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Juan Blasi

Bio: Juan Blasi is an academic researcher from University of Barcelona. The author has contributed to research in topics: Synaptic vesicle & Synaptobrevin. The author has an hindex of 31, co-authored 85 publications receiving 5025 citations. Previous affiliations of Juan Blasi include University of Salamanca & Bellvitge University Hospital.


Papers
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Journal ArticleDOI
09 Sep 1993-Nature
TL;DR: It is demonstrated that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.
Abstract: Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.

1,171 citations

Journal ArticleDOI
TL;DR: It is concluded that HPC‐1/syntaxin, a membrane protein present in axonal and synaptic membranes, is involved in exocytotic membrane fusion.
Abstract: The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

541 citations

Journal ArticleDOI
TL;DR: BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli, and 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.

432 citations

Journal ArticleDOI
TL;DR: It is demonstrated that major pools of syntaxin 1 and SNAP-25 recycle with SVs, concluding that t- SNAREs participate in SV recycling in what may be functionally distinct forms.
Abstract: Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.

339 citations

Journal ArticleDOI
TL;DR: Tetanus toxin and botulinal toxins are potent inhibitors of neuronal exocytosis and could serve in the future as tools to study membrane trafficking events, or even higher brain functions such as behaviour and learning.

320 citations


Cited by
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Journal ArticleDOI
TL;DR: A fascinating picture of these robust nanomachines is emerging, which seems to be conserved and adaptable for fusion reactions as diverse as those involved in cell growth, membrane repair, cytokinesis and synaptic transmission.
Abstract: Since the discovery of SNARE proteins in the late 1980s, SNAREs have been recognized as key components of protein complexes that drive membrane fusion. Despite considerable sequence divergence among SNARE proteins, their mechanism seems to be conserved and is adaptable for fusion reactions as diverse as those involved in cell growth, membrane repair, cytokinesis and synaptic transmission. A fascinating picture of these robust nanomachines is emerging.

2,424 citations

Journal ArticleDOI
20 Mar 1998-Cell
TL;DR: Recombinant v- and t- SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes, leading to spontaneous fusion of the docked membranes at physiological temperature.

2,374 citations

Journal ArticleDOI
17 Nov 2006-Cell
TL;DR: A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins and, with the exception of the V-ATPase, contains numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.

2,030 citations

Journal ArticleDOI
05 Nov 1993-Cell
TL;DR: It is reported that in the absence of SNAP and NSF, these three SNAREs form a stable complex that can also bind synaptotagmin, suggesting that synapttagmin operates as a "clamp" to prevent fusion from proceeding in the absent of a signal.

1,873 citations

Journal ArticleDOI
23 Jan 2009-Science
TL;DR: The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion and are spectacularly apparent in the exquisite speed and precision of synaptic exocytosis.
Abstract: The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.

1,862 citations