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Biomedical subjects

Michael Beyermann

Publications and source records attributed to Michael Beyermann.

At least 19 recordsLinked to original sources
PubMed2006

Mass-spectrometric identification of a novel angiotensin peptide in human plasma.

OBJECTIVE: Angiotensin peptides play a central role in cardiovascular physiology and pathology. Among these peptides, angiotensin II (Ang II) has been investigated most intensively. However, further angiotensin peptides such as Ang 1-7, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients. METHODS AND RESULTS: Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an angiotensin octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala1 instead of Asp1. Des[Asp1]-[Ala1]-Ang II, in the following named Angiotensin A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp1. Ang A has the same affinity to the AT1 receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD 123319, suggesting a lower intrinsic activity at the AT1 receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A/Ang II was higher in end-stage renal failure patients. CONCLUSIONS: Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.

Aged↗
PubMed2006

Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells.

The cAMP/protein kinase A (PKA)-dependent insertion of water channel aquaporin-2 (AQP2)-bearing vesicles into the plasma membrane in renal collecting duct principal cells (AQP2 shuttle) constitutes the molecular basis of arginine vasopressin (AVP)-regulated water reabsorption. cAMP/PKA signaling systems are compartmentalized by A kinase anchoring proteins (AKAP) that tether PKA to subcellular sites and by phosphodiesterases (PDE) that terminate PKA signaling through hydrolysis of localized cAMP. In primary cultured principal cells, AVP causes focal activation of PKA. PKA and cAMP-specific phosphodiesterase-4D (PDE4D) are located on AQP2-bearing vesicles. The selective PDE4 inhibitor rolipram increases AKAP-tethered PKA activity on AQP2-bearing vesicles and enhances the AQP2 shuttle and thereby the osmotic water permeability. AKAP18delta, which is located on AQP2-bearing vesicles, directly interacts with PDE4D and PKA. In response to AVP, PDE4D and AQP2 translocate to the plasma membrane. Here PDE4D is activated through PKA phosphorylation and reduces the osmotic water permeability. Taken together, a novel, compartmentalized, and physiologically relevant cAMP-dependent signal transduction module on AQP2-bearing vesicles, comprising anchored PDE4D, AKAP18delta, and PKA, has been identified.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

Depsipeptide methodology for solid-phase peptide synthesis: circumventing side reactions and development of an automated technique via depsidipeptide units.

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.

Amides↗
PubMed2006

The corticotropin-releasing factor receptor type 2a contains an N-terminal pseudo signal peptide.

The corticotropin-releasing factor receptor type 2a (CRF(2(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 CRF(1) receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF(2(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 Asn(13) 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.

Amino Acid Substitution↗
PubMed2006

High-affinity AKAP7delta-protein kinase A interaction yields novel protein kinase A-anchoring disruptor peptides.

PKA (protein kinase A) is tethered to subcellular compartments by direct interaction of its regulatory subunits (RI or RII) with AKAPs (A kinase-anchoring proteins). AKAPs preferentially bind RII subunits via their RII-binding domains. RII-binding domains form structurally conserved amphipathic helices with unrelated sequences. Their binding affinities for RII subunits differ greatly within the AKAP family. Amongst the AKAPs that bind RIIalpha subunits with high affinity is AKAP7delta [AKAP18delta; K(d) (equilibrium dissociation constant) value of 31 nM]. An N-terminally truncated AKAP7delta mutant binds RIIalpha subunits with higher affinity than the full-length protein presumably due to loss of an inhibitory region [Henn, Edemir, Stefan, Wiesner, Lorenz, Theilig, Schmidtt, Vossebein, Tamma, Beyermann et al. (2004) J. Biol. Chem. 279, 26654-26665]. In the present study, we demonstrate that peptides (25 amino acid residues) derived from the RII-binding domain of AKAP7delta bind RIIalpha subunits with higher affinity (K(d)=0.4+/-0.3 nM) than either full-length or N-terminally truncated AKAP7delta, or peptides derived from other RII binding domains. The AKAP7delta-derived peptides and stearate-coupled membrane-permeable mutants effectively disrupt AKAP-RII subunit interactions in vitro and in cell-based assays. Thus they are valuable novel tools for studying anchored PKA signalling. Molecular modelling indicated that the high affinity binding of the amphipathic helix, which forms the RII-binding domain of AKAP7delta, with RII subunits involves both the hydrophobic and the hydrophilic faces of the helix. Alanine scanning (25 amino acid peptides, SPOT technology, combined with RII overlay assays) of the RII binding domain revealed that hydrophobic amino acid residues form the backbone of the interaction and that hydrogen bond- and salt-bridge-forming amino acid residues increase the affinity of the interaction.

A Kinase Anchor Proteins↗
PubMed2006

N-terminal proteolysis of the endothelin B receptor abolishes its ability to induce EGF receptor transactivation and contractile protein expression in vascular smooth muscle cells.

OBJECTIVE: The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ETB receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs expressing either the full-length ETB receptor or an N-terminally truncated ETB receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ETB receptor, IRL1620 (an ETB-selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22alpha, and alpha-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of Gi proteins. In contrast, in VSMCs expressing N-terminally truncated ETB receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. CONCLUSIONS: This study is the first to show that stimulation of full-length ETB receptors promotes expression of contractile proteins and may thus participate in the differentiation of VSMCs.

Animals↗
PubMed2006

High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies.

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 (15)N and (13)C 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.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Probing the ligand-binding specificity and analyzing the folding state of SPOT-synthesized FBP28 WW domain variants.

The WW domains are known as the smallest naturally occurring, monomeric, triple-stranded, antiparallel beta-sheet domains. Hence, we chose the FBP28 WW domain as a model to investigate the stability of the beta-sheet structure at the amino acid level in the context of its function (ligand binding). The structure-function relationship was investigated through a complete substitution analysis of the FBP28 WW domain, with variants synthesized as a cellulose-bound peptide array. The functionality of the FBP28 WW domain variants was examined by probing the peptide array for ligand binding. In addition, selected FBP28 WW domain variants were investigated by CD measurements to determine the stability of the antiparallel beta-sheet structure. We discuss the correlation between structure stability and functionality for the FBP28 WW domain, as well as the effect of ligand-induced structure stabilization.

Carrier Proteins↗
PubMed2006

Evaluation of the titanium dioxide approach for MS analysis of phosphopeptides.

The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account.

Animals↗
PubMed2006

Endothelin A and endothelin B receptors differ in their ability to stimulate ERK1/2 activation.

Endothelin-1 (ET-1) acts on two different G protein-coupled receptors, namely the endothelin A (ET(A)) and the endothelin B (ET(B)) receptors. Both receptor subtypes show differences in their tissue expression and signal transduction. In the present study, we compared the ability of ET(A) and ET(B) receptors to stimulate extracellular signal-regulated kinase 1/2 (ERK1/2). In addition, we analyzed the role of the extracellular N terminus for ERK1/2 activation, because the ET(B) receptor undergoes an agonist-dependent N-terminal proteolysis. ET-1 stimulation of HEK293 cells stably expressing the ET(A) receptor induced a monophasic, but sustained ERK1/2 activation, whereas the ET(B) receptor showed a biphasic ERK1/2 activation. A truncated mutant ET(B) receptor, lacking the proteolytically cleaved N terminus (delta2-64 ET(B)) revealed only a monophasic and transient ERK1/2 activation. Treatment of HEK293 delta2-64 ET(B) cell clones with ET-1 and a synthetic NT27-64 peptide, corresponding to the N-terminally cleaved fragment of the ET(B) receptor and ET-1, did not restore the biphasic activation of ERK1/2. A chimeric ET(B) receptor in which the N terminus was replaced by the N terminus of the ET(A) receptor elicited biphasic ERK1/2 activation. The presented data suggest that an intact N terminus of the ET(B) receptor is necessary for the second phase of ERK1/2 activation. However, it appears that the length of the N terminus rather than a specific sequence motif is required for biphasic ERK1/2 activation.

Cell Line↗
PubMed2005

Photoaffinity cross-linking of the corticotropin-releasing factor receptor type 1 with photoreactive urocortin analogues.

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 CRF(1) 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.

Animals↗
PubMed2005

The signal peptide of the rat corticotropin-releasing factor receptor 1 promotes receptor expression but is not essential for establishing a functional receptor.

Approximately 5-10% of the GPCRs (G-protein-coupled receptors) contain N-terminal signal peptides that are cleaved off during receptor insertion into the ER (endoplasmic reticulum) membrane by the signal peptidases of the ER. The reason as to why only a subset of GPCRs requires these additional signal peptides is not known. We have recently shown that the signal peptide of the human ET(B)-R (endothelin B receptor) does not influence receptor expression but is necessary for the translocation of the receptor's N-tail across the ER membrane and thus for the establishment of a functional receptor [Köchl, Alken, Rutz, Krause, Oksche, Rosenthal and Schülein (2002) J. Biol. Chem. 277, 16131-16138]. In the present study, we show that the signal peptide of the rat CRF-R1 (corticotropin-releasing factor receptor 1) has a different function: a mutant of the CRF-R1 lacking the signal peptide was functional and displayed wild-type properties with respect to ligand binding and activation of adenylate cyclase. However, immunoblot analysis and confocal laser scanning microscopy revealed that the mutant receptor was expressed at 10-fold lower levels than the wild-type receptor. Northern-blot and in vitro transcription translation analyses precluded the possibility that the reduced receptor expression is due to decreased transcription or translation levels. Thus the signal peptide of the CRF-R1 promotes an early step of receptor biogenesis, such as targeting of the nascent chain to the ER membrane and/or the gating of the protein-conducting translocon of the ER membrane.

Amino Acid Sequence↗
PubMed2005

Arrestin-independent internalization and recycling of the urotensin receptor contribute to long-lasting urotensin II-mediated vasoconstriction.

Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (-log EC50, mol/L:9.0+/-0.1). A second application of UII after 30 minutes elicited a reduced contraction (36+/-4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII ((125)I-UII), approximately 70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [t(h)]: 5.6+/-0.2 minutes), but recycled quantitatively within 60 minutes (t(h) 31.9+/-2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction.

Animals↗
PubMed2005

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39).

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.

Animals↗
PubMed2005

Ca2+ spikes in the flagellum control chemotactic behavior of sperm.

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.

Animals↗
PubMed2005

Novel interaction partners of the CD2BP2-GYF domain.

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.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions.

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 Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) 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.

Amino Acid Sequence↗