SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane–bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events

An overview of the synaptic vesicle lipid composition

This work was funded by a Ramon y Cajal grant to A.P-L. (RYC2018-023837-I) and by a grant from the National Institutes of Health to R.J. (P01 GM072694) .Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steadystate levels. The protein machineries responsible for exo-and endocytosis have been extensively investigated. In contrast, less is known about the role of lipids in synaptic transmission and how the lipid composition of SVs is affected by dynamic exo-endocytotic cycling. Here we summarize the current knowledge about the composition, organization, and function of SV membrane lipids. We also cover lipid biogenesis and maintenance during the synaptic vesicle cycle.Spanish Government RYC2018-023837-IUnited States Department of Health & Human Services National Institutes of Health (NIH) - USA P01 GM07269

Der Patentlebenszyklus: Methodische L�sungsans�tze der externen Technologieanalyse

Die Technologielebenszyklusanalyse stellt ein geeignetes Instrument f�r die Absch�tzung der Chancen und Risiken innerhalb eines Technologiefeldes und die Ermittlung der Technologieattraktivit�t dar. Durch die Operationalisierung des Technologielebenszyklus als Anzahl der Patentanmeldungen oder Patenterteilungen ber der Zeit kann das Instrumentarium patentstatistischer Analysen eine verl�ssliche Grundlage f�r die externe Technologieanalyse bilden. Die zentrale Problemstellung in der Analysepraxis ist die Abgrenzung des Technologiefeldes, die die Anwendbarkeit der Patentlebenszyklusanalyse bisher einschr�nkt. Der vorliegende Beitrag verdeutlicht am Beispiel des Herzschrittmachers, wie die Abgrenzungsprobleme des Technologiefeldes und der Lebenszyklusphasen gel�st werden k�nnen. Summary: The technology life cycle analysis is an ideal method for estimating the opportunities and threats within a technology field and for determining the attractiveness of a technology. By defining the technology life cycle as the number of patent applications or patents granted over time, the patent analysis tool provides a reliable basis for the external technology forecasting. The main issue in practical analyses is the isolation of the technology field, which limits the way in which patent life cycle analysis can be applied. By discussing the example of the cardiac pacemaker, this paper explains the manner in which a technology field can be isolated and in which the life cycle phases can be identified.Technologielebenszyklus, Patentlebenszyklus, Technologielebenszyklusanalyse, Patentlebenszyklusanalyse, Technologiefeldabgrenzung, Patentanmeldungen, Patenterteilungen

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

AbstractEnzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78ng/ml to 77pg/ml (or 74–0.1fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins

Technikkostenschätzung für die CO2-Emissionsminderung bei Pkw

TECHNIKKOSTENSCHÄTZUNG FÜR DIE CO2-EMISSIONSMINDERUNG BEI PKW Technikkostenschätzung für die CO2-Emissionsminderung bei Pkw / Herbener, Reinhard (Rights reserved) ( -

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex