Biomedical subjects

Olaf Jahn

Publications and source records attributed to Olaf Jahn.

13 recordsLinked to original sources

PubMed2006

Characterization of the Munc13-calmodulin interaction by photoaffinity labeling.

Sensing of and response to transient increases in the residual presynaptic Ca2+ levels are important adaptive mechanisms that define the short-term plasticity characteristics of neurons. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins have emerged as key regulators of these adaptive mechanisms. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+ -dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. Here, we used Munc13-derived model peptides in photoaffinity labeling (PAL) experiments to demonstrate the stoichiometric and Ca2+ -dependent CaM binding of the other members of the Munc13 family, bMunc13-2 and Munc13-3, via structurally distinct non-conserved binding sites. A PAL-based Ca2+ titration assay revealed that all Munc13 isoforms can form a complex with CaM already at low Ca2+ concentrations just above resting levels, underscoring the Ca2+ sensor/effector function of this interaction in short-term synaptic plasticity phenomena.

Amino Acid Sequence↗

PubMed2006

Technical innovations for the automated identification of gel-separated proteins by MALDI-TOF mass spectrometry.

The combination of gel-based two-dimensional protein separations with protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the workhorse for the large-scale analyses of proteomes. Such high-throughput proteomic approaches require automation of all post-separation steps and the in-gel digest of proteins especially is often the bottleneck in the protein identification workflow. With the objective of reaching the same high performance of manual low-throughput in-gel digest procedures, we have developed a novel stack-type digestion device and implemented it into a commercially available robotic liquid handling system. This modified system is capable of performing in-gel digest, extraction of proteolytic peptides, and subsequent sample preparation for MALDI-MS without any manual intervention, but with a performance at least identical to manual procedures as indicated on the basis of the sequence coverage obtained by peptide mass fingerprinting. For further refinement of the automated protein identification workflow, we have also developed a motor-operated matrix application device to reproducibly obtain homogenous matrix preparation of high quality. This matrix preparation was found to be suitable for the automated acquisition of both peptide mass fingerprint and fragment ion spectra from the same sample spot, a prerequisite for high confidence protein identifications on the basis of peptide mass and sequence information. Due to the implementation of the stack-type digestion device and the motor-operated matrix application device, the entire platform works in a reliable, cost-effective, and sensitive manner, yielding high confidence protein identifications even for samples in the concentration range of as low as 100 fmol protein per gel plug.

Automation↗

PubMed2006

Identification and characterization of a stress-inducible and a constitutive small heat-shock protein targeted to the matrix of plant peroxisomes.

Small heat-shock proteins (sHsps) are widespread molecular chaperones for which a peroxisomal localization has not yet been reported. The Arabidopsis (Arabidopsis thaliana) genome encodes two sHsps with putative peroxisomal targeting signals type 1 or 2 (PTS1 or PTS2). As demonstrated by double-labeling experiments using full-length fusion proteins with enhanced yellow fluorescent protein and deletion constructs lacking the putative targeting domains, AtHsp15.7 (At5g37670) and AtAcd31.2 (At1g06460) are targeted to the peroxisome matrix by a functional PTS1 (SKL>) and a functional PTS2 (RLx5HF), respectively. The peroxisomal localization of AtAcd31.2 was further confirmed by isolation of leaf peroxisomes from Arabidopsis by two successive sucrose density gradients, protein separation by one- and two-dimensional gel electrophoresis, and mass spectrometric protein identification. When AtHsp15.7 and AtAcd31.2 were heterologously expressed in yeast (Saccharomyces cerevisiae) and directed to the cytosol by deletion of the PTSs, both sHsps were able to complement the morphological phenotype of yeast mutants deficient in the cytosolic homologs ScHsp42 or ScHsp26. According to expression studies by reverse transcription-PCR, AtAcd31.2 is constitutively expressed, whereas AtHsp15.7 is hardly expressed under normal conditions but strongly induced by heat and oxidative stress, the latter of which was triggered by the catalase inhibitor 3-aminotriazole or the herbicide methyl viologen applied by watering of whole plants or infiltration of rosette leaves. Thus, plants are exceptional among eukaryotes in employing sHsps in the peroxisome matrix to prevent unspecific aggregation of partially denatured proteins under both physiological and stress conditions.

Amino Acid Sequence↗

PubMed2005

The role of CRF receptors in anxiety and depression: implications of the novel CRF1 agonist cortagine.

Corticotropin-releasing factor (CRF), a 41 amino acid peptide exhibits its actions through two pharmacologically distinct CRF receptor subtypes CRF(1) and CRF(2). Regulation of the relative contribution of the two CRF receptors to central CRF activity may be essential in coordinating physiological responses to stress. To facilitate the analysis of their differential involvement, we recently developed a CRF(1)-selective agonist cortagine by synthesis of chimeric peptides derived from human/rat CRF, ovine CRF, and sauvagine. Cortagine was analyzed in behavioral experiments using male wild type and CRF(2)-deficient C57BL/6J mice for its action on anxiety- and depression-like behaviors. In contrast to the current hypothesis that increased CRF(1) activity facilitates the expression of anxiety- and depression-like behavior, cortagine combines anxiogenic properties with antidepressant effects. In this article, we show that antidepressant effects are partially mediated by CRF(1) of the dorsal hippocampus. Possible pathways responsible for the paradoxical antidepressant effects observed after CRF(1) activation are discussed.

Animals↗

PubMed2005

Unchanged survival rates of 14-3-3gamma knockout mice after inoculation with pathological prion protein.

The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3gamma-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (epsilon, beta, zeta, and eta). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and alpha-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3gamma is unlikely to play a causal role in CJD and related diseases.

14-3-3 Proteins↗

PubMed2005

Corticotropin-releasing factor binding protein--a ligand trap?

The actions of the neuropeptide corticotropin-releasing factor (CRF) are modulated by a CRF binding protein (CRFBP). In view of the memory-enhancing effects of CRF, the release of endogenous CRF from CRFBP by CRFBP inhibitors has been suggested as a therapeutical strategy for the treatment of cognitive deficits. This mini-review will summarize recent advances in the field with a focus on the pharmaceutical potential of CRFBP inhibitors.

Amino Acid Sequence↗

PubMed2004

Corticotropin-releasing factor receptor 1 and central heart rate regulation in mice during expression of conditioned fear.

The present study was performed to 1) determine heart rate (HR) effects mediated through central corticotropin-releasing factor receptor subtypes 1 (CRF(1)) investigate and 2 (CRF(2)) and 2) to the contribution of endogenous CRF to baseline HR and its fear-induced adjustment in freely moving mice. CRF ligands were injected into both lateral ventricles (i.c.v.) 15 min before the presentation of a conditioned auditory fear stimulus (CS). Initial behavioral results suggest an ovine CRF (oCRF)-mediated enhanced baseline fear and mildly enhanced conditioned auditory fear. In contrast, i.c.v. injection of oCRF (35-210 ng/mouse) dose-dependently decreased baseline HR, increased HR variability, and attenuated the CS-induced tachycardia. This effect is suggested to depend on a combined activation of sympathetic and parasympathetic activity referred to as enhanced sympathovagal antagonism. An extreme bradycardia was elicited by oCRF injection into the lower brainstem. All HR effects were probably mediated by CRF(1) because injection of the CRF(2)-selective agonist mouse urocortin II was ineffective, and the baseline bradycardia by i.c.v. CRF was preserved in CRF(2)-deficient mice. Injection of various CRF receptor antagonists including the CRF(2)-selective antisauvagine-30 did not affect the conditioned HR response. This finding suggests that endogenous CRF does not contribute to the fear-mediated tachycardia. Thus, the hypothesis of an involvement of CRF in HR responses of mice to acute aversive stimulation is rejected. Pharmacological evidence points at the involvement of CRF(1) in enhanced sympathovagal antagonism, a pathological state contributing to elevated cardiac risk, whereas the physiological role of the brain CRF system in cardiovascular regulation remains to be determined.

Animals↗

PubMed2004

Calmodulin and Munc13 form a Ca2+ sensor/effector complex that controls short-term synaptic plasticity.

The efficacy of synaptic transmission between neurons can be altered transiently during neuronal network activity. This phenomenon of short-term plasticity is a key determinant of network properties; is involved in many physiological processes such as motor control, sound localization, or sensory adaptation; and is critically dependent on cytosolic [Ca2+]. However, the underlying molecular mechanisms and the identity of the Ca2+ sensor/effector complexes involved are unclear. We now identify a conserved calmodulin binding site in UNC-13/Munc13s, which are essential regulators of synaptic vesicle priming and synaptic efficacy. Ca2+ sensor/effector complexes consisting of calmodulin and Munc13s regulate synaptic vesicle priming and synaptic efficacy in response to a residual [Ca2+] signal and thus shape short-term plasticity characteristics during periods of sustained synaptic activity.

Animals↗

PubMed2004

Cortagine, a specific agonist of corticotropin-releasing factor receptor subtype 1, is anxiogenic and antidepressive in the mouse model.

Two subtypes of the corticotropin-releasing factor (CRF) receptor, CRF(1) and CRF(2), differentially modulate brain functions such as anxiety and memory. To facilitate the analysis of their differential involvement, we developed a CRF(1)-specific peptidic agonist by synthesis of chimeric peptides derived from human/rat CRF, ovine CRF (oCRF), and sauvagine (Svg). High affinity to the CRF-binding protein was prevented by introduction of glutamic acid in the binding site of the ligand. The resulting chimeric peptide, [Glu(21),Ala(40)][Svg(1-12)]x[human/rat CRF(14-30)]x[Svg(30-40)], named cortagine, was analyzed pharmacologically in cell culture by using human embryonic kidney-293 cells transfected with cDNA coding for CRF(1) or CRF(2), in autoradiographic experiments, and in behavior experiments using male C57BL/6J mice for its modulatory action on anxiety- and depression-like behaviors with the elevated plus-maze test and the forced swim test (FST), respectively. We observed that cortagine was more selective than oCRF, frequently used as CRF(1)-specific agonist, in stimulating the transfected cells to release cAMP. Cortagine's specificity was demonstrated in autoradiographic experiments by its selective binding to CRF(1) of brain sections of the mouse. After injection into the brain ventricles, it enhanced anxiety-like behavior on the elevated plus-maze at a lower dose than oCRF. Whereas at high doses, oCRF injected into the lateral intermediate septum containing predominantly CRF(2) increased anxiety-like behavior as CRF(2)-specific agonists do, cortagine did not. In contrast to its anxiogenic actions, cortagine reduced significantly the immobility time in the FST as described for antidepressive drugs. Thus, cortagine combines anxiogenic properties with antidepressive effects in the FST.

Amino Acid Sequence↗

PubMed2004

Three-amino acid motifs of urocortin II and III determine their CRF receptor subtype selectivity.

Corticotropin-releasing factor (CRF) and the CRF-like peptide urocortin I (UcnI) exert their activity through two different CRF receptors, CRF1 and CRF2. Recently, UcnII and UcnIII have been discovered as potential endogenous agonists selective for CRF2 known to be involved in brain functions such as learning and anxiety, as well as in cardiovascular functions. A structure-affinity relationship study using chimeric peptides was designed to characterize mouse UcnII (mUcnII) and mUcnIII further and to investigate the structural basis of their receptor subtype selectivity. In the framework of this study, mUcnII (IC50 = 4.4 nM) but not mUcnIII was identified as high-affinity ligand for the rat CRF binding protein. Such affinity had previously not been observed for the human version of this protein. On the basis of secondary structure predictions, it was hypothesized that the amino acid motifs Pro-Ile-Gly of mUcnII and Pro-Thr-Asn of mUcnIII decrease alpha-helicity and thereby impair binding to CRF1. In support of this hypothesis, binding affinity to CRF1 of the chimeric peptides [Pro11Ile12Gly13]h/rCRF, [Pro11Thr12Asn13]h/rCRF, and the corresponding rUcnI analogs was found to be decreased by three orders of magnitude, whereas binding affinity to CRF2 was much less affected. The dramatic decrease in binding affinity to CRF1 correlated with a decrease in alpha-helicity as indicated by the data of circular dichroism spectroscopy.

Amino Acid Sequence↗

PubMed2003

Characterization of peptide-protein interactions using photoaffinity labeling and LC/MS.

The combination of photoaffinity labeling (PAL) with modern mass spectrometric techniques is a powerful approach for the characterization of peptide-protein interactions. Depending on the analytical strategy applied, a PAL experiment can provide different levels of information ranging from the identification of interaction partners to the structural characterization of ligand-binding sites. On the basis of LC/MS data generated in the framework of the identification of the binding site of the neuropeptide corticotropin-releasing factor (CRF) on its binding protein (CRFBP), the key role of LC/MS in the characterization of photoadducts on different structural levels was demonstrated. Covalent photoadducts of rat CRFBP (rCRFBP) were obtained by PAL with different mono- and bifunctional benzophenone photoprobes designed on the basis of the sequence of the synthetic CRF fragment human/rat CRF(6-33) which binds to CRFBP with high affinity. In view of the stoichiometry, LC/MS analysis revealed that the photoadducts consisted of one molecule of photoprobe and one molecule of rCRFBP. For a further characterization of the photoadducts on the oligopeptide level, enzymatic digests of unlabeled rCRFBP and of the respective photoadduct were compared by peptide mapping monitored with LC/MS. Thereby, it was found that the photoprobe that contained the photophore at its N-terminus labeled the amino acid sequence rCRFBP(34-38), whereas the photoprobe that contained the photophore at its C-terminus labeled rCRFBP(12-26). On the basis of the characterization of the photoadduct formed by rCRFBP and the bifunctional photoprobe that contained photophores on both termini, semiquantitative comparison of different enzymatic digests was accomplished by application of the mass-selective multiple ion chromatogram strategy.

Amino Acid Sequence↗

PubMed2002

The binding protein of corticotropin-releasing factor: ligand-binding site and subunit structure.

Corticotropin-releasing factor (CRF), recognized as an important stress factor, binds to a CRF receptor and a CRF-binding protein (CRFBP) that represents a reservoir of endogenous CRF. Although CRFBP was observed to dimerize, at least in part, the ligand was found to be exclusively bound to the monomer-as indicated by photoaffinity labeling. We localized the CRF binding site by using photoaffinity labeling in combination with different mass spectrometric techniques. The amino acid residues Arg-23 and Arg-36 of CRFBP were identified as the sites of photoincorporation of monofunctional and bifunctional photoprobes designed on the basis of the amino acid sequence of human/rat CRF(6-33). It was, therefore, concluded that the sequence of amino acid residues 23-36 of CRFBP is involved in ligand binding. Our data are in support of an antiparallel alignment of the photoprobe with the amino acid residues 23-36 of the CRFBP monomer.

Amino Acid Sequence↗

PubMed2002

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands.

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor. It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.

Amino Acid Sequence↗