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Abstract: Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with 15N and 13C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general. [Copyright &y& Elsevier]

The Ca2+-free form of calmodulin (CaM), apocalmodulin (ApoCaM), regulates a variety of target proteins including nitric oxide synthase II (NOS-II). The CaM-binding site of NOS-II can bind ApoCaM with high affinity. Substitution of hydrophobic amino acids by charged amino acids at crucial positions 3, 9 and 13 within the CaM-binding motif did not abolish the ApoCaM interaction that occurred with significant affinity, though the affinity of the interaction was decreased remarkably. Isothermal titration calorimetry revealed that interaction of ApoCaM and synthetic NOS-II peptides was driven entropically. [Copyright &y& Elsevier]

Examines the role of nitric oxide synthase I peptides in the target recognition of apocalmodulin. Increase in the level of hydrophobic amino acids; Details on the binding of wild-type and mutant peptides to calcium-calmodulin; Absorption of peptide bonds.

The binding of peptides or proteins to a bilayer membrane is often coupled with a random coil→α-helix transition. Knowledge of the energetics of this membrane-induced folding event is essential for the understanding of the mechanism of membrane activity. In a recent study [Wieprecht et al., J. Mol. Biol. 294 (1999) 785–794], we have developed an approach which allows an analysis of the energetics of membrane-induced folding. We have systematically varied the helix content of the amphipathic peptide magainin-2-amide by synthesizing analogs where two adjacent amino acid residues were substituted by their corresponding d-enantiomers and have measured their binding to small unilamellar vesicles (SUVs). Correlation of the binding parameters with the helicities allowed the evaluation of the thermodynamic parameters of helix formation. Since SUVs (30 nm in diameter) are characterized by a non-ideal lipid packing due to their high membrane curvature, we have now extended our studies to large unilamellar vesicles (LUVs) (100 nm in diameter) with a lipid packing close to planar membranes. While the free energy of binding was similar for SUVs and LUVs, the binding enthalpies and entropies were distinctly different for the two membrane systems. The thermodynamic parameters of the coil–helix transition were nevertheless not affected by the vesicle size. Helix formation at the membrane surface of LUVs (SUVs) was characterized by an enthalpy change of −0.8 (−0.7) kcal/mol per residue, an entropy change of−2.3 (−1.9) cal/mol K per residue, and a free energy change of −0.12 (−0.14) kcal/mol per residue. Helix formation accounted for ∼50% of the free energy of binding underlining its major role as a driving force for membrane-binding. [Copyright &y& Elsevier]

Identifies glutamic-acid-rich proteins (GARP) as multivalent proteins that interact with the key players of cGMP signalling, phosphodiesterase and guanylate cyclase, and with a retina-specific ATP-binding cassette transporter (ABCR).

Investigates the thermodynamics and structure of the binding of antibacterial magainin peptides to electrically neutral membranes. Reduction of extent of peptide binding through addition of cholesterol; Enthalpy-entropy compensation mechanism; Membrane perturbation effect.

Determines the secondary structure of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. Design of model peptides; Nuclear magnetic resonance spectroscopy; Hydrogen exchange.

Presents the stepwise automated solid phase synthesis of the naturally occurring peptides called peptaibols using amino acid fluorides. Direct resin loading of terminal carbon alcohols; Final peptide resin cleavage of the ether bond; Racemization of chiral amino acids; Purification and characterization of products.

Describes the use of Fmoc amino acid fluorides in solid-state synthesis of medium-sized peptides. Rapid-acting acylating agents; High reactivity in the coupling of stearically hindered amino acid residues; Identification of side products with rationalization of their formation.

Investigates the location of an amphipathic alpha-helix in peptides using reversed phase high performance liquid chromatography (HPLC) retention behavior of d-amino acid analogs.

