Henning Otto

Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors α-, β-, and γ-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant α-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in α-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.

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.

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains

The proteins synaptobrevin (VAMP), SNAP‐25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co‐reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.

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.

Heat-resistant inhibitors of protein kinase C from bovine brain

Bovine brain cytosol is shown to contain two heat-resistant inhibitors of protein kinase C, with the following characteristics: 1. One protein kinase C inhibitor can be easily purified to homogeneity. Evidence is presented that this polypeptide of Mr 19,000 is calmodulin. It inhibits protein kinase C with an EC50 of about 2.5 microM and the inhibition is Ca2+-independent. It inhibits only intact protein kinase C. Removal of the regulatory domain of protein kinase C, by limited proteolysis with trypsin, abolishes the inhibition. 2. Another protein kinase C inhibitory activity has been partially purified. Its Mr is low (Mr 600-700, as estimated by gel chromatography). It is not digested by proteases, is hydrophilic, acid- and alkali-resistant, acts Ca2+-independently, and, in contrast to calmodulin, inhibits even the catalytic fragment of protein kinase C after removal of the regulatory domain by limited proteolysis. This inhibition is, at least partially, due to a competition with ATP. Besides protein kinase C, calcium/calmodulin-dependent protein kinase II is inhibited to a similar extent. cAMP-dependent protein kinase is not affected.

Fluorescent dendrimers with a peptide cathepsin B cleavage site for drug delivery applications

The synthesis of a multifunctionally equipped first generation (G1) dendrimer carrying a pentapeptide with a cathepsin[space]B cleavage site, chelating ligands for Pt2+-complexation, and a dansyl fluorescence marker is described and an investigation of its cellular uptake as well as intracellular localization by confocal fluorescence microscopy reported.

Identification of tyrosine-phosphorylation sites in the nuclear membrane protein emerin

Although several proteins undergo tyrosine phosphorylation at the nuclear envelope, we achieved, for the first time, the identification of tyrosine‐phosphorylation sites of a nuclear‐membrane protein, emerin, by applying two mass spectrometry‐based techniques. With a multiprotease approach combined with highly specific phosphopeptide enrichment and nano liquid chromatography tandem mass spectrometry analysis, we identified three tyrosine‐phosphorylation sites, Y‐75, Y‐95, and Y‐106, in mouse emerin. Stable isotope labeling with amino acids in cell culture revealed phosphotyrosines at Y‐59, Y‐74, Y‐86, Y‐161, and Y‐167 of human emerin. The phosphorylation sites Y‐74/Y‐75 (human/mouse emerin), Y‐85/Y‐86, Y‐94/Y‐95, and Y‐105/Y‐106 are located in regions previously shown to be critical for interactions of emerin with lamin A, actin or the transcriptional regulators GCL and Btf, while the residues Y‐161 and Y‐167 are in a region linked to binding lamin‐A or actin. Tyrosine Y‐94/Y‐95 is located adjacent to a five‐residue motif in human emerin, whose deletion has been associated with X‐linked Emery–Dreifuss muscle dystrophy.

GTP-binding proteins in bovine brain nuclear membranes

Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.

A Novel Form of 6-Phosphofructokinase IDENTIFICATION AND FUNCTIONAL RELEVANCE OF A THIRD TYPE OF SUBUNIT IN PICHIA PASTORIS

Classically, 6-phosphofructokinases are homo- and hetero-oligomeric enzymes consisting of α subunits and α/β subunits, respectively. Herein, we describe a new form of 6-phosphofructokinase (Pfk) present in several Pichia species, which is composed of three different types of subunit, α, β, and γ. The sequence of the γ subunit shows no similarity to classic Pfk subunits or to other known protein sequences. In-depth structural and functional studies revealed that the γ subunit is a constitutive component of Pfk from Pichia pastoris (PpPfk). Analyses of the purified PpPfk suggest a heterododecameric assembly from the three different subunits. Accordingly, it is the largest and most complex Pfk identified yet. Although, the γ subunit is not required for enzymatic activity, the γ subunit-deficient mutant displays a decreased growth on nutrient limitation and reduced cell flocculation when compared with the P. pastoris wild-type strain. Subsequent characterization of purified Pfks from wild-type and γ subunit-deficient strains revealed that the allosteric regulation of the PpPfk by ATP, fructose 2,6-bisphosphate, and AMP is fine-tuned by the γ subunit. Therefore, we suggest that the γ subunit contributes to adaptation of P. pastoris to energy resources.

Chapter 2 Ligand-binding studies — theory and experimental techniques

Publisher Summary Binding study methods are designed to characterize the binding of a ligand to the binding site of a receptor. Binding assays provide information about the concentration of binding sites, the ligands affinity to the binding site, or the kinetics and thermodynamics of the binding process. They provide insight into intramolecular dynamics in that cooperativity in binding is revealed. Therefore, binding assays are useful to quantitate receptors and to identify and characterize either a set of ligands that bind a receptor or vice versa and the receptors that are able to bind a defined ligand. This chapter discusses the basic features of binding studies. It demonstrates the basic types of experiments and their limitations for the simplest situations and illustrates the mathematical relations, their graphical presentation, the influence of nonspecific binding, and the influence of cooperative binding. The binding of the ligand should be assigned to a defined biological effect. In a functional assay, this effect should appear at a range of the ligand concentration that is quantitatively correlated to the affinity of the ligand in a binding assay.