PubMed Health⌕ Search

Biomedical subjects

F Hucho

Publications and source records attributed to F Hucho.

At least 19 recordsLinked to original sources
PubMed2006

Intracellular domain of nicotinic acetylcholine receptor: the importance of being unfolded.

Bioinformatics methods with subsequent verification by experimental data were applied to the structural investigation of the intracellular loop of the delta-subunit of the nicotinic acetylcholine receptor (nAChR). Three complementary methods were used: prediction of secondary structure elements, prediction of ordered/disordered protein regions and prediction of short functional binding motifs. The output of five different algorithms was used for the secondary structure construction. Most of the intracellular domain is predicted to be unfolded. The predictions correlate well with the experimental data of limited proteolysis and NMR performed on the mostly monomeric fraction of heterologously expressed Torpedo intracellular domain protein. Twelve functional binding motifs within the disordered regions of the nAChR intracellular domain are predicted. Identification of proteins that interact with the intracellular domain will provide a better understanding of protein-protein interactions involved in nAChR assembly, trafficking and clustering.

Amino Acid Sequence↗
PubMed2005

Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1.

The transmission of pain signalling involves the cytoskeleton, but mechanistically this is poorly understood. We recently demonstrated that the capsaicin receptor TRPV1, a non-selective cation channel expressed by nociceptors that is capable of detecting multiple pain-producing stimuli, directly interacts with the tubulin cytoskeleton. We hypothesized that the tubulin cytoskeleton is a downstream effector of TRPV1 activation. Here we show that activation of TRPV1 results in the rapid disassembly of microtubules, but not of the actin or neurofilament cytoskeletons. TRPV1 activation mainly affects dynamic microtubules that contain tyrosinated tubulins, whereas stable microtubules are apparently unaffected. The C-terminal fragment of TRPV1 exerts a stabilizing effect on microtubules when over-expressed in F11 cells. These findings suggest that TRPV1 activation may contribute to cytoskeleton remodelling and so influence nociception.

Actins↗
PubMed2004

Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications.

This review covers recent data on interactions of nicotinic acetylcholine receptors (AChR) with snake venom proteins (alpha- and kappa-neurotoxins, 'weak' toxins recently shown to act on AChRs), as well as with peptide alpha-conotoxins from Conus snails. Mutations of AChRs and toxins, X-ray/nuclear magnetic resonance structures of alpha-neurotoxin bound to AChR fragments, and the X-ray structure of the acetylcholine-binding protein were used by several groups to build models for the alpha-neurotoxin-AChR complexes. Application of snake toxins and alpha-conotoxins for pharmacological distinction of muscle, neuronal and neuronal-like AChR subtypes and for other medical purposes is briefly discussed.

Animals↗
PubMed2004

Identification and characterization of a Ca2+ -sensitive interaction of the vanilloid receptor TRPV1 with tubulin.

The vanilloid receptor TRPV1 plays a well-established functional role in the detection of a range of chemical and thermal noxious stimuli, such as those associated with tissue inflammation and the resulting pain. TRPV1 activation results in membrane depolarization, but may also trigger intracellular Ca2+ -signalling events. In a proteomic screen for proteins associated with the C-terminal sequence of TRPV1, we identified beta-tubulin as a specific TRPV1-interacting protein. We demonstrate that the TRPV1 C-terminal tail is capable of binding tubulin dimers, as well as of binding polymerized microtubules. The interaction is Ca2+ -sensitive, and affects microtubule properties, such as microtubule sensitivity towards low temperatures and nocodazole. Our data thus provide compelling evidence for the interaction of TRPV1 with the cytoskeleton. The Ca2+ -sensitivity of this interaction suggests that the microtubule cytoskeleton at the cell membrane may be a downstream effector of TRPV1 activation.

Animals↗
PubMed2001

Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear membrane.

The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.

Animals↗
PubMed2001

First tryptophan-containing weak neurotoxin from cobra venom.

With the purpose of studying structure-function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated alpha-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.

Amino Acid Sequence↗
PubMed2001

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.

Animals↗
PubMed2001

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line.

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.

Animals↗
PubMed2000

Location of the polyamine binding site in the vestibule of the nicotinic acetylcholine receptor ion channel.

To map the structure of a ligand-gated ion channel, we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, (125)I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies, we conclude that the site of (125)I-MR44 cross-linking is contained in the sequence alpha His-186 to alpha Leu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of (125)I-MR44. The site where the hydrophobic head group of (125)I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nicotinic acetylcholine receptor. In binding and photocross-linking experiments, the luminal noncompetitive inhibitors ethidium and triphenylmethylphosphonium were found to compete with (125)I-MR44. We conclude that the polyamine moiety of (125)I-MR44 interacts with the high affinity noncompetitive inhibitor site deep in the channel of the nicotinic acetylcholine receptor, while the aromatic ring of this compound binds in the upper part of the ion channel (i.e. in the vestibule) to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by (125)I-MR44 should therefore be accessible from the luminal side of the vestibule.

Binding Sites↗
PubMed2000

CREB-binding protein/p300 activates MyoD by acetylation.

The myogenic protein MyoD requires two nuclear histone acetyltransferases, CREB-binding protein (CBP)/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells. In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle-specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo.

Acetylation↗
PubMed2000

Binding properties of agonists and antagonists to distinct allosteric states of the nicotinic acetylcholine receptor are incompatible with a concerted model.

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.

Acetylcholine↗
PubMed2000

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.

Journal Article↗
PubMed2000

Analysis of the subcellular distribution of protein kinase Calpha using PKC-GFP fusion proteins.

One important factor for the determination of the specific functions of protein kinase C (PKC) isoforms is their specific subcellular localization. In NIH 3T3 fibroblasts phorbol esters induce translocation of PKCalpha to the plasma membrane and the nucleus. In order to investigate PKCalpha's subcellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKCalpha and the green fluorescent protein (GFP). Purified GFP-PKCalpha from baculovirus-infected insect cells undergoes nuclear accumulation without any further stimuli in digitonin-permeabilized cells. Interestingly, permeabilization appears to be a trigger for PKCalpha's nuclear translocation, since the fusion protein also translocates to the nucleus in transiently transfected cells following permeabilization. This suggests that PKCalpha has a high nuclear binding capacity even in the case of large protein amounts. In contrast to endogenous PKCalpha, overexpressed GFP-PKCalpha as well as overexpressed PKCalpha itself translocates mainly to the plasma membrane and only to a smaller extent to the nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKCalpha enabled determination of motifs involved PKCalpha's subcellular distribution: A25E and K368R point mutations of PKCalpha showed enhanced affinity for the plasma membrane, whereas sequences within the regulatory domain probably confer PKCalpha's nuclear accumulation.

3T3 Cells↗
PubMed2000

Muscarinic toxin-like proteins from cobra venom.

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.

Amino Acid Sequence↗
PubMed2000

Structure-activity relationship and site of binding of polyamine derivatives at the nicotinic acetylcholine receptor.

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues. The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel conformation resulting in labeling yields for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively. Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high-affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.

Binding Sites↗