Abstract: The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications. [Copyright &y& Elsevier]

Abstract: Protein interaction domains (PIDs) play a critical role in signal transduction. One PID of great interest is the PDZ domain, a 100 amino-acid-residue domain. Most PDZ domains recognize short, C-terminal peptide motives. In the heterodimer of the nNOS-PDZ domain and the α-syntrophin-PDZ domain, however, one PDZ domain forms a β-finger that binds to the other PDZ domain. We show here that cyclic peptides derived from the β-finger of the nNOS-PDZ domain can bind the syntrophin-PDZ domain in the same manner as the whole domain. The structure of three “finger-peptides” of different size has been determined and the binding investigated using calorimetry and NMR-titration experiments. [Copyright &y& Elsevier]

We developed and characterized efficient, remarkably water-soluble photolabile protecting groups for thiols based on 2-nitrobenzyl and (coumarin-4-yl)methyl chromophores, among them two new ones. The protecting groups allow, due to their different absorption maxima, wavelength-selective photocleavage of binary mixtures of cysteine derivatives protected at the thiol function with various photolabile protecting groups by irradiation with light. The concept was also functional with the two different S-protected cysteine residues in derivatives of the model peptide resact. Selective photolysis could be achieved for the peptides Ac°-Cys1(BCMACMOC),Cys8(7,8BCMCMOC)-resact and Ac0-Cys1(C4MNB),Cys8(BCMACMOC)-resact by irradiation with light of ⩾402 nm or ⩾436 nm wavelength, respectively, followed by irradiation at χ ⩾ 325 nm. [ABSTRACT FROM AUTHOR]

Abstract: Liposomes and micellar carriers equipped with targeting and cellular uptake mediating peptides have attracted attention for numerous applications. The optimization of the carrier requires an understanding of how their properties influence target cell recognition and uptake. We developed a dipalmitoylated apolipoprotein E-derived peptide, named P2A2 as promising vector to mediate cellular uptake of potential micellar and liposomal carriers. Confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) were used to get insight into the internalization mediated by carboxyfluoresceine-labeled P2fA2 and the all-D amino acid analogue P2fa2 into brain capillary endothelial cells. Both peptide micelles and liposomes entered cells via endocytosis. Cell surface heparan sulfate proteoglycans (HSPGs) were involved in the internalization process of peptide-bearing liposomes characterized by a diameter of 100 nm, a low surface density of 100 peptide molecules per vesicle and a helical conformation of the vector. In contrast, peptide micelles characterized by a diameter of about 10 nm, a high peptide density caused by 19 associated molecules and a high conformational flexibility of the vector sequence did not address HSPG. Unspecific interactions between the carriers and membrane constituents predominate the two uptake processes but stereospecific components seem to be involved. Both routes differ with respect to transport efficiency. The results provide a prospective basis to optimize liposomes and micelles as drug delivery systems. [Copyright &y& Elsevier]

The GYF domain of CD2BP2 serves as an adapter that recognizes proline-rich sequences in intracellular proteins. Although the T cell adhesion molecule CD2 and the core splicing protein SmB/B′ were previously shown to interact with CD2BP2-GYF, we are now using a general approach to identify putative GYF domain target sites within the human proteome. The phage display-derived recognition motif for CD2BP2-GYF is PPG(W/F/Y/M/L). SPOT analysis confirmed that the GYF domain interacts with peptides from human proteins containing the consensus site. Epitope mapping by NMR spectroscopy performed for several peptides revealed a conserved binding surface. A direct interaction of the CD2BP2-GYF domain with the novel protein interaction partners PI31 and NPWBP was verified by yeast two-hybrid analysis. [ABSTRACT FROM AUTHOR]

Abstract: The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled 31P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from 2H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment. [Copyright &y& Elsevier]

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucnl analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes I and 2(α) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2--C6), the disulfide arrangement C2--C5 and C4--C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT. [ABSTRACT FROM AUTHOR]

Photolysis of model peptides containing α-carboxy-2-nitrobenzyl (CNB) or α-carboxy-4,5-dimethoxy-2-nitrobenzyl (CDMNB) protected cysteines in aqueous solution gives the expected 2-nitrobenzyl-type photocleavage and can be accompanied by photodecarboxylation depending on structural aspects. [ABSTRACT FROM AUTHOR]

Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognitionmotifs. As proofof- principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasiveMDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity. [ABSTRACT FROM AUTHOR]

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity. [ABSTRACT FROM AUTHOR]

Summary: Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click-chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function. [ABSTRACT FROM AUTHOR]

