Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1′ binding subsite
TL;DR: Type A botulinum neurotoxin (botox A) displayed an unusual requirement for arginine as the P1′ inhibitor residue, demonstrating that the S1′ binding subsite of botox A is dissimilar to those of most other zinc metalloproteases.
About: This article is published in FEBS Letters.The article was published on 1998-09-11 and is currently open access. It has received 116 citations till now. The article focuses on the topics: Substrate analog & Protease.
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TL;DR: This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication, thereby achieving an exquisite toxicity.
Abstract: Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.
376 citations
TL;DR: The remarkable success of botulinum toxin as a therapeutic agent has created a new field of investigation in microbiology, and the clostridia produce more protein toxins than any other bacterial genus and are a rich reservoir of toxins for research and medicinal uses.
Abstract: Toxins are increasingly being used as valuable tools for analysis of cellular physiology, and some are used medicinally for treatment of human diseases. In particