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F Hucho

Publications and source records attributed to F Hucho.

At least 37 records · Page 2Linked to original sources
PubMed2000

Structural organization of nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.

Allosteric Regulation↗
PubMed1999

Design, synthesis, and biological evaluation of symmetrically and unsymmetrically substituted methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists.

The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) muscarinic acetylcholine receptors. All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M(2) and M(3) receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7-25 microM range while not showing any antagonism for muscarinic receptors up to 10 microM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12 methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M(2) receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M(2) receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the receptor.

Animals↗
PubMed1999

Identification of phosphorylation sites in native lamina-associated polypeptide 2 beta.

Lamina-associated polypeptide 2 beta (LAP 2 beta), an integral protein of the inner nuclear membrane, appears to be involved in the spatial organization of the interface between nucleoplasma, lamina, and nuclear envelope. Its ability to interact with other proteins and the structural integrity of the nuclear envelope is probably regulated by phosphorylation. Here, we report nonmitotic LAP 2 beta phosphorylation sites that are phosphorylated in the native protein when purified from nuclear envelopes of mouse neuroblastoma Neuro2a cells. Five phosphorylation sites were detected by nano-electrospray mass spectrometric analysis of tryptic LAP 2 beta peptides using parent ion scans specific for phosphopeptides. By mass spectrometric sequencing of these peptides, we identified as phosphorylated residues Thr 74, Thr 159, Ser 176, and Ser 179. Two of the phosphorylation sites, Thr 74 (within a region known to bind chromatin) and Thr 159, are part of consensus sequences of proline-directed kinases. Ser 179 is part of a consensus site for protein kinase C which is able to highly phosphorylate LAP 2 beta in vitro. Three phosphorylation sites, Thr 159, Ser 176, and Ser 179, are located within a stretch of 20 amino acids, thereby forming a highly phosphorylated protein domain which may integrate signaling by multiple protein kinases. Additionally, we identified for the first time at the protein level the LAP 2 splice variant LAP 2 epsilon in nuclear envelopes.

Amino Acid Sequence↗
PubMed1999

Butyrylcholinesterase is complexed with transferrin in chicken serum.

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.

Amino Acid Sequence↗
PubMed1999

Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

Amino Acid Sequence↗
PubMed1999

Analysis of ectatomin action on cell membranes.

Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four alpha-helix bundle. At concentrations of 0.05-0.1 microm, ectatomin forms channels in cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 x 10-7 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L-type calcium current was studied. Calcium currents (ICa) in isolated rat cardiac ventricular myocytes were measured using the whole-cell perforated patch-clamp technique. It was shown that ectatomin at concentrations of 0.01-10 nm inhibited ICa after a latency of few seconds. ICa was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of ICa by isoproterenol, an agonist of beta-adrenoreceptors, or forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol- and forskolin-sensitive components of ICa. The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 microm of forskolin, whereas subsequent isoproterenol application did not produce the same effect.

Amino Acid Sequence↗
PubMed1999

How do acetylcholine receptor ligands reach their binding sites?

The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with alpha-neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor's binding sites. Together, our data suggest (a) that alpha-neurotoxins approach their binding sites from the membrane-facing periphery of the receptor's extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.

Animals↗
PubMed1998

Downstream targets of urokinase-type plasminogen-activator-mediated signal transduction.

We have investigated cellular signalling events induced by urokinase-type plasminogen activator (uPA) independent of its proteolytic activity. Treatment of the human fibrosarcoma cell line HT 1080 with diisopropylphosphorofluoridate-inactivated uPA (Dip-F-uPA) triggers a cascade of intracellular signals which are mediated by the specific cell surface receptor for uPA (uPAR). We have found that anti-uPAR Ig precipitate the src-type protein tyrosine kinases fyn, hck and lck, which belong to a family of structurally and functionally related effectors participating in signalling from antigen and cytokine receptors. Of the three uPAR-associated kinases, only hck is activated by uPA, whereas no changes in the activities of either fyn or lck could be detected by an in vitro immune complex kinase assay. We identified p38 and extracellular-signal-regulated kinase 2 from the mitogen-activated protein kinase family as downstream components of a set of consecutive signalling molecules which teleologically alter the program of gene expression. Exposure of cells to uPA results in a significant increase in c-fos mRNA that is partially due to an elevated rate of gene transcription. Presumably, the activation of the c-fos gene leads to the subsequent formation of the transcription factor activator protein-1 (AP-1), since accumulation of c-fos mRNA is followed by induction of target genes sensitive to AP-1 such as plasminogen activator inhibitor type 2 (PAI-2). These results provide new insights into proteolysis-independent cytokine-like effects of uPA.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23.

Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.

Animals↗
PubMed1998

The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.

Amino Acid Sequence↗
PubMed1998

Reverse-phase chromatography isolation and MALDI mass spectrometry of the acetylcholine receptor subunits.

A procedure for purifying the Torpedo californica nicotinic acetylcholine receptor subunits is proposed which involves preparative SDS-PAGE followed by reverse-phase HPLC on a C4 column in an acetonitrile-isopropanol system. By this method, the alpha-subunit can be completely separated from the 43-kDa protein which migrates very close to it on SDS-PAGE, and the delta-subunit can be isolated free from the beta-subunit of Na+, K(+)-ATPase comigrating with it on SDS-PAGE. The purity of all acetylcholine receptor subunits thus obtained was verified by Edman degradation and MALDI mass-spectrometric analysis which could be performed quite easily on the HPLC-purified samples. In general, we observed a good correlation between the experimentally determined molecular masses and those calculated from the amino acid sequences and when known, posttranslational modifications (glycosylation and phosphorylation) of individual receptor subunits. Transfer of the isolated receptor subunits into 1% octyl-beta-D-glucopyranoside generates samples suitable for functional studies and enzymatic proteolysis or deglycosylation.

Amidohydrolases↗
PubMed1998

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal.

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

3T3 Cells↗
PubMed1997

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking.

Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an "unconventional allosteric protein" which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3'-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly "freezes" the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.

Acetylcholine↗
PubMed1997

Large-conductance cation channels in the envelope of nuclei from rat cerebral cortex.

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K+ is 166 pS. The channels are selective for cations with PK/PNa = 2. They are neither permeable to, nor gated by Ca2+. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles.

Animals↗
PubMed1997

The glycosylated matrix protein of Borna disease virus is a tetrameric membrane-bound viral component essential for infection.

Borna disease virus (BDV) is representative of the family of Bornaviridae in the order Mononegavirales (negative-stranded, non-segmented, enveloped RNA viruses). It is the causal agent for Borna disease, characterized as an encephalomyelitis (typical form) in a wide variety of domestic animals (from rodents to birds). Recent information shows the involvement of BDV in the pathogenesis of some human psychiatric disorders. The 8.9-kb viral antigenome codes for five major ORF. The third ORF codes for a 16-kDa protein (matrix protein) that is posttranslationally modified, yielding an N-linked glycoprotein. Our data show that the glycosylated matrix protein exists as a stable tetrameric structure detectable either by electrospray ionization or matrix-assisted laser-desorption ionization mass spectrometry. Under native conditions, the tetramer, with a relative molecular mass of 68 kDa, was isolated from a sediment-free brain suspension of a BDV-infected horse. The 68-kDa entity is stable in the presence of ionic and nonionic detergents but dissociates into subunits when heated. We found that the tetrameric matrix protein inhibits in vitro BDV infection in a dose-dependent manner. In contrast to inhibition of BDV infection with hydrophobic carbohydrate derivatives and protein-bound glycoconjugates, the glycosylated matrix protein is a very potent inhibitor of BDV infection, indicating that this protein represents an essential virus-specific membrane component for viral attachment.

Animals↗
PubMed1997

Nuclear localization of protein kinase C alpha and its association with nuclear components in neuro-2a neuroblastoma cells.

Using activity measurements and Western blotting, we demonstrated that PKC alpha is constitutively present in nuclei of Neuro-2a neuroblastoma cells. Confocal microscopy revealed that PKC alpha is present in the nucleoplasm and that this localization does not change after stimulation with phorbol ester. However, as revealed by extraction experiments, phorbol ester leads to a firmer association of PKC alpha with nuclear components. Our findings suggest that PKC alpha not only associates with lipids but also with proteins inside the nucleus. The presence of active PKC alpha inside the nucleus allows the enzyme to phosphorylate not only proteins at the nuclear envelope but also proteins in the nucleoplasm.

Animals↗
PubMed1997

Interactions of the nicotinic acetylcholine receptor transmembrane segments with the lipid bilayer in native receptor-rich membranes.

Proper ion channel function of the nicotinic acetylcholine receptor (nAChR) requires the interaction of the protein with distinct lipid species present in the receptor's membrane microenvironment. Two classes of lipid binding sites present at the protein-membrane interface have been postulated: annular binding sites primarily occupied by phospholipids and non-annular binding sites mainly occupied by cholesterol [Jones & McNamee (1988) Biochemistry 27, 2364-2374]. We investigated the binding of these lipids to the nAChR and potential dynamics of these interactions during events associated with signal transduction by electron spin resonance spectroscopy (ESR) using spin-labeled analogues of phospholipids, androstane, and stearic acid. Protein-lipid interactions were characterized in receptor-rich membranes prepared from Torpedo californica electric tissue preserving the native lipid environment of the nAChR. We found a strong preference of the receptor for the phosphatidylserine (PS) analogue as compared to the other probes. Up to 57% of PS were perturbed by the membrane protein, while the fraction of motionally restricted lipid for the other analogues was on the order of 30%. After removal of the extramembrane portions of the membrane-bound receptor, we observed a loss of binding sites for the spin-labeled analogue of androstane and for stearic acid, but not for phospholipids and sphingomyelin analogues. Our results demonstrate the existence of topologically distinct lipid binding sites for different lipid species. In the case of cholesterol, extramembrane portions of the receptor are involved, whereas the transmembrane segments meet the requirements for the binding of phospholipids. Tyrosine phosphorylation of the nAChR did not affect protein-lipid interactions in samples of intact nAChR. Similarly, no significant changes were observed in the presence of carbamoylcholine at concentrations that caused rapid and quantitative desensitization of the nAChR.

Animals↗