Activation of recombinantly expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

Hahn K, Hertle Y, Bloess S, Kottke T, Hellweg T, Fischer von Mollard G. Activation of recombinantly expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate. Molecules. 2017;22(12): 2272.l-Amino acid oxidases (l-AAO) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS) stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site

The SNARE protein Vti1b is recruited to the sites of BCR activation but is redundant for antigen internalisation, processing and presentation

In order to fulfil the special requirements of antigen-specific activation and communication with other immune cells, B lymphocytes require finely regulated endosomal vesicle trafficking. How the endosomal machinery is regulated in B cells remains largely unexplored. In our previous proximity proteomic screen, we identified the SNARE protein Vti1b as one of the strongest candidates getting accumulated to the sites of early BCR activation. In this report, we follow up on this finding and investigate the localisation and function of Vti1b in B cells. We found that GFP-fused Vti1b was concentrated at the Golgi complex, around the MTOC, as well as in the Rab7(+) lysosomal vesicles in the cell periphery. Upon BCR activation with soluble antigen, Vti1b showed partial localization to the internalized antigen vesicles, especially in the periphery of the cell. Moreover, upon BCR activation using surface-bound antigen, Vti1b polarised to the immunological synapse, colocalising with the Golgi complex, and with lysosomes at actin foci. To test for a functional role of Vti1b in early B cell activation, we used primary B cells isolated from Vit1b-deficient mouse. However, we found no functional defects in BCR signalling, immunological synapse formation, or processing and presentation of the internalized antigen, suggesting that the loss of Vti1b in B cells could be compensated by its close homologue Vti1a or other SNAREs

The Saccharomyces cerevisiae v-SNARE Vti1p Is Required for Multiple Membrane Transport Pathways to the Vacuole

The interaction between v-SNAREs on transport vesicles and t-SNAREs on target membranes is required for membrane traffic in eukaryotic cells. Here we identify Vti1p as the first v-SNARE protein found to be required for biosynthetic traffic into the yeast vacuole, the equivalent of the mammalian lysosome. Certain vti1-ts yeast mutants are defective in alkaline phosphatase transport from the Golgi to the vacuole and in targeting of aminopeptidase I from the cytosol to the vacuole. VTI1 interacts genetically with the vacuolar t-SNARE VAM3, which is required for transport of both alkaline phosphatase and aminopeptidase I to the vacuole. The v-SNARE Nyv1p forms a SNARE complex with Vam3p in homotypic vacuolar fusion; however, we find that Nyv1p is not required for any of the three biosynthetic pathways to the vacuole. v-SNAREs were thought to ensure specificity in membrane traffic. However, Vti1p also functions in two additional membrane traffic pathways: Vti1p interacts with the t-SNAREs Pep12p in traffic from the TGN to the prevacuolar compartment and with Sed5p in retrograde traffic to the cis-Golgi. The ability of Vti1p to mediate multiple fusion steps requires additional proteins to ensure specificity in membrane traffic

The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex

Antonin W, Riedel D, Fischer von Mollard G. The SNARE Vti1a-beta is localized to small synaptic vesicles and participates in a novel SNARE complex. JOURNAL OF NEUROSCIENCE. 2000;20(15):5724-5732.Specific soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins are required for different membrane transport steps. The SNARE Vti1a has been colocalized with Golgi markers and Vti1b with Golgi and the trans-Golgi network or endosomal markers in fibroblast cell lines. Here we study the distribution of Vti1a and Vti1b in brain. Vti1b was found in synaptic vesicles but was not enriched in this organelle. A brain-specific splice variant of Vti1a was identified that had an insertion of seven amino acid residues next to the putative SNARE-interacting helix. This Vti1a-beta was enriched in small synaptic vesicles and clathrin-coated vesicles isolated from nerve terminals. Vti1a-beta also copurified with the synaptic vesicle R-SNARE synaptobrevin during immunoisolation of synaptic vesicles and endosomes. Therefore, both synaptobrevin and Vti1a-beta are integral parts of synaptic vesicles throughout their life cycle. Vti1a-beta was part of a SNARE complex in nerve terminals, which bound N-ethylmaleimide-sensitive factor and alpha-SNAP. This SNARE complex was different from the exocytic SNARE complex because Vti1a-beta was not coimmunoprecipitated with syntaxin 1 or SNAP-25. These data suggest that Vti1a-beta does not function in exocytosis but in a separate SNARE complex in a membrane fusion step during recycling or biogenesis of synaptic vesicles

A SMALL GTP-BINDING PROTEIN DISSOCIATES FROM SYNAPTIC VESICLES DURING EXOCYTOSIS

Fischer von Mollard G, SUDHOF TC, JAHN R. A SMALL GTP-BINDING PROTEIN DISSOCIATES FROM SYNAPTIC VESICLES DURING EXOCYTOSIS. NATURE. 1991;349(6304):79-81.LOW-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic1-3. In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively4-6. It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state7, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling. In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals8,9. Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis. We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2+-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation. These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane

Rational design of dinuclear complexes binding at two neighboring phosphate esters of DNA

Glaser T, Fischer von Mollard G, Anselmetti D. Rational design of dinuclear complexes binding at two neighboring phosphate esters of DNA. INORGANICA CHIMICA ACTA. 2016;452(SI):62-72.This microreview summarizes a study aiming at the development of a novel family of metal complexes that binds to the phosphate esters of the DNA backbone inspired by the cytotoxicity of the anticancer drug cisplatin and by the phosphate ester cleaving reactivity of metalloenzymes and their biomimetic model complexes. A rational design is presented that is based on the requirements to establish a molecular recognition for the phosphate esters of the DNA backbone and to suppress binding to the less exposed nucleobases. Two phosphate binding sites should be preoriented and fixed by a rigid backbone to the distance of two neighboring phosphate ester in the DNA backbone of 6-7 angstrom. Sterical demand close to the phosphate binding sites should inhibit coordination to the nucleobases. This was molecularly translated into an unprecedented family of dinuclear complexes based on 1,8-naphthalenediol ligands with sterically demanding chelate arms in 2,7-position. The synthesis of the ligand and its first dinuclear Cull complex is described. The binding of this complex to DNA has been studied by biochemical ensemble methods and biophysical single-molecule methods. The incubation of DNA with the Cull complex results in interstrand interactions forming aggregates that prevent the DNA to enter the gel in the electrophoresis experiments. AFM experiments show an increase of the DNA diameter and local entanglements of the DNA by intrastrand interactions. The stretching of a single DNA molecule by optical tweezers exhibits distinct force peaks upon treatment with the complex necessary to break the intrastrand interactions. Torsional measurements of a single DNA molecule by magnetic tweezers showed a shortening of the effective DNA length due to the intrastrand interactions. Incubation with the Cull complex suggests inhibition of DNA synthesis in polymerase chain reaction experiments and strong cytotoxicity to human HeLa cancer cells, both at lower concentration than with cisplatin. A coherent model is provided that explains all experimental observations by the intended binding of the Cull complex to two neighboring phosphates of the DNA backbone and the formation of intra- or interstrand interactions by pi-pi interactions of the outward oriented and freely exposed naphthalene rings. (C) 2016 Elsevier B.V. All rights reserved

ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome

Chidambaram S, Zimmermann J, Fischer von Mollard G. ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome. JOURNAL OF CELL SCIENCE. 2008;121(3):329-338