Helixinduktion: Die synthetische tricyclische Aminosäure ProM ‐ 5, die formal durch stereoselektive Einführung einer Vinylidenbrücke in eine Diprolineinheit entsteht, ist ein leistungsstarkes Gerüst für die Nukleierung der α ‐ Helix ‐ Bildung in einer linearen Peptidkette. Dieses Verhalten könnte bei der Entwicklung neuer Proteomimetika zur Modulation von Proteinwechselwirkungen genutzt werden. [ABSTRACT FROM AUTHOR]

Secondary structure induction: The synthetic tricyclic amino acid ProM‐5, which is formally created by stereoselective introduction of a vinylidene bridge into a di‐proline unit, acts as a powerful scaffold to nucleate α‐helix formation in a linear peptide chain. This might be exploited in the development of new proteomimetics for the modulation of protein interactions. [ABSTRACT FROM AUTHOR]

Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence. [ABSTRACT FROM AUTHOR]

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca2+ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2-0.6 s before a Ca2+ response was produced. Additional molecules delivered during a Ca2+response reset the cell by causing a pronounced Ca2+ drop that terminated the response; this reset was followed by a new Ca2+ rise. After stimulation, sperm adapted their sensitivity following the Weber-Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well. [ABSTRACT FROM AUTHOR]

The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches. [ABSTRACT FROM AUTHOR]

The specific inhibition of the biosynthesis of target proteins is a relatively novel strategy in pharmacology and is based mainly on antisense approaches (e.g. antisense oligonucleotides or RNA interference). Recently, a novel class of substances was described acting at a later step of protein biosynthesis. The cyclic heptadepsipeptides CAM741 and cotransin were shown to inhibit selectively the biosynthesis of a small subset of secretory proteins by preventing stable insertion of the nascent chains into the Sec61 translocon complex at the endoplasmic reticulum membrane (Besemer, J., Harant, H., Wang, S., Oberhauser, B., Marquardt, K., Foster, C. A., Schreiner, E. P., de Vries, J. E., Dascher- Nadel, C., and Lindley, I. J. (2005) Nature 436, 290-293; Garrison, J. L., Kunkel, E. J., Hegde, R. S., and Taunton, J. (2005) Nature 436, 285-289). These peptides act in a signal sequence-discriminatory manner, which explains their selectivity. Here, we have analyzed the cotransin sensitivity of various G protein-coupled receptors in transfected HEK 293 cells. We show that the biosynthesis of the human endothelin B receptor (ETBR) is highly sensitive to cotransin, in contrast to that of the other G protein-coupled receptors analyzed. Using a novel biosynthesis assay based on fusions with the photoconvertible Kaede protein, we show that the IC50 value of cotransin action on ETBR biosynthesis is 5.4 μm and that ETBR signaling could be completely blocked by treating cells with 30 μm cotransin. Taken together, our data add an integral membrane protein, namely the ETBR, to the small group of cotransin-sensitive proteins. [ABSTRACT FROM AUTHOR]

The family of angiotensin peptides has been steadily growing in recent years. Most are fragments of angiotensin II (Ang II) with different affinities to the known angiotensin receptors. Here, we describe a novel endogenous Ang II-like octapeptide in plasma from healthy humans and patients with end-stage renal failure, which acts as a stronger agonist at Mas receptors than Ang 1-7. Chromatographic purification and structural analysis by matrix-assisted laser desorption/ ionization time-of-flight/time-of-flight (MALDI-TOF/ TOF) revealed an Ang II-like octapeptide, angioprotectin, with the sequence Pro-Glu-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Pro¹ and Glu² instead of Asp¹ and Arg². Pro-Glu-Val-Tyr-Ile-His-Pro-Phe in angioprotectin is most likely generated enzymatically from Ang II. Angioprotectin antagonized the contractile actions of Ang II on rat aortic rings. The physiological antagonism of vasoconstrictor actions of Ang II by angioprotectin is mediated by the Mas receptor. Angioprotectin has a stronger affinity to the Mas receptor than Ang-1-7. Plasma concentrations were ~15% of plasma Ang II concentrations in healthy volunteers and up to 50% in patients with renal failure. A commercially available Ang II antibody did not discriminate between angioprotectin and Ang II; thus, angioprotectin can contribute to Ang II concentrations measured by antibody-based assays. This novel peptide is likely to be a relevant component of the human renin-angiotensin-system. [ABSTRACT FROM AUTHOR]

Abstract: G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems. [Copyright &y& Elsevier]

Following ectodomain shedding by p-secretase, successive proteolytic cleavages within the transmembrane sequence (TMS) of the amyloid precursor protein (APP) catalyzed by y-secretase result in the release of amyloid-β3 (A13) peptides of variable length. Aβ peptides with 42 amino acids appear to be the key pathogenic species in Alzheimer's disease, as they are believed to initiate neuronal degeneration. Sulindac sulfide, which is known as a potent y-secretase modulator (GSM), selectively reduces Aβ342 production in favor of shorter Aβ species, such as Ap38. By studying APP-TMS dimerization we previously showed that an attenuated interaction similarly decreased Aβ42 levels and concomitantly increased Aβ38 levels. However, the precise molecular mechanism by which GSMs modulate Aβ production is still unclear. In this study, using a reporter gene-based dimerization assay, we found that APP-TMS dimers are destabilized by sulindac sulfide and related A1342-lowering compounds in a concentration-dependent manner. By surface plasmon resonance analysis and NMR spectroscopy, we show that sulindac sulfide and novel sulindac-derived compounds directly bind to the A13 sequence. Strikingly, the attenuated APP-TMS inter- action by GSM5 correlated strongly with AI342-lowering activity and binding strength to the Aβ sequence. Molecular docking analyses suggest that certain GSMs bind to the GxxxG dimerization motif in the APP-TMS. We conclude that these GSMs decrease Aβ42 levels by modulating APP-TMS interactions. This effect specifically emphasizes the importance of the dimeric APP-TMS as a promising drug target in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The aggregation of the amyloid-β (Aβ) peptide plays a pivotal role in the pathogenesis of Alzheimer's disease, as soluble oligomers are intimately linked to neuronal toxicity and inhibition of hippocampal long-term potentiation (LTP). In the C-terminal region of Aβ there are three consecutive GxxxG dimerization motifs, which we could previously demonstrate to play a critical role in the generation of Aβ. Here, we show that glycine 33 (G33) of the central GxxxG interaction motif within the hydrophobic Aβ sequence is important for the aggregation dynamics of the peptide. Aβ peptides with alanine or isoleucine substitutions of G33 displayed an increased propensity to form higher oligomers, which we could attribute to conformational changes. Importantly, the oligomers of G33 variants were much less toxic than Aβ42 wild type (WT), in vitro and in vivo. Also, whereas Aβ42 WT is known to inhibit LTP, Aβ42 G33 variants had lost the potential to inhibit LTP. Our findings reveal that conformational changes induced by G33 substitutions unlink toxicity and oligomerization of Aβ on the molecular level and suggest that G33 is the key amino acid in the toxic activity of Aβ. Thus, a specific toxic conformation of Aβ exists, which represents a promising target for therapeutic interventions. [ABSTRACT FROM AUTHOR]

βArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with βarrestin1. Asp26 and Asp29 in the N-terminal domain of βarrestin1 are critical for its binding to MEK1, whereas Arg47 and Arg49 in the N-terminal domain of MEK1 are critical for its binding to βarrestin1. Wildtype FLAG-tagged βarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A1D29A mutant does not. ERK-dependent phosphorylation at Ser412 was compromised in the D26A/D29A-βarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated βarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked βarrestinl/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-βarrestin1 in compromising the ERK-dependent phosphorylation of βarrestin1. In addition, the MEK disruptor peptide promoted the ability of βarrestinl to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the β2-adrenergic receptor. [ABSTRACT FROM AUTHOR]

The functional capacity of the transcriptional regulatory CCAAT/enhancer-binding protein-β (C/EBPβ) is governed by protein interactions and post-translational protein modifications. In a proteome-wide interaction screen, the histone-lysine N-methyltransferase, H3 lysine 9-specific 3 (G9a), was found to directly interact with the C/EBPβ transactivation domain (TAD). Binding between G9a and C/EBPβ was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation. Metabolic labeling showed that C/EBPβ is post-translationally modified by methylation in vivo.Aconserved lysine residue in the C/EBPβ TAD served as a substrate for G9a-mediated methylation. G9a, but not a methyltransferase-defective G9a mutant, abrogated the transactivation potential of wild type C/EBPβ. A C/EBPβ TAD mutant that contained a lysine-to-alanine exchange was resistant to G9a-mediated inhibition. Moreover, the same mutation conferred super-activation of a chromatin-embedded, endogenous C/EBPβ target gene. Our data identify C/EBPβ as a direct substrate of G9a-mediated post-translational modification that alters the functional properties of C/EBPβ during gene regulation. [ABSTRACT FROM AUTHOR]

Abstract: Catabolic processes play a crucial role in the steady state of the amyloid-β peptide (Aβ). Neprilysin (NEP) and angiotensin-converting enzyme (ACE), two transmembranal enzymes with greatest importance in peptide pharmacology, are known to play a role in Aβ catabolism. This paper focuses on the N-terminal part of Aβ. This region contains the three amino acid residues that determine the differences between human (hAβ) and murine Aβ (mAβ). Moreover, the N-terminal part of Aβ contains the zinc-binding site of the molecule. Consequently, all hydrolytic attacks on this part of the Alzheimer peptide should be of exceptional interest. We investigated domain-selective forms of ACE in HPLC-monitored peptide degradation studies and used mass spectrometry for product analyses. We found that ACE-evoked a hydrolysis of the N-terminal part of m- and hAβ. The hAβ sequence hAβ (4–15) was found to be a better substrate for ACE compared to the corresponding murine form. Moreover, we localized the corresponding cleavage sites in the N-terminal part of Aβ as well as in the full-length molecule and identified new sites of endopeptidolytic attack by ACE. Finally, we demonstrate that both catalytic domains of mACE have similar hydrolytic activity on the N-terminal part of Aβ. Our results show that ACE besides its typical function as a dipeptidyl-carboxypeptidase has also unequivocal endopeptidolytic activities. [Copyright &y& Elsevier]

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of -secretory APP increased, soluble β-cleaved APP levels decreased. Because Aβ40 and Aβ42 decreased to similar levels as soluble β-cleaved APP, we conclude either that β-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains. [ABSTRACT FROM AUTHOR]

Mutant membrane proteins are frequently retained in the early secretory pathway by a quality control system, thereby causing disease. An example are mutants of the vasopressin V2 receptor (V2R) leading to nephrogenic diabetes insipidus. Transport-defective V2Rs fall into two classes: those retained exclusively in the endoplasmic reticulum (ER) and those reaching post-ER compartments such as the ER/Golgi intermediate compartment. Although numerous chemical or pharmacological chaperones that rescue the transport of ER-retained membrane proteins are known, substances acting specifically in post-ER compartments have not been described as yet. Using the L62P (ER-retained) and Y205C (reaching post-ER compartments) mutants of the V2R as a model, we show here that the cell-penetrating peptide penetratin and its synthetic analog KLAL rescue the transport of the Y205C mutant, In contrast, the location of the L62P mutant is not influenced by either peptide because the peptides are unable to enter the ER. We also show data indicating that the peptide-mediated transport rescue is associated with an increase in cytosolic Ca2+ concentrations. Thus, we describe a new class of substances influencing protein transport specifically in post-ER compartments. [ABSTRACT FROM AUTHOR]

The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93–131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations. [ABSTRACT FROM AUTHOR]

The corticotropin-releasing factor receptor type 2a (CRF2(a) receptor) belongs to the family of G protein-coupled receptors. The receptor possesses a putative N-terminal signal peptide that is believed to be cleaved-off after mediating the endoplasmic reticulum targeting/insertion process, like the corresponding sequence of the homologous CRF1 receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF2(a) receptor and show that it is surprisingly completely incapable of mediating endoplasmic reticulum targeting, despite meeting all sequence criteria for a functional signal by prediction algorithms. Moreover, it is uncleaved and forms part of the mature receptor protein. Replacement of residue Asn13 by hydrophobic or positively charged residues converts the sequence into a fully functional and cleaved signal peptide demonstrating that conventional signal peptide functions are inhibited by a single amino acid residue. Deletion of the domain leads to an increase in the amount of immature, intracellularly retained receptors demonstrating that the sequence has adopted a new function in receptor trafficking through the early secretory pathway. Taken together, our results identify a novel hydrophobic receptor domain in the family of the heptahelical G protein-coupled receptors and the first pseudo signal peptide of a eukaryotic membrane protein. Our data also show that the extreme N termini of the individual CRF receptor subtypes differ substantially. [ABSTRACT FROM AUTHOR]

The depsipeptide technique is a recently developed method for peptide synthesis which is applicable to difficult sequences when the synthetic difficulty arises because of aggregation phenomena. In the present work, application of the depsipeptide method to extremely difficult sequences has been demonstrated and a serious side reaction involving diketopiperazine formation uncovered and subsequently avoided by the appropriate use of the Bsmoc protecting group. Many other aspects of the technique have been investigated, such as the stability of the depsi units during assembly and workup procedures, the completeness of the O-acylation step, the occurrence of epimerization of the amino acid activated during O-acylation, and the nature of side products formed. In addition, the method was modified so as to allow for completely automated syntheses of long-chain depsipeptides without the need for any interruption by manual esterification procedures. Finally, the synthesis efficiency of the new depsipeptide technique was shown to be comparable to that of the well-known pseudoproline technique. [ABSTRACT FROM AUTHOR]

Interaction of natural peptide ligands with class 2 GPCRs, which are targets of biologically important hormones such as glucagon, secretin, and corticotropin-releasing factor (CRF), occurs with a common orientation, in that the ligand C-terminus binds to the extracellular receptor N-terminus, whereas the ligand N-terminus binds to the receptor juxtamembrane domain. N-Terminal truncation, by eight amino acids in the case of CRF, leads to antagonists, suggesting those residues constitute the receptor activating sequence. Here, we identified by photoaffinity cross-linking using p-benzoyl-L-phenylalanine (Bpa) analogues of urocortin (Ucn) the most affine CRF receptor agonist, interaction domains of CRF1 receptor with Bpa residues at exclusive positions. Specific cleavage patterns of the corresponding ligand-receptor complexes, obtained using several cleavage methods in combination with SDS-PAGE for fragment size determination, showed that a Bpa group located N-terminally or in position 12 binds at the second and such in position 17 or 22 at the first extracellular receptor loop. Our results indicate that the very N-terminal ligand residues (1-11), which are responsible for receptor activation, are oriented to the juxtamembrane domain by interaction of amino acid residues 12, 17, and 22. Our findings contradict a recently proposed interaction model derived from ligand interaction with a soluble receptor N-terminus, indicating that conclusions drawn from such a reduced system may be of limited value to understand the interaction with the full-length receptor. [ABSTRACT FROM AUTHOR]

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming (‘turn-and-run’). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years. [ABSTRACT FROM AUTHOR]

Abstract: N→O acyl migrations at serine or threonine residues in peptides or proteins have previously been observed upon treatment with strong acids. Here we show that the extent of such N→O shifts depends on the peptide sequence and even in the presence of moderately acidic trifluoroacetic acid, as during Fmoc or Bsmoc-based solid phase peptide synthesis, may give rise to large amounts of depsipeptide by-products. [Copyright &y& Elsevier]

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK)293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC50 values as measured by stimulation of [35S]GTPγS binding. Contrary to the Iow potency response, the high potency response was of lower GTPγS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [35S]GTPγS-bound Gαs and Gαi subunits it is concluded that the high and low potency [35S]GTPγS binding stimulation reflected coupling to Gs and Gi proteins, respectively, only Gs coupling being homologously desensitized. Immunoprecipitation of [35S]GTPγS-bound Gαq/11 revealed additional coupling to Gq/11, which also was homologously desensitized. Although Gαq/11 coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of Gi in addition to Gq/11 in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to Gs- and Gq/11-mediated signaling steps and desensitization and another leading to Gi-mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins. [ABSTRACT FROM AUTHOR]

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ETA) and endothelin B (ETB) receptors. Although both receptor subtypes are co-ex- pressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ETB- specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc- tagged ETA receptor and CFP (ETAmyc·CFP) and a fusion protein consisting of an ETB receptor and YFP (ETB·YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ETB and myc-tagged ETA receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ETAmyc·CFP and ETB·YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-l-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ETB receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A·dynamin I or incubation with 450 mM sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ETAmyc·CFP/ETBflag·YFP receptors revealed a slower sequestration of the ETBflag·YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ETBflag·YFP receptors alone. The data suggest that ETA and ETB receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ETB-selective stimulation. [ABSTRACT FROM AUTHOR]

Arginine vasopressin (AVP) increases the water permeability of renal collecting duct principal cells by inducing the fusion of vesicles containing the water channel aquaporin-2 (AQP2) with the plasma membrane (AQP2 shuttle). This event is initiated by activation of vasopressin V2 receptors, followed by an elevation of cAMP and the activation of protein kinase A (PKA). The tethering of PKA to subcellular compartments by protein kinase A anchoring proteins (AKAPs) is a prerequisite for the AQP2 shuttle. During the search for AKAP(s) involved in the shuttle, a new splice variant of AKAP18, AKAP18δ, was identified. AKAP18δ functions as an AKAP in vitro and in vivo. In the kidney, it is mainly expressed in principal cells of the inner medullary collecting duct, closely resembling the distribution of AQP2. It is present in both the soluble and particulate fractions derived from renal inner medullary tissue. Within the particulate fraction, AKAP18δ was identified on the same intracellular vesicles as AQP2 and PKAδ AVP not only recruited AQP2, but also AKAP18δ to the plasma membrane. The elevation of cAMP caused the dissociation of AKAP18δ and PKA. The data suggest that AKAP18δ is involved in the AQP2 shuttle. [ABSTRACT FROM AUTHOR]

Analyzes the solution structure and function of the 'tandem inactivation domain' of the neuronal A-type potassium channel Kv1.4 using nuclear magnetic resonance spectroscopy with patch clamping recordings in whole cell configuration and simulations of neuronal spiking behavior. Promotion of rapid inactivation of Kv1.4 crucial for the channel function in short term plasticity.

A highly efficient and simple Fmoc-based preparation of peptide αthioesters is presented. After Fmoc/t-butyl solid-phase synthesis on 2-chlorotrityl resin the C-terminal carboxylic group of the protected peptide is directly converted to the corresponding thioester. The method leads to very high yields, shows a low level of epimerization and can be easily applied also for the preparation of long peptide αthioesters as demonstrated for the 41 amino acid N-terminal fragment of pro-neuropeptide Y (proNPY 1–40). [Copyright &y& Elsevier]

The signalling pathway and the behavioural strategy underlying chemotaxis of sperm are poorly understood. We have studied the cellular events and motor responses that mediate chemotaxis of sperm from the sea urchin Arbacia punctulata. Here we show that resact, a chemoattractant peptide, initiates a rapid and transient rise in the concentration of cyclic GMP, followed by a transient influx of Ca[sup 2+]. The binding of a single resact molecule elicits a Ca[sup 2+] response, and 50-100 bound molecules saturate the response. The ability to register single molecules is reminiscent of the single-photon sensitivity of rod photoreceptors. Both resact and cyclic nucleotides cause a turn or brief tumbling in the swimming path of sperm. We conclude that a cGMP-mediated increase in the Ca[sup 2+] concentration induces the primary motor response of sperm to the chemoattractant. [ABSTRACT FROM AUTHOR]

This study documents the identity of a calcium‐ regulated membrane guanylate cyclase transduction system in the photoreceptor‐bipolar synaptic region. The guanylate cyclase is the previously characterized ROS‐GC1 from the rod outer segments and its modulator is S100β. S100β senses increments in free Ca2+ and stimulates the cyclase. Specificity of photoreceptor guanylate cyclase activation by S100β is validated by the identification of two S100β‐regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are specific for S100β and not for another regulator of ROS‐GC1, guanylate cyclase‐activating protein 1. One site comprises amino acids (aa) Gly962–Asn981, the other, aa Ile1030–Gln1041. The former represents the binding site. The latter binds S100β only marginally, yet it is critical for control of maximal cyclase activity. The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane‐bound guanylate cyclase is regulated by small Ca2+‐sensor proteins. [ABSTRACT FROM AUTHOR]