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Eberhard Krause

Publications and source records attributed to Eberhard Krause.

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

Role of lipid rafts in membrane delivery of renal epithelial Na+-K+-ATPase, thick ascending limb.

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the alpha-subunit of Na(+)-K(+)-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The beta-subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.

Animals↗
PubMed2006

A K+-selective cGMP-gated ion channel controls chemosensation of sperm.

Eggs attract sperm by chemical factors, a process called chemotaxis. Sperm from marine invertebrates use cGMP signalling to transduce incident chemoattractants into changes in the Ca2+ concentration in the flagellum, which control the swimming behaviour during chemotaxis. The signalling pathway downstream of the synthesis of cGMP by a guanylyl cyclase is ill-defined. In particular, the ion channels that are involved in Ca2+ influx and their mechanisms of gating are not known. Using rapid voltage-sensitive dyes and kinetic techniques, we record the voltage response that is evoked by the chemoattractant in sperm from the sea urchin Arbacia punctulata. We show that the chemoattractant evokes a brief hyperpolarization followed by a sustained depolarization. The hyperpolarization is caused by the opening of K+-selective cyclic-nucleotide-gated (CNG) channels in the flagellum. Ca2+ influx commences at the onset of recovery from hyperpolarization. The voltage threshold of Ca2+ entry indicates the involvement of low-voltage-activated Ca(v) channels. These results establish a model of chemosensory transduction in sperm whereby a cGMP-induced hyperpolarization opens Ca(v) channels by a 'recovery-from-inactivation' mechanism and unveil an evolutionary kinship between transduction mechanisms in sperm and photoreceptors.

Animals↗
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

Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579.

RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.

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

Quantitative proteomics analysis of detergent-resistant membranes from chemical synapses: evidence for cholesterol as spatial organizer of synaptic vesicle cycling.

Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exoendocytic cycling within the nerve terminal that at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomics analysis of detergent-resistant membranes (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography-tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exoendocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this, SV membrane and endocytic proteins displayed a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localized with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover SV proteins formed cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exoendocytic vesicle cycling at synapses.

Amino Acid Sequence↗
PubMed2006

Switch-peptides to trigger the peptide guided assembly of poly(ethylene oxide)-peptide conjugates into tape structures.

A strong beta-sheet forming peptide was conjugated to PEO and utilized to guide the structure formation process toward well-defined, tape-like structures with millimeters in length, about 2 mum width, and approximately 50 nm height. The aggregation tendency of the peptide was temporarily suppressed for ease of synthesis by the integration of multiple switch-peptide defect segments into the peptide backbone. A subsequent rearrangement in the defects re-establishes the native peptide backbone and triggers the assembly by switching the aggregation properties on.

Microscopy, Electron, Scanning↗
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

Proteomic analysis of adaptor protein 1A coats selectively assembled on liposomes.

Coat components localize to specific membrane domains, where they sort selected transmembrane proteins. To study how clathrin coats are stabilized on such domains and to identify the protein networks involved, we combined proteomic screens and in vitro liposome-based assays that recapitulate the fidelity of protein sorting in vivo. Our study identifying approximately 40 proteins on AP-1A-coated liposomes revealed that AP-1A coat assembly triggers the concomitant recruitment of Rac1, its effectors, and the Wave/Scar complex as well as that of Rab11 and Rab14. The coordinated recruitment of these different machineries requires a mosaic of membrane components comprising the GTPase ADP-ribosylation factor 1, sorting signals in selected transmembrane proteins, and phosphatidylinositol 4-phosphate. These results demonstrate that the combinatorial use of low-affinity binding sites present on the same membrane domain accounts not only for a selective coat assembly but also for the coordinated assembly of selected machineries required for actin polymerization and subsequent membrane fusion.

Actins↗
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

Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation.

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.

Animals↗
PubMed2006

Solid-phase supported polymer synthesis of sequence-defined, multifunctional poly(amidoamines).

A novel synthesis route toward multifunctional, sequence-defined polyamides is described. A fully automated, solid-phase supported polymer synthesis was developed and utilized to obtain linear poly(amidoamine) segments (PAAs) that exhibit the absence of molecular weight and chemical distribution. This was achieved by an alternating assembly of diacids and diamines, using a forced step-growth mechanism, and driving each coupling step to completion. Within the monodisperse PAA segment, functionalities can be precisely positioned along the polymer chain allowing local control of the chain properties. The versatility of the approach was demonstrated by the conjugation of the monodisperse PAA segment toward an oligopeptide, leading to a single component block copolymer as verified by mass spectrometry. Moreover, two different poly(ethylene oxide)-PAA conjugates were synthesized utilizing the direct, solid-phase supported route. By varying the PAA repeat unit, the cationic nature of the PAA segment was adjusted, demonstrating the potential of the approach. The products were characterized by means of 1H NMR and matrix-assisted laser desorption mass spectrometry (MALDI-TOF-MS) methods, which confirmed the chemical structures conclusively.

Cations↗
PubMed2006

Identification of evolutionary conserved mouse sperm surface antigens by human antisperm antibodies (ASA) from infertile patients.

PROBLEM: The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. METHODS OF STUDY: The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. RESULTS: ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. CONCLUSIONS: Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model.

Animals↗
PubMed2005

Uptake, tissue distribution and accumulation of microcystin-RR in Corydoras paleatus, Jenynsia multidentata and Odontesthes bonariensis. A field and laboratory study.

The uptake and accumulation of microcystin-RR (MC-RR) in fish was investigated under laboratory conditions and in wild fish. Jenynsia multidentata and Corydoras paleatus were exposed for 24h to 50mug/L MC-RR dissolved in water. After exposure, liver, gill, brain, intestine, gall bladder, blood and muscle were analyzed for MC-RR by HPLC and analysis confirmed by LC-ESI-TOF-MS spectrometry. Furthermore, wild individuals of Odontesthes bonariensis were sampled from the eutrophic, cyanobacteria-containing San Roque reservoir, and analyzed for the presence of MC-RR in liver, gill, intestine, and muscle. MC-RR was found in liver, gills, and muscle of all exposed and wild fish, while in C. paleatus MC-RR was also present in the intestine. Moreover, we found presence of MC-RR in brain of J. multidentata. Results indicate that MC-RR uptake might occur at two different organs: intestine and gills, through either feeding (including drinking) or respiratory activities. This suggests that MC-RR is taken into the blood stream after absorption, and distributed to different tissues. The liver showed the major bioaccumulation of MC-RR in both experimentally exposed and wild individuals, with muscle of wild fish showing relative high amounts of this toxin in comparison with those exposed in the laboratory; though MC-RR was present in muscle of fish exposed for 24h. The amount of MC-RR in muscle of O. bonariensis exceeded the value suggested by WHO to be safe, thus causing a health risk to persons consuming fish as a result of chronic exposure to microcystin. Gills also showed bioaccumulation of MC-RR, raising questions on the mechanism involved in the possible uptake of MC-RR through gills as well as on its accumulation in this organ. Although MC-LR has been reported in brain of fish, this is the first report confirming the presence of MC-RR in this organ, which means that both toxins are able to cross the blood-brain barrier. These findings also raise questions on the probable neurotoxicity of microcystins.

Animals↗
PubMed2005

Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.

Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.

Animals↗
PubMed2005

Actin remodeling requires ERM function to facilitate AQP2 apical targeting.

This study provides the first evidence that actin reorganization during AQP2 vesicular trafficking to the plasma membrane requires the functional involvement of ERM (ezrin/radixin/moesin) proteins cross-linking actin filaments with plasma membrane proteins. We report that forskolin stimulation was associated with a redistribution of moesin from intracellular sites to the cell cortex and with a concomitant enrichment of moesin in the particulate fraction in renal cells. Introduction of a peptide reproducing a short sequence of moesin within the binding site for F-actin induced all the key effects of forskolin stimulation, including a decrease in F-actin, translocation of endogenous moesin, and AQP2 translocation. A straightforward explanation for these effects is the ability of the peptide to uncouple moesin from its putative effector. This modifies the balance between the active and inactive forms of moesin. Extraction with Triton X-100, which preserves cytoskeletal associated proteins, showed that forskolin stimulation or peptide introduction reduced the amount of phophorylated moesin, a molecular modification known to stabilize moesin in an active state. Our data point to a dual role of moesin in AQP2 trafficking: it might modulate actin depolymerization and it participates in the reorganization of F-actin-containing cytoskeletal structures close to the fusion sites of the AQP2-bearing vesicles.

Actin Cytoskeleton↗