A synthetic organelle approach to probe SNARE-mediated membrane fusion in a bacterial host.

In vivo and in vitro assays, particularly reconstitution using artificial membranes, have established the role of synaptic soluble N-Ethylmaleimide-sensitive attachment protein receptors (SNAREs) VAMP2, Syntaxin-1A, and SNAP-25 in membrane fusion. However, using artificial membranes requires challenging protein purifications that could be avoided in a cell-based assay. Here, we developed a synthetic biological approach based on the generation of membrane cisternae by the integral membrane protein Caveolin in Escherichia coli and coexpression of SNAREs.

Detection of VAMP Proteolysis by Tetanus and Botulinum Neurotoxin Type B In Vivo with a Cleavage-Specific Antibody.

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A.

Ability of human SNAP-23 to generate high molecular weight SDS-resistant ternary SNARE complexes is influenced by C-terminal coil content.

Using protein complex formation assay, ability of SNAP-25 isoforms to generate SDS-resistant ternary SNARE complexes with Syntaxin-1 and VAMP-2 was investigated. Major SNAP-25 family proteins were found to generate heat-resistant ternary complexes with varying efficiency. Compared to human SNAP-25, its non-neuronal counterparts SNAP-23 and SNAP-29 formed lower amounts of ternary complexes.

Engineering an Effective Human SNAP-23 Cleaving Botulinum Neurotoxin A Variant.

Botulinum neurotoxin (BoNT) serotype A inhibits neurotransmitter release by cleaving SNAP-25 and represents an established pharmaceutical for treating medical conditions caused by hyperactivity of cholinergic nerves. Oversecretion from non-neuronal cells is often also the cause of diseases. Notably, excessive release of inflammatory messengers is thought to contribute to diseases such as chronic obstructive pulmonary disease, asthma, diabetes etc.

Role of the Sec22b-E-Syt complex in neurite growth and ramification.

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here, we show that Sec22b interacts with members of the extended synaptotagmin (E-Syt) family of ER lipid transfer proteins (LTPs), and this interaction depends on the longin domain of Sec22b.

Developing a Comprehensive, Adaptive, and International Biosafety and Biosecurity Program for Advanced Biotechnology: The iGEM Experience.

Introduction: The international synthetic biology competition iGEM (formally known as the international Genetically Engineered Machines competition) has a dedicated biosafety and biosecurity program.

Method: A review of specific elements of the program and a series of concrete examples illustrate how experiences in implementing the program have helped improved policy, including an increasing diversity of sources for genetic parts and organisms, keeping pace with technical developments, considering pathways toward future environmental release, addressing antimicrobial resistance, and testing the efficacy of current biosecurity arrangements.

Results: iGEM's program is forward-leaning, in that it addresses both traditional (pathogen-based) and emerging risks both in terms of new technologies and new risks.

Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q⁻K Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities.

In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes.

Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA.

The extreme toxicity of botulinum neurotoxins (BoNTs) relies on their specific cleavage of SNARE proteins, which eventually leads to muscle paralysis. One newly identified mosaic toxin, BoNT/HA (aka H or FA), cleaves VAMP-2 at a unique position between residues L54 and E55, but the molecular basis underlying VAMP-2 recognition of BoNT/HA remains poorly characterized. Here, we report a ∼2.

A Cell Line for Detection of Botulinum Neurotoxin Type B.

Botulinum neurotoxins (BoNTs) type A and type B are commonly used as biopharmaceutics for neurological diseases, uniquely allowing months-long paralysis of target muscles. Their exquisite neuronal specificity is conferred by a multistep process of binding, internalization, cytosolic escape and cleavage of the neuron-specific proteins, SNAP-25 and vesicle-associated membrane proteins (VAMPs), ultimately to inhibit secretion of neurotransmitters. Currently the mouse lethality bioassay is the only available method for quality control testing of VAMP-cleaving botulinum products.

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission.

Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C S51T/R52N/N53P (BoNT/C α-51) and BoNT/C L200W/M221W/I226W (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C in vivo.

The first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain.

The genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus.

Hsp90 is involved in the entry of clostridial neurotoxins into the cytosol of nerve terminals.

Botulinum and tetanus neurotoxins are the most toxic substances known and form the growing family of clostridial neurotoxins. They are composed of a metalloprotease light chain (L), linked via a disulfide bond to a heavy chain (H). H mediates the binding to nerve terminals and the membrane translocation of L into the cytosol where their substrates, the three SNARE proteins, are localised.

Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells.

Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C.

A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route.

Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved.

Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism.

Botulinum neurotoxins (BoNTs) form a growing family of metalloproteases with a unique specificity either for VAMP, SNAP25 or syntaxin. The BoNTs are grouped in seven different serotypes indicated by letters from A to G. These neurotoxins enter the cytosol of nerve terminals via a 100 kDa chain which binds to the presynaptic membrane and assists the translocation of a 50 kDa metalloprotease chain.

Two complementary approaches for intracellular delivery of exogenous enzymes.

Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.

The thioredoxin reductase--Thioredoxin redox system cleaves the interchain disulphide bond of botulinum neurotoxins on the cytosolic surface of synaptic vesicles.

Botulinum neurotoxins (BoNTs) are Janus toxins, as they are at the same time the most deadly substances known and one of the safest drugs used in human therapy. They specifically block neurotransmission at peripheral nerves through the proteolysis of SNARE proteins, i.e.

Thioredoxin and its reductase are present on synaptic vesicles, and their inhibition prevents the paralysis induced by botulinum neurotoxins.

Botulinum neurotoxins consist of a metalloprotease linked via a conserved interchain disulfide bond to a heavy chain responsible for neurospecific binding and translocation of the enzymatic domain in the nerve terminal cytosol. The metalloprotease activity is enabled upon disulfide reduction and causes neuroparalysis by cleaving the SNARE proteins. Here, we show that the thioredoxin reductase-thioredoxin protein disulfide-reducing system is present on synaptic vesicles and that it is functional and responsible for the reduction of the interchain disulfide of botulinum neurotoxin serotypes A, C, and E.

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC).

Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells.

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal-associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA-transfection reagents.

Neutralisation of specific surface carboxylates speeds up translocation of botulinum neurotoxin type B enzymatic domain.

Botulinum neurotoxins translocate their enzymatic domain across vesicular membranes. The molecular triggers of this process are unknown. Here, we tested the possibility that this is elicited by protonation of conserved surface carboxylates.

Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons.

The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood-brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC.