenas. Using aequorin luminescence to measure in vitro Ca2+ mobilization we demonstrated receptor-ligand pairings of CRZ and RPCH. CRZR-activated cell signaling in a dose-dependent manner (EC50 0.75 nM) and comparative studies with insect CRZ peptides suggest that the C-terminus of this peptide is important in receptor-ligand interaction. RPCH interacted with RPCHR with extremely high sensitivity (EC50 20 pM). Neither receptor bound GnRH, nor the AKH/CRZ-related peptide. Transcript distributions of both receptors indicate that CRZR expression was, unexpectedly, restricted to the Y-organs (YO). Application of CRZ peptide to YO had no effect on ecdysteroid biosynthesis, excepting a modest stimulation in early post-molt. CRZ had no effect on heart activity, blood glucose levels, lipid mobilization or pigment distribution in chromatophores, a scenario that reflected the distribution of its mRNA. Apart from the well-known activity of RPCH as a chromatophorotropin, it also indirectly elicited hyperglycemia (which was eyestalk-dependent). RPCHR mRNA was also expressed in the ovary, indicating possible roles in reproduction. The anatomy of CRZ and RPCH neurons in the nervous system is described in detail by immunohistochemistry and in situ hybridization. Each peptide has extensive but non-overlapping distribution in the CNS, and neuroanatomy suggests that both are possibly released from the post-commissural organs. This study is one of the first to deorphanize a GPCR in a crustacean and to provide evidence for hitherto unknown and diverse functions of these evolutionarily-related neuropeptides. PMID:29379412
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Radford, Jonathan C; Terhzaz, Selim; Cabrero, Pablo; Davies, Shireen-A; Dow, Julian A T
2004-12-01
Identification of the Anopheles gambiae leucokinin gene from the completed A. gambiae genome revealed that this insect species contains three leucokinin peptides, named Anopheles leucokinin I-III. These peptides are similar to those identified in two other mosquito species, Aedes aegypti and Culex salinarius. Additionally, Anopheles leucokinin I displays sequence similarity to Drosophila melanogaster leucokinin. Using a combination of computational and molecular approaches, a full-length cDNA for a candidate leucokinin-like receptor was isolated from A. stephensi, a close relative of A. gambiae. Alignment of the known leucokinin receptors--all G protein-coupled receptors (GPCRs)--with this receptor, identified some key conserved regions within the receptors, notably transmembrane (TM) domains I, II, III, VI and VII. The Anopheles leucokinins and receptor were shown to be a functional receptor-ligand pair. All three Anopheles leucokinins caused a dose-dependent rise in intracellular calcium ([Ca2+]i) when applied to S2 cells co-expressing the receptor and an aequorin transgene, with a potency order of I>II>III. Drosophila leucokinin was also found to activate the Anopheles receptor with a similar EC50 value to Anopheles leucokinin I. However, when the Anopheles peptides were applied to the Drosophila receptor, only Anopheles leucokinin I and II elicited a rise in [Ca2+]i. This suggests that the Anopheles receptor has a broader specificity for leucokinin ligands than the Drosophila receptor. Antisera raised against the Anopheles receptor identified a doublet of approx. 65 and 72 kDa on western blots, consistent with the presence of four N-glycosylation sites within the receptor sequence, and the known glycosylation of the receptor in Drosophila. In Anopheles tubules, as in Drosophila, the receptor was localised to the stellate cells. Thus we provide the first identification of Anopheles mosquito leucokinins (Anopheles leucokinins) and a cognate leucokinin receptor, characterise their interaction and show that Dipteran leucokinin signalling is closely conserved between Drosophila and Anopheles.
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Kojima, Shu-Ichi; Kojima, Ken; Fujita, Tomoe
2017-03-15
The effect of a 5-HT 3 receptor-selective agonist SR57227A was investigated on the outflow of 5-hydroxytryptamine (5-HT) from isolated muscle layer-free mucosal preparations of guinea-pig colon. The mucosal preparations were incubated in vitro and the outflow of 5-HT from these preparations was determined by high-performance liquid chromatography with electrochemical detection. SR57227A (100μM) produced a tetrodotoxin-resistant and sustained increase in the outflow of 5-HT from the mucosal preparations. The SR57227A-evoked sustained 5-HT outflow was completely inhibited by the 5-HT 3 receptor antagonist ramosetron (1μM). The neuropeptide Y 1 receptor antagonist BIBO3304 (100nM) partially inhibited the SR57227A-evoked sustained 5-HT outflow, but the Y 2 receptor antagonist BIIE0246 (1μM) or the glucagon-like peptide-1 (GLP-1) receptor antagonist exendin-(9-39) (1μM), showed a minimal effect on the SR57227A-evoked sustained 5-HT outflow. In the presence of BIBO3304 (100nM) and exendin-(9-39) (1μM), SR57227A (100μM) failed to produce a sustained increase in the outflow of 5-HT. The Y 1 receptor agonist [Leu 31 , Pro 34 ]-neuropeptide Y (10nM), but not GLP-1-(7-36) amide (100nM), produced a sustained increase in the outflow of 5-HT. We found that 5-HT 3 receptor-triggered 5-HT release from guinea-pig colonic mucosa is mediated by the activation of 5-HT 3 receptors located at endocrine cells (enterochromaffin cells and peptide YY (PYY)-containing endocrine cells). The activation of both Y 1 and GLP-1 receptors appears to be required for the maintenance of 5-HT 3 receptor-triggered 5-HT release. It is therefore considered that 5-HT 3 receptors located at colonic mucosa play a crucial role in paracrine signaling between enterochromaffin cells and PYY-containing endocrine cells. Copyright © 2017 Elsevier B.V. All rights reserved.
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David, Marion; Lécorché, Pascaline; Masse, Maxime; Faucon, Aude; Abouzid, Karima; Gaudin, Nicolas; Varini, Karine; Gassiot, Fanny; Ferracci, Géraldine; Jacquot, Guillaume; Vlieghe, Patrick
2018-01-01
Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells—or organs that express the LDLR. PMID:29485998
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Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition
Hubers, Scott A.; Brown, Nancy J.
2016-01-01
Heart failure affects approximately 5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the FDA approved the first of a new class of drugs for the treatment of heart failure; valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses two of the pathophysiologic mechanisms of heart failure - activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared to enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacologic properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension. PMID:26976916
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Zhang, Xue-Wei; Chen, Shi-Lu
2018-05-11
The initial step of methanogenesis is the fixation of CO 2 to formyl-methanofuran (formyl-MFR) catalyzed by formyl-MFR dehydrogenase, which can be divided into two half reactions. Herein, the second half reaction catalyzed by FwdA (formyl-methanofuran dehydrogenase subunit A), i.e., from formate to formyl-methanofuran, has been investigated using density functional theory and a chemical model based on the X-ray crystal structure. The calculations indicate that, compared with other well-known di-zinc hydrolases, the FwdA reaction employs a reverse mechanism, including the nucleophilic attack of MFR amine on formate carbon leading to a tetrahedral gem-diolate intermediate, two steps of proton transfer from amine to formate moieties assisted by the Asp385, and the CO bond dissociation to form the formyl-MFR product. The second step of proton transfer from the amine moiety to the Asp385 is rate-limiting with an overall barrier of 21.2 kcal/mol. The two zinc ions play an important role in stabilizing the transition states and intermediates, in particular the negative charge at the formate moiety originated from the nucleophilic attack of the MFR amine. The work here appends a crucial piece in the methanogenic mechanistics and advances the understanding of the global carbon cycle. Copyright © 2018 Elsevier Inc. All rights reserved.
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Ding, H; Hayashida, K; Suto, T; Sukhtankar, D D; Kimura, M; Mendenhall, V; Ko, M C
2015-01-01
Background and Purpose Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor agonists display a promising analgesic profile in preclinical studies. However, supraspinal N/OFQ produced hyperalgesia in rodents and such effects have not been addressed in primates. Thus, the aim of this study was to investigate the effects of centrally administered ligands on regulating pain and itch in non-human primates. In particular, nociceptive thresholds affected by intracisternal N/OFQ were compared with those of morphine and substance P, known to provide analgesia and mediate hyperalgesia, respectively, in humans. Experimental Approach Intrathecal catheters were installed to allow intracisternal and lumbar intrathecal administration in awake and unanaesthetized rhesus monkeys. Nociceptive responses were measured using the warm water tail-withdrawal assay. Itch scratching responses were scored from videotapes recording behavioural activities of monkeys in their home cages. Antagonist studies were conducted to validate the receptor mechanisms underlying intracisternally elicited behavioural responses. Key Results Intracisternal morphine (100 nmol) elicited more head scratches than those after intrathecal morphine. Distinct dermatomal scratching locations between the two routes suggest a corresponding activation of supraspinal and spinal μ receptors. Unlike intracisternal substance P, which induced hyperalgesia, intracisternal N/OFQ (100 nmol) produced antinociceptive effects mediated by NOP receptors. Neither peptide increased scratching responses. Conclusions and Implications Taken together, these results demonstrated differential actions of ligands in the primate supraspinal region in regulating pain and itch. This study not only improves scientific understanding of the N/OFQ-NOP receptor system in pain processing but also supports the therapeutic potential of NOP-related ligands as analgesics. PMID:25752320
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Regulation and signaling of human bombesin receptors and their biological effects.
Weber, H Christian
2009-02-01
This review will highlight recent advances in the understanding of molecular mechanisms by which mammalian bombesin receptors are regulated and which intracellular signaling pathways have been characterized to mediate agonist-dependent receptor biological effects. Mammalian bombesin receptors have been demonstrated to be involved in a larger array of physiological and pathophysiological conditions than previously reported. Pharmacological experiments in vitro and in vivo as well as utilization of animals genetically deficient of the gastrin-releasing peptide receptor demonstrated roles in memory and fear behavior, lung development and injury, small intestinal cell repair, autocrine tumor growth, and mediating signals for pruritus and penile reflexes. Intracellular signaling studies predominantly of the gastrin-releasing peptide receptor owing to its frequent overexpression in some human malignancies showed that PI3 kinase activation is an important mechanism of cell proliferation. Tumor cell treatment including gastrin-releasing peptide receptor antagonists combined with inhibition of epidermal growth factor receptor resulted in an additive effect on blocking cell proliferation. Novel molecular mechanisms of the orphan bombesin receptor subtype-3 and gastrin-releasing peptide receptor gene regulation have been elucidated. Inhibition of gastrin-releasing peptide receptor signaling in human malignancies represents an attractive target for pharmacological treatment. Novel functions of bombesin related peptides have been identified including processes in the central nervous system, lung and intestinal tract.
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Zhang, Xiao-min; Yan, Xun-yi; Zhang, Bin; Yang, Qian; Ye, Mao; Cao, Wei; Qiang, Wen-bin; Zhu, Li-jun; Du, Yong-lan; Xu, Xing-xing; Wang, Jia-sheng; Xu, Fei; Lu, Wei; Qiu, Shuang; Yang, Wei; Luo, Jian-hong
2015-01-01
The N-methyl-D-aspartate receptor (NMDAR) in adult forebrain is a heterotetramer mainly composed of two GluN1 subunits and two GluN2A and/or GluN2B subunits. The synaptic expression and relative numbers of GluN2A- and GluN2B-containing NMDARs play critical roles in controlling Ca2+-dependent signaling and synaptic plasticity. Previous studies have suggested that the synaptic trafficking of NMDAR subtypes is differentially regulated, but the precise molecular mechanism is not yet clear. In this study, we demonstrated that Bip, an endoplasmic reticulum (ER) chaperone, selectively interacted with GluN2A and mediated the neuronal activity-induced assembly and synaptic incorporation of the GluN2A-containing NMDAR from dendritic ER. Furthermore, the GluN2A-specific synaptic trafficking was effectively disrupted by peptides interrupting the interaction between Bip and GluN2A. Interestingly, fear conditioning in mice was disrupted by intraperitoneal injection of the interfering peptide before training. In summary, we have uncovered a novel mechanism for the activity-dependent supply of synaptic GluN2A-containing NMDARs, and demonstrated its relevance to memory formation. PMID:26088419
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Seebach, Dieter; Lukaszuk, Aneta; Patora-Komisarska, Krystyna; Podwysocka, Dominika; Gardiner, James; Ebert, Marc-Olivier; Reubi, Jean Claude; Cescato, Renzo; Waser, Beatrice; Gmeiner, Peter; Hübner, Harald; Rougeot, Catherine
2011-05-01
The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β(2) - and of the C-terminal amino acid residue by a β(3) -homo-amino acid moiety (β(2) hXaa and β(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.
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Peptides as modifiers of Na+-induced pinocytosis in starved Amoeba proteus.
Josefsson, J O; Johansson, P
1985-01-01
Low concentrations of six peptide hormones; glucagon, vasoactive intestinal peptide, substance P, angiotensin II, lysine-vasopressin, arginine-vasopressin, and the chemotactic peptide fMet-Leu-Phe, activated the capacity for pinocytosis in starved Amoeba proteus. Competitive inhibitors of the chemotactic peptide in leucocytes inhibited activation by fMet-Leu-Phe, suggesting that its action in the amoeba is mediated by specific receptors. The opioid peptides, beta-endorphin, dynorphin (1-13) and leu-enkephalin abolished through a naloxone-sensitive mechanism activation by hormones and several other activating agents. Also, low concentrations of beef and pork insulin inhibited activation by peptide hormones. An insulin analogue of low potency in mammalian cells was inactive in the amoeba. These results support the hypothesis that besides opioid receptors, there may be insulin receptors and possibly receptors for several other peptide hormones in Amoeba proteus.
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Cheung, Adrian Wai-Hing; Qi, Lida; Gore, Vijay; Chu, Xin-Jie; Bartkovitz, David; Kurylko, Grazyna; Swistok, Joseph; Danho, Waleed; Chen, Li; Yagaloff, Keith
2005-12-15
Two libraries of hMC4R agonists, X-Y-DPhe(7)-Arg(8)-2-Nal(9)-Z-NH(2) and X-Y-DPhe(7)-Arg(8)-Trp(9)-Z-NH(2), totaling 185 peptides were prepared using Irori radiofrequency tagging technology and Argonaut Quest 210 Synthesizer, where X stands for N-caps, Y for His(6) surrogates and Z for Gly(10) surrogates. As a result of this study, His-modified pentapeptides with Trp were found to be more hMC4R potent than the corresponding 2-Nal analogs, novel N-caps and Gly surrogates were identified and 19 new peptides which are potent hMC4R agonists (EC(50) 1-15nM) and selective against hMC1R were discovered.
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Naranda, Tatjana; Wong, Kenneth; Kaufman, R. Ilene; Goldstein, Avram; Olsson, Lennart
1999-01-01
Applying a homology search method previously described, we identified a sequence in the extracellular dimerization site of the erythropoietin receptor, distant from the hormone binding site. A peptide identical to that sequence was synthesized. Remarkably, it activated receptor signaling in the absence of erythropoietin. Neither the peptide nor the hormone altered the affinity of the other for the receptor; thus, the peptide does not bind to the hormone binding site. The combined activation of signal transduction by hormone and peptide was strongly synergistic. In mice, the peptide acted like the hormone, protecting against the decrease in hematocrit caused by carboplatin. PMID:10377456
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Chrysant, Steven G
2017-07-01
LCZ-696, sacubitril/valsartan, is a dual-acting molecule consisting of the angiotensin II (Ang II) receptor blocker valsartan and the neprilysin (neutral endopeptidase) inhibitor AHU-377 with significant beneficial effects in patients with hypertension and heart failure (HF). Several recent studies have demonstrated a higher effectiveness of LCZ-696 compared to valsartan in the treatment of hypertension and HF. The rationale for the development and the Food and Drug Administration approval of LCZ-696 was based on the concept of an additive effect of the Ang II receptor blocker valsartan and the neutral endopeptidase (neprilysin) inhibitor AHU-377 for the treatment of hypertension and HF. The synergism from these drugs arises from the vasodilating effects of valsartan through its blockade of Ang II type 1 receptor and the action of natriuretic peptides atrial natriuretic peptide and B-type natriuretic peptide (BNP) by preventing their catabolism with neprilysin resulting in increase of cyclic guanosine monophosphate. This action of neprilysin is associated with increased natriuresis, diuresis, and systemic vasodilation, since these peptides have been shown to have potent diuretic, natriuretic, and vasodilating effects. In addition, it reduces the levels of N terminal pro-BNP. Therefore, administration of LCZ-696 results in significant reduction of wall stress from pressure and volume overload of the left ventricle as demonstrated by the reduction of N terminal pro-BNP, both significant constituents of hypertension and HF, and it is safe, well tolerated and is almost free of cough and angioedema. Copyright © 2017 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.
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Structural analysis of the interaction of IGF I with the IGF types 1 and 2 and insulin receptors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cascieri, M.A.; Chicchi, G.G.; Hayes, N.S.
1987-05-01
A synthetic gene for human IGF I has been synthesized which directs the synthesis and secretion of fully active human IGF I (rIGF I) from yeast. rIGF I inhibits binding of /sup 125/I-IGF I to type 1 IGF receptors from human placenta (IGF-R1, IC50 = 4 nM), binding of /sup 125/I-insulin to insulin receptors (IR, IC50 = 881 nM), binding of /sup 125/I-MSA to type 2 IGF receptors from rat liver (IGF-R2, IC50 = 80 nM), and binding of /sup 125/I-IGF I to crude human serum binding protein (hBP, IC50 = 0.42 nM). rIGF I is equipotent to human IGFmore » I in stimulating glucose transport in murine BC3H1 cells and in stimulating DNA synthesis in rat A10 cells. Site directed mutagenesis of the synthetic gene is being used to characterize the structural requirements for binding to these receptors. IGF I (FFY) B(23-25) is equipotent to rIGF I at the IGF-R1 (6.9 nM), the IGF-R2 (36 nM), and the IR (841 nM) and is less potent at the hBP (1.7 nM). In contrast, IGF I(SFY) B(23-25) is 20-fold less potent than rIGF I at the IGF-R1 and is 10-fold less potent than rIGF I at hBP. This peptide is greater than 10-fold less active at the IGF-R2 and the IR. This peptide is a full agonist in the cell assays but 20-50 fold less potent than rIGF I. These data are consistent with the hypothesis that the F to S change destabilizes the tertiary structure of IGF I.« less
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Zhang, Y; Simpson-Durand, C D; Standifer, K M
2015-01-01
Single-prolonged stress (SPS), a rat model of post-traumatic stress disorder (PTSD), also induces long-lasting hyperalgesia associated with hypocortisolism and elevated nociceptin/orphanin FQ (N/OFQ) levels in serum and CSF. Here, we determined the effect of JTC-801 (N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride), a nociceptin/orphanin FQ peptide (NOP) receptor antagonist, on symptoms of pain and anxiety in rats after SPS exposure, and examined N/OFQ-NOP receptor system changes. Male Sprague Dawley rats received JTC-801 (6 mg kg(-1) i.p., once daily) during days 7-21 of SPS. The ability of JTC-801 to inhibit N/OFQ-stimulated [(35) S]-GTPγS binding was confirmed in rat brain membranes. Anxiety-like behaviour and pain sensitivity were monitored by changes in elevated plus maze performance and withdrawal responses to thermal and mechanical stimuli. Serum corticosterone and N/OFQ content in CSF, serum and brain tissues were determined by radioimmunoassay; NOP receptor protein and gene expression in amygdala, hippocampus and periaqueductal grey (PAG) were examined by immunoblotting and real-time PCR respectively. JTC-801 treatment reversed SPS-induced mechanical allodynia, thermal hyperalgesia, anxiety-like behaviour and hypocortisolism. Elevated N/OFQ levels in serum, CSF, PAG and hippocampus at day 21 of SPS were blocked by JTC-801; daily JTC-801 treatment also reversed NOP receptor protein and mRNA up-regulation in amygdala and PAG. JTC-801 reversed SPS-induced anxiety- and pain-like behaviours, and NOP receptor system up-regulation. These findings suggest that N/OFQ plays an important role in hyperalgesia and allodynia maintenance after SPS. NOP receptor antagonists may provide effective treatment for co-morbid PTSD and pain. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.
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Zhang, Y; Simpson-Durand, C D; Standifer, K M
2015-01-01
BACKGROUND AND PURPOSE Single-prolonged stress (SPS), a rat model of post-traumatic stress disorder (PTSD), also induces long-lasting hyperalgesia associated with hypocortisolism and elevated nociceptin/orphanin FQ (N/OFQ) levels in serum and CSF. Here, we determined the effect of JTC-801 (N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride), a nociceptin/orphanin FQ peptide (NOP) receptor antagonist, on symptoms of pain and anxiety in rats after SPS exposure, and examined N/OFQ-NOP receptor system changes. EXPERIMENTAL APPROACH Male Sprague Dawley rats received JTC-801 (6 mg kg−1 i.p., once daily) during days 7–21 of SPS. The ability of JTC-801 to inhibit N/OFQ-stimulated [35S]-GTPγS binding was confirmed in rat brain membranes. Anxiety-like behaviour and pain sensitivity were monitored by changes in elevated plus maze performance and withdrawal responses to thermal and mechanical stimuli. Serum corticosterone and N/OFQ content in CSF, serum and brain tissues were determined by radioimmunoassay; NOP receptor protein and gene expression in amygdala, hippocampus and periaqueductal grey (PAG) were examined by immunoblotting and real-time PCR respectively. KEY RESULTS JTC-801 treatment reversed SPS-induced mechanical allodynia, thermal hyperalgesia, anxiety-like behaviour and hypocortisolism. Elevated N/OFQ levels in serum, CSF, PAG and hippocampus at day 21 of SPS were blocked by JTC-801; daily JTC-801 treatment also reversed NOP receptor protein and mRNA up-regulation in amygdala and PAG. CONCLUSION AND IMPLICATIONS JTC-801 reversed SPS-induced anxiety- and pain-like behaviours, and NOP receptor system up-regulation. These findings suggest that N/OFQ plays an important role in hyperalgesia and allodynia maintenance after SPS. NOP receptor antagonists may provide effective treatment for co-morbid PTSD and pain. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24666365
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USDA-ARS?s Scientific Manuscript database
To date, some biological activities have been confirmed as different named peptides, however, most FXPRLamide peptides are still poorly understood although these peptides are found in all insects. So, the study of receptors for these peptides is particularly important. Receptors of FXPRLamide peptid...
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Wang, Hao; Anthony, Desiree; Yatmaz, Selcuk; Wijburg, Odilia; Satzke, Catherine; Levy, Bruce; Vlahos, Ross; Bozinovski, Steven
2017-09-15
Formyl peptide receptor 2/lipoxin A 4 (LXA 4 ) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with Streptococcus pneumoniae and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4-6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs. © 2017 The Author(s).
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High Dynamic Range Characterization of the Trauma Patient Plasma Proteome
DOE Office of Scientific and Technical Information (OSTI.GOV)
Liu, Tao; Qian, Weijun; Gritsenko, Marina A.
2006-06-08
While human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein, we describe a strategy that combines immunoaffinity subtraction and chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with 2D-LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this ''divide-and-conquer'' strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) thatmore » covered 3654 nonredundant proteins. Numerous low-abundance proteins were identified, exemplified by 78 ''classic'' cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally, a total of 2910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients, which provides a foundation for future high-throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.« less
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Elevation of Proenkephalin 143-183 in Cerebrospinal Fluid in Moyamoya Disease.
Yokoyama, Kinya; Maruwaka, Mikio; Yoshikawa, Kazuhiro; Araki, Yoshio; Okamoto, Sho; Sumitomo, Masaki; Kawamura, Akino; Sakamoto, Yusuke; Shimizu, Kenzo; Izumi, Takashi; Wakabayashi, Toshihiko
2018-01-01
In moyamoya disease (MMD), the causes of differences in clinical features between children and adults and of the dramatic temporal changes in moyamoya vessels are poorly understood. We previously discovered elevated levels of m/z 4588 and m/z 4473 peptides in cerebrospinal fluid (CSF) in patients with MMD. This study examined the amino acid sequences of these peptides and quantified in specimens. The m/z 4588 and m/z 4473 peptides in CSF from patients with MMD were purified and concentrated by high-performance liquid chromatography and ultrafiltration. Liquid chromatography coupled with tandem mass spectrometry analysis was performed to identify the amino acid sequences of these peptides. We quantified these peptides in samples using sandwich enzyme-linked immunosorbent assay, and concentrations in CSF were compared between MMD (n = 40, 19 male; median age, 37 years) and non-MMD intracranial disease (n = 40, 19 male; median age, 39 years) as controls. These peptides were identified as proenkephalin 143-183 (PENK 143-183). The concentration of PENK 143-183 was significantly greater in patients with MMD (median, 8,270 pmol/L) than control patients (median, 3,760 pmol/L; P < 0.001) and decreased in an age-dependent manner in MMD (r = -0.57; P < 0.001). The area under the receiver operating characteristic curve in children (age <18 years) was 0.885 (95% confidence interval 0.741-1). The correlation between proenkephalin concentration and temporal changes in moyamoya vessels was suggested. Proenkephalin 143-183 in CSF may offer a helpful diagnostic biomarker in pediatric MMD. The effect of enkephalin peptides through opioid growth factor receptor or delta opioid receptor might be associated with the pathophysiology of MMD. Copyright © 2017 Elsevier Inc. All rights reserved.
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Development of second generation peptides modulating cellular adiponectin receptor responses
NASA Astrophysics Data System (ADS)
Otvos, Laszlo; Knappe, Daniel; Hoffmann, Ralf; Kovalszky, Ilona; Olah, Julia; Hewitson, Tim; Stawikowska, Roma; Stawikowski, Maciej; Cudic, Predrag; Lin, Feng; Wade, John; Surmacz, Eva; Lovas, Sandor
2014-10-01
The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM - low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.
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Frączak, Oliwia; Lasota, Anika; Kosson, Piotr; Leśniak, Anna; Muchowska, Adriana; Lipkowski, Andrzej W; Olma, Aleksandra
2015-04-01
Biphalin, a synthetic opioid octapeptide with a palindromic sequence has high analgesic activity. Biphalin displays a strong affinity for μ and δ-opioid receptors, and a significant to κ-receptor. The paper reports the synthesis of novel analogs of biphalin containing β(3)-homo-amino acid residues at the 4,4' positions and a hydrazine or 1,2-phenylenediamine linker. The potency and selectivity of the peptides were evaluated by a competitive receptor-binding assay in rat brain homogenate using [(3)H]DAMGO (a μ ligand) and [(3)H]DELT (a δ ligand). Analogs with β(3)-h-p-NO2Phe in positions 4 and 4' are the most active compounds. Selectivity depends on the degree of freedom between the two pharmacophore moieties. Analogs with a hydrazine linker show noticeable binding selectivity to μ receptors (IC50(μ)=0.72nM; IC50(δ)=4.66nM), while the peptides with a 1,2-phenylenediamine linker show slight δ selectivity (IC50(μ)=10.97nM; IC50(δ)=1.99nM). Tyr-d-Ala-Gly-β(3)-h-p-NO2PheNHNH-β(3)-h-p-NO2Phe (1) and (Tyr-d-Ala-Gly-β(3)-h-p-NO2PheNH)2 (2) produced greater antinociceptive effect compared to morphine after i.t. administration. Copyright © 2015 Elsevier Inc. All rights reserved.
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Fricker, Lloyd D.
2010-01-01
Peptides are known to play many important physiological roles in signaling. A large number of peptides have been detected in mouse brain extracts using mass spectrometry-based peptidomics studies, and 850 peptides have been identified. Half of these peptides are derived from secretory pathway proteins and many are known bioactive neuropeptides which activate G protein-coupled receptors; these are termed “classical neuropeptides.” In addition, 427 peptides were identified that are derived from non-secretory pathway proteins; the majority are cystosolic, and the remainder are mitochondrial, nuclear, lysosomal, or membrane proteins. Many of these peptides represent the N- or C-terminus of the protein, rather than internal fragments, raising the possibility that they are formed by selective processing rather than protein degradation. In addition to consideration of the cleavage site required to generate the intracellular peptides, their potential functions are discussed. Several of the cytosolic peptides were previously found to interact with receptors and/or otherwise influence cellular activity; examples include hemophins, hemopressins, diazepam binding inhibitor, and hippocampal cholinergic neurostimulating peptide. The possibility that these peptides are secreted from cells and function in cell-cell signaling is discussed. If these intracellular peptides can be shown to be secreted in levels sufficient to produce a biological effect, they would appropriately be called “non-classical neuropeptides” by analogy with non-classical neurotransmitters such as nitric oxide and anandamide. It is also possible that intracellular peptides function as “microproteins” and modulate protein-protein interactions; evidence for this function is discussed, along with future directions that are needed to establish this and other possible functions for peptides. PMID:20428524
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Bardelli, A; Longati, P; Williams, T A; Benvenuti, S; Comoglio, P M
1999-10-08
Interaction of the hepatocyte growth factor (HGF) with its receptor, the Met tyrosine kinase, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the Met receptor tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs HGF-dependent Met phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits HGF-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The Met tail peptide inhibits the closely related Ron receptor but does not significantly affect the epidermal growth factor, platelet-derived growth factor, or vascular endothelial growth factor receptor activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the Met receptor, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Booe, Jason M.; Walker, Christopher S.; Barwell, James
Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less
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Booe, Jason M.; Walker, Christopher S.; Barwell, James; ...
2015-05-14
Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind relatedmore » GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. Lastly, the structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes.« less
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Li, Haiou; Lu, Liyao; Chen, Rong; Quan, Lijun; Xia, Xiaoyan; Lü, Qiang
2014-01-01
Structural information related to protein-peptide complexes can be very useful for novel drug discovery and design. The computational docking of protein and peptide can supplement the structural information available on protein-peptide interactions explored by experimental ways. Protein-peptide docking of this paper can be described as three processes that occur in parallel: ab-initio peptide folding, peptide docking with its receptor, and refinement of some flexible areas of the receptor as the peptide is approaching. Several existing methods have been used to sample the degrees of freedom in the three processes, which are usually triggered in an organized sequential scheme. In this paper, we proposed a parallel approach that combines all the three processes during the docking of a folding peptide with a flexible receptor. This approach mimics the actual protein-peptide docking process in parallel way, and is expected to deliver better performance than sequential approaches. We used 22 unbound protein-peptide docking examples to evaluate our method. Our analysis of the results showed that the explicit refinement of the flexible areas of the receptor facilitated more accurate modeling of the interfaces of the complexes, while combining all of the moves in parallel helped the constructing of energy funnels for predictions.
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Vigna, S R
2001-02-01
The agonist activity of substance P (SP) is a function of the C-terminal domain of the peptide. A C-terminal SP fragment (SP(6-11)) and analog (septide) and neurokinin A (NKA; a related tachykinin with a divergent N-terminal amino acid sequence) were found to be full neurokinin-1 receptor (NK-1R) agonists, but were not able to desensitize the receptor maximally as much as SP. Substance P caused 95.6 +/- 0.9% maximal desensitization of the NK-1R whereas SP(6-11), septide, and NKA(only)caused 74 +/- 3.5, 50.6 +/- 8, and 71.5 +/- 4.4% maximal desensitization, respectively (mean +/- SEM; P < 0.001 vs SP). When a series of SP C-terminal fragment peptides were tested for their NK-1R desensitizing activity, it was found that SP(5-11)and SP(6-11)caused significantly less maximal NK-1R desensitization than SP. SP N-terminal fragment peptides had no effect on the ability of SP(6-11)to compete with(3)H-SP binding, generate an IP(3)response, or cause NK-1R desensitization when tested with or without SP(6-11). SP, SP(6-11), septide, and NKA all maximally stimulated 8-9-fold increases in NK-1R phosphorylation. When attached to the C-terminal domain of SP responsible for NK-1R binding and agonism, the N-terminus of SP is responsible for 25-50% of homologous desensitization and this may occur via a mechanism other than NK-1R phosphorylation. Copyright 2001 Harcourt Publishers Ltd.
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Transcriptional regulation of the human mitochondrial peptide deformylase (PDF).
Pereira-Castro, Isabel; Costa, Luís Teixeira da; Amorim, António; Azevedo, Luisa
2012-05-18
The last years of research have been particularly dynamic in establishing the importance of peptide deformylase (PDF), a protein of the N-terminal methionine excision (NME) pathway that removes formyl-methionine from mitochondrial-encoded proteins. The genomic sequence of the human PDF gene is shared with the COG8 gene, which encodes a component of the oligomeric golgi complex, a very unusual case in Eukaryotic genomes. Since PDF is crucial in maintaining mitochondrial function and given the atypical short distance between the end of COG8 coding sequence and the PDF initiation codon, we investigated whether the regulation of the human PDF is affected by the COG8 overlapping partner. Our data reveals that PDF has several transcription start sites, the most important of which only 18 bp from the initiation codon. Furthermore, luciferase-activation assays using differently-sized fragments defined a 97 bp minimal promoter region for human PDF, which is capable of very strong transcriptional activity. This fragment contains a potential Sp1 binding site highly conserved in mammalian species. We show that this binding site, whose mutation significantly reduces transcription activation, is a target for the Sp1 transcription factor, and possibly of other members of the Sp family. Importantly, the entire minimal promoter region is located after the end of COG8's coding region, strongly suggesting that the human PDF preserves an independent regulation from its overlapping partner. Copyright © 2012 Elsevier Inc. All rights reserved.
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NASA Astrophysics Data System (ADS)
Giblin, M. F.; Sieckman, G. L.; Owen, N. K.; Hoffman, T. J.; Forte, L. R.; Volkert, W. A.
2005-12-01
The human Escherichia coli heat-stable enterotoxin (STh, amino acid sequence N1SSNYCCELCCNPACTGCY19) binds specifically to the guanylate cyclase C (GC-C) receptor, which is present in high density on the apical surface of normal intestinal epithelial cells as well as on the surface of human colon cancer cells. In the current study, two STh analogs were synthesized and evaluated in vitro and in vivo. Both analogs shared identical 6-19 core sequences, and had N-terminal pendant DOTA moieties. The analogs differed in the identity of a 6 amino acid peptide sequence intervening between DOTA and the 6-19 core. In one analog, the peptide was an RGD-containing sequence found in human fibronectin (GRGDSP), while in the other this peptide sequence was randomly scrambled (GRDSGP). The results indicated that the presence of the human fibronectin sequence in the hybrid peptide did not affect tumor localization in vivo.
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MATSUBAYASHI, Yoshikatsu
2018-01-01
The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone–receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation. PMID:29434080
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Matsubayashi, Yoshikatsu
2018-01-01
The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to-cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post-translational modification. This review summarizes how we discovered these peptide hormone-receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation.
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FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa.
Shen, Jiangsheng; Meldrum, Allison; Poole, Keith
2002-06-01
Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.
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Zhang, Rui-San; He, Zhen; Jin, Wei-Dong; Wang, Rui
2016-10-01
The cannabinoid system plays an important role in memory processes, many studies have indicated that cannabinoid receptor ligands have ability to modulate memory in rodents. A nonapeptide hemopressin (Hp) derived from rat brain, acts as a peptide antagonist or selective inverse peptide agonist of cannabinoid 1 (CB1) receptor. N-terminally extended forms of Hp isolated from mouse brain, (m)RVD-hemopressin(α) (RVD) and (m)VD-hemopressin(α) (VD) also bind CB1 receptor, however, as peptide agonists. Here, we investigated the roles of Hp, RVD, and VD on memory in mice using novel object recognition (NOR) and object location recognition (OLR) tasks. In normal young mice, intracerebroventricular (i.c.v.) infusion of Hp before training not only improved memory formation, but also prolonged memory retention in the tasks, these effects could be inhibited by RVD or VD at the same dose and intraperitoneal (i.p.) injection of a small molecule agonist of CB1 receptor WIN55, 212-2 15min before administration of Hp inhibited the memory-improving effect of Hp. In addition, under the same experimental conditions, i.c.v. RVD or VD displayed memory-impairing effects, which could be prevented by Hp (i.c.v.) or AM251 (i.p.), a small molecule antagonist of CB1 receptor. Infusion of amyloid-β (1-42) (Aβ1-42) 14days before training resulted in impairment of memory in mice which could be used as animal model of Alzheimer's disease (AD). In these mice, RVD or VD (i.c.v.) reversed the memory impairment induced by Aβ1-42, and the effects of RVD and VD could be suppressed by Hp (i.c.v.) or AM251 (2mg/kg, i.p.). Separate administration of Hp had no effect in Aβ1-42-treated mice. The above results suggested that Hp, RVD and VD, as CB1 receptor peptide ligands, may be potential drugs to treatment of the memory deficit-involving disease, just as AD. Copyright © 2016 Elsevier Inc. All rights reserved.
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Structural Insight into Recognition of Plant Peptide Hormones by Receptors.
Zhang, Heqiao; Han, Zhifu; Song, Wen; Chai, Jijie
2016-11-07
Secreted signaling peptides or peptide hormones play crucial roles in plant growth and development through coordination of cell-cell communication. Perception of peptide hormones in plants generally relies on membrane-localized receptor kinases (RKs). Progress has recently been made in structural elucidation of interactions between posttranslationally modified peptide hormones and RKs. The structural studies suggest conserved receptor binding and activation mechanisms of this type of peptide hormones involving their conserved C-termini. Here, we review these structural data and discuss how the conserved mechanisms can be used to match peptide-RK pairs. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.
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Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen
2010-01-01
GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.
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Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen
2010-01-01
GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722
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Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.
Yang, Zhenlin; Han, Shuo; Keller, Max; Kaiser, Anette; Bender, Brian J; Bosse, Mathias; Burkert, Kerstin; Kögler, Lisa M; Wifling, David; Bernhardt, Guenther; Plank, Nicole; Littmann, Timo; Schmidt, Peter; Yi, Cuiying; Li, Beibei; Ye, Sheng; Zhang, Rongguang; Xu, Bo; Larhammar, Dan; Stevens, Raymond C; Huster, Daniel; Meiler, Jens; Zhao, Qiang; Beck-Sickinger, Annette G; Buschauer, Armin; Wu, Beili
2018-04-01
Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y 1 , Y 2 , Y 4 and Y 5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y 1 receptor (Y 1 R) 4 . A number of peptides and small-molecule compounds have been characterized as Y 1 R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y 1 R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y 1 R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y 1 R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y 1 R can enable structure-based drug discovery that targets NPY receptors.
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Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli.
Ballermann, B J; Hoover, R L; Karnovsky, M J; Brenner, B M
1985-01-01
Isolated rat renal glomeruli and cultured glomerular mesangial and epithelial cells were examined for atrial natriuretic peptide (ANP) receptors, and for ANP-stimulated cyclic guanosine monophosphate (cGMP) generation. In glomeruli from normal rats, human (1-28) 125I-ANP bound to a single population of high affinity receptors with a mean equilibrium dissociation constant of 0.46 nM. Human (1-28) ANP markedly stimulated cGMP generation, but not cAMP generation in normal rat glomeruli. Analogues of ANP that bound to the glomerular ANP receptor with high affinity stimulated cGMP accumulation, whereas the (13-28) ANP fragment, which failed to bind to the receptor, was devoid of functional activity. Cell surface receptors for ANP were expressed on cultured glomerular mesangial but not epithelial cells, and appreciable ANP-stimulated cGMP accumulation was elicited only in mesangial cells. Approximately 12,000 ANP receptor sites were present per mesangial cell, with an average value for the equilibrium dissociation constant of 0.22 nM. Feeding of a low-salt diet to rats for 2 wk resulted in marked up regulation of the glomerular ANP receptor density to a mean of 426 fmol/mg protein, compared with 116 fmol/mg in rats given a high-salt diet. A modest reduction in the affinity of glomerular ANP receptors was also observed in rats fed the low-salt diet. ANP-stimulated cGMP generation in glomeruli did not change with alterations in salt intake. We conclude that high salt feeding in the rat results in reduced glomerular ANP receptor density relative to values in salt restricted rats. Furthermore, the mesangial cell is a principal target for ANP binding in the glomerulus. Images PMID:3001139
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McKeen, E S; Feniuk, W; Humphrey, P P
1994-01-01
1. The motor effects of somatostatin-14 (SRIF), and several SRIF peptide analogues were investigated on the rat isolated distal colon. The objective of these studies was to characterize the receptor mediating the contractile action of SRIF by comparing the relative agonist potencies of a range of SRIF analogues. 2. SRIF (1 nM-1 microM) produced concentration-dependent contractions with an EC50 value of approximately 10 nM. Contractile responses induced by SRIF were insensitive to atropine (1 microM) or naloxone (1 microM) but abolished by tetrodotoxin (1 microM). Somatostatin-28 (SRIF28), also induced concentration-dependent contractions and was equipotent with SRIF. Phosphoramidon (1 microM) and amastatin (10 microM) did not increase the potency of either SRIF or SRIF28. 3. The SRIF peptide analogues, octreotide, SRIF25, seglitide, angiopeptin and CGP23996 (1 nM-1 microM) produced contractile responses in the rat distal colon, each having similar potency and maximal activity relative to SRIF. The SSTR2 receptor-selective hexapeptide, BIM23027 (0.1 nM-1 microM), and the SRIF stereoisomer, D-Trp8-SRIF (0.1 nM-1 microM), were the most potent agonists examined being approximately 12 and 7 times more potent than SRIF, respectively. In contrast, the SSTR5 receptor-selective analogue, L362,855, was approximately 120 times weaker than SRIF, whilst the SSTR3 receptor-selective analogue, BIM23056, was inactive at concentrations up to 3 microM. 4. The putative SRIF receptor antagonist, (cyclo(7-aminoheptanoyl Phe-D-Trp-Lys-Thr[Bzl]))(CPP) (1 microM), had no agonist activity and had no effect on contractions induced by SRIF. 5. The contractile actions of BIM23027 and seglitide were subject to pronounced desensitization.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7834217
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Rhodium-catalyzed C-H functionalization with N-acylsaccharins.
Wu, Hongxiang; Liu, Tingting; Cui, Ming; Li, Yue; Jian, Junsheng; Wang, Hui; Zeng, Zhuo
2017-01-18
A rhodium-catalyzed C-H functionalization with activated amides by decarbonylation has been developed. Notably, this is the first C-H arylation employing N-acylsaccharins as coupling partners to give biaryls in good to excellent yields. The highlight of the work is the high tolerance of functional groups such as formyl, ester, and vinyl and the use of a removable directing group.
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Chigaev, Alexandre; Smagley, Yelena; Sklar, Larry A
2011-05-17
Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation. Using fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation. We conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.
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Jiang, Yingchun; Xi, Xinping; Ge, Lilin; Yang, Nan; Hou, Xiaojuan; Ma, Jie; Ma, Chengbang; Wu, Yuxin; Guo, Xiaoxiao; Li, Renjie; Zhou, Mei; Wang, Lei; Chen, Tianbao; Shaw, Chris
2014-02-01
While bradykinin has been identified in the skin secretions from several species of amphibian, bradykinin-related peptides (BRPs) are more common constituents. These peptides display a plethora of primary structural variations from the type peptide which include single or multiple amino acid substitutions, N- and/or C-terminal extensions and post-translational modifications such as proline hydroxylation and tyrosine sulfation. Such modified peptides have been reported in species from many families, including Bombinatoridae, Hylidae and Ranidae. The spectrum of these peptides in a given species is thought to be reflective of its predator profile from different vertebrate taxa. Here we report the isolation of BRPs and parallel molecular cloning of their respective biosynthetic precursor-encoding cDNAs from the skin secretions of the Mexican leaf frog (Pachymedusa dacnicolor), the Central American red-eyed leaf frog (Agalychnis callidryas) and the South American orange-legged leaf frog (Phyllomedusa hypochondrialis). Additionally, the eight different BRPs identified were chemically synthesized and screened for bioactivity using four different mammalian smooth muscle preparations and their effects and rank potencies were found to be radically different in these with some acting preferentially through bradykinin B1-type receptors and others through B(2)-type receptors. Copyright © 2013 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Goeransson, Anna-Lena, E-mail: anngo@ifm.liu.se; Nilsson, K. Peter R., E-mail: petni@ifm.liu.se; Kagedal, Katarina, E-mail: katarina.kagedal@liu.se
2012-04-20
Highlights: Black-Right-Pointing-Pointer Identification of toxic prefibrillar A{beta} species. Black-Right-Pointing-Pointer Fluorescence measurements using a combined set of fluorophores. Black-Right-Pointing-Pointer Morphology studies using transmission electron microscopy. -- Abstract: The formation of amyloid-{beta} peptide (A{beta}) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of A{beta}. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of A{beta}-derived peptides possessing different levels of neurotoxic activity,more » using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various A{beta} aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those A{beta} peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the A{beta} peptide to form nontoxic versus toxic species.« less
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Millard, Christopher J; Ludeman, Justin P; Canals, Meritxell; Bridgford, Jessica L; Hinds, Mark G; Clayton, Daniel J; Christopoulos, Arthur; Payne, Richard J; Stone, Martin J
2014-11-04
Trafficking of leukocytes in immune surveillance and inflammatory responses is activated by chemokines engaging their receptors. Sulfation of tyrosine residues in peptides derived from the eosinophil chemokine receptor CCR3 dramatically enhances binding to cognate chemokines. We report the structural basis of this recognition and affinity enhancement. We describe the structure of a CC chemokine (CCL11/eotaxin-1) bound to a fragment of a chemokine receptor: residues 8–23 of CCR3, including two sulfotyrosine residues. We also show that intact CCR3 is sulfated and sulfation enhances receptor activity. The CCR3 sulfotyrosine residues form hydrophobic, salt bridge and cation-p interactions with residues that are highly conserved in CC chemokines. However, the orientation of the chemokine relative to the receptor N terminus differs substantially from those observed for two CXC chemokines, suggesting that initial binding of the receptor sulfotyrosine residues guides subsequent steps in receptor activation, thereby influencing the receptor conformational changes and signaling.
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P2X(7) is a scavenger receptor for apoptotic cells in the absence of its ligand, extracellular ATP.
Gu, Ben J; Saunders, Bernadette M; Petrou, Steven; Wiley, James S
2011-09-01
Phagocytosis of apoptotic cells is essential during development and tissue remodeling. Our previous study has shown that the P2X(7) receptor regulates phagocytosis of nonopsonized particles and bacteria. In this study, we demonstrate that P2X(7) also mediates phagocytosis of apoptotic lymphocytes and neuronal cells by human monocyte-derived macrophages under serum-free conditions. ATP inhibited this process to a similar extent as observed with cytochalasin D. P2X(7)-transfected HEK-293 cells acquired the ability to phagocytose apoptotic lymphocytes. Injection of apoptotic thymocytes into the peritoneal cavity of wild-type mice resulted in their phagocytosis by macrophages, but injection of ATP prior to thymocytes markedly decreased this uptake. In contrast, ATP failed to inhibit phagocytosis of apoptotic thymocytes in vivo by P2X(7)-deficient peritoneal macrophages. The surface expression of P2X(7) on phagocytes increased significantly during phagocytosis of either beads or apoptotic cells. A peptide screen library containing 24 biotin-conjugated peptides mimicking the extracellular domain of P2X(7) was used to evaluate the binding profile to beads, bacteria, and apoptotic cells. One peptide showed binding to all particles and cell membrane lipids. Three other cysteine-containing peptides uniquely bound the surface of apoptotic cells but not viable cells, whereas substitution of alanine for cysteine abolished peptide binding. Several thiol-reactive compounds including N-acetyl-L-cysteine abolished phagocytosis of apoptotic SH-SY5Y cells by macrophages. These data suggest that the P2X(7) receptor in its unactivated state acts like a scavenger receptor, and its extracellular disulphide bonds play an important role in direct recognition and engulfment of apoptotic cells.
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GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis
Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko
2015-01-01
Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910
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GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.
Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko
2015-01-02
Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. © 2014 The Authors.
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A role for hippocampal gastrin-releasing peptide receptors in extinction of aversive memory.
Luft, Tatiana; Flores, Debora G; Vianna, Monica R M; Schwartsmann, Gilberto; Roesler, Rafael; Izquierdo, Ivan
2006-06-26
Although the gastrin-releasing peptide receptor has been implicated in memory consolidation, previous studies have not examined whether it is involved in extinction. Here we show that gastrin-releasing peptide receptor blockade in the hippocampus disrupts extinction of aversive memory. Male rats were trained in inhibitory avoidance conditioning and then returned repeatedly to the training context without shock on a daily basis for 3 days. Infusion of a gastrin-releasing peptide receptor antagonist or the protein synthesis inhibitor anisomycin into the dorsal hippocampus immediately after the first extinction session blocked extinction. These drugs did not affect performance in subsequent sessions when the first extinction session (1 day after training) was omitted. The results indicate that hippocampal gastrin-releasing peptide receptors are involved in memory extinction.
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Fotsch, Christopher; Smith, Duncan M; Adams, Jeffrey A; Cheetham, Janet; Croghan, Michael; Doherty, Elizabeth M; Hale, Clarence; Jarosinski, Mark A; Kelly, Michael G; Norman, Mark H; Tamayo, Nuria A; Xi, Ning; Baumgartner, James W
2003-07-21
The solution structure of a potent melanocortin receptor agonist, Ac-Nle-cyclo[Asp-Pro-DPhe-Arg-Trp-Lys]-NH(2) (1) was calculated using distance restraints determined from 1H NMR spectroscopy. Eight of the lowest energy conformations from this study were used to identify non-peptide cores that mimic the spatial arrangement of the critical tripeptide region, DPhe-Arg-Trp, found in 1. From these studies, compound 2a, containing the cis-cyclohexyl core, was identified as a functional agonist of the melanocortin-4 receptor (MC4R) with an IC(50) and EC(50) below 10 nM. Compound 2a also showed 36- and 7-fold selectivity over MC3R and MC1R, respectively, in the binding assays. Subtle changes in cyclohexane stereochemistry and removal of functional groups led to analogues with lower affinity for the MC receptors.
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Torres-Reverón, Annelyn; Palermo, Karylane; Hernández-López, Anixa; Hernández, Siomara; Cruz, Myrella L.; Thompson, Kenira J.; Flores, Idhaliz; Appleyard, Caroline B.
2016-01-01
Studies have examined how endometriosis interacts with the nervous system, but little attention has been paid to opioidergic systems, which are relevant to pain signaling. We used the autotransplantation rat model of endometriosis and allowed to progress for 60 days. The brain was collected and examined for changes in endogenous opioid peptides, mu opioid receptors (MORs), and the N-methyl-d-aspartate subunit receptor (NR1) in the periaqueductal gray (PAG), since both of these receptors can regulate PAG activity. No changes in endogenous opioid peptides in met- and leu-enkephalin or β-endorphin levels were observed within the PAG. However, MOR immunoreactivity was significantly decreased in the ventral PAG in the endometriosis group. Endometriosis reduced by 20% the number of neuronal profiles expressing MOR and reduced by 40% the NR1 profiles. Our results suggest that endometriosis is associated with subtle variations in opioidergic and glutamatergic activity within the PAG, which may have implications for pain processing. PMID:27089914
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Hermans, Emmanuel; Geurts, Muriel; Maloteaux, Jean-Marie
1997-01-01
The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. The agonist-induced binding of [35S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3±0.9 nM). The stimulation of [35S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC50 value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21±6 nM). The NT-induced [35S]-GTPγS binding was competitively inhibited by SR48692 (pA2 value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]-GTPγS. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor. PMID:9283723
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Pereira, Naveen L; Redfield, Margaret M; Scott, Christopher; Tosakulwong, Nirubol; Olson, Timothy M; Bailey, Kent R; Rodeheffer, Richard J; Burnett, John C
2014-01-01
To evaluate the impact of a functional genetic variant in the natriuretic peptide clearance receptor, NPR3, on circulating natriuretic peptides (NPs) and myocardial structure and function in the general community. NPR3 plays an important role in the clearance of NPs and through direct signaling mechanisms modulates smooth muscle cell function and cardiac fibroblast proliferation. A NPR3 nonsynonymous single nucleotide polymorphism (SNP) rs2270915, resulting in a N521D substitution in the intracellular catalytic domain that interacts with Gi could affect receptor function. Whether this SNP is associated with alterations in NPs levels and altered cardiac structure and function is unknown. DNA samples of 1931 randomly selected residents of Olmsted County, Minnesota were genotyped. Plasma NT-proANP1-98, ANP1-28, proBNP1-108, NT-proBNP1-76, BNP1-32 and BNP3-32 levels were measured. All subjects underwent comprehensive echocardiography. Genotype frequencies for rs2270915 were as follows: (A/A 60%, A/G 36%, G/G 4%). All analyses performed were for homozygotes G/G versus wild type A/A plus the heterozygotes A/G. Diastolic dysfunction was significantly more common (p = 0.007) in the homozygotes G/G (43%) than the A/A+A/G (28%) group. Multivariate regression adjusted for age, sex, body mass index and hypertension demonstrated rs2270915 to be independently associated with diastolic dysfunction (odds ratio 1.94, p = 0.03). There was no significant difference in NPs levels between the 2 groups suggesting that the clearance function of the receptor was not affected. A nonsynonymous NPR3 SNP is independently associated with diastolic dysfunction and this association does not appear to be related to alterations in circulating levels of natriuretic peptides.
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Lindner, Simon; Fiedler, Luise; Wängler, Björn; Bartenstein, Peter; Schirrmacher, Ralf; Wängler, Carmen
2018-05-29
Radiolabeled heterobivalent peptidic ligands (HBPLs), being able to address different receptors, are highly interesting tumor imaging agents as they can offer multiple advantages over monovalent peptide receptor ligands. However, few examples of radiolabeled HBPLs have been described so far. One promising approach is the combination of gastrin-releasing peptide receptor (GRPR)- and vasoactive intestinal peptide receptor subtype 1 (VPAC 1 R)-targeting peptides into one single radioligand since gastrinomas, prostate and breast cancer have been shown to concomitantly or complementarily overexpress both receptors. Here we report the design and synthesis of different HBPLs, comprising a GRPR-binding (BBN 7-14 ) and a VPAC 1 R-targeting (PACAP-27) peptide. The heterodimers were varied with regard to the distance between the peptide binders and the steric rigidity of the systems. We radiolabeled the HBPLs 19-23 as well as their monomeric reference standards 26 and 27 with 68 Ga, achieving radiochemical yields and purities of 95-99% and non-optimized molar activities of 25-61 GBq/μmol. We tested the stability of the radioligands and further evaluated them in vitro regarding their uptake in different prostate carcinoma cell lines (PC-3, DU-145 and VCaP cells). We found that the heterobivalent substances [ 68 Ga]19 - [ 68 Ga]23 showed comparable uptakes into the tumor cells to those of the respective monomers [ 68 Ga]26 and [ 68 Ga]27, indicating that both peptides are still able to address their target receptors. Furthermore, the obtained results indicate that in case of overall low receptor densities, heterobivalent peptides surpass peptide monomers in tumor cell uptake. Most importantly, it could be shown by blocking studies that both peptide parts of the HBPL [ 68 Ga]19 contributed to tumor cell uptake in VCaP cells, expressing both receptor types. Thus, we describe here the first examples of HBPLs being able to address the GRPR as well as the VPAC 1 R and have the potential to - by several mechanisms - improve tumor targeting for several malignancies compared to monospecific peptides. Copyright © 2018 Elsevier Masson SAS. All rights reserved.
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Shepard, Blythe D.; Natarajan, Niranjana; Protzko, Ryan J.; Acres, Omar W.; Pluznick, Jennifer L.
2013-01-01
Olfactory receptors (ORs) are G protein-coupled receptors that detect odorants in the olfactory epithelium, and comprise the largest gene family in the genome. Identification of OR ligands typically requires OR surface expression in heterologous cells; however, ORs rarely traffic to the cell surface when exogenously expressed. Therefore, most ORs are orphan receptors with no known ligands. To date, studies have utilized non-cleavable rhodopsin (Rho) tags and/or chaperones (i.e. Receptor Transporting Protein, RTP1S, Ric8b and Gαolf) to improve surface expression. However, even with these tools, many ORs still fail to reach the cell surface. We used a test set of fifteen ORs to examine the effect of a cleavable leucine-rich signal peptide sequence (Lucy tag) on OR surface expression in HEK293T cells. We report here that the addition of the Lucy tag to the N-terminus increases the number of ORs reaching the cell surface to 7 of the 15 ORs (as compared to 3/15 without Rho or Lucy tags). Moreover, when ORs tagged with both Lucy and Rho were co-expressed with previously reported chaperones (RTP1S, Ric8b and Gαolf), we observed surface expression for all 15 receptors examined. In fact, two-thirds of Lucy-tagged ORs are able to reach the cell surface synergistically with chaperones even when the Rho tag is removed (10/15 ORs), allowing for the potential assessment of OR function with only an 8-amino acid Flag tag on the mature protein. As expected for a signal peptide, the Lucy tag was cleaved from the mature protein and did not alter OR-ligand binding and signaling. Our studies demonstrate that widespread surface expression of ORs can be achieved in HEK293T cells, providing promise for future large-scale deorphanization studies. PMID:23840901
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Raveh, Barak; London, Nir; Zimmerman, Lior; Schueler-Furman, Ora
2011-04-29
Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. © 2011 Raveh et al.
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Raveh, Barak; London, Nir; Zimmerman, Lior; Schueler-Furman, Ora
2011-01-01
Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions. PMID:21572516
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Evaluating Ga-68 Peptide Conjugates for Targeting VPAC Receptors: Stability and Pharmacokinetics.
Kumar, Pardeep; Tripathi, Sushil K; Chen, C P; Wickstrom, Eric; Thakur, Mathew L
2018-05-25
In recent years, considerable progress has been made in the use of gallium-68 labeled receptor-specific peptides for imaging oncologic diseases. The objective was to examine the stability and pharmacokinetics of [ 68 Ga]NODAGA and DOTA-peptide conjugate targeting VPAC [combined for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)] receptors on tumor cells. A VPAC receptor-specific peptide was chosen as a model peptide and conjugated to NODAGA and DOTA via solid-phase synthesis. The conjugates were characterized by HPLC and MALDI-TOF. Following Ga-68 chelation, the radiochemical purity of Ga-68 labeled peptide conjugate was determined by radio-HPLC. The stability was tested against transmetallation using 100 nM Fe 3+ /Zn 2+ /Ca 2+ ionic solution and against transchelation using 200 μM DTPA solution. The ex vivo and in vivo stability of the Ga-68 labeled peptide conjugate was tested in mouse plasma and urine. Receptor specificity was determined ex vivo by cell binding assays using human breast cancer BT474 cells. Positron emission tomography (PET)/X-ray computed tomography (CT) imaging, tissue distribution, and blocking studies were performed in mice bearing BT474 xenografts. The chemical and radiochemical purity was greater than 95 % and both conjugates were stable against transchelation and transmetallation. Ex vivo stability at 60 min showed that the NODAGA-peptide-bound Ga-68 reduced to 42.1 ± 3.7 % (in plasma) and 37.4 ± 2.9 % (in urine), whereas the DOTA-peptide-bound Ga-68 was reduced to 1.2 ± 0.3 % (in plasma) and 4.2 ± 0.4 % (in urine) at 60 min. Similarly, the in vivo stability for [ 68 Ga]NODAGA-peptide was decreased to 2.1 ± 0.2 % (in plasma) and 2.2 ± 0.4 % (in urine). For [ 68 Ga]DOTA-peptide, it was decreased to 1.4 ± 0.3 % (in plasma) and 1.2 ± 0.4 % (in urine) at 60 min. The specific BT474 cell binding was 53.9 ± 0.8 % for [ 68 Ga]NODAGA-peptide, 25.8 ± 1.4 % for [ 68 Ga]-DOTA-peptide, and 18.8 ± 2.5 % for [ 68 Ga]GaCl 3 at 60 min. Inveon microPET/CT imaging at 1 h post-injection showed significantly (p < 0.05) higher tumor to muscle (T/M) ratio for [ 68 Ga]NODAGA-peptide (3.4 ± 0.3) as compared to [ 68 Ga]DOTA-peptide (1.8 ± 0.6). For [ 68 Ga]GaCl 3 and blocked mice, their ratios were 1.5 ± 0.6 and 1.5 ± 0.3 respectively. The tissue distributions data were similar to the PET imaging data. NODAGA is superior to DOTA in terms of radiolabeling kinetics. The method of radiolabeling was reproducible and yielded higher specific activity. Although both agents have relatively low in vivo stability, PET/CT imaging studies delineated BC tumors with [ 68 Ga]NODAGA-peptide, but not with [ 68 Ga]DOTA-peptide.
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Endothelin-converting enzyme 2 differentially regulates opioid receptor activity
Gupta, A; Fujita, W; Gomes, I; Bobeck, E; Devi, L A
2015-01-01
BACKGROUND AND PURPOSE Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization. EXPERIMENTAL APPROACH The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24990314
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Small-molecule agonists for the glucagon-like peptide 1 receptor
Knudsen, Lotte Bjerre; Kiel, Dan; Teng, Min; Behrens, Carsten; Bhumralkar, Dilip; Kodra, János T.; Holst, Jens J.; Jeppesen, Claus B.; Johnson, Michael D.; de Jong, Johannes Cornelis; Jorgensen, Anker Steen; Kercher, Tim; Kostrowicki, Jarek; Madsen, Peter; Olesen, Preben H.; Petersen, Jacob S.; Poulsen, Fritz; Sidelmann, Ulla G.; Sturis, Jeppe; Truesdale, Larry; May, John; Lau, Jesper
2007-01-01
The peptide hormone glucagon-like peptide (GLP)-1 has important actions resulting in glucose lowering along with weight loss in patients with type 2 diabetes. As a peptide hormone, GLP-1 has to be administered by injection. Only a few small-molecule agonists to peptide hormone receptors have been described and none in the B family of the G protein coupled receptors to which the GLP-1 receptor belongs. We have discovered a series of small molecules known as ago-allosteric modulators selective for the human GLP-1 receptor. These compounds act as both allosteric activators of the receptor and independent agonists. Potency of GLP-1 was not changed by the allosteric agonists, but affinity of GLP-1 for the receptor was increased. The most potent compound identified stimulates glucose-dependent insulin release from normal mouse islets but, importantly, not from GLP-1 receptor knockout mice. Also, the compound stimulates insulin release from perfused rat pancreas in a manner additive with GLP-1 itself. These compounds may lead to the identification or design of orally active GLP-1 agonists. PMID:17213325
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Preprotein mature domains contain translocase targeting signals that are essential for secretion.
Chatzi, Katerina E; Sardis, Marios Frantzeskos; Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G; Karamanou, Spyridoula; Economou, Anastassios
2017-05-01
Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as pre proteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. © 2017 Chatzi et al.
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Preprotein mature domains contain translocase targeting signals that are essential for secretion
Tsirigotaki, Alexandra; Koukaki, Marina; Šoštarić, Nikolina; Konijnenberg, Albert; Sobott, Frank; Kalodimos, Charalampos G.; Karamanou, Spyridoula
2017-01-01
Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion. PMID:28404644
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Novoa, Alexandre; Van Dorpe, Sylvia; Wynendaele, Evelien; Spetea, Mariana; Bracke, Nathalie; Stalmans, Sofie; Betti, Cecilia; Chung, Nga N; Lemieux, Carole; Zuegg, Johannes; Cooper, Matthew A; Tourwé, Dirk; De Spiegeleer, Bart; Schiller, Peter W; Ballet, Steven
2012-11-26
The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.
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Novoa, Alexandre; Van Dorpe, Sylvia; Wynendaele, Evelien; Spetea, Mariana; Bracke, Nathalie; Stalmans, Sofie; Betti, Cecilia; Chung, Nga N.; Lemieux, Carole; Zuegg, Johannes; Cooper, Matthew A.; Tourwé, Dirk; De Spiegeleer, Bart; Schiller, Peter W.; Ballet, Steven
2012-01-01
The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-D-Arg-Phe-Lys-NH2 ([Dmt1]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt1]-DALDA and to investigate the Phe3 side chain flexibility, the final amide bond was N-methylated and Phe3 was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (i.p. and s.c.) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity and transport properties. Strikingly, while [Dmt1]-DALDA and its N-methyl analogue, Dmt-D-Arg-Phe-NMeLys-NH2, showed a long-lasting antinociceptive effect (>7h), the peptides with D-Cit2 generate potent antinociception more rapidly (maximal effect at 1h post-injection) but also lose their analgesic activity faster, when compared to [Dmt1]-DALDA and [Dmt1,NMeLys4]-DALDA. PMID:23102273
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Dingess, Kelly A; de Waard, Marita; Boeren, Sjef; Vervoort, Jacques; Lambers, Tim T; van Goudoever, Johannes B; Hettinga, Kasper
2017-10-18
Variations in endogenous peptide profiles, functionality, and the enzymes responsible for the formation of these peptides in human milk are understudied. Additionally, there is a lack of knowledge regarding peptides in donor human milk, which is used to feed preterm infants when mother's own milk is not (sufficiently) available. To assess this, 29 human milk samples from the Dutch Human Milk Bank were analyzed as three groups, preterm late lactation stage (LS) (n = 12), term early (n = 8) and term late LS (n = 9). Gestational age (GA) groups were defined as preterm (24-36 weeks) and term (≥37 weeks). LS was determined as days postpartum as early (16-36 days) or late (55-88 days). Peptides, analyzed by LC-MS/MS, and parent proteins (proteins from matched peptide sequences) were identified and quantified, after which peptide functionality and the enzymes responsible for protein cleavage were determined. A total of 16 different parent proteins were identified from human milk, with no differences by GA or LS. We identified 1104 endogenous peptides, of which, the majority were from the parent proteins β-casein, polymeric immunoglobulin receptor, α s1 -casein, osteopontin, and κ-casein. The absolute number of peptides differed by GA and LS with 30 and 41 differing sequences respectively (p < 0.05) Odds likelihood tests determined that 32 peptides had a predicted bioactive functionality, with no significant differences between groups. Enzyme prediction analysis showed that plasmin/trypsin enzymes most likely cleaved the identified human milk peptides. These results explain some of the variation in endogenous peptides in human milk, leading to future targets that may be studied for functionality.
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Recke, Andreas; Regensburger, Ann-Katrin; Weigold, Florian; Müller, Antje; Heidecke, Harald; Marschner, Gabriele; Hammers, Christoph M; Ludwig, Ralf J; Riemekasten, Gabriela
2018-01-01
Systemic sclerosis (SSc) is a severe chronic autoimmune disease with high morbidity and mortality. Sera of patients with SSc contain a large variety of autoantibody (aab) reactivities. Among these are functionally active aab that bind to G protein-coupled receptors (GPCR) such as C-X-C motif chemokine receptor 3 (CXCR3) and 4 (CXCR4). Aab binding to the N-terminal portion of these two GPCRs have been shown to be associated with slower disease progression in SSc, especially deterioration of lung function. Aabs binding to GPCRs exhibit functional activities by stimulating or inhibiting GPCR signaling. The specific functional activity of aabs crucially depends on the epitopes they bind to. To identify the location of important epitopes on CXCR3 recognized by aabs from SSc patients, we applied an array of 36 overlapping 18-20mer peptides covering the entire CXCR3 sequence, comparing epitope specificity of SSc patient sera ( N = 32, with positive reactivity with CXCR3) to healthy controls ( N = 30). Binding of SSc patient and control sera to these peptides was determined by ELISA. Using a Bayesian model approach, we found increased binding of SSc patient sera to peptides corresponding to intracellular epitopes within CXCR3, while the binding signal to extracellular portions of CXCR3 was found to be reduced. Experimentally determined epitopes showed a good correspondence to those predicted by the ABCpred tool. To verify these results and to translate them into a novel diagnostic ELISA, we combined the peptides that represent SSc-associated epitopes into a single ELISA and evaluated its potential to discriminate SSc patients ( N = 31) from normal healthy controls ( N = 47). This ELISA had a sensitivity of 0.61 and a specificity of 0.85. Our data reveals that SSc sera preferentially bind intracellular epitopes of CXCR3, while an extracellular epitope in the N-terminal domain that appears to be target of aabs in healthy individuals is not bound by SSc sera. Based upon our results, we could devise a novel ELISA concept that may be helpful for monitoring of SSc patients.
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Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang
2017-12-01
Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.
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Characterization of C-type natriuretic peptide receptors in human mesangial cells.
Zhao, J; Ardaillou, N; Lu, C Y; Placier, S; Pham, P; Badre, L; Cambar, J; Ardaillou, R
1994-09-01
Our aim was to examine whether the human glomerulus was a target for C-type natriuretic peptide (CNP) and how A, B and C receptors of natriuretic peptides (ANPR-A, ANPR-B, ANPR-C) were distributed in glomerular mesangial and epithelial cells. CNP stimulated cyclic GMP production in cultured human mesangial and epithelial cells with similar threshold concentrations (1 nM) and maximum effects (basal value x 30 at 1 microM). In contrast, atrial natriuretic peptide (ANP) was only stimulatory in epithelial cells. [125I] CNP bound specifically to mesangial cells with a Kd of 0.47 nM and Bmax of 42 fmol/mg. Equilibrium of binding was obtained after four to five hours at +4 degrees C and nonspecific binding represented 10 to 20% of total binding. HS142-1 (100 micrograms/ml), a specific inhibitor of ANPR-A and ANPR-B, suppressed 90% of CNP-dependent cyclic GMP production whereas it had little effect on [125I]-CNP binding, suggesting that C receptors were largely predominant in mesangial cells. No biological effect of CNP on mesangial cells, including change in basal or angiotensin II-induced contractility and inhibition of basal or serum-dependent proliferation, could be demonstrated. Similar results were obtained with 8-bromo-cyclic GMP and sodium nitroprusside. Intraglomerular localization of ANPR-A, ANPR-B and ANPR-C mRNA was studied using reverse transcriptase-polymerase chain reaction with amplification of their corresponding cDNA by different primers. Amplification products were identified on gel electrophoresis by their predicted sizes and sequencing. ANPR-A, ANPR-B and ANPR-C mRNA were present in epithelial cells whereas only ANPR-B and ANPR-C mRNA were detected in mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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USDA-ARS?s Scientific Manuscript database
The objective was to determine the effects of naloxone, an opioid peptide receptor antagonist on circulating concentrations of luteinizing hormone (LH) and testosterone in boars characterized as having high (n = 8) or low libido (n = 8) based on the willingness to mount an artificial sow and allow s...
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Ulfig, Agnes; Fröbel, Julia; Lausberg, Frank; Blümmel, Anne-Sophie; Heide, Anna Katharina; Müller, Matthias; Freudl, Roland
2017-06-30
The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in their binding to the Tat translocase, but some facets of this interaction remain unclear. Here, we investigated the role of the hydrophobic (h-) region of the Escherichia coli trimethylamine N -oxide reductase (TorA) signal peptide in TatBC receptor binding in vivo and in vitro We show that besides the RR motif, a minimal, functional h-region in the signal peptide is required for Tat-dependent export in Escherichia coli Furthermore, we identified mutations in the h-region that synergistically suppressed the export defect of a TorA[KQ]-30aa-MalE Tat reporter protein in which the RR motif was replaced with a lysine-glutamine pair. Strikingly, all suppressor mutations increased the hydrophobicity of the h-region. By systematically replacing a neutral residue in the h-region with various amino acids, we detected a positive correlation between the hydrophobicity of the h-region and the translocation efficiency of the resulting reporter variants. In vitro cross-linking of residues located in the periplasmically-oriented part of the TatBC receptor to TorA[KQ]-30aa-MalE reporter variants harboring a more hydrophobic h-region in their signal peptides confirmed that unlike in TorA[KQ]-30aa-MalE with an unaltered h-region, the mutated reporters moved deep into the TatBC-binding cavity. Our results clearly indicate that, besides the Tat motif, the h-region of the Tat signal peptides is another important binding determinant that significantly contributes to the productive interaction of Tat precursor proteins with the TatBC receptor complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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The Aryl hydrocarbon receptor (AhR) is a ligand-activated, transcription factor with a basic region/helix (bHLH) motif. hR has been sequenced and the functional domains defined and there is information on the formation of complexes with other peptides and interactions with DNA, a...
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NASA Astrophysics Data System (ADS)
Michel, André; Villeneuve, Gérald; DiMaio, John
1991-12-01
The molecular basis underlying the divergent receptor selectivity of two cyclic opioid peptides Tyr-c[ N δ- d-Orn2-Gly-Phe-Leu-] (c-ORN) and [ d-Pen2, l-Cys5]-enkephalinamide (c-PEN) was investigated using a molecular modeling approach. Ring closure and conformational searching procedures were used to determine low-energy cyclic backbone conformers. Following reinsertion of amino acid side chains, the narcotic alkaloid 7α-[(1R)-1-methyl-1-hydroxy-3-phenylpropyl]-6,14-endoethenotetrahydro oripavine (PEO) was used as a flexible template for bimolecular superpositions with each of the determined peptide ring conformers using the coplanarity and cocentricity of the phenolic rings as the minimum constraint. A vector space of PEO, accounting for all possible orientations for the C21-aromatic ring of PEO served as a geometrical locus for the aromatic ring of the Phe4 residue in the opioid peptides. Although a vast number of polypeptide conformations satisfied the criteria of the opiate pharmacophore, they could be grouped into three classes differing in magnitude and sign of the torsional angle values of the tyrosyl side chain. Only class III conformers for both c-ORN and c-PEN, having tyramine dihedral angles χ1 =-150° ± 30° and χ2=-155° ± 20°, had significant structural and conformational properties that were mutually compatible while respecting the PEO vector space. Comparison of these properties in the context of the divergent receptor selectivity of the studied opioid peptides suggests that the increased distortion of the peptide backbone in the closure region of c-PEN together with the pendant β,β-dimethyl group, combine to generate a steric volume which is absent in c-ORN and that may be incompatible with a restrictive topography of the μ receptor. The nature and stereo-chemistry of substituents adjacent to the closure region of the peptides could also modulate receptor selection by interacting with a charged (δ) or neutral (μ) subsite.
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Functional somatostatin receptors on a rat pancreatic acinar cell line
DOE Office of Scientific and Technical Information (OSTI.GOV)
Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.
1988-07-01
Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less
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Hoffmann, Julia; Schneider, Carola; Heinbockel, Lena; Brandenburg, Klaus; Reimer, Rudolph; Gabriel, Gülsah
2014-04-01
Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy. Copyright © 2014 Elsevier B.V. All rights reserved.
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Liu, Yaquan; Tian, Fang; Zhi, Dejuan; Wang, Haiqing; Zhao, Chunyan; Li, Hongyu
2017-02-01
Thrombopoietin (TPO) acts in promoting the proliferation of hematopoietic stem cells and by initiating specific maturation events in megakaryocytes. Now, TPO-mimetic peptides with amino acid sequences unrelated to TPO are of considerable pharmaceutical interest. In the present paper, four new TPO mimetic peptides that bind and activate c-Mpl receptor have been identified, synthesized and tested by Dual-Luciferase reporter gene assay for biological activities. The molecular modeling research was also approached to understand key molecular mechanisms and structural features responsible for peptide binding with c-Mpl receptor. The results presented that three of four mimetic peptides showed significant activities. In addition, the molecular modeling approaches proved hydrophobic interactions were the driven positive forces for binding behavior between peptides and c-Mpl receptor. TPO peptide residues in P7, P13 and P7' positions were identified by the analysis of hydrogen bonds and energy decompositions as the key ones for benefiting better biological activities. Our data suggested the synthesized peptides have considerable potential for the future development of stable and highly active TPO mimetic peptides. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts
Eggink, Laura L.; Chen, Min
2007-01-01
Chlorophyll (Chl) b serves an essential function in accumulation of light-harvesting complexes (LHCs) in plants. In this article, this role of Chl b is explored by considering the properties of Chls and the ligands with which they interact in the complexes. The overall properties of the Chls, not only their spectral features, are altered as consequences of chemical modifications on the periphery of the molecules. Important modifications are introduction of oxygen atoms at specific locations and reduction or desaturation of sidechains. These modifications influence formation of coordination bonds by which the central Mg atom, the Lewis acid, of Chl molecules interacts with amino acid sidechains, as the Lewis base, in proteins. Chl a is a versatile Lewis acid and interacts principally with imidazole groups but also with sidechain amides and water. The 7-formyl group on Chl b withdraws electron density toward the periphery of the molecule and consequently the positive Mg is less shielded by the molecular electron cloud than in Chl a. Chl b thus tends to form electrostatic bonds with Lewis bases with a fixed dipole, such as water and, in particular, peptide backbone carbonyl groups. The coordination bonds are enhanced by H-bonds between the protein and the 7-formyl group. These additional strong interactions with Chl b are necessary to achieve assembly of stable LHCs. PMID:17505910
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Bruchas, Michael R.; Calo', Girolamo; Cox, Brian M.; Zaveri, Nurulain T.
2016-01-01
The NOP receptor (nociceptin/orphanin FQ opioid peptide receptor) is the most recently discovered member of the opioid receptor family and, together with its endogenous ligand, N/OFQ, make up the fourth members of the opioid receptor and opioid peptide family. Because of its more recent discovery, an understanding of the cellular and behavioral actions induced by NOP receptor activation are less well developed than for the other members of the opioid receptor family. All of these factors are important because NOP receptor activation has a clear modulatory role on mu opioid receptor-mediated actions and thereby affects opioid analgesia, tolerance development, and reward. In addition to opioid modulatory actions, NOP receptor activation has important effects on motor function and other physiologic processes. This review discusses how NOP pharmacology intersects, contrasts, and interacts with the mu opioid receptor in terms of tertiary structure and mechanism of receptor activation; location of receptors in the central nervous system; mechanisms of desensitization and downregulation; cellular actions; intracellular signal transduction pathways; and behavioral actions with respect to analgesia, tolerance, dependence, and reward. This is followed by a discussion of the agonists and antagonists that have most contributed to our current knowledge. Because NOP receptors are highly expressed in brain and spinal cord and NOP receptor activation sometimes synergizes with mu receptor-mediated actions and sometimes opposes them, an understanding of NOP receptor pharmacology in the context of these interactions with the opioid receptors will be crucial to the development of novel therapeutics that engage the NOP receptor. PMID:26956246
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Invertebrate Gonadotropin-Releasing Hormone-Related Peptides and Their Receptors: An Update
Sakai, Tsubasa; Shiraishi, Akira; Kawada, Tsuyoshi; Matsubara, Shin; Aoyama, Masato; Satake, Honoo
2017-01-01
Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproductive functions via the hypothalamus, pituitary, and gonad axis, namely, HPG axis in vertebrates. GnRHs and their receptors (GnRHRs) are likely to be conserved in invertebrate deuterostomes and lophotrochozoans. All vertebrate and urochordate GnRHs are composed of 10 amino acids, whereas protostome, echinoderm, and amphioxus GnRH-like peptides are 11- or 12-residue peptide containing two amino acids after an N-terminal pyro-Glu. In urochordates, Halocynthia roretzi GnRH gene encodes two GnRH peptide sequences, whereas two GnRH genes encode three different GnRH peptides in Ciona intestinalis. These findings indicate the species-specific diversification of GnRHs. Intriguingly, the major signaling pathway for GnRHRs is intracellular Ca2+ mobilization in chordates, echinoderms, and protostomes, whereas Ciona GnRHRs (Ci-GnRHRs) are endowed with multiple GnRHergic cAMP production pathways in a ligand-selective manner. Moreover, the ligand-specific modulation of signal transduction via heterodimerization among Ci-GnRHR paralogs suggests the species-specific development of fine-tuning of gonadal functions in ascidians. Echinoderm GnRH-like peptides show high sequence differences compared to those of protostome counterparts, leading to the difficulty in classification of peptides and receptors. These findings also show both the diversity and conservation of GnRH signaling systems in invertebrates. The lack of the HPG axis in invertebrates indicates that biological functions of GnRHs are not release of gonadotropins in current invertebrates and common ancestors of vertebrates and invertebrates. To date, authentic or putative GnRHRs have been characterized from various echinoderms and protostomes as well as chordates and the mRNAs have been found to be distributed not only reproductive organs but also other tissues. Collectively, these findings further support the notion that invertebrate GnRHs have biological roles other than the regulation of reproductive functions. Moreover, recent molecular phylogenetic analysis suggests that adipokinetic hormone (AKH), corazonin (CRZ), and AKH/CRZ-related peptide (ACP) belong to the GnRH superfamily but has led to the different classifications of these peptides and receptors using different datasets including the number of sequences and structural domains. In this review, we provide current knowledge of, and perspectives in, molecular basis and evolutionary aspects of the GnRH, AKH, CRZ, and ACP. PMID:28932208
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Smilkov, Katarina; Janevik, Emilija; Guerrini, Remo; Pasquali, Micol; Boschi, Alessandra; Uccelli, Licia; Di Domenico, Giovanni; Duatti, Adriano
2014-09-01
New (188)Re and (99m)Tc peptide conjugates with substance- P (SP) were prepared and biologically evaluated. The radiopharmaceuticals have been labelled with the [M≡N](2+) (M=(99m)Tc, (188)Re) core using a combination of π-donor tridentate and π-acceptor monodentate ancillary ligands. The new radiopharmaceuticals have been prepared through a two-step reaction by simultaneous addition of the tridentate and monodentate ligands to a vial containing a preformed [M≡N](2+) core. The tridentate ligand was formed by linking two cysteine residues to the terminal arginine of the undecapeptide SP, whereas the monodentate ligand was a tertiary phosphine. The preparation of the corresponding Re-188 derivative required developing a more complex chemical procedure to obtain the [Re≡N](2+) core in satisfactory yields. Characterization of the resulting products was obtained by chromatographic methods. Biological evaluation was performed for both Tc-99m and Re-188 derivatives by in-vitro studies on isolated cells expressing NK1-receptors. In-vivo imaging in mice was carried out using a small-animal YAP(S)PET tomograph. New Tc-99m and Re-188 peptide radiopharmaceuticals with SP have been prepared in high-yield and with high-specific activity. Both Tc-99m and Re-188 peptide radioconjugates exhibit high affinity for NK1 receptors, thus giving further evidence to the empirical rule that structurally related Tc-99m and Re-188 radiopharmaceuticals exhibit identical biological properties. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Simulations of a Membrane-Anchored Peptide: Structure, Dynamics, and Influence on Bilayer Properties
Jensen, Morten Ø.; Mouritsen, Ole G.; Peters, Günther H.
2004-01-01
A three-dimensional structure of a model decapeptide is obtained by performing molecular dynamics simulations of the peptide in explicit water. Interactions between an N-myristoylated form of the folded peptide anchored to dipalmitoylphosphatidylcholine fluid phase lipid membranes are studied at different applied surface tensions by molecular dynamics simulations. The lipid membrane environment influences the conformational space explored by the peptide. The overall secondary structure of the anchored peptide is found to deviate at times from its structure in aqueous solution through reversible conformational transitions. The peptide is, despite the anchor, highly mobile at the membrane surface with the peptide motion along the bilayer normal being integrated into the collective modes of the membrane. Peptide anchoring moderately alters the lateral compressibility of the bilayer by changing the equilibrium area of the membrane. Although membrane anchoring moderately affects the elastic properties of the bilayer, the model peptide studied here exhibits conformational flexibility and our results therefore suggest that peptide acylation is a feasible way to reinforce peptide-membrane interactions whereby, e.g., the lifetime of receptor-ligand interactions can be prolonged. PMID:15189854
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Renal receptors for atrial and C-type natriuretic peptides in the rat.
Brown, J; Zuo, Z
1992-07-01
Receptors for alpha-atrial natriuretic peptide (alpha-ANP) and C-type natriuretic peptide [CNP-(1-22)] were quantified in kidneys from adult Wistar rats by in vitro autoradiography. 125I-labeled alpha-ANP (100 pM) bound reversibly to glomeruli, outer medullary vasa recta, and inner medulla with an apparent dissociation constant (Kd) of 3-6 nM. The presence of 10 microM des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANP-(4- 23) (C-ANP), a specific ligand of the ANPR-C subtype of alpha-ANP receptor, inhibited approximately 50% of the glomerular binding of 125I-alpha-ANP, and this moiety of glomerular binding was also inhibited by CNP-(1-22) with an apparent inhibitory constant (Ki) of 10.47 +/- 7.59 nM. C-ANP and CNP-(1-22) showed little affinity for the medullary binding sites of alpha-ANP. 125I-[Tyr0]CNP-(1-22) (110 pM) bound solely to glomeruli and was competitively displaced by increasing concentrations of [Tyr0]CNP-(1-22) with an apparent Kd of 1.42 +/- 0.48 nM. Binding of increasing concentrations (25 pM to 1 nM) of 125I-[Tyr0]CNP-(1-22) in the presence or absence of 1 microM [Tyr0]CNP-(1-22) also demonstrated a high affinity (Kd of 0.41 +/- 0.07 nM) for the glomerular binding of 125I-[Tyr0]CNP-(1-22). Bound 125I-[Tyr0]CNP-(1-22) could be displaced by excess alpha-ANP and excess CNP-(1-22), both with high affinities. The glomerular binding of 125I-[Tyr0]CNP-(1-22) was also prevented by 10 microM C-ANP. Guanosine 3',5'-cyclic monophosphate produced by isolated glomeruli was measured by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)
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Bednarek, Maria A; MacNeil, Tanya; Tang, Rui; Fong, Tung M; Cabello, M Angeles; Maroto, Marta; Teran, Ana
2007-05-01
Alpha-melanotropin, Ac-Ser(1)-Tyr-Ser-Met-Glu-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2)(1), is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of alphaMSH, including a broadly used and more potent the NDP-alphaMSH peptide, Ac-Ser(1)-Tyr-Ser-Nle(4)-Glu-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2), are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-alphaMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand alphaMSH, are exemplified by compound 7, Ac-Ser(1)-Tyr-Ser-Met-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2) (Oic: octahydroindole-2-COOH, 4,4'-Bip: 4,4'-biphenylalanine, Pip: pipecolic acid), shortly NODBP-alphaMSH, which has an IC(50)=0.74 nM (binding assay) and EC(50)=0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-alphaMSH: Ac-Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9) -NH(2) (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8) segment was found to be critical for high agonist potency, while the C-terminal Trp(9) residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.
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Lee, Sang-Min; Hay, Debbie L; Pioszak, Augen A
2016-04-15
Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Influence of the anti-inflammatory compound flosulide on granulocyte function.
Zimmerli, W; Sansano, S; Wiesenberg-Böttcher, I
1991-10-24
Polymorphonuclear leukocytes (PMN) are involved in inflammatory reactions. It is thought that oxygen-derived free radicals released from activated PMN may participate in tissue damage during inflammation. We have shown that flosulide (6-(2,4-difluorophenoxy)-5-methylsulfonylamino-1-indanone ), a novel highly potent anti-inflammatory compound, inhibits superoxide production induced by N-formyl-Met-Leu-Phe (FMLP), C5a and PMA without impairing bacterial killing or chemotaxis. Flosulide (10(-5)-10(-7) M) was more potent in inhibiting the FMLP-induced respiratory burst of PMN than the structurally related compound nimesulide. FMLP-induced superoxide generation was also inhibited by two human flosulide metabolites. A good correlation between this in vitro effect and in vivo anti-inflammatory potency in rat adjuvant arthritis was found for flosulide and its metabolites. Indomethacin, piroxicam and ibuprofen did not inhibit the respiratory burst at 10(-5) M. FMLP receptor number was decreased by 36% in the presence of 10(-5) M flosulide. However, a 250-fold molar excess of flosulide could not displace labeled FMLP from the receptor. Inhibition of degranulation of primary and secondary granules was a common effect of all anti-inflammatory compounds tested. At a concentration of 10(-5) M, all drugs inhibited degranulation to about the same degree, independent of their in vivo anti-inflammatory activity.
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LS, Cohen; B, Arshava; A, Neumoin; JM, Becker; P, Güntert; O, Zerbe; Naider, F
2011-01-01
Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 2 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG)[1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol(TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information. PMID:21791199
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Han, Xing-Fa; Li, Jun-Li; Zhou, Yu-Qin; Ren, Xiao-Hua; Liu, Gong-Cheng; Cao, Xiao-Han; Du, Xiao-Gang; Zeng, Xian-Yin
2016-01-01
GnRH sterilization vaccines have been developed for various practical and clinical reasons. However, conjugation of GnRH peptide to carrier protein has many drawbacks, hampering the further commercialization of GnRH vaccines. In this study, a new nonconjugated GnRH vaccine, D-Lys6-GnRH-tandem-dimer peptide (TDK), emulsified in Specol adjuvant was investigated for its immunocastration efficacy in young male rats. Prepubertal male rats were randomly allocated into three groups (n = 12): control (no treatment), surgically castrated or immunized against 100 μg TDK in Specol adjuvant at 6 weeks of age (with a booster 8 weeks later). Blood samples (for antibody titers and hormone concentrations) were collected at 2-week intervals until rats were killed (18 weeks of age). Compared to intact controls, active immunization against TDK reduced (P < 0.05) serum concentrations of testosterone, inhibin B, LH and FSH, prevented the onset of spermatogenesis at puberty. Furthermore, mRNA expressions of GnRH receptor, LH-β and FSH-β in the pituitary, LH receptor, FSH receptor, inhibin α, βA and βB subunit in the testes were decreased in immunocastrated rats compared to intact controls (P < 0.05). These results demonstrate for the first time that GnRH-tandem-dimer peptide emulsified in Specol is a promising veterinary sterilization medicine.
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Cruz, Luis J; Rueda, Felix; Cordobilla, Begoña; Simón, Lorena; Hosta, Leticia; Albericio, Fernando; Domingo, Joan Carles
2011-02-07
Dendritic cells (DCs) are increasingly being explored as cellular vaccines for tumor immunotherapy, since they provide an effective system of antigen presentation both in vitro and in vivo. An additional advantage of this cell type is that it is possible to target specific antigens through the activation of receptors, such as FcR (the receptor for the IgG Fc fragment) and TLR (toll-like Receptor). Thus, the uptake capacity of DCs can be improved, thereby increasing antigen presentation. This, in turn, would lead to an enhanced immune response, and, in some instances, the tolerance/anergy of immune effector cells present in cancer patients could be reverted. Here we studied various nanotargeting systems, including liposomes and gold nanoparticles of a peptide-based immunotherapeutic vaccine for the treatment of androgen-responsive prostate cancer. Building blocks of the immunogenic peptide consisted of the luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH) peptide (B- and T-cell epitope), in tandem with a T-helper epitope corresponding to the 830-844 region of tetanus toxoid. Three new peptides with several modifications at the N-terminal (palmitoyl, acetyl, and FITC) were synthesized. These peptides also contained a Cys as C-terminal residue to facilitate grafting onto gold nanoparticles. To target different antigen formulations to human DCs, the Fc was activated with a cross-linking spacer to generate a free thiol group and thus facilitate conjugation onto gold nanoparticles, liposomes, and peptide. Our results show that gold nanoparticles and liposomes targeted to FcRs of human DCs are effective antigen delivery carriers and induce a strong immune response with respect to nontargeted LHRH-TT-nanoparticle conjugates and a superior response to that of naked antigens. In addition, dual labeling using gold and FITC-peptide allowed DC tracking by flow cytometry as well as transmission electron microscopy. Nanoparticles were observed to show a homogeneous distribution throughout the cytoplasm. These results open up a new approach to the development of a novel strategy for cancer vaccines.
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Li, Zheng; Wang, Ji-Wei; Wang, Wei-Zhi; Zhi, Xiao-Fei; Zhang, Qun; Li, Bo-Wen; Wang, Lin-Jun; Xie, Kun-Ling; Tao, Jin-Qiu; Tang, Jie; Wei, Song; Zhu, Yi; Xu, Hao; Zhang, Dian-Cai; Yang, Li; Xu, Ze-Kuan
2016-10-01
Natriuretic peptide receptor A (NPRA), the major receptor for atrial natriuretic peptide (ANP), has been implicated in tumorigenesis; however, the role of ANP-NPRA signaling in the development of gastric cancer remains unclear. Immunohistochemical analyses indicated that NPRA expression was positively associated with gastric tumor size and cancer stage. NPRA inhibition by shRNA induced G2/M cell cycle arrest, cell death, and autophagy in gastric cancer cells, due to accumulation of reactive oxygen species (ROS). Either genetic or pharmacologic inhibition of autophagy led to caspase-dependent cell death. Therefore, autophagy induced by NPRA silencing may represent a cytoprotective mechanism. ROS accumulation activated c-Jun N-terminal kinase (JNK) and AMP-activated protein kinase (AMPK). ROS-mediated activation of JNK inhibited cell proliferation by disturbing cell cycle and decreased cell viability. In addition, AMPK activation promoted autophagy in NPRA-downregulated cancer cells. Overall, our results indicate that the inhibition of NPRA suppresses gastric cancer development and targeting NPRA may represent a promising strategy for the treatment of gastric cancer. Copyright © 2016 Elsevier Inc. All rights reserved.
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Wootten, Denise; Reynolds, Christopher A; Smith, Kevin J; Mobarec, Juan C; Furness, Sebastian G B; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M
2016-10-15
Class B GPCRs can activate multiple signalling effectors with the potential to exhibit biased agonism in response to ligand stimulation. Previously, we highlighted key TM domain polar amino acids that were crucial for the function of the GLP-1 receptor, a key therapeutic target for diabetes and obesity. Using a combination of mutagenesis, pharmacological characterisation, mathematical and computational molecular modelling, this study identifies additional highly conserved polar residues located towards the TM helical boundaries of Class B GPCRs that are important for GLP-1 receptor stability and/or controlling signalling specificity and biased agonism. This includes (i) three positively charged residues (R3.30 227 , K4.64 288 , R5.40 310 ) located at the extracellular boundaries of TMs 3, 4 and 5 that are predicted in molecular models to stabilise extracellular loop 2, a crucial domain for ligand affinity and receptor activation; (ii) a predicted hydrogen bond network between residues located in TMs 2 (R2.46 176 ), 6 (R6.37 348 ) and 7 (N7.61 406 and E7.63 408 ) at the cytoplasmic face of the receptor that is important for stabilising the inactive receptor and directing signalling specificity, (iii) residues at the bottom of TM 5 (R5.56 326 ) and TM6 (K6.35 346 and K6.40 351 ) that are crucial for receptor activation and downstream signalling; (iv) residues predicted to be involved in stabilisation of TM4 (N2.52 182 and Y3.52 250 ) that also influence cell signalling. Collectively, this work expands our understanding of peptide-mediated signalling by the GLP-1 receptor. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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Li, Caina; Yang, Miaomiao; Hou, Guojiang; Liu, Shuainan; Huan, Yi; Yu, Dongan; Sun, Sujuan; Liu, Quan; Yan, Shousheng; Shen, Zhufang
2017-09-01
GW002 is a recombinant protein engineered by fusing the C-terminal region of human glucagon-like peptide-1 (GLP-1) to the N-terminal region of human serum albumin (HSA) with a peptide linker. This study aims to evaluate its anti-diabetic effects both in vitro and in vivo. The GLP-1 receptor-dependent luciferase reporter plasmid was transiently transfected in NIT-1 cells to calculate the half-maximal concentration (EC 50 ) for GLP-1 receptor activation, and normal ICR mice and diabetic KKAy mice were acutely injected with GW002 (1, 3, 9 mg/kg) subcutaneously to evaluate the hypoglycemic action, while the diabetic KKAy and db/db mice were treated with GW002 once daily for 7 weeks to evaluate the effects on glucose metabolism. The results showed that GW002 activated GLP-1 receptor in NIT-1 cells with higher EC 50 versus exendin-4 (46.7 vs. 7.89 nM), and single subcutaneous injection of GW002 at doses of 1, 3 and 9 mg/kg efficiently restrained the glycemia variation after oral glucose loading in ICR mice for at least 4 d, as well as reducing the non-fasting blood glucose in KKAy mice for about 2 d, while repeated injections of GW002 significantly improved abnormal glycaemia, hemoglobin (Hb)A1c levels, oral glucose intolerance and β-cell function in diabetic db/db mice. These results suggested that GW002 showed prolonged hypoglycemic action by activating its cognate receptor and provided efficient control of glucose metabolism. Thus GW002 may be a potential treatment for the management of type 2 diabetes.
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Xia, Pengpeng; Quan, Guomei; Yang, Yi; Zhao, Jing; Wang, Yiting; Zhou, Mingxu; Hardwidge, Philip R; Zhu, Jianzhong; Liu, Siguo; Zhu, Guoqiang
2018-02-26
The binding of F4 + enterotoxigenic Escherichia coli (ETEC) and the specific receptor on porcine intestinal epithelial cells is the initial step in F4 + ETEC infection. Porcine aminopeptidase N (APN) is a newly discovered receptor for F4 fimbriae that binds directly to FaeG adhesin, which is the major subunit of the F4 fimbriae variants F4ab, F4ac, and F4ad. We used overlapping peptide assays to map the APN-FaeG binding sites, which has facilitated in the identifying the APN-binding amino acids that are located in the same region of FaeG variants, thereby limiting the major binding regions of APN to 13 peptides. To determine the core sequence motif, a panel of FaeG peptides with point mutations and FaeG mutants were constructed. Pull-down and binding reactivity assays using piglet intestines determined that the amino acids G159 of F4ab, N209 and L212 of F4ac, and A200 of F4ad were the critical residues for APN binding of FaeG. We further show using ELISA and confocal microscopy assay that amino acids 553-568, and 652-670 of the APN comprise the linear epitope for FaeG binding in all three F4 fimbriae variants.
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van der Wal, Steffen; de Korne, Clarize M; Sand, Laurens L G; van Willigen, Danny M; Hogendoorn, Pancras C W; Szuhai, Karoly; van Leeuwen, Fijs W B; Buckle, Tessa
2018-06-04
Availability of a receptor for theranostic pre-targeting approaches was assessed using a novel "click" chemistry-based de-activatable fluorescence-quenching concept. Efficacy was evaluated in a cell-based model system that exhibits both membranous (available) and internalized (unavailable) receptor-fractions of the clinically relevant receptor chemokine receptor 4 (CXCR4). Proof of concept was based on a de-activatable tracer consisting out of a CXCR4 specific peptide functionalized with a Cy5 dye comprising a chemo-selective azide handle (N3-Cy5-AcTZ14011). Reaction with a Cy7 quencher dye (Cy7-DBCO) resulted in optically silent Cy7-["click"]-Cy5-AcTZ14011. In situ a >90% FRET-based reduction of signal intensity of N3-Cy5-AcTZ14011 (KD 222.4 ± 25.2 nM) was seen within minutes after quencher addition. In cells, discrimination between the membranous and internalized receptor-fraction could be made through quantitative assessment of quenching/internalization kinetics. As such, using this approach screening of membrane receptors and their applicability in receptor-(pre-)targeted theranostics can become straightforward. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Wu, Xin; Zhang, Jie-Ting; Liu, Jue; Yang, Si; Chen, Tao; Chen, Jian-Guo; Wang, Fang
2015-11-01
Calcitonin gene-related peptide (CGRP) is a 37 amino acid neuropeptide, which plays a critical role in the central nervous system. CGRP binds to G protein-coupled receptors, including CGRP1, which couples positively to adenylyl cyclase (AC) and protein kinase A (PKA) activation. CGRP and CGRP1 receptors are enriched in central nucleus of the amygdala (CeA), the main part of the amygdala, which regulates conditioned fear memories. Here, we reported the importance of CGRP and CGRP1 receptor for synaptic plasticity in the CeA and the extinction of fear memory in rats. Our electrophysiological and behavioral in vitro and in vivo results showed exogenous application of CGRP induced an immediate and lasting long-term potentiation in the basolateral nucleus of amygdala-CeA pathway, but not in the lateral nucleus of amygdala-CeA pathway, while bilateral intra-CeA infusion CGRP (0, 5, 13 and 21 μM/side) dose dependently enhanced fear memory extinction. The effects were blocked by CGRP1 receptor antagonist (CGRP8-37 ), N-methyl-d-aspartate receptors antagonist MK801 and PKA inhibitor H89. These results demonstrate that CGRP can lead to long-term potentiation of basolateral nucleus of amygdala-CeA pathway through a PKA-dependent postsynaptic mechanism that involved N-methyl-d-aspartate receptors and enhance the extinction of fear memory in rats. Together, the results strongly support a pivotal role of CGRP in the synaptic plasticity of CeA and extinction of fear memory. Calcitonin gene-related peptide (CGRP) plays an essential role in synaptic plasticity in the amygdala and fear memory. We found that CGRP-induced chemical long-term potentiation (LTP) in a dose-dependent way in the BLA-CeA (basolateral and central nucleus of amygdala, respectively) pathway and enhanced fear memory extinction in rats through a protein kinase A (PKA)-dependent postsynaptic mechanism that involved NMDA receptors. These results support a pivotal role of CGRP in amygdala. © 2015 International Society for Neurochemistry.
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NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids.
Simeth, Nadja A; Bause, Manuel; Dobmeier, Michael; Kling, Ralf C; Lachmann, Daniel; Hübner, Harald; Einsiedel, Jürgen; Gmeiner, Peter; König, Burkhard
2017-01-01
Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8-13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr 11 in NT(8-13). Additionally, Arg 8 , Arg 9 , and Ile 12 of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (K i (NTS2): 29nM, K i (NTS1): 35,000nM) was observed for the peptide analog 17R trans . Copyright © 2016 Elsevier Ltd. All rights reserved.
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Identification of new pyrrole alkaloids from the fruits of Lycium chinense.
Youn, Ui Joung; Lee, Joo Yun; Kil, Yun-Seo; Han, Ah-Reum; Chae, Chong Hak; Ryu, Shi Yong; Seo, Eun-Kyoung
2016-03-01
Three new minor pyrrole alkaloids, 3-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]pentanedioic acid (1), (2R)-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl]-1-methoxy-1-oxobutanoic acid (2), and methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-4-methylpentanoate (3) were isolated from the fruits of Lycium chinense Miller (Solanaceae), along with the known compound, methyl (2R)-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(phenyl)propanoate (4). The structures of 1-4 were elucidated by analysis of their 1D- and 2D-NMR and HRMS data. The absolute configurations of 2-4, possessing a stereogenic center in each structure, were determined by comparison of their experimental electronic circular dichroism (ECD) with those of calculated ECD values.
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Jin, Dao-Zhong; Xue, Bing; Mao, Li-Min; Wang, John Q
2015-10-22
Metabotropic and ionotropic glutamate receptors are closely clustered in postsynaptic membranes and are believed to interact actively with each other to control excitatory synaptic transmission. Metabotropic glutamate receptor 5 (mGluR5), for example, has been well documented to potentiate ionotropic NMDA receptor activity, although underlying mechanisms are poorly understood. In this study, we investigated the role of mGluR5 in regulating trafficking and subcellular distribution of NMDA receptors in adult rat striatal neurons. We found that the mGluR1/5 agonist DHPG concentration-dependently increased NMDA receptor GluN1 and GluN2B subunit expression in the surface membrane. Meanwhile, DHPG reduced GluN1 and GluN2B levels in the intracellular compartment. The effect of DHPG was blocked by an mGluR5 selective antagonist MTEP but not by an mGluR1 selective antagonist 3-MATIDA. Pretreatment with an inhibitor or a specific inhibitory peptide for synapse-enriched Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) also blocked the DHPG-stimulated redistribution of GluN1 and GluN2B. In addition, DHPG enhanced CaMKIIα activity and elevated GluN2B phosphorylation at a CaMKII-sensitive site (serine 1303). These results demonstrate that mGluR5 regulates trafficking of NMDA receptors in striatal neurons. Activation of mGluR5 appears to induce rapid trafficking of GluN1 and GluN2B to surface membranes through a signaling pathway involving CaMKII. Copyright © 2015 Elsevier B.V. All rights reserved.
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NASA Astrophysics Data System (ADS)
Bugaj, Joseph E.; Achilefu, Samuel I.; Dorshow, Richard B.; Rajagopalan, Raghavan
2001-04-01
We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst2) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide- dye conjugates are effective as contrast agents for optical imaging of tumors.
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Role of PSMA in Aberrant Cell Cycle Progression in Prostate Cancer
2004-12-01
playing a role in nutrient uptake, and a peptidase involved in signal transduction in prostate epithelial cells. Insights into possible functions of PSMA...should improve the diagnostic and therapeutic values of this clinically important molecule. prostate cancer; receptor; peptidase ; endocytosis 2 OVERVIEW... peptidase activities hydrolyze gamma-peptide bonds between N-acetylaspartate and glutamate in the abundant neuropeptide N-acetylaspartylglutamate
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Chung, Connie; Wu, Wei-Hua; Chen, Bo-Shiun
2015-01-01
The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, β, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission. PMID:26229101
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Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94.
Cheent, Kuldeep S; Jamil, Khaleel M; Cassidy, Sorcha; Liu, Mengya; Mbiribindi, Berenice; Mulder, Arend; Claas, Frans H J; Purbhoo, Marco A; Khakoo, Salim I
2013-10-15
Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94-NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor-ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A(+) NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR(+) and NKG2A(+) NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I-bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I.
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Novel thiophene-based cycloruthenated compounds: synthesis, characterization, and reactivity.
Cuesta, Luciano; Maluenda, Irene; Soler, Tatiana; Navarro, Rafael; Urriolabeitia, Esteban P
2011-01-03
The reactions between a series of thiophene-based imines with [(η(6)-C(6)H(6))RuCl(μ-Cl)](2), in a basic medium, and in MeCN give a family of ruthenacycles of stoichiometry [Ru(C^N)(NCMe)(4)]PF(6) (C^N = orthometalated thiopheneimine). In these species, the C-H activation process is produced in most cases at the thiophene ring. When two C-H bonds are competing (thiophene vs aryl), the cyclometalation can be driven regioselectively to the thiophene unit or to the aryl ring as a function of the location of the iminic C=N bond. Cyclometalation can also be oriented to positions 2 or 3 of the thiophene depending on the situation of the imine in the heterocycle (3 or 2, respectively). In all studied cases, the η(6)-C(6)H(6) ligand was substituted by acetonitrile. The X-ray structures of two representative complexes have been determined. These thiophene-based metallacycles react with iodine under very mild conditions affording, after hydrolysis, substituted 3-iodo-2-formyl(benzo)thiophenes or substituted 2-iodo-3-formyl(benzo)thiophenes, as a function of the organometallic precursor.
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Paquet, J -L; Luccarini, J -M; Fouchet, C; Defrêne, E; Loillier, B; Robert, C; Bélichard, P; Cremers, B; Pruneau, D
1999-01-01
The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1-oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8–6.6 fold and 7.0–16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6–23 fold in physiological HBSS compared to low ionic strength TES binding buffer. BK (0.01–3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10−7 M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents. PMID:10204994
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Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.
Pomorska, Dorota K; Gach, Katarzyna; Janecka, Anna
2014-01-01
The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.
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Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks
Schäffer, Lauge; Brissette, Renee E.; Spetzler, Jane C.; Pillutla, Renuka C.; Østergaard, Søren; Lennick, Michael; Brandt, Jakob; Fletcher, Paul W.; Danielsen, Gillian M.; Hsiao, Ku-Chuan; Andersen, Asser S.; Dedova, Olga; Ribel, Ulla; Hoeg-Jensen, Thomas; Hansen, Per Hertz; Blume, Arthur J.; Markussen, Jan; Goldstein, Neil I.
2003-01-01
Insulin is thought to elicit its effects by crosslinking the two extracellular α-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases. PMID:12684539
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Cosentino, Marco; Luini, Alessandra; Bombelli, Raffaella; Corasaniti, Maria T; Bagetta, Giacinto; Marino, Franca
2014-08-01
Bergamot (Citrus aurantium L. subsp. bergamia) essential oil (BEO) is used in folk medicine as an antiseptic and anthelminthic and to facilitate wound healing. Evidence indicates that BEO has substantial antimicrobial activity; however its effects on immunity have never been examined. We studied the effects of BEO on reactive oxygen species (ROS) production in human polymorphonuclear leukocytes (PMN) and the role of Ca(2+) in the functional responses evoked by BEO in these cells. Results show that BEO increased intracellular ROS production in human PMN, an effect that required the contribution of extracellular (and, to a lesser extent, of intracellular) Ca(2+) . Bergamot essential oil also significantly increased ROS production induced by the chemotactic peptide N-formyl-Met-Leu-Phe and reduced the response to the protein kinase C activator phorbol myristate acetate. In conclusion, this is the first report showing the ability of BEO to increase ROS production in human PMN. This effect could both contribute to the activity of BEO in infections and in tissue healing as well as underlie an intrinsic proinflammatory potential. The relevance of these findings for the clinical uses of BEO needs careful consideration. Copyright © 2014 John Wiley & Sons, Ltd.
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Réglier-Poupet, H; Hakim, J; Gougerot-Pocidalo, M A; Elbim, C
1998-12-01
Cytokines such as IL-10, IL-4, IL-13 and TGF-beta play a major role in the regulation of immune responses and are considered as anti-inflammatory agents mainly due to their actions on monocytes. These cytokines are also known to participate in the regulation of PMN activities. However, few and contradictory results have been reported on their direct and priming effects on the PMN oxidative burst, which is essential for killing bacteria. We used a flow cytometry method to study the effects of these cytokines on the PMN oxidative burst; we also used whole blood to avoid PMN activation related to isolation procedures and in order to simulate the in vivo situation more closely. None of the cytokines tested had direct or priming effects on PMN H2O2 production. We also show for the first time that these cytokines do not modulate TNF priming of the PMN oxidative burst in response to N-formyl peptides (fMLP). These results show that the anti-bacterial activity of PMN, in terms of the PMN respiratory burst, is not down regulated by these anti-inflammatory cytokines in whole blood.
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Becker, Jason R.; Chatterjee, Sneha; Robinson, Tamara Y.; Bennett, Jeffrey S.; Panáková, Daniela; Galindo, Cristi L.; Zhong, Lin; Shin, Jordan T.; Coy, Shannon M.; Kelly, Amy E.; Roden, Dan M.; Lim, Chee Chew; MacRae, Calum A.
2014-01-01
Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways. PMID:24353062
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Huang, Xing-Fen; Wu, Qing-Lai; He, Jian-Shi; Huang, Zhi-Zhen
2015-04-21
A novel DCC reaction between aromatic aldehydes or ketones and H-phosphonates has been developed for the synthesis of p-formyl or p-acylphenylphosphonates. The synthetic method has excellent para regioselectivities, good yields, and broad substrate scopes and is more benign to the environment. The DCC reaction also tolerates many functional groups, and results in a series of new p-formyl and p-acylphenylphosphonates, which should be important building blocks for the synthesis of versatile arylphosphonate derivatives.
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NASA Astrophysics Data System (ADS)
Shen, Duanwen; Liang, Kexiang; Ye, Yunpeng; Tetteh, Elizabeth; Achilefu, Samuel
2006-02-01
Numerous studies have shown that basic Tat peptide (48-57) internalized non-specifically in cells and localized in the nucleus. However, localization of imaging agents in cellular nucleus is not desirable because of the potential mutagenesis. When conjugated to the peptides that undergo receptor-mediated endocytosis, Tat peptide could target specific cells or pathologic tissue. We tested this hypothesis by incorporating a somatostatin receptor-avid peptide (octreotate, Oct) and two different fluorescent dyes, Cypate 2 (Cy2) and fluorescein 5'-carboxlic acid (5-FAM), into the Tat-peptide sequence. In addition to the Cy2 or 5-FAM-labeled Oct conjugated to Tat peptide (Tat) to produce Tat-Oct-Cypate2 or Tat-Oct-5-FAM, we also labeled the Tat the Tat peptide with these dyes (Tat-Cy2 and Tat-5-FAM) to serve as positive control. A somatostatin receptor-positive pancreatic tumor cell line, AR42J, was used to assess cell internalization. The results show that Tat-5-FAM and Tat-Cypate2 localized in both nucleus and cytoplasm of the cells. In contrast to Tat-Oct-Cypate2, which localized in both the cytoplasm and nucleus, Tat-Oct-5-FAM internalized in the cytoplasm but not in the nucleus of AR42J cells. The internalizations were inhibited by adding non-labeled corresponding peptides, suggesting that the endocytoses of each group of labeled and the corresponding unlabeled compounds occurred through a common pathway. Thus, fluorescent probes and endocytosis complex between octreotate and somatostatin receptors in cytoplasm could control nuclear internalization of Tat peptides.
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Goh, William; Yamamoto, Sandra Y; Thompson, Karen S; Bryant-Greenwood, Gillian D
2013-08-01
This study was designed to show whether placental relaxin (RLN), its receptor (RXFP1), or insulin-like peptide 4 (INSL4) might have altered expression in patients with placenta accreta. The baseline expression of their genes through gestation (n = 34) was quantitated in the placental basal plate (BP) and villous trophoblast (TR), and compared to their expression in placenta accreta (n = 6). The proteins were also immunolocalized and quantitated in the accreta tissues. The messenger RNAs (mRNAs) of matrix metalloproteinase 9, -2, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were also measured. Results demonstrated that the BP and TR expressed low levels of RLN/RXFP1 and INSL4 through gestation. In accreta, increased RLN gene and protein in BP were associated with antepartum bleeding whereas INSL4 expression decreased throughout the TR. There were no changes in mRNAs for MMPs, but TIMP-1 was increased only in the invasive TR.
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Yamamoto, Sandra Y.; Thompson, Karen S.; Bryant-Greenwood, Gillian D.
2013-01-01
This study was designed to show whether placental relaxin (RLN), its receptor (RXFP1), or insulin-like peptide 4 (INSL4) might have altered expression in patients with placenta accreta. The baseline expression of their genes through gestation (n = 34) was quantitated in the placental basal plate (BP) and villous trophoblast (TR), and compared to their expression in placenta accreta (n = 6). The proteins were also immunolocalized and quantitated in the accreta tissues. The messenger RNAs (mRNAs) of matrix metalloproteinase 9, -2, and tissue inhibitors of matrix metalloproteinase (TIMP)-1 were also measured. Results demonstrated that the BP and TR expressed low levels of RLN/RXFP1 and INSL4 through gestation. In accreta, increased RLN gene and protein in BP were associated with antepartum bleeding whereas INSL4 expression decreased throughout the TR. There were no changes in mRNAs for MMPs, but TIMP-1 was increased only in the invasive TR. PMID:23302396
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Springer, Jeremy; Azer, John; Hua, Rui; Robbins, Courtney; Adamczyk, Andrew; McBoyle, Sarah; Bissell, Mary Beth; Rose, Robert A
2012-05-01
Natriuretic peptides (NPs) are best known for their ability to regulate blood vessel tone and kidney function whereas their electrophysiological effects on the heart are less clear. Here, we measured the effects of BNP and CNP on sinoatrial node (SAN) and atrial electrophysiology in isolated hearts as well as isolated SAN and right atrial myocytes from mice. BNP and CNP dose-dependently increased heart rate and conduction through the heart as indicated by reductions in R-R interval, P wave duration and P-R interval on ECGs. In conjunction with these ECG changes BNP and CNP (100 nM) increased spontaneous action potential frequency in isolated SAN myocytes by increasing L-type Ca(2+) current (I(Ca,L)) and the hyperpolarization-activated current (I(f)). BNP had no effect on right atrial myocyte APs in basal conditions; however, in the presence of isoproterenol (10nM), BNP increased atrial AP duration and I(Ca,L). Quantitative gene expression and immunocytochemistry data show that all three NP receptors (NPR-A, NPR-B and NPR-C) are expressed in the SAN and atrium. The effects of BNP and CNP on SAN and right atrial myocytes were maintained in mutant mice lacking functional NPR-C receptors and blocked by the NPR-A antagonist A71915 indicating that BNP and CNP function through their guanylyl cyclase-linked receptors. Our data also show that the effects of BNP and CNP are completely absent in the presence of the phosphodiesterase 3 inhibitor milrinone. Based on these data we conclude that NPs can increase heart rate and electrical conduction by activating the guanylyl cyclase-linked NPR-A and NPR-B receptors and inhibiting PDE3 activity. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Cryptic bioactivity capacitated by synthetic hybrid plant peptides
Hirakawa, Yuki; Shinohara, Hidefumi; Welke, Kai; Irle, Stephan; Matsubayashi, Yoshikatsu; Torii, Keiko U.; Uchida, Naoyuki
2017-01-01
Evolution often diversifies a peptide hormone family into multiple subfamilies, which exert distinct activities by exclusive interaction with specific receptors. Here we show that systematic swapping of pre-existing variation in a subfamily of plant CLE peptide hormones leads to a synthetic bifunctional peptide that exerts activities beyond the original subfamily by interacting with multiple receptors. This approach provides new insights into the complexity and specificity of peptide signalling. PMID:28165456
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Heterogeneity of prejunctional NPY receptor-mediated inhibition of cardiac neurotransmission
Serone, Adrian P; Wright, Christine E; Angus, James A
1999-01-01
Neuropeptide Y (NPY) has been proposed as the candidate inhibitory peptide mediating interactions between sympathetic and vagal neurotransmission in several species, including man. Here, we have defined the NPY receptors involved in modulation of cardiac autonomic neurotransmission using receptor-selective agonists and antagonists in the rabbit and guinea-pig isolated right atria.In isolated atrial preparations, sympathetically-mediated tachycardia (ST; with atropine 1 μM) or vagally-mediated bradycardia (VB; with propranolol 0.1–1 μM) in response to electrical field stimulation (EFS, 1–4 pulses) were tested 0–30 min after incubation with single concentrations of vehicle, NPY (0.01–10 μM), the Y2 receptor agonist N-Acetyl-[Leu28,31]NPY(24–36) (termed N-A[L]NPY(24–36)) or the Y1 receptor agonist [Leu31,Pro34]NPY (LP). The effect of NPY on the concentration-chronotropic response curves to isoprenaline and bethanechol were also assessed.Guinea-pig atria: NPY and N-A[L]NPY(24–36) caused concentration-dependent inhibition of VB and ST to EFS. Both peptides caused maximal inhibition of VB and ST within 10 min incubation and this remained constant. LP caused a concentration-dependent, transient inhibition of ST which was antagonized by the Y1-receptor antagonist GR231118 (0.3 μM), with apparent competitive kinetics. Rabbit atria: NPY (1 or 10 μM) had no effect on VB at any time point, but both NPY and LP caused a transient (∼10 min) inhibition of sympathetic tachycardia. This inhibition could be prevented by 0.3 μM GR231118. N-A[L]NPY(24–36) had no effect on ST. NPY had no effect on the response to β-adrenoceptor stimulation by isoprenaline nor muscarinic-receptor stimulation by bethanechol in either species.Thus, in the guinea-pig, NPY causes a stable inhibition of both VB and ST to EFS via Y2 receptors and transient inhibition of ST via Y1 receptors. In contrast in the rabbit, NPY has no effect on the cardiac vagus and prejunctional inhibition of ST is transient and mediated by a Y1-like receptor (rather than Y2). Therefore it would be surprising if NPY plays a functional role in modulation of cardiac neurotransmission in the rabbit. PMID:10369461
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N-octanoylated ghrelin peptide inhibits bovine oocyte meiotic resumption.
Xu, X L; Bai, J H; Feng, T; Xiao, L L; Song, Y Q; Xiao, Y X; Liu, Y
2018-07-01
Studies have shown that ghrelin plays an important role in the mammalian reproductive system, including the central, gonadal levels, and also during in vitro maturation of oocytes; however, the functions of ghrelin in bovine oocyte meiosis require further investigation. We aimed to evaluate the effects of an n-octanoylated ghrelin peptide on oocyte meiotic resumption and the developmental competence of mature oocytes in vitro. design: The expression of GHRL (encoding ghrelin) mRNA and its receptor (the growth hormone secretagogue receptor, GHSR) in the cumulus-oocyte complex (COCs), denuded oocytes (DOs), and cumulus cells (CCs) was assessed using quantitative real-time reverse transcription PCR (qRT-PCR), and the effects of the n-octanoylated ghrelin peptide on meiotic resumption were studied at four different doses (0, 10, 50, and 100 ng/mL) in a 6 h culture system. qRT-PCR analysis showed that GHRL and GHSR mRNAs were expressed in all tested samples; however, GHRL was predominantly expressed in DOs, and GHSR was predominantly expressed in CCs. Germinal vesicle breakdown was inhibited significantly by 50 ng/mL ghrelin compared with that in the negative control (P < 0.05). Further studies showed that n-octanoylated ghrelin increased the levels of cAMP and cGMP in the CCs and DOs, which inhibited the meiotic resumption of bovine oocytes. And the inhibitory role in the developmental competence of mature oocytes were also included, ghrelin could significantly improve the cleavage rate (P < 0.05) and blastocyst rate (P < 0.05). N-octanoylated ghrelin maintained bovine oocytes meiotic arrest and further improved their developmental competence; therefore, n-octanoylated ghrelin could be considered as a potential pharmaceutical inhibitor of meiosis for the in vitro maturation of bovine oocytes. Copyright © 2018 Elsevier Inc. All rights reserved.
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Molecular Recognition of Corticotropin releasing Factor by Its G protein-coupled Receptor CRFR1
DOE Office of Scientific and Technical Information (OSTI.GOV)
Pioszak, Augen A.; Parker, Naomi R.; Suino-Powell, Kelly
2009-01-15
The bimolecular interaction between corticotropin-releasing factor (CRF), a neuropeptide, and its type 1 receptor (CRFR1), a class B G-protein-coupled receptor (GPCR), is crucial for activation of the hypothalamic-pituitary-adrenal axis in response to stress, and has been a target of intense drug design for the treatment of anxiety, depression, and related disorders. As a class B GPCR, CRFR1 contains an N-terminal extracellular domain (ECD) that provides the primary ligand binding determinants. Here we present three crystal structures of the human CRFR1 ECD, one in a ligand-free form and two in distinct CRF-bound states. The CRFR1 ECD adopts the alpha-beta-betaalpha fold observedmore » for other class B GPCR ECDs, but the N-terminal alpha-helix is significantly shorter and does not contact CRF. CRF adopts a continuous alpha-helix that docks in a hydrophobic surface of the ECD that is distinct from the peptide-binding site of other class B GPCRs, thereby providing a basis for the specificity of ligand recognition between CRFR1 and other class B GPCRs. The binding of CRF is accompanied by clamp-like conformational changes of two loops of the receptor that anchor the CRF C terminus, including the C-terminal amide group. These structural studies provide a molecular framework for understanding peptide binding and specificity by the CRF receptors as well as a template for designing potent and selective CRFR1 antagonists for therapeutic applications.« less
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NASA Astrophysics Data System (ADS)
Said, Sami I.; Berisha, Hasan I.; Pakbaz, Hedayatollah
1996-05-01
Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the ``adult respiratory distress syndrome,'' and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.
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Vandormael, Bart; Fourla, Danai-Dionysia; Gramowski-Voss, Alexandra; Kosson, Piotr; Weiss, Dieter G; Schröder, Olaf H-U; Lipkowski, Andrzej; Georgoussi, Zafiroula; Tourwé, Dirk
2011-11-24
Novel dermorphin tetrapeptides are described in which Tyr(1) is replaced by Dmt(1), where d-Ala(2) and Gly(4) are N-methylated, and where Phe(3)-Gly(4) residue is substituted by the constrained Aba(3)-Gly(4) peptidomimetic. Most of these peptidic ligands displayed binding affinities in the nanomolar range for both μ- and δ-opioid receptors but no detectable affinity for the κ-opioid receptor. Measurements of cAMP accumulation, phosphorylation of extracellular signal-regulated kinase (ERK1/2) in HEK293 cells stably expressing each of these receptors individually, and functional screening in primary neuronal cultures confirmed the potent agonistic properties of these peptides. The most potent ligand H-Dmt-NMe-d-Ala-Aba-Gly-NH(2) (BVD03) displayed mixed μ/δ opioid agonist properties with picomolar functional potencies. Functional electrophysiological in vitro assays using primary cortical and spinal cord networks showed that this analogue possessed electrophysiological similarity toward gabapentin and sufentanil, which makes it an interesting candidate for further study as an analgesic for neuropathic pain.
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Structural basis for bifunctional peptide recognition at human δ-opioid receptor
Fenalti, Gustavo; Zatsepin, Nadia A.; Betti, Cecilia; ...
2015-02-16
Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH 2 (DIPP-NH 2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. In summary, the observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.
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Urokinase and its Receptors in Chronic Kidney Disease
Zhang, Guoqiang; Eddy, Allison A.
2011-01-01
Since the recognition that plasminogen activator inhibitor-1 (PAI-1) is a powerful profibrotic molecule, there has been considerable interest in deciphering the extent to which this effect is mediated by its ability to inhibit serine proteases with downstream effects on fibrogenesis. This review will summarize current knowledge about the serine protease urokinase-type plasminogen activator and its high affinity receptor uPAR/CD87 as it pertains to chronic kidney disease (CKD) progression. An emerging theme is that the effects of PAI-1 and uPAR appear to be organ- and site-specific. Normal kidney tubules produce a large quantity of uPA that is secreted into the urinary space. Activity levels increase during CKD presumably due to new sources of production by macrophages and fibroblasts. By activating hepatocyte growth factor and degrading fibrinogen uPA may have anti-fibrotic effects. However CKD severity after experimental ureteral obstruction is not altered by endogenous uPA deficiency. Beneficial effects of exogenous uPA have been reported in experimental models of fibrosis in the lung and liver but CKD awaits exploration. Absent in normal kidneys uPAR is expressed by both renal parenchymal cells and inflammatory cells in a variety of pathological states. Such expression appears beneficial based on studies performed in uPAR-deficient mice. The uPAR promotes bacterial clearance in infectious diseases. In CKD uPAR expression is associated with high uPA activity but its most important effect appears to be due to scavenging activities and effects on cell recruitment and migration. Although uPAR itself is a non-signaling receptor, it interacts with a variety of co-receptors to modify cellular behavior. Best known are interactions with the low-density lipoprotein receptor-related protein (LRP-1) that lead to PAI-1 endocytosis and degradation, and interactions with several integrins to regulate matrix-dependent cell migration. Contacts with the receptor for the complement C5a component and the interleukin −6 receptor gp130 are examples of other recently recognized interactions. In addition to uPA, vitronectin and high molecular weight kininogen are alternate uPAR ligands that could be implicated in CKD progression. uPAR may also be shed from cell membranes. This soluble form (suPAR) has been detected in plasma and urine and is known to be a chemoattractant for leukocytes that express the formyl-peptide-receptor-like receptor 1/lipoxin A4 receptor. In addition to uPAR several other receptors, including some of the uPAR co-receptors, may also bind directly to uPA and activate cell signaling pathways. The roles of these newer uPAR ligands and uPA receptors are just beginning to be investigated. Since many of them are expressed in the kidney, their potential participation in CKD pathogenesis will be of interest. PMID:18508599
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Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui
2005-03-01
For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.
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Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S
2017-07-25
Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.
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Yang, Jianquan; Guo, Haixun; Gallazzi, Fabio; Berwick, Marianne; Padilla, R Steven; Miao, Yubin
2009-08-19
The purpose of this study was to determine whether Arg-Gly-Asp (RGD)-conjugated α-melanocyte stimulating hormone (α-MSH) hybrid peptide could be employed to target melanocortin-1 (MC1) receptor for potential melanoma therapy. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys(3,4,10), DPhe(7), Arg(11)]α-MSH(3-13) {(Arg(11))CCMSH} to generate RGD-Lys-(Arg(11))CCMSH hybrid peptide. The MC1 receptor binding affinity of RGD-Lys-(Arg(11))CCMSH was determined in B16/F1 melanoma cells. The internalization and efflux, melanoma targeting and pharmacokinetic properties and single photon emission computed tomography/CT (SPECT/CT) imaging of (99m)Tc-RGD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma cells and melanoma-bearing C57 mice. Clonogenic cytotoxic effect of RGD-Lys-(Arg(11))CCMSH was examined in B16/F1 melanoma cells. RGD-Lys-(Arg(11))CCMSH displayed 2.1 nM MC1 receptor binding affinity. (99m)Tc-RGD-Lys-(Arg(11))CCMSH showed rapid internalization and extended retention in B16/F1 cells. The cellular uptake of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was MC1 receptor-mediated. (99m)Tc-RGD-Lys-(Arg(11))CCMSH exhibited high tumor uptake (14.83 ± 2.94% ID/g 2 h postinjection) and prolonged tumor retention (7.59 ± 2.04% ID/g 24 h postinjection) in B16/F1 melanoma-bearing mice. Nontarget organ uptakes were generally low except for the kidneys. Whole-body clearance of (99m)Tc-RGD-Lys-(Arg(11))CCMSH was rapid, with approximately 62% of the injected radioactivity cleared through the urinary system by 2 h postinjection. Flank melanoma tumors were clearly imaged by small animal SPECT/CT using (99m)Tc-RGD-Lys-(Arg(11))CCMSH as an imaging probe 2 h postinjection. Single treatment (3 h incubation) with 100 nM of RGD-Lys-(Arg(11))CCMSH significantly (p < 0.05) decreased the clonogenic survival of B16/F1 cells by 65% compared to the untreated control cells. Favorable melanoma targeting property of (99m)Tc-RGD-Lys-(Arg(11))CCMSH and remarkable cytotoxic effect of RGD-Lys-(Arg(11))CCMSH in B16/F1 cells warranted the further evaluation of (188)Re-labeled α-MSH hybrid peptides as novel therapeutic peptides for melanoma treatment once the strategies of amino acid coinjection or structural modification of peptide sequence substantially reduce the renal uptake.
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Plant peptide hormone signalling.
Motomitsu, Ayane; Sawa, Shinichiro; Ishida, Takashi
2015-01-01
The ligand-receptor-based cell-to-cell communication system is one of the most important molecular bases for the establishment of complex multicellular organisms. Plants have evolved highly complex intercellular communication systems. Historical studies have identified several molecules, designated phytohormones, that function in these processes. Recent advances in molecular biological analyses have identified phytohormone receptors and signalling mediators, and have led to the discovery of numerous peptide-based signalling molecules. Subsequent analyses have revealed the involvement in and contribution of these peptides to multiple aspects of the plant life cycle, including development and environmental responses, similar to the functions of canonical phytohormones. On the basis of this knowledge, the view that these peptide hormones are pivotal regulators in plants is becoming increasingly accepted. Peptide hormones are transcribed from the genome and translated into peptides. However, these peptides generally undergo further post-translational modifications to enable them to exert their function. Peptide hormones are expressed in and secreted from specific cells or tissues. Apoplastic peptides are perceived by specialized receptors that are located at the surface of target cells. Peptide hormone-receptor complexes activate intracellular signalling through downstream molecules, including kinases and transcription factors, which then trigger cellular events. In this chapter we provide a comprehensive summary of the biological functions of peptide hormones, focusing on how they mature and the ways in which they modulate plant functions. © 2015 Authors; published by Portland Press Limited.
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[Coumarins of Anemone raddeana Regel and their biological activity].
Ren, Feng-Zhi; Chen, Shu-Hong; Zheng, Zhi-Hui; Zhang, Xue-Xia; Li, Li-Hong; Dong, Ai-Hua
2012-02-01
To study the coumarins of Anemone raddeana Regel, the compounds were separated by silica gel column chromatography and HPLC. Their structures were identified by their physicochemical property and spectral analysis. Two new compounds were isolated and identified as 4, 7-dimethoxyl-5-methyl-6-hydroxy coumarin (1) and 4, 7-dimethoxyl-5-formyl-6-hydroxycoumarin (2). The bioassays indicated that compounds 1 and 2 could significantly inhibit the proliferation of cancer cell, and showed the agonist effect on the transactivity of retinoic acid receptor-alpha (RARalpha). In addition, the two compounds had inhibitory effect against human leukocyte elastase (HLE).
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Development of a (68)Ga-peptide tracer for PET GnRH1-imaging.
Zoghi, Masoumeh; Jalilian, Amir R; Niazi, Ali; Johari-Daha, Fariba; Alirezapour, Behrouz; Ramezanpour, Saeed
2016-07-01
Total synthesis, quality control and preclinical evaluation of [(68)Ga]-DOTA-triptorelin ([(68)Ga]-DOTA-TRP) is reported as a possible PET radiotracer for GnRH receptor imaging. DOTA-TRP was totally synthesized in two steps and after characterization went through radiolabelling optimization studies followed by tracer stability. The biodistribution of the tracer in normal male rats and 4T1 tumour-bearing mice was performed in 120 min after i.v. injection. The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 μg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.
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Blois, Anna; Holmsen, Holm; Martino, Guglielmo; Corti, Angelo; Metz-Boutigue, Marie-Hélène; Helle, Karen B
2006-03-15
Vasostatin-I (CgA1-76) is a naturally occurring and biologically active N-terminal peptide derived from chromogranin A (CgA), produced and secreted at high concentrations by neuroendocrine tissues and also from a range of neuroendocrine tumors. This study aims to examine the hypothesis that in the absence of classical protein receptors CgA1-76 may, like its two derived peptides CgA1-40 and CgA47-66, perturb the lipid microenvironment of other membrane receptors, as a basis for the largely inhibitory activities of these CgA peptides. The nature of the interactions between phospholipids and vasostatin-derived fragments was studied in the Langmuir film balance apparatus at 37 degrees C. The synthetic peptides CgA1-40 and CgA47-66 and a recombinant fragment (VS-I) containing vasostatin-I (Ser-Thr-Ala-CgA1-78) were compared for their effects on monolayers of phosphatidylcholine and phosphatidylethanolamine from pig brain and defined species of phosphatidylserine. Marked differences in surface pressure-area isotherms and phase-transition plateaus were apparent with the three classes of phospholipids on VS-I, CgA1-40 and CgA47-66 in physiological buffer or pure water. The results indicate that VS-I and CgA47-66 at 5-10 nM concentrations may engage in electrostatic as well as hydrophobic interactions with membrane-relevant phospholipids at physiological conditions, VS-I in particular enhancing the fluidity of saturated species of phosphatidylserine.
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Gastrin Receptor-Avid Peptide Conjugates
Hoffman, Timothy J.; Volkert, Wynn A.; Li, Ning; Sieckman, Gary; Higginbotham, Chrys-Ann
2005-07-26
A compound for use as a therapeutic or diagnostic radiopharmaceutical includes a group capable of complexing a medically useful metal attached to a moiety which is capable of binding to a gastrin releasing peptide receptor. A method for treating a subject having a neoplastic disease includes administering to the subject an effective amount of a radiopharmaceutical having a metal chelated with a chelating group attached to a moiety capable of binding to a gastrin releasing peptide receptor expressed on tumor cells with subsequent internalization inside of the cell. A method of forming a therapeutic or diagnostic compound includes reacting a metal synthon with a chelating group covalently linked with a moiety capable of binding a gastrin releasing peptide receptor.
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Gastrin receptor-avid peptide conjugates
Hoffman, Timothy J.; Volkert, Wynn A.; Li, Ning; Sieckman, Gary; Higginbotham, C. A.
2001-01-01
A compound for use as a therapeutic or diagnostic radiopharmaceutical includes a group capable of complexing a medically useful metal attached to a moiety which is capable of binding to a gastrin releasing peptide receptor. A method for treating a subject having a neoplastic disease includes administering to the subject an effective amount of a radiopharmaceutical having a metal chelated with a chelating group attached to a moiety capable of binding to a gastrin releasing peptide receptor expressed on tumor cells with subsequent internalization inside of the cell. A method of forming a therapeutic or diagnostic compound includes reacting a metal synthon with a chelating group covalently linked with a moiety capable of binding a gastrin releasing peptide receptor.
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Gastrin receptor-avid peptide conjugates
Hoffman, Timothy J.; Volkert, Wynn A.; Sieckman, Gary; Smith, Charles J.; Gali, Hariprasad
2006-06-13
A compound for use as a therapeutic or diagnostic radiopharmaceutical includes a group capable of complexing a medically useful metal attached to a moiety which is capable of binding to a gastrin releasing peptide receptor. A method for treating a subject having a neoplastic disease includes administering to the subject an effective amount of a radiopharmaceutical having a metal chelated with a chelating group attached to a-moiety capable of binding to a gastrin releasing peptide receptor expressed on tumor cells with subsequent internalization inside of the cell. A method of forming a therapeutic or diagnostic compound includes reacting a metal synthon with a chelating group covalently linked with a moiety capable of binding a gastrin releasing peptide receptor.
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Gastrin receptor-avid peptide conjugates
Hoffman, Timothy J.; Volkert, Wynn A.; Li, Ning; Sieckman, Gary; Higginbotham, Chrys-Ann
2006-12-12
A compound for use as a therapeutic or diagnostic radiopharmaceutical includes a group capable of complexing a medically useful metal attached to a moiety which is capable of binding to a gastrin releasing peptide receptor. A method for treating a subject having a neoplastic disease includes administering to the subject an effective amount of a radiopharmaceutical having a metal chelated with a chelating group attached to a moiety capable of binding to a gastrin releasing peptide receptor expressed on tumor cells with subsequent internalization inside of the cell. A method of forming a therapeutic or diagnostic compound includes reacting a metal synthon with a chelating group covalently linked with a moiety capable of binding a gastrin releasing peptide receptor.
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Identification and quantification of human kidney atrial natriuretic peptide receptors.
Kahana, L; Yechiely, H; Mecz, Y; Lurie, A
1995-04-01
The present study determined 125I-label atrial natriuretic peptide (ANP) binding sites in human kidney glomerular and papillary membranes. The membranes were prepared from non-malignant renal tissue obtained at nephrectomy of patients with renal carcinoma. To evaluate the proportion of ANP receptor classes ANP-R1 (ANPR-A, -B) versus ANP-R2 (ANPR-C), competitive binding studies were performed using [125I]-ANP in the presence of increasing concentrations of ANP or an internally ring-deleted analog, des(Gln116, Ser117, Gly118, Leu119, Gly120)ANP(102-121), called C-ANP, which binds selectively to ANPR-C receptors. Analysis of the competitive binding curve with ANP in glomerular membranes suggested the presence of one group of high-affinity receptors with dissociation constant Kd = 26 +/- 12 pmol/l and density Bmax = 101 +/- 47 nmol/kg protein. A decrease of 10-30% in Bmax with no change in Kd was obtained in the presence of excess (10(-6) mol/l) C-ANP, suggesting the existence of a small amount of a second class of receptors, the ANPR-C class. The densities of ANPR-A, -B versus ANPR-C receptors in human glomeruli, calculated from competitive inhibition experiments, were 75 +/- 42 and 22 +/- 16 nmol/kg protein (N = 8). Autoradiography of the sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions showed two bands: a highly labeled 130kD band and a weakly labeled 66 kD band, both displaced by ANP. Only the 66-kD band was displaced by the C-ANP analog. Human papilla membrane, as shown by competition binding studies and SDS gel electrophoresis, presented only one class of receptors with Kd = 40 +/- 23 pmol/l (mean +/- SD, N = 3) and Bmax = 17 +/- 6.3 nmol/kg protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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Yang, Yunlong; Nachman, Ronald J; Pietrantonio, Patricia V
2015-05-01
We identified the first pyrokinin receptor (Rhimi-PKR) in Chelicerata and analyzed structure-activity relationships of cognate ligand neuropeptides and their analogs. Based on comparative and phylogenetic analyses, this receptor, which we cloned from larvae of the cattle tick Rhipicephalus microplus (Acari: Ixodidae), is the ortholog of the insect pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN)/diapause hormone (DH) neuropeptide family receptor. Rhimi-PKR functional analyses using calcium bioluminescence were performed with a developed stable recombinant CHO-K1 cell line. Rhimi-PKR was activated by four endogenous PKs from the Lyme disease vector, the tick Ixodes scapularis (EC50s range: 85.4 nM-546 nM), and weakly by another tick PRX-amide peptide, periviscerokinin (PVK) (EC50 = 24.5 μM). PK analogs with substitutions of leucine, isoleucine or valine at the C-terminus for three tick PK peptides, Ixosc-PK1, Ixosc-PK2, and Ixosc-PK3, retained their potency on Rhimi-PKR. Therefore, Rhimi-PKR is less selective and substantially more tolerant than insect PK receptors of C-terminal substitutions of leucine to isoleucine or valine, a key structural feature that serves to distinguish insect PK from PVK/CAP2b receptors. In females, ovary and synganglion had the highest Rhimi-PKR relative transcript abundance followed by the rectal sac, salivary glands, Malpighian tubules, and midgut. This is the first pharmacological analysis of a PK/PBAN/DH-like receptor from the Chelicerata, which will now permit the discovery of the endocrinological roles of this neuropeptide family in vectors of vertebrate pathogens. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Budiawan, Hendra; Salavati, Ali; Kulkarni, Harshad R; Baum, Richard P
2014-01-01
The overall survival rate of non-radioiodine avid differentiated (follicular, papillary, medullary) thyroid carcinoma is significantly lower than for patients with iodine-avid lesions. The purpose of this study was to evaluate toxicity and efficacy (response and survival) of peptide receptor radionuclide therapy (PRRT) in non-radioiodine-avid or radioiodine therapy refractory thyroid cancer patients. Sixteen non-radioiodine-avid and/or radioiodine therapy refractory thyroid cancer patients, including follicular thyroid carcinoma (n = 4), medullary thyroid carcinoma (n = 8), Hürthle cell thyroid carcinoma (n = 3), and mixed carcinoma (n = 1) were treated with PRRT by using 90Yttrium and/or 177Lutetium labeled somatostatin analogs. 68Ga somatostatin receptor PET/CT was used to determine the somatostatin receptor density in the residual tumor/metastatic lesions and to assess the treatment response. Hematological profiles and renal function were periodically examined after treatment. By using fractionated regimen, only mild, reversible hematological toxicity (grade 1) or nephrotoxicity (grade 1) were seen. Response assessment (using EORTC criteria) was performed in 11 patients treated with 2 or more (maximum 5) cycles of PRRT and showed disease stabilization in 4 (36.4%) patients. Two patients (18.2%) showed partial remission, in the remaining 5 patients (45.5%) disease remained progressive. Kaplan-Meier analysis resulted in a mean survival after the first PRRT of 4.2 years (95% CI, range 2.9-5.5) and median progression free survival of 25 months (inter-quartiles: 12-43). In non-radioiodine-avid/radioiodine therapy refractory thyroid cancer patients, PRRT is a promising therapeutic option with minimal toxicity, good response rate and excellent survival benefits. PMID:24380044
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WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei
2013-01-01
Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237
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Vasoactive receptors in abdominal blood vessels of the dogfish shark, Squalus acanthias.
Evans, D H
2001-01-01
Previous studies have demonstrated that the ventral aorta of the dogfish shark, Squalus acanthias, responds to a variety of cell-signaling agents. To investigate the generality of vasoactive receptors in the shark vasculature, in particular a conductance artery (anterior mesenteric) and vein (posterior intestinal), I measured the effect of acetylcholine, endothelin, nitric oxide, natriuretic peptides, and prostaglandins on tension in isolated rings from these vessels. Both vessels responded to these agents, and responses to receptor-specific ligands for endothelin and natriuretic peptide receptors suggest that B-type endothelin receptors are expressed in both vessels and that the artery expresses both A- and B-type natriuretic peptide receptors; however, the vein (like the ventral aorta) expresses only the B-type natriuretic peptide receptor. My data suggest that a suite of signaling systems is ubiquitous in both arteries and veins in at least this elasmobranch species. Their role in hemodynamics and osmoregulation (perfusion of gill and rectal gland) remains to be determined.
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Vats, Ishwar Dutt; Chaudhary, Snehlata; Sharma, Ahuti; Nath, Mahendra; Pasha, Santosh
2010-07-25
The physiological role of NPFF/FMRFa family of peptides appears to be complex and exact mechanism of action of these peptides is not yet completely understood. In same line of scrutiny, another analog of YGGFMKKKFMRFamide (YFa), a chimeric peptide of met-enkephalin and FMRFamide, was rationally designed and synthesized which contain D-alanine and p-Cl-phenylalanine residues at 2nd and 4th positions, respectively i.e., Y-(D-Ala)-G-(p-Cl-Phe)-MKKKFMRFamide ([D-Ala(2), p-Cl-Phe(4)]YFa) in order to achieve improved bioavailability and blood brain barrier penetration. Therefore, present study investigates the possible antinociceptive effect of [D-Ala(2), p-Cl-Phe(4)]YFa on intra-peritoneal (i.p.) administration using tail-flick test in rats followed by its opioid receptor(s) specificity using mu, delta and kappa receptor antagonists. Further, its antinociceptive effect was examined during 6 days of chronic i.p. treatment and assessed effect of this treatment on differential expression of opioid receptors. [D-Ala(2), p-Cl-Phe(4)]YFa in comparison to parent peptide YFa, induce significantly higher dose dependent antinociception in rats which was mediated by all three opioid receptors (mu, delta and kappa). Importantly, it induced comparable antinociception in rats throughout the chronic i.p. treatment and significantly up-regulated the overall expression (mRNA and protein) of mu, delta and kappa opioid receptors. Therefore, pharmacological and molecular behavior of [D-Ala(2), p-Cl-Phe(4)]YFa demonstrate that incorporation of D-alanine and p-Cl-phenylalanine residues at appropriate positions in chimeric peptide leads to altered opioid receptor selectivity and enhanced antinociceptive potency, relative to parent peptide. (c) 2010 Elsevier B.V. All rights reserved.
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2013-01-01
Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds. PMID:23650868
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Tachykinin receptors in the circular muscle of the guinea-pig ileum.
Maggi, C. A.; Patacchini, R.; Giachetti, A.; Meli, A.
1990-01-01
1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1707710
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Deaton, J D; Guerrero, T; Howard, T H
1992-01-01
In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin independent. PMID:1337290
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Deaton, J D; Guerrero, T; Howard, T H
1992-12-01
In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin independent.
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Colbert, Karen N.; Hattendorf, Douglas A.; Weiss, Thomas M.; ...
2013-07-15
In neurons, soluble N-ethylmaleimide–sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1–24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Inmore » addition, we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a–Syx1a complex.« less
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Osteogenic properties of a short BMP-2 chimera peptide.
Falcigno, Lucia; D'Auria, Gabriella; Calvanese, Luisa; Marasco, Daniela; Iacobelli, Roberta; Scognamiglio, Pasqualina L; Brun, Paola; Danesin, Roberta; Pasqualin, Matteo; Castagliuolo, Ignazio; Dettin, Monica
2015-09-01
Bone morphogenetic proteins (BMPs) play a key role in bone and cartilage formation. For these properties, BMPs are employed in the field of tissue engineering to induce bone regeneration in damaged tissues. To overcome drawbacks due to the use of entire proteins, synthetic peptides derived from their parent BMPs have come out as promising molecules for biomaterial design. On the structural ground of the experimental BMP-2 receptor complexes reported in the literature, we designed three peptides, reproducing the BMP-2 region responsible for the binding to the type II receptor, ActRIIB. These peptides were characterized by NMR, and the structural features of the peptide-receptor binding interface were highlighted by docking experiments. Peptide-receptor binding affinities were analyzed by means of ELISA and surface plasmon resonance techniques. Furthermore, cellular assays were performed to assess their osteoinductive properties. A chimera peptide, obtained by combining the sequence portions 73-92 and 30-34 of BMP-2, shows the best affinity for ActRIIB in the series and represents a good starting point for the design of new compounds able to reproduce osteogenic properties of the parent BMP-2. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.
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Esapa, Christopher T; Piret, Sian E; Nesbit, M Andrew; Loh, Nellie Y; Thomas, Gethin; Croucher, Peter I; Brown, Matthew A; Brown, Steve D M; Cox, Roger D; Thakker, Rajesh V
2016-01-01
Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis.
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Nesbit, M. Andrew; Loh, Nellie Y.; Thomas, Gethin; Croucher, Peter I.; Brown, Matthew A.; Brown, Steve D. M.; Cox, Roger D.; Thakker, Rajesh V.
2016-01-01
Non-syndromic kyphosis is a common disorder that is associated with significant morbidity and has a strong genetic involvement; however, the causative genes remain to be identified, as such studies are hampered by genetic heterogeneity, small families and various modes of inheritance. To overcome these limitations, we investigated 12 week old progeny of mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) using phenotypic assessments including dysmorphology, radiography, and dual-energy X-ray absorptiometry. This identified a mouse with autosomal recessive kyphosis (KYLB). KYLB mice, when compared to unaffected littermates, had: thoraco-lumbar kyphosis, larger vertebrae, and increased body length and increased bone area. In addition, female KYLB mice had increases in bone mineral content and plasma alkaline phosphatase activity. Recombination mapping localized the Kylb locus to a 5.5Mb region on chromosome 15A1, which contained 51 genes, including the natriuretic peptide receptor 3 (Npr3) gene. DNA sequence analysis of Npr3 identified a missense mutation, Tyr209Asn, which introduced an N-linked glycosylation consensus sequence. Expression of wild-type NPR3 and the KYLB-associated Tyr209Asn NPR3 mutant in COS-7 cells demonstrated the mutant to be associated with abnormal N-linked glycosylation and retention in the endoplasmic reticulum that resulted in its absence from the plasma membrane. NPR3 is a decoy receptor for C-type natriuretic peptide (CNP), which also binds to NPR2 and stimulates mitogen-activated protein kinase (MAPK) signaling, thereby increasing the number and size of hypertrophic chondrocytes. Histomorphometric analysis of KYLB vertebrae and tibiae showed delayed endochondral ossification and expansion of the hypertrophic zones of the growth plates, and immunohistochemistry revealed increased p38 MAPK phosphorylation throughout the growth plates of KYLB vertebrae. Thus, we established a model of kyphosis due to a novel NPR3 mutation, in which loss of plasma membrane NPR3 expression results in increased MAPK pathway activation, causing elongation of the vertebrae and resulting in kyphosis. PMID:27959934
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Luk, J K; Wong, E F; Sun, A; Wong, N L
1994-12-01
The effects of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on renal medullary thick ascending limb (mTAL) have not been fully understood. The aim of this study is to examine the second-messenger responses of rat mTAL to ANF, BNP, and CNP. Characterizations of the ANF, BNP, and CNP receptors in mTAL were also performed by radioligand studies. Results showed that ANF and BNP were both capable of eliciting cyclic guanosine monophosphate (cGMP) responses in mTAL. Conversely, no cGMP response was observed upon stimulation by CNP in mTAL. The presence of ANF receptors was demonstrated by radioligand studies. One receptor site was found, and the Kd and maximum binding capacity were 4.0 +/- 0.45 nmol/L and 277.8 +/- 47.7 fmol/mg protein, respectively. BNP receptors were also found in mTAL, and ANF and BNP were sharing the same receptor. On the contrary, no CNP receptor could be shown by radioligand studies. These results suggest that guanylyl cyclase-coupled receptors (atrial natriuretic peptide receptor-A [ANPR-A]) specific for ANF and BNP are present in rat mTAL, while those for CNP (ANPR-B) are absent. ANF and BNP but not CNP act on mTAL to control water excretion.
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Plasma urotensin in human systolic heart failure.
Ng, Leong L; Loke, Ian; O'Brien, Russell J; Squire, Iain B; Davies, Joan E
2002-12-03
Human urotensin II (UTN) has potent vasoactive and cardiostimulatory effects, acting on the G protein-linked receptor GPR14. Myocardial UTN expression is upregulated in heart failure, and UTN stimulates myocardial expression of the natriuretic peptides. We investigated plasma UTN levels in heart failure (HF; left ventricular systolic dysfunction) in comparison with plasma N-terminal pro-brain natriuretic peptide (N-BNP) levels. N-BNP and UTN were measured in plasma from 126 patients with HF and 220 age- and sex-matched controls. Both peptides were elevated in plasma of HF patients and were correlated (r(s)=0.35, P<0.001). In contrast to N-BNP, there was no relationship of plasma UTN with New York Heart Association (NYHA) class. Although plasma N-BNP showed a positive relationship with age and female sex, there was no such age-dependent change in plasma UTN, and control women had lower levels compared with control men. Receiver operating characteristic curves for the diagnosis of HF had areas of 0.90 and 0.86 for N-BNP and UTN, respectively (P<0.001 for both). Receiver operating characteristic curve area for diagnosis of NYHA class I HF with UTN was better than that with N-BNP. Plasma UTN is elevated in HF, which suggests a pathophysiological role for this peptide. Plasma UTN may be a useful alternative to N-BNP in the diagnosis of HF, inasmuch as its levels are elevated irrespective of age, sex, or NYHA class.
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Ma, Jie; Luo, Yu; Ge, Lilin; Wang, Lei; Zhou, Mei; Zhang, Yingqi; Duan, Jinao; Chen, Tianbao; Shaw, Chris
2014-01-01
The defensive skin secretions of many amphibians are a rich source of bradykinins and bradykinin-related peptides (BRPs). Members of this peptide group are also common components of reptile and arthropod venoms due to their multiple biological functions that include induction of pain, effects on many smooth muscle types, and lowering systemic blood pressure. While most BRPs are bradykinin receptor agonists, some have curiously been found to be exquisite antagonists, such as the maximakinin gene-related peptide, kinestatin-a specific bradykinin B2-receptor antagonist from the skin of the giant fire-bellied toad, Bombina maxima. Here, we describe the identification, structural and functional characterization of a heptadecapeptide (DYTIRTRLHQGLSRKIV), named ranakinestatin-PPF, from the skin of the Chinese ranid frog, Pelophylax plancyi fukienensis, representing a prototype of a novel class of bradykinin B2-receptor specific antagonist. Using a preconstricted preparation of rat tail arterial smooth muscle, a single dose of 10(-6)M of the peptide effectively inhibited the dose-dependent relaxation effect of bradykinin between 10(-11)M and 10(-5)M and subsequently, this effect was pharmacologically-characterized using specific bradykinin B1- (desArg-HOE140) and B2-receptor (HOE140) antagonists; the data from which demonstrated that the antagonism of the novel peptide was mediated through B2-receptors. Ranakinestatin-PPF-thus represents a prototype of an amphibian skin peptide family that functions as a bradykinin B2-receptor antagonist herein demonstrated using mammalian vascular smooth muscle.
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Calcitonin and Amylin Receptor Peptide Interaction Mechanisms
Lee, Sang-Min; Hay, Debbie L.; Pioszak, Augen A.
2016-01-01
Receptor activity-modifying proteins (RAMP1–3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8–37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity. PMID:26895962
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Circular dichroism study of the carbohydrate-modified opioid peptides
NASA Astrophysics Data System (ADS)
Horvat, Štefica; Otvos, Laszlo; Urge, Laszlo; Horvat, Jaroslav; Čudić, Mare; Varga-Defterdarović, Lidija
1999-09-01
The conformational preferences of enkephalins and the related glycoconjugates in which free or protected carbohydrate moieties were linked to the opioid peptides through an ether, ester or amide bond were investigated by circular dichroism spectroscopy in water, trifluoroethanol and water-trifluoroethanol mixtures. The analysis of the spectra revealed that the conformation of the enkephalin molecule is very sensitive to slight changes in the peptide structure around the C-terminal region. It was found that the type II β-turn structures are populated in N-terminal tetrapeptide enkephalin fragment, while leucine-enkephalin amide feature a type I (III) β-turn structure in solution. Incorporation of the sugar moiety into opioid peptide compound did not significantly influence the overall conformation of the peptide backbone, although minor intensity changes may reflect shifts in the population of the different turn systems. These small structural alterations can be responsible for the receptor-subtype selectivity of the various carbohydrate-modified enkephalin analogs.
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Ohnishi, Toshiyuki; Sakamoto, Kotaro; Asami-Odaka, Asano; Nakamura, Kimie; Shimizu, Ayako; Ito, Takashi; Asami, Taiji; Ohtaki, Tetsuya; Inooka, Hiroshi
2017-01-29
Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors. Copyright © 2016 Elsevier Inc. All rights reserved.
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Song, B; Marvizón, J C G
2005-01-01
Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn mediate analgesia, inhibition of spinal opioid release could contribute to the hyperalgesic actions of spinal N-methyl-D-aspartate receptors.
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SONG, B.; MARVIZÓN, J. C. G.
2006-01-01
Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the μ-opioid receptor, we measured μ-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced μ-opioid receptor internalization in half of the μ-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-d-aspartate (IC50=2 μM), and N-methyl-d-aspartate antagonists prevented this effect. μ-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-d-aspartate receptor activation. N-methyl-d-aspartate did not affect μ-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-d-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-d-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase μ-opioid receptor internalization in the absence of N-methyl-d-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked μ-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-d-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since μ-opioid receptors in the dorsal horn mediate analgesia, inhibition of spinal opioid release could contribute to the hyperalgesic actions of spinal N-methyl-d-aspartate receptors. PMID:16203108
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Gougat, Jean; Ferrari, Bernard; Sarran, Lionel; Planchenault, Claudine; Poncelet, Martine; Maruani, Jeanne; Alonso, Richard; Cudennec, Annie; Croci, Tiziano; Guagnini, Fabio; Urban-Szabo, Katalin; Martinolle, Jean-Pierre; Soubrié, Philippe; Finance, Olivier; Le Fur, Gérard
2004-05-01
The biochemical and pharmacological properties of a novel non-peptide antagonist of the bradykinin (BK) B(1) receptor, SSR240612 [(2R)-2-[((3R)-3-(1,3-benzodioxol-5-yl)-3-[[(6-methoxy-2-naphthyl)sulfonyl]amino]propanoyl)amino]-3-(4-[[2R,6S)-2,6-dimethylpiperidinyl]methyl]phenyl)-N-isopropyl-N-methylpropanamide hydrochloride] were evaluated. SSR240612 inhibited the binding of [(3)H]Lys(0)-des-Arg(9)-BK to the B(1) receptor in human fibroblast MRC5 and to recombinant human B(1) receptor expressed in human embryonic kidney cells with inhibition constants (K(i)) of 0.48 and 0.73 nM, respectively. The compound selectivity for B(1) versus B(2) receptors was in the range of 500- to 1000-fold. SSR240612 inhibited Lys(0)-desAr(9)-BK (10 nM)-induced inositol monophosphate formation in human fibroblast MRC5, with an IC(50) of 1.9 nM. It also antagonized des-Arg(9)-BK-induced contractions of isolated rabbit aorta and mesenteric plexus of rat ileum with a pA(2) of 8.9 and 9.4, respectively. Antagonistic properties of SSR240612 were also demonstrated in vivo. SSR240612 inhibited des-Arg(9)-BK-induced paw edema in mice (3 and 10 mg/kg p.o. and 0.3 and 1 mg/kg i.p.). Moreover, SSR240612 reduced capsaicin-induced ear edema in mice (0.3, 3 and 30 mg/kg p.o.) and tissue destruction and neutrophil accumulation in the rat intestine following splanchnic artery occlusion/reperfusion (0.3 mg/kg i.v.). The compound also inhibited thermal hyperalgesia induced by UV irradiation (1 and 3 mg/kg p.o.) and the late phase of nociceptive response to formalin in rats (10 and 30 mg/kg p.o.). Finally, SSR240612 (20 and 30 mg/kg p.o.) prevented neuropathic thermal pain induced by sciatic nerve constriction in the rat. In conclusion, SSR240612 is a new, potent, and orally active specific non-peptide bradykinin B(1) receptor antagonist.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Boyd, N.D.; White, C.F.; Leeman, S.E.
A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine (L-Phe(pBz)) in place of the Phe{sup 8} residue of SP. (Phe{sup 8}(OpBz))SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the {sup 125}I-labeled Bolton-Hunter conjugate of (Phe{sup 8}(pBz))SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites with an affinity K{sub D} = 0.4 nM. The binding of {sup 125}I-(Phe{sup 8}(pBz))SP was inhibited competitivelymore » by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70{percent} of the specifically bound {sup 125}I-(Phe{sup 8}(pBz))SP underwent covalent linkage to two polypeptides of M{sub r} = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on {sup 125}I-(Phe{sup 8}(pBz))SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies. The highly specific and remarkably efficient photolabeling achieved with {sup 125}I-(Phe{sup 8}(pBz))SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor.« less
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Targeted silver nanoparticles for ratiometric cell phenotyping
NASA Astrophysics Data System (ADS)
Willmore, Anne-Mari A.; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N.; Ruoslahti, Erkki; Braun, Gary B.; Teesalu, Tambet
2016-04-01
Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo. Electronic supplementary information (ESI) available: TEM images of isotopic AgNPs, cell antibody staining, coadministration ICP-MS data, and biotin control particle ICP-MS data. See DOI: 10.1039/C5NR07928D
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Schröder, W; Lambert, D G; Ko, M C; Koch, T
2014-01-01
Despite high sequence similarity between NOP (nociceptin/orphanin FQ opioid peptide) and opioid receptors, marked differences in endogenous ligand selectivity, signal transduction, phosphorylation, desensitization, internalization and trafficking have been identified; underscoring the evolutionary difference between NOP and opioid receptors. Activation of NOP receptors affects nociceptive transmission in a site-specific manner, with antinociceptive effects prevailing after peripheral and spinal activation, and pronociceptive effects after supraspinal activation in rodents. The net effect of systemically administered NOP receptor agonists on nociception is proposed to depend on the relative contribution of peripheral, spinal and supraspinal activation, and this may depend on experimental conditions. Functional expression and regulation of NOP receptors at peripheral and central sites of the nociceptive pathway exhibits a high degree of plasticity under conditions of neuropathic and inflammatory pain. In rodents, systemically administered NOP receptor agonists exerted antihypersensitive effects in models of neuropathic and inflammatory pain. However, they were largely ineffective in acute pain while concomitantly evoking severe motor side effects. In contrast, systemic administration of NOP receptor agonists to non-human primates (NHPs) exerted potent and efficacious antinociception in the absence of motor and sedative side effects. The reason for this species difference with respect to antinociceptive efficacy and tolerability is not clear. Moreover, co-activation of NOP and μ-opioid peptide (MOP) receptors synergistically produced antinociception in NHPs. Hence, both selective NOP receptor as well as NOP/MOP receptor agonists may hold potential for clinical use as analgesics effective in conditions of acute and chronic pain. PMID:24762001
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Folate-Dependent Purine Nucleotide Biosynthesis in Humans.
Baggott, Joseph E; Tamura, Tsunenobu
2015-09-01
Purine nucleotide biosynthesis de novo (PNB) requires 2 folate-dependent transformylases-5'-phosphoribosyl-glycinamide (GAR) and 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) transformylases-to introduce carbon 8 (C8) and carbon 2 (C2) into the purine ring. Both transformylases utilize 10-formyltetrahydrofolate (10-formyl-H4folate), where the formyl-carbon sources include ring-2-C of histidine, 3-C of serine, 2-C of glycine, and formate. Our findings in human studies indicate that glycine provides the carbon for GAR transformylase (exclusively C8), whereas histidine and formate are the predominant carbon sources for AICAR transformylase (C2). Contrary to the previous notion, these carbon sources may not supply a general 10-formyl-H4folate pool, which was believed to equally provide carbons to C8 and C2. To explain these phenomena, we postulate that GAR transformylase is in a complex with the trifunctional folate-metabolizing enzyme (TFM) and serine hydroxymethyltransferase to channel carbons of glycine and serine to C8. There is no evidence for channeling carbons of histidine and formate to AICAR transformylase (C2). GAR transformylase may require the TFM to furnish 10-formyl-H4folate immediately after its production from serine to protect its oxidation to 10-formyldihydrofolate (10-formyl-H2folate), whereas AICAR transformylase can utilize both 10-formyl-H2folate and 10-formyl-H4folate. Human liver may supply AICAR to AICAR transformylase in erythrocytes/erythroblasts. Incorporation of ring-2-C of histidine and formate into C2 of urinary uric acid presented a circadian rhythm with a peak in the morning, which corresponds to the maximum DNA synthesis in the bone marrow, and it may be useful in the timing of the administration of drugs that block PNB for the treatment of cancer and autoimmune disease. © 2015 American Society for Nutrition.
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Characterization of Three Venom Peptides from the Spitting Spider Scytodes thoracica
Ariki, Nathanial K.; Muñoz, Lisa E.; Armitage, Elizabeth L.; Goodstein, Francesca R.; George, Kathryn G.; Smith, Vanessa L.; Vetter, Irina; Herzig, Volker; King, Glenn F.; Loening, Nikolaus M.
2016-01-01
We present the solution-state NMR structures and preliminary functional characterizations of three venom peptides identified from the spitting spider Scytodes thoracica. Despite little sequence identity to other venom peptides, structural characterization reveals that these peptides contain an inhibitor cystine knot motif common to many venom peptides. These are the first structures for any peptide or protein from spiders of the Scytodidae family. Many venom peptides target neuronal ion channels or receptors. However, we have not been able to determine the target of these Scytodes peptides so we can only state with certainty the channels and receptors that they do not target. PMID:27227898
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Ton, Thi My Uyen; Tejo, Ciputra; Tania, Stefani; Chang, Joyce Wei Wei; Chan, Philip Wai Hong
2011-06-17
A method for the amidation of aldehydes with PhI=NTs/PhI=NNs as the nitrogen source and an inexpensive iron(II) chloride + pyridine as the in situ formed precatalyst under mild conditions at room temperature or microwave assisted conditions is described. The reaction was operationally straightforward and accomplished in moderate to excellent product yields (20-99%) and with complete chemoselectivity with the new C-N bond forming only at the formylic C-H bond in substrates containing other reactive functional groups. By utilizing microwave irradiation, comparable product yields and short reaction times of 1 h could be accomplished. The mechanism is suggested to involve insertion of a putative iron-nitrene/imido group to the formylic C-H bond of the substrate via a H-atom abstraction/radical rebound pathway mediated by the precatalyst [Fe(py)(4)Cl(2)] generated in situ from reaction of FeCl(2) with pyridine.
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Structural, ac conductivity and dielectric properties of 3-formyl chromone
NASA Astrophysics Data System (ADS)
Ali, H. A. M.
2017-07-01
The structure for the powder of 3-formyl chromone was examined by X-ray diffraction technique in the 2θ° range ( 4° - 60° . The configuration of Al/3-formyl chromone/Al samples was designed. The electrical and dielectric properties were studied as a function of frequency (42- 5 × 106 Hz) and temperature (298-408K). The ac conductivity data of bulk of 3-formyl chromone varies as a power law with the frequency at different temperatures. The predominant mechanism for ac conduction was deduced. The ac conductivity shows a thermally activated process at different frequencies. The dielectric constant and dielectric loss were determined using the capacitance and dissipation factor measurements at different temperatures. The dielectric loss shows a peak of relaxation time that shifted to higher frequency with an increase in the temperature. The activation energy of the relaxation process was estimated.
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Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Prossnitz, Eric R; Sklar, Larry A; Miao, Yubin
2009-11-01
The purpose of this study was to examine the effect of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting and pharmacokinetics of radiolabeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (alpha-MSH) peptide. A novel lactam bridge-cyclized alpha-MSH peptide, Ac-GluGlu-CycMSH[DOTA] {Ac-Glu-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Lys(DOTA)]}, was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. DOTA was directly attached to the alpha-amino group of Lys in the cyclic ring, while the N-terminus of the peptide was acetylated to generate Ac-GluGlu-CycMSH[DOTA]. The MC1 receptor binding affinity of Ac-GluGlu-CycMSH[DOTA] was determined in B16/F1 melanoma cells. Melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In were determined in B16/F1 melanoma-bearing C57 mice and compared to that of 111In-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] (111In-DOTA-GlyGlu-CycMSH; DOTA was coupled to the N-terminus of the peptide). Ac-GluGlu-CycMSH[DOTA] displayed 0.6 nM MC1 receptor binding affinity in B16/F1 cells. Ac-GluGlu-CycMSH[DOTA]-111In was readily prepared with greater than 95% radiolabeling yield. Ac-GluGlu-CycMSH[DOTA]-111In exhibited high tumor uptake (11.42 +/- 2.20% ID/g 2 h postinjection) and prolonged tumor retention (9.42 +/- 2.41% ID/g 4 h postinjection) in B16/F1 melanoma-bearing C57 mice. The uptake values for nontarget organs were generally low (<1.3% ID/g) except for the kidneys 2, 4, and 24 h postinjection. DOTA position exhibited profound effect on melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In, providing a new insight into the design of lactam bridge-cyclized peptide for melanoma imaging and therapy.
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Ismail, Sadek; Dubois-Vedrenne, Ingrid; Laval, Marie; Tikhonova, Irina G; D'Angelo, Romina; Sanchez, Claire; Clerc, Pascal; Gherardi, Marie-Julie; Gigoux, Véronique; Magnan, Remi; Fourmy, Daniel
2015-10-15
How incretins regulate presence of their receptors at the cell surface and their activity is of paramount importance for the development of therapeutic strategies targeting these receptors. We have studied internalization of the human Glucose-Insulinotropic Polypeptide receptor (GIPR). GIP stimulated rapid robust internalization of the GIPR, the major part being directed to lysosomes. GIPR internalization involved mainly clathrin-coated pits, AP-2 and dynamin. However, neither GIPR C-terminal region nor β-arrestin1/2 was required. Finally, N-acetyl-GIP recognized as a dipeptidyl-IV resistant analogue, fully stimulated cAMP production with a ∼15-fold lower potency than GIP and weakly stimulated GIPR internalization and desensitization of cAMP response. Furthermore, docking N-acetyl-GIP in the binding site of modeled GIPR showed slighter interactions with residues of helices 6 and 7 of GIPR compared to GIP. Therefore, incomplete or partial activity of N-acetyl-GIP on signaling involved in GIPR desensitization and internalization contributes to the enhanced incretin activity of this peptide. Copyright © 2015. Published by Elsevier Ireland Ltd.
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Regulation of lung branching morphogenesis by bombesin-like peptides and neutral endopeptidase.
Aguayo, S M; Schuyler, W E; Murtagh, J J; Roman, J
1994-06-01
The expression of bombesin-like peptides (BLPs) by pulmonary neuroendocrine cells is transiently upregulated during lung development. A functional role for BLPs is supported by their ability to stimulate lung growth and maturation both in vitro and in vivo during the late stages of lung development. In addition, the cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP), which inactivates BLPs and other regulatory peptides, is also expressed by developing lungs and modulates the stimulatory effects of BLPs on lung growth and maturation. We hypothesized that, in addition to expressing BLPs and CD10/NEP, embryonic lungs must express BLP receptors, and that BLPs may also regulate processes that occur during early lung development such as branching morphogenesis. Using reverse transcriptase-polymerase chain reaction and oligonucleotide primers designed for amplifying a BLP receptor originally isolated from Swiss 3T3 mouse fibroblasts, we found that embryonic mouse lungs express a similar BLP receptor mRNA during the pseudoglandular stage of lung development when branching morphogenesis take place. Subsequently, we evaluated the effects of ligands for this BLP receptor using embryonic mouse lungs in an in vitro model of lung branching morphogenesis. We found that, in comparison with control lungs, treatment with bombesin (1 to 100 nM) resulted in a modest increase in clefts or branching points. In contrast, embryonic mouse lungs treated with the BLP analog [Leu13-psi(CH2NH)Leu14]bombesin (1 microM), which also binds to this BLP receptor but has predominantly antagonistic effects, demonstrated fewer branching points.(ABSTRACT TRUNCATED AT 250 WORDS)
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Tucholski, Janusz; Simmons, Micah S; Pinner, Anita L; McMillan, Laurence D; Haroutunian, Vahram; Meador-Woodruff, James H
2013-08-21
Dysfunctional glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. Abnormal expressions in schizophrenia of ionotropic glutamate receptors (iGluRs) and the proteins that regulate their trafficking have been found to be region and subunit specific in brain, suggesting that abnormal trafficking of iGluRs may contribute toward altered glutamatergic neurotransmission. The post-translational modification N-glycosylation of iGluR subunits can be used as a proxy for their intracellular localization. Receptor complexes assemble in the lumen of the endoplasmic reticulum, where N-glycosylation begins with the addition of N-linked oligomannose glycans, and is subsequently trimmed and replaced by more elaborate glycans while trafficking through the Golgi apparatus. Previously, we found abnormalities in N-glycosylation of the GluR2 AMPA receptor subunit in schizophrenia. Here, we investigated N-glycosylation of N-methyl-D-aspartate and kainate (KA) receptor subunits in the dorsolateral prefrontal cortex from patients with schizophrenia and a comparison group. We used enzymatic deglycosylation with two glycosidases: endoglycosidase H (Endo H), which removes immature high mannose-containing sugars, and peptide-N-glycosidase F (PNGase F), which removes all N-linked sugars. The NR1, NR2A, NR2B, GluR6, and KA2 subunits were all sensitive to treatment with Endo H and PNGase F. The GluR6 KA receptor subunit was significantly more sensitive to Endo H-mediated deglycosylation in schizophrenia, suggesting a larger molecular mass of N-linked high mannose and/or hybrid sugars on GluR6. This finding, taken with our previous work, suggests that a cellular mechanism underlying abnormal glutamate neurotransmission in schizophrenia may involve abnormal trafficking of both AMPA and KA receptors.
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Characterization of a novel non-peptide vasopressin V1 receptor antagonist (OPC-21268) in the rat.
Burrell, L M; Phillips, P A; Stephenson, J; Risvanis, J; Hutchins, A M; Johnston, C I
1993-08-01
A non-peptide, orally effective, vasopressin (AVP) V1 receptor antagonist 1-(1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl)-3,4-dihydro-2(1H)-quinolinone (OPC-21268) has recently been described. This paper reports the in-vitro and in-vivo characterization of OPC-21268 binding to vasopressin receptors in rat liver and kidney. OPC-21268 caused a concentration-dependent displacement of the selective V1 receptor antagonist radioligand, 125I-labelled [d(CH2)5,sarcosine7]AVP to V1 receptors in both rat liver and kidney medulla membranes. The concentration of OPC-21268 that displaced 50% of specific AVP binding (IC50) was 40 +/- 3 nmol/l for liver V1 and 15 +/- 2 nmol/l for kidney V1 receptors (mean +/- S.E.M.; n = 3). OPC-21268 had little effect on the selective V2 antagonist radioligand [3H]desGly-NH2(9)]d(CH2)5,D-Ile2,Ile4] AVP binding to V2 receptors in renal medulla membranes (IC50 > 0.1 mmol/l). After oral administration to rats, OPC-21268 was an effective V1 antagonist in a time- and dose-dependent manner. Binding kinetic studies showed that OPC-21268 acted as a competitive antagonist at the liver V1 receptor in vitro and in vivo, in addition to its in-vitro competitive effects at the renal V1 receptor. OPC-21268 shows promise as an orally active V1 antagonist.
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Neurotensin receptors in human neoplasms: high incidence in Ewing's sarcomas.
Reubi, J C; Waser, B; Schaer, J C; Laissue, J A
1999-07-19
Receptors for regulatory peptides, such as somatostatin or vasoactive intestinal peptide (VIP), expressed at high density by neoplastic cells, can be instrumental for tumor diagnosis and therapy. Little is known about the expression of neurotensin receptors in human tumors. In the present study, 464 human neoplasms of various types were investigated for their neurotensin receptor content by in vitro receptor autoradiography on tissue sections using 125I-[Tyr3]-neurotensin as radioligand. Neurotensin receptors were identified and localized in tumor cells of 11/17 Ewing's sarcomas, 21/40 meningiomas, 10/23 astrocytomas, 5/13 medulloblastomas, 7/24 medullary thyroid cancers and 2/8 small cell lung cancers. They were rarely found in non-small cell lung cancers and breast carcinomas; they were absent in prostate, ovarian, renal cell and hepatocellular carcinomas, neuroendocrine gut tumors, pituitary adenomas, schwannomas, neuroblastomas and lymphomas. When present, the receptors bound with nanomolar affinity neurotensin and acetyl-neurotensin-(8-13), with lower affinity neuromedin N, diethylenetriamine penta-acetic acidneurotensin-(8-13) and SR 48692, but not neurotensin-(1-11). They were all of the NT1 type, without high affinity for levocabastine. Further, in 2 receptor-positive Ewing's sarcomas, neurotensin mRNA was detected by in situ hybridization techniques. Since neurotensin is known to stimulate cell proliferation, the presence of neurotensin receptors in human neoplasia may be of biological relevance, possibly as an integrative part of an autocrine feedback mechanism of tumor growth stimulation.
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Biomimetic oligosaccharide and peptide surfactant polymers designed for cardiovascular biomaterials
NASA Astrophysics Data System (ADS)
Ruegsegger, Mark Andrew
A common problem associated with cardiovascular devices is surface induced thrombosis initiated by the rapid, non-specific adsorption of plasma proteins onto the biomaterial surface. Control of the initial protein adsorption is crucial to achieve the desired longevity of the implanted biomaterial. The cell membrane glycocalyx acts as a non-thrombogenic interface through passive (dense oligosaccharide structures) and active (ligand/receptor interactions) mechanisms. This thesis is designed to investigate biomimicry of the cell glycocalyx to minimize non-specific protein adsorption and promote specific ligand/receptor interactions. Biomimetic macromolecules were designed through the molecular-scale engineering of polymer surfactants, utilizing a poly(vinyl amine) (PVAm) backbone to which hydrophilic (dextran, maltose, peptide) and hydrophobic alkyl (hexanoyl or hexanal) chains are simultaneously attached. The structure was controlled through the molar feed ratio of hydrophobic-to-hydrophilic groups, which also provided control of the solution and surface-active properties. To mimic passive properties, a series of oligomaltose surfactants were synthesized with increasing saccharide length (n = 2, 7, 15 where n is number of glucose units) to investigate the effect of coating height on protein adsorption. The surfactants were characterized by infra red (IR) and nuclear magnetic resonance (NMR) spectroscopies for structural properties and atomic force microscopy (AFM) and contact angle goniometry for surface activity. Protein adsorption under dynamic flow (5 dyn/cm2) was reduced by 85%--95% over the bare hydrophobic substrate; platelet adhesion dropped by ˜80% compared to glass. Peptide ligands were incorporated into the oligosaccharide surfactant to promote functional activity of the passive coating. The surfactants were synthesized to contain 0%, 25%, 50%, 75%, and 100% peptide ligand density and were stable on hydrophobic surfaces. The peptide surface density was calculated to be 0.86 ligands/nm2 for PVAm(Pep)(100%), as determined by total internal reflection fluorescence (TIRF) spectroscopy. Similar cell growth was observed on the 100% peptide surfactant as for the fibronectin control, and no cell growth was seen on the 0% peptide. Increasing cell viability was observed for the surfaces with increasing peptide density. These results indicate much promise for surfactant polymers in surface modification and the capability to mimic the passive and active properties of the cell glycocalyx.
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Cadet, Patrick; Mantione, Kirk J; Stefano, George B
2003-05-15
Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.
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Biosynthesis and processing of the mannose receptor in human macrophages.
Lennartz, M R; Cole, F S; Stahl, P D
1989-02-05
The biosynthesis and processing of the human mannose receptor has been studied in monocyte-derived macrophages. Adherent cells were labeled for 60 min with Trans35S (a mixture of 35S-labeled methionine and cysteine), chased, and subjected to immunoprecipitation by antibody raised against the human placental receptor. The antibody immunoprecipitated a single protein of molecular mass 162 kDa; precipitation of the labeled receptor could be inhibited by placental receptor. The results presented demonstrate that the receptor is synthesized as a 154-kDa precursor which is processed to 162 kDa in 90 min. The precursor is a glycoprotein bearing endoglycosidase H-sensitive oligosaccharides; the 162-kDa form is endoglycosidase H-resistant but peptide:N-glycanase-sensitive. Desialylation of the mannose receptor with neuraminidase generates a protein which is recognized by peanut agglutinin, a lectin that specifically binds desialylated O-linked oligosaccharides. Thus, the human macrophage mannose receptor bears both N- and O-linked oligosaccharide chains. Newly synthesized mannose receptor exhibits a half-life of 33 h as determined by pulse-chase studies. This indicates that on the average, each molecule of receptor recycles between the cell surface and endosomes hundreds of times before degradation.
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Garcia, Maria Fernanda; Zhang, Xiuli; Gallazzi, Fabio; Fernandez, Marcelo; Moreno, Maria; Gambini, Juan Pablo; Porcal, Williams; Cabral, Pablo; Quinn, Thomas P
2015-01-01
Several radiolabeled alpha-melanocyte stimulating hormone (α-MSH) analogs have been studied for their abilities to target melanoma tumor cells through specific recognition and binding to the melanocortin receptor 1 (MCR1). In this work, a lactam bridgecyclized α-MSH analog was labeled with (99m) via the hydrazinonicotinamide (HYNIC) chelator and characterized for its melanoma tumor targeting properties. The bifunctional chelating agent HYNIC-Boc was attached to the N-terminus of the MSH peptide followed by the lactam cyclization, resulting in the HYNIC-cyc-MSH analog. The lactam cyclized peptide displayed high affinity and specificity for MC1-receptors present on B16/F1 melanoma tumor cells, exhibiting an IC50 of 6.48 nM. HYNIC-cyc-MSH was radiolabeled with (99m)Tc using two common co-ligands, tricine and EDDA. In vitro, the radiochemical stability, cell binding and efflux properties were similar between the peptides radiolabeled with tricine and EDDA as co-ligands. In vivo, biodistribution studies (n=4) demonstrated that (99m)Tc- HYNIC-cyc-MSH/tricine had superior tumor to muscle and tumor to blood ratios than (99m)Tc-HYNIC-cyc-MSH/EDDA at early time points. Planar gamma imaging of melanoma bearing mice showed that 99mTc-HYNIC-cyc-MSH/tricine was able to clearly visualize tumors, underscoring the potential utility of (99m)Tc labeled lactam cyclized MSH molecules as melanoma imaging agents.
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Mikami, M; Llewellyn-Jones, C G; Stockley, R A
1998-08-14
Neutrophils isolated from patients with chronic bronchitis and emphysema have been shown to have enhanced responses to formyl peptides when assessed in vitro compared to age, sex matched controls. It is currently unclear whether the observed differences are due to a 'priming' effect by a second agent in vivo, or whether this is a primary difference in the neutrophils. We have studied the effects of interleukin-8, which is thought to be one of the major pro-inflammatory cytokines in chronic lung disease and granulocyte macrophage colony stimulating factor (GMCSF), in order to assess their effects on neutrophil chemotaxis and connective tissue degradation. In addition, we have assessed the effect of preincubation of these agents with neutrophils for 30 min followed by stimulation with F-Met-Leu-Phe (FMLP) to investigate any possible 'priming' effect that may be relevant to our clinical data. We report suppression of neutrophil chemotaxis to FMLP following incubation of the neutrophils with both IL-8 and GMCSF. However, we have observed an additive effect of IL-8 and FMLP for neutrophil degranulation leading to fibronectin degradation. The results suggest that IL-8 does not 'prime' neutrophils for subsequent FMLP stimulation as observed in vivo. Although the results for GMCSF were similar for the chemotactic response, the agent also had a synergistic effect on connective tissue degradation. However, it is concluded that neither agent could explain the enhanced neutrophil responses seen in our patients.
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Jorgensen, Rasmus; Kubale, Valentina; Vrecl, Milka; Schwartz, Thue W; Elling, Christian E
2007-07-01
The glucagon-like peptide (GLP)-1 receptor is a promising target for the treatment of type 2 diabetes and obesity, and there is great interest in characterizing the pharmacology of the GLP-1 receptor and its ligands. In the present report, we have applied bioluminescence resonance energy transfer assays to measure agonist-induced recruitment of betaarrestins and G-protein-coupled receptor kinase (GRK) 2 to the GLP-1 receptor in addition to traditional measurements of second messenger generation. The peptide hormone oxyntomodulin is described in the literature as a full agonist on the glucagon and GLP-1 receptors. Surprisingly, despite being full agonists in GLP-1 receptor-mediated cAMP accumulation, oxyntomodulin and glucagon were observed to be partial agonists in recruiting betaarrestins and GRK2 to the GLP-1 receptor. We suggest that oxyntomodulin and glucagon are biased ligands on the GLP-1 receptor.
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Kwon, Koo Chul; Ko, Ho Kyung; Lee, Jiyun; Lee, Eun Jung; Kim, Kwangmeyung; Lee, Jeewon
2016-08-01
Human ferritin heavy-chain nanoparticle (hFTH) is genetically engineered to present tumor receptor-binding peptides (affibody and/or RGD-derived cyclic peptides, named 4CRGD here) on its surface. The affibody and 4CRGD specifically and strongly binds to human epidermal growth factor receptor I (EGFR) and human integrin αvβ3, respectively, which are overexpressed on various tumor cells. Through in vitro culture of EGFR-overexpressing adenocarcinoma (MDA-MB-468) and integrin-overexpressing glioblastoma cells (U87MG), it is clarified that specific interactions between receptors on tumor cells and receptor-binding peptides on engineered hFTH is critical in active tumor cell targeting. After labeling with the near-infrared fluorescence dye (Cy5.5) and intravenouse injection into MDA-MB-468 or U87MG tumor-bearing mice, the recombinant hFTHs presenting either peptide or both of affibody and 4CRGD are successfully delivered to and retained in the tumor for a prolonged period of time. In particular, the recombinant hFTH presenting both affibody and 4CRGD notably enhances in vivo detection of U87MG tumors that express heterogeneous receptors, integrin and EGFR, compared to the other recombinant hFTHs presenting either affibody or 4CRGD only. Like affibody and 4CRGD used in this study, other multiple tumor receptor-binding peptides can be also genetically introduced to the hFTH surface for actively targeting of in vivo tumors with heterogenous receptors. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing*
Gupta, Achla; Gomes, Ivone; Wardman, Jonathan; Devi, Lakshmi A.
2014-01-01
Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2. PMID:24847082
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Stimulation of ANP secretion by 2-Cl-IB-MECA through A(3) receptor and CaMKII.
Yuan, Kuichang; Bai, Guang Yi; Park, Woo Hyun; Kim, Sung Zoo; Kim, Suhn Hee
2008-12-01
Adenosine is a potent mediator of myocardial protection against hypertrophy via A(1) or A(3) receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A(3) receptor on ANP release. We studied the effects of the A(3) receptor on atrial functions and its modification in hypertrophied atria. A selective A(3) receptor agonist, 2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A(3) receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30muM 2-CI-IB-MECA were not affected. The 100muM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA), A(3) receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA>IB-MECA=HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca(2+) channels, sarcoplasmic reticulum Ca(2+)-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A(3) receptor activation and positive inotropy by intracellular Ca(2+) regulation via the ryanodine receptor and CaMKII.
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Sun, Wei; Li, Lian; Li, Li-jia; Yang, Qing-qing; Zhang, Zhi-rong; Huang, Yuan
2017-01-01
Active tumor-targeting approaches using specific ligands have drawn considerable attention over the years. However, a single ligand often fails to simultaneously target the cancer cell surface and subcellular organelles, which limits the maximum therapeutic efficacy of delivered drugs. We describe a polymeric delivery system modified with the G3-C12 peptide for sequential dual targeting. In this study, galectin-3-targeted G3-C12 peptide was conjugated onto the N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer for the delivery of D(KLAKLAK)2 (KLA) peptide. G3-C12-HPMA-KLA exhibited increased receptor-mediated internalization into galectin-3-overexpressing PC-3 cells. Furthermore, G3-C12 peptide also directed HPMA-KLA conjugates to mitochondria. This occurred because the apoptosis signal triggered the accumulation of galectin-3 in mitochondria, and the G3-C12 peptide that specifically bound to galectin-3 was trafficked along with its receptor intracellularly. As a result, G3-C12-HPMA-KLA disrupted the mitochondrial membrane, increased the generation of reactive oxygen species (ROS) and induced cytochrome c release, which ultimately resulted in enhanced cytotoxicity. An in vivo study revealed that the G3-C12 peptide significantly enhanced the tumor accumulation of the KLA conjugate. In addition, G3-C12-HPMA-KLA exhibited the best therapeutic efficacy and greatly improved the animal survival rate. Our work demonstrates that G3-C12 is a promising ligand with dual-targeting functionality. PMID:28065935
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Babinska, A; Clement, C C; Swiatkowska, M; Szymanski, J; Shon, A; Ehrlich, Y H; Kornecki, E; Salifu, M O
2014-07-01
Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 receptor molecule (F11R, aka JAM-A/Junctional adhesion molecule-A), were designed, prepared, and examined as potential candidates for the development of anti-atherosclerotic and anti-thrombotic therapeutic drugs. A sequence at the N-terminal of F11R together with another sequence located in the first Ig-loop of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned in the active-site was used to generate peptide-candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine-inflammed endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events. © 2014 Wiley Periodicals, Inc.
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Aldrich, Jane V; Kulkarni, Santosh S; Senadheera, Sanjeewa N; Ross, Nicolette C; Reilley, Kate J; Eans, Shainnel O; Ganno, Michelle L; Murray, Thomas F; McLaughlin, Jay P
2011-09-05
An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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The C21-formyl group in chlorophyll f originates from molecular oxygen.
Garg, Harsh; Loughlin, Patrick C; Willows, Robert D; Chen, Min
2017-11-24
Chlorophylls (Chls) are the most important cofactors for capturing solar energy to drive photosynthetic reactions. Five spectral types of Chls have been identified to date, with Chl f having the most red-shifted absorption maximum because of a C2 1 -formyl group substitution of Chl f However, the biochemical provenance of this formyl group is unknown. Here, we used a stable isotope labeling technique ( 18 O and 2 H) to determine the origin of the C2 1 -formyl group of Chl f and to verify whether Chl f is synthesized from Chl a in the cyanobacterial species Halomicronema hongdechloris. In the presence of either H 2 18 O or 18 O 2 , the origin of oxygen atoms in the newly synthesized chlorophylls was investigated. The pigments were isolated with HPLC, followed by MS analysis. We found that the oxygen atom of the C2 1 -formyl group originates from molecular oxygen and not from H 2 O. Moreover, we examined the kinetics of the labeling of Chl a and Chl f from H. hongdechloris grown in 50% D 2 O-seawater medium under different light conditions. When cells were shifted from white light D 2 O-seawater medium to far-red light H 2 O-seawater medium, the observed deuteration in Chl f indicated that Chl(ide) a is the precursor of Chl f Taken together, our results advance our understanding of the biosynthesis pathway of the chlorophylls and the formation of the formyl group in Chl f . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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Sex peptides and MIPs can activate the same G protein-coupled receptor
USDA-ARS?s Scientific Manuscript database
In many animal species, copulation elicits a number of physiological and behavioral changes in the female partner. In Drosophila melanogaster, the main molecular effector of these physiological responses has been identified as sex peptide (SP). The sex peptide receptor (SPR) has been characterized a...
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Lee, Heeseung; Ko, Mei-Chuan
2015-06-29
How neuropeptides in the primate spinal cord regulate itch and pain is largely unknown. Here we elucidate the sensory functions of spinal opioid-related peptides and gastrin-releasing peptide (GRP) in awake, behaving monkeys. Following intrathecal administration, β-endorphin (10-100 nmol) and GRP (1-10 nmol) dose-dependently elicit the same degree of robust itch scratching, which can be inhibited by mu-opioid peptide (MOP) receptor and GRP receptor (BB2) antagonists, respectively. Unlike β-endorphin, which produces itch and attenuates inflammatory pain, GRP only elicits itch without affecting pain. In contrast, enkephalins (100-1000 nmol) and nociceptin-orphanin FQ (3-30 nmol) only inhibit pain without eliciting itch. More intriguingly, dynorphin A(1-17) (10-100 nmol) dose-dependently attenuates both β-endorphin- and GRP-elicited robust scratching without affecting pain processing. The anti-itch effects of dynorphin A can be reversed by a kappa-opioid peptide (KOP) receptor antagonist nor-binaltorphimine. These nonhuman primate behavioral models with spinal delivery of ligands advance our understanding of distinct functions of neuropeptides for modulating itch and pain. In particular, we demonstrate causal links for itch-eliciting effects by β-endorphin-MOP receptor and GRP-BB2 receptor systems and itch-inhibiting effects by the dynorphin A-KOP receptor system. These studies will facilitate transforming discoveries of novel ligand-receptor systems into future therapies as antipruritics and/or analgesics in humans.
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Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand.
Laufer, R; Gilon, C; Chorev, M; Selinger, Z
1986-08-05
We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.
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Song, Wen; Liu, Li; Wang, Jizong; Wu, Zhen; Zhang, Heqiao; Tang, Jiao; Lin, Guangzhong; Wang, Yichuan; Wen, Xing; Li, Wenyang; Han, Zhifu; Guo, Hongwei; Chai, Jijie
2016-06-01
Peptide-mediated cell-to-cell signaling has crucial roles in coordination and definition of cellular functions in plants. Peptide-receptor matching is important for understanding the mechanisms underlying peptide-mediated signaling. Here we report the structure-guided identification of root meristem growth factor (RGF) receptors important for plant development. An assay based on a signature ligand recognition motif (Arg-x-Arg) conserved in a subfamily of leucine-rich repeat receptor kinases (LRR-RKs) identified the functionally uncharacterized LRR-RK At4g26540 as a receptor of RGF1 (RGFR1). We further solved the crystal structure of RGF1 in complex with the LRR domain of RGFR1 at a resolution of 2.6 Å, which reveals that the Arg-x-Gly-Gly (RxGG) motif is responsible for specific recognition of the sulfate group of RGF1 by RGFR1. Based on the RxGG motif, we identified additional four RGFRs. Participation of the five RGFRs in RGF-induced signaling is supported by biochemical and genetic data. We also offer evidence showing that SERKs function as co-receptors for RGFs. Taken together, our study identifies RGF receptors and co-receptors that can link RGF signals with their downstream components and provides a proof of principle for structure-based matching of LRR-RKs with their peptide ligands.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kumar G.; Swaminathan S.; Kumaran, D.
Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC{sub 50} of 0.9 {micro}M and a K{sub i} ofmore » 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.« less
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Kumar, Gyanendra; Kumaran, Desigan; Ahmed, S Ashraf; Swaminathan, Subramanyam
2012-05-01
Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC(50) of 0.9 µM and a K(i) of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features. © 2012 International Union of Crystallography
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Tachykinin receptors in the circular muscle of the guinea-pig ileum.
Maggi, C A; Patacchini, R; Giachetti, A; Meli, A
1990-12-01
1. We have studied the mechanical response of circular strips of the guinea-pig ileum to tachykinins and characterized the receptors involved by means of receptor-selective agonists. 2. The strips responded to both substance P (SP) and neurokinin A (NKA), as well as to [Pro9]-SP sulphone (selective NK1-receptor agonist), [beta Ala8]-NKA(4-10) (selective NK2-receptor agonist) and [MePhe7]-neurokinin B (selective NK3-receptor agonist). The ED50s of the various peptides (calculated as the concentration of agonist which produced 50% of the response to 10 microM carbachol) were similar, in the range of 40-200 nM, i.e. no clearcut rank order of potency was evident. 3. The response to a submaximal (10 nM) concentration of SP or NKA was unaffected in the presence of peptidase inhibitors (thiorphan, captopril and bestatin, 1 microM each). 4. The response to the NK1-agonist was totally atropine-resistant, but was reduced (about 30% inhibition) by tetrodotoxin. The response to the NK3-receptor agonist was halved by atropine and abolished by tetrodotoxin. The response to the NK2-agonist was unaffected by either atropine or tetrodotoxin. 5. The response to the selective NK2-agonist was unchanged after desensitization of NK1- or NK3-receptors. 6. The response to the NK2-selective agonist was strongly inhibited by [Tyr5, D-Trp6,8,9, Arg10]-NKA(4-10) (MEN 10,207) a selective NK2-receptor antagonist which did not modify the response to the NK1-selective agonist. 7. Our findings indicate that all the three known types of tachykinin receptors mediate the contractile response of the circular muscle of the guinea-pig ileum to peptides of this family. The response to activation of NK3-receptors is totally neurogenic and partially mediated by endogenous acetylcholine, the response to activation of NK1-receptors is partly neurogenic and largely myogenic and the response to activation of NK2-receptors is totally myogenic.
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Wyatt, M. A.; Jarvie, E.; Feniuk, W.; Humphrey, P. P.
1996-01-01
1. The aim of this study was to determine the location and functional characteristics of the somatostatin (SRIF) receptor type(s) which mediate inhibition of acid secretion in rat isolated gastric mucosa. 2. Gastrin (1 nM-1 microM), dimaprit (10 microM-300 microM) and isobutyl methylxanthine (IBMX, 1 microM-100 microM) all caused concentration-dependent increases in acid output. Responses to gastrin were almost completely inhibited by ranitidine (10 microM) at a concentration which abolished the secretory response to dimaprit. In contrast, responses to IBMX were not changed by ranitidine suggesting that IBMX acts directly on the parietal cell and not indirectly by releasing histamine from enterochromaffin-like (ECL) cells. 3. SRIF-14 (1 nM-1 microM) had no effect on basal acid output, but inhibited acid output produced by gastrin, dimaprit and IBMX in a concentration-dependent manner with respective EC50 values of 46, 54 and 167 nM. The peptidase inhibitors, amastatin (10 microM) and phosphoramidon (1 microM), had no effect on SRIF-induced inhibition of dimaprit stimulated gastric acid secretion. 4. The inhibitory effect of a range of SRIF analogues on gastrin-, dimaprit- and IBMX-induced acid secretion was also studied. Irrespective of the secretagogue used to increase acid output, the rank order of potencies was similar (BIM-23027 = seglitide = octreotide > SRIF-14 = SRIF-28 > L-362,855). The linear peptide BIM-23056 was devoid of agonist or antagonist activity in concentrations up to 1 microM. 5. The sst2 receptor selective peptides, BIM-23027, seglitide and octreotide were the most potent inhibitors of gastrin-, dimaprit- and IBMX-induced acid secretion suggesting that SRIF receptors resembling the recombinant sst2 receptors are involved. Furthermore, since dimaprit and IBMX stimulate gastric acid secretion independently of histamine release, sst2 receptor-mediated inhibition must occur at the level of the parietal cell itself. PMID:8922739
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Wyatt, M A; Jarvie, E; Feniuk, W; Humphrey, P P
1996-11-01
1. The aim of this study was to determine the location and functional characteristics of the somatostatin (SRIF) receptor type(s) which mediate inhibition of acid secretion in rat isolated gastric mucosa. 2. Gastrin (1 nM-1 microM), dimaprit (10 microM-300 microM) and isobutyl methylxanthine (IBMX, 1 microM-100 microM) all caused concentration-dependent increases in acid output. Responses to gastrin were almost completely inhibited by ranitidine (10 microM) at a concentration which abolished the secretory response to dimaprit. In contrast, responses to IBMX were not changed by ranitidine suggesting that IBMX acts directly on the parietal cell and not indirectly by releasing histamine from enterochromaffin-like (ECL) cells. 3. SRIF-14 (1 nM-1 microM) had no effect on basal acid output, but inhibited acid output produced by gastrin, dimaprit and IBMX in a concentration-dependent manner with respective EC50 values of 46, 54 and 167 nM. The peptidase inhibitors, amastatin (10 microM) and phosphoramidon (1 microM), had no effect on SRIF-induced inhibition of dimaprit stimulated gastric acid secretion. 4. The inhibitory effect of a range of SRIF analogues on gastrin-, dimaprit- and IBMX-induced acid secretion was also studied. Irrespective of the secretagogue used to increase acid output, the rank order of potencies was similar (BIM-23027 = seglitide = octreotide > SRIF-14 = SRIF-28 > L-362,855). The linear peptide BIM-23056 was devoid of agonist or antagonist activity in concentrations up to 1 microM. 5. The sst2 receptor selective peptides, BIM-23027, seglitide and octreotide were the most potent inhibitors of gastrin-, dimaprit- and IBMX-induced acid secretion suggesting that SRIF receptors resembling the recombinant sst2 receptors are involved. Furthermore, since dimaprit and IBMX stimulate gastric acid secretion independently of histamine release, sst2 receptor-mediated inhibition must occur at the level of the parietal cell itself.
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Silva, Roberta N; Oliveira, Lilian C G; Parise, Carolina B; Oliveira, Juliana R; Severino, Beatrice; Corvino, Angela; di Vaio, Paola; Temussi, Piero A; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Luiz; Juliano, Maria A
2017-05-01
Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R ↓ R-ACC and Z-R ↓ R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates. Copyright © 2017 Elsevier B.V. All rights reserved.
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Contractile effects and binding properties of endothelins/sarafotoxins in the guinea pig ileum.
Wollberg, Z; Bdolah, A; Galron, R; Sokolovsky, M; Kochva, E
1991-05-30
Seven of the eight known isopeptides of the endothelin/sarafotoxin (ET/SRTX) family were tested on the isolated guinea pig ileum and found to cause a concentration-dependent increase in basal tone. The rate or the amplitude of the spontaneous rhythmic contractions of the ileal smooth muscle were essentially not affected by any of the peptides. The maximum contraction elicited by vasoactive intestinal contractor (VIC) was slightly stronger than that induced by endothelin-1 (ET-1) or sarafotoxin-b (SRTX-b), and significantly stronger than the maximal contractions elicited by sarafotoxin-a (SRTX-a), sarafotoxin-c (SRTX-c), or endothelin-3 (ET-3). Sarafotoxin-d (SRTX-d) caused, essentially, no contraction but a rather marked relaxation. The potencies of the various peptides to induce the increase in tension, in terms of EC50 values (cumulative effective concentrations that induce half-maximum response), ranged between 6 and 95 nM depending on the peptide. VIC, ET-1, SRTX-b and SRTX-a had similar potencies and were significantly more potent than SRTX-c and ET-3. A high concentration of SRTX-b elicited no additional response when applied to the organ bath after one of the other peptides had shown a maximal effect. Binding experiments with ileal membranes revealed similar binding properties for the various peptides. Competition with iodinated SRTX-b showed no meaningful differences between the various peptides. It is concluded that all the ET/SRTX peptides compete for the same receptor subtype in the ileum. In terms of efficacy, VIC can be considered as a full agonist of this receptor, SRTX-d is probably an antagonist, while all the other peptides behave as partial agonists.
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[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.
Buku, A; Condie, B A; Price, J A; Mezei, M
2005-09-01
An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Pennington, M.W.
We have determined that ShN I, a 48-residue type 2 sea anemone toxin, delays the inactivation of the Na channel in lobster olfactory somas. The receptor for ShN I was identified in vesicle preparations of neuronal tissues from both crustaceans and mammals; however, the K{sub D} values for the former is more than 1,000 fold lower for the later. The binding of ({sup 125}I)-ShN I to this receptor was determined to be unaffected by Anemonia sulcata II, depolarization of the membrane, or veratridine. ShN I was unable to displace ({sup 125}I)-Androctonus austrialis Hector II, whereas unlabeled AaH II and Asmore » II displaced the labeled scorpion toxin from rat brain synaptosomes. This is the first characterization of a new Na channel receptor site which specifically binds type 2 anemone toxins. To study the interactions that specific amino acid residues of ShN I have with this receptor, we developed a strategy using solid phase peptide synthesis. Prior to the synthesis of analogs to ShN I, we assembled the native ShN I sequence and reoxidized the three intramolecular disulfide bonds. Chemical, physical, and pharmacological characterization of the purified synthetic ShN I showed it to be indistinguishable from the natural toxin.« less
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Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP).
Koller, K J; Lowe, D G; Bennett, G L; Minamino, N; Kangawa, K; Matsuo, H; Goeddel, D V
1991-04-05
The natriuretic peptides are hormones that can stimulate natriuretic, diuretic, and vasorelaxant activity in vivo, presumably through the activation of two known cell surface receptor guanylyl cyclases (ANPR-A and ANPR-B). Although atrial natriuretic peptide (ANP) and, to a lesser extent, brain natriuretic peptide (BNP) are efficient activators of the ANPR-A guanylyl cyclase, neither hormone can significantly stimulate ANPR-B. A member of this hormone family, C-type natriuretic peptide (CNP), potently and selectively activated the human ANPR-B guanylyl cyclase. CNP does not increase guanosine 3',5'-monophosphate accumulation in cells expressing human ANPR-A. The affinity of CNP for ANPR-B is 50- or 500-fold higher than ANP or BNP, respectively. This ligand-receptor pair may be involved in the regulation of fluid homeostasis by the central nervous system.
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Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor*
Lawrence, Callum F.; Margetts, Mai B.; Menting, John G.; Smith, Nicholas A.; Smith, Brian J.; Ward, Colin W.; Lawrence, Michael C.
2016-01-01
Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe701 and Phe705. The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor. PMID:27281820
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Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin
2011-01-01
The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide. PMID:21421725
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Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin
2011-04-01
The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.
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2016-01-01
Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B1-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B1 (AFB1–FAPY) adduct. The AFB1–FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′, and 5′-XY-3′ sequences (X = AFB1–FAPY; Y = 7-deaza-dG) demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions. Structural analysis of the adduct in the 5′-XA-3′ sequence indicates the preference of the E isomer of the formamide group, attributed to formation of a hydrogen bond between the formyl oxygen and the N6 exocyclic amino group of the 3′-neighbor adenine. While the 5′-XA-3′ sequence exhibits the E isomer, the 5′-XC-3′ sequence exhibits a 7:3 E:Z ratio at equilibrium at 283 K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N4-dC exocyclic amino group of the 3′-neighbor cytosine. The 5′-XT-3′ and 5′-XY-3′ sequences cannot form such a hydrogen bond between the formyl oxygen and the 3′-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and β anomers and the potential to exhibit atropisomers about the C5–N5 bond do not depend upon sequence. In each of the four DNA sequences, the AFB1–FAPY adduct maintains the β deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB1 moiety that is intercalated above the 5′-face of the damaged guanine. This enforces the Ra axial conformation for the C5–N5 bond. PMID:25587868
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Li, Liang; Brown, Kyle L; Ma, Ruidan; Stone, Michael P
2015-02-16
Aflatoxin B(1) (AFB(1)), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B(1)-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (AFB(1)−FAPY) adduct. The AFB(1)−FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′, and 5′-XY-3′ sequences (X = AFB(1)−FAPY; Y = 7-deaza-dG)demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions.Structural analysis of the adduct in the 5′-XA-3′ sequence indicates the preference of the E isomer of the formamide group,attributed to formation of a hydrogen bond between the formyl oxygen and the N(6) exocyclic amino group of the 3′-neighboradenine. While the 5′-XA-3′ sequence exhibits the E isomer, the 5′-XC-3′ sequence exhibits a 7:3 E:Z ratio at equilibrium at 283K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N(4)-dC exocyclic amino group of the 3′-neighbor cytosine. The 5′-XT-3′ and 5′-XY-3′ sequences cannot form such a hydrogen bond between the formyl oxygen and the 3′-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and β anomers and the potential to exhibit atropisomers about the C5−N(5) bond do not depend upon sequence. In each of the four DNA sequences, the AFB(1)−FAPY adduct maintains the β deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB(1) moiety that is intercalated above the 5′-face of the damaged guanine. This enforces the Ra axialc onformation for the C5−N(5) bond.
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D'Andrea, Luca D; Testa, Irma; Panico, Mariarosaria; Di Stasi, Rossella; Caracò, Corradina; Tarallo, Laura; Arra, Claudio; Barbieri, Antonio; Romanelli, Alessandra; Aloj, Luigi
2008-01-01
The development of receptor targeting radiolabeled ligands has gained much interest in recent years for diagnostic and therapeutic applications in nuclear medicine. Cholecystokinin (CCK) receptors have been shown to be overexpressed in a subset of neuroendocrine and other tumors. We are evaluating binding and biodistribution properties of a CCK8 peptide derivative labeled with (99m)Tc(I)-tricarbonyl. The CCK8 peptide was modified at its N-terminus by adding to its N-terminus two lysine-histidine modules (KH), where histidine is coupled to the side chain of the lysine ((KH)(2)-CCK8). (99m)Tc(I)-tricarbonyl was generated with the IsoLinktrade mark kit. A431 cells stably transfected with a cDNA encoding for the human CCK2 receptor were utilized to determine binding affinity, internalization, and retention of the labeled peptide, in comparison with wild-type A431 cells. A nude mouse tumor model was obtained by generating A431-CCK2R and A431-control tumors in opposite flanks of the animals. High specific activity labeling with (99m)Tc was achieved. In A431-CCK2R cells, specific saturable binding was observed as well as evident internalization of the radiolabeled peptide after binding. Biodistribution experiments showed rapid, specific localization of (KH)(2)-CCK8 on A431-CCK2R xenografts compared with control tumors, although absolute uptake values were not markedly higher compared with background activity. Clearance of unbound radioactivity was both urinary and hepatobiliary. In imaging experiments, while targeting to CCK2R positive tumors could be appreciated, there was poor contrast between target and nontarget areas. (KH)(2)-CCK8 shows adequate in vitro and in vivo properties for CCK2R targeting although improvement of biodistribution warrant further development. (c) 2008 Wiley Periodicals, Inc.
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Balsera, Beatriz; Mulet, José; Sala, Salvador; Sala, Francisco; de la Torre-Martínez, Roberto; González-Rodríguez, Sara; Plata, Adrián; Naesens, Lieve; Fernández-Carvajal, Asia; Ferrer-Montiel, Antonio; Criado, Manuel; Pérez de Vega, María Jesús; González-Muñiz, Rosario
2018-01-01
α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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Maryanoff, Bruce E; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Derian, Claudia K
2003-03-01
Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.
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Statnick, Michael A; Chen, Yanyun; Ansonoff, Michael; Witkin, Jeffrey M; Rorick-Kehn, Linda; Suter, Todd M; Song, Min; Hu, Charlie; Lafuente, Celia; Jiménez, Alma; Benito, Ana; Diaz, Nuria; Martínez-Grau, Maria Angeles; Toledo, Miguel A; Pintar, John E
2016-02-01
Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide, is the endogenous ligand of the ORL1/nociceptin-opioid-peptide (NOP) receptor. N/OFQ appears to regulate a variety of physiologic functions including stimulating feeding behavior. Recently, a new class of thienospiro-piperidine-based NOP antagonists was described. One of these molecules, LY2940094 has been identified as a potent and selective NOP antagonist that exhibited activity in the central nervous system. Herein, we examined the effects of LY2940094 on feeding in a variety of behavioral models. Fasting-induced feeding was inhibited by LY2940094 in mice, an effect that was absent in NOP receptor knockout mice. Moreover, NOP receptor knockout mice exhibited a baseline phenotype of reduced fasting-induced feeding, relative to wild-type littermate controls. In lean rats, LY2940094 inhibited the overconsumption of a palatable high-energy diet, reducing caloric intake to control chow levels. In dietary-induced obese rats, LY2940094 inhibited feeding and body weight regain induced by a 30% daily caloric restriction. Last, in dietary-induced obese mice, LY2940094 decreased 24-hour intake of a high-energy diet made freely available. These are the first data demonstrating that a systemically administered NOP receptor antagonist can reduce feeding behavior and body weight in rodents. Moreover, the hypophagic effect of LY2940094 is NOP receptor dependent and not due to off-target or aversive effects. Thus, LY2940094 may be useful in treating disorders of appetitive behavior such as binge eating disorder, food choice, and overeating, which lead to obesity and its associated medical complications and morbidity. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
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Je, Byoung Il; Xu, Fang; Wu, Qingyu; Liu, Lei; Meeley, Robert; Gallagher, Joseph P; Corcilius, Leo; Payne, Richard J; Bartlett, Madelaine E; Jackson, David
2018-03-15
Meristems contain groups of indeterminate stem cells, which are maintained by a feedback loop between CLAVATA ( CLV ) and WUSCHEL ( WUS ) signaling. CLV signaling involves the secretion of the CLV3 peptide and its perception by a number of Leucine-Rich-Repeat (LRR) receptors, including the receptor-like kinase CLV1 and the receptor-like protein CLV2 coupled with the CORYNE (CRN) pseudokinase. CLV2, and its maize ortholog FASCIATED EAR2 (FEA2) appear to function in signaling by CLV3 and several related CLV3/EMBRYO-SURROUNDING REGION (CLE) peptide ligands. Nevertheless, how signaling specificity is achieved remains unknown. Here we show that FEA2 transmits signaling from two distinct CLE peptides, the maize CLV3 ortholog ZmCLE7 and ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1) through two different candidate downstream effectors, the alpha subunit of the maize heterotrimeric G protein COMPACT PLANT2 (CT2), and ZmCRN. Our data provide a novel framework to understand how diverse signaling peptides can activate different downstream pathways through common receptor proteins. © 2018, Je et al.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia
Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145more » binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.« less
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Synergistic inhibition of natural killer cells by the nonsignaling molecule CD94
Cheent, Kuldeep S.; Jamil, Khaleel M.; Cassidy, Sorcha; Liu, Mengya; Mbiribindi, Berenice; Mulder, Arend; Claas, Frans H. J.; Purbhoo, Marco A.; Khakoo, Salim I.
2013-01-01
Peptide selectivity is a feature of inhibitory receptors for MHC class I expressed by natural killer (NK) cells. CD94–NKG2A operates in tandem with the polymorphic killer cell Ig-like receptors (KIR) and Ly49 systems to inhibit NK cells. However, the benefits of having two distinct inhibitory receptor–ligand systems are not clear. We show that noninhibitory peptides presented by HLA-E can augment the inhibition of NKG2A+ NK cells mediated by MHC class I signal peptides through the engagement of CD94 without a signaling partner. Thus, CD94 is a peptide-selective NK cell receptor, and NK cells can be regulated by nonsignaling interactions. We also show that KIR+ and NKG2A+ NK cells respond with differing stoichiometries to MHC class I down-regulation. MHC-I–bound peptide functions as a molecular rheostat controlling NK cell function. Selected peptides which in isolation do not inhibit NK cells can have different effects on KIR and NKG2A receptors. Thus, these two inhibitory systems may complement each other by having distinct responses to bound peptide and surface levels of MHC class I. PMID:24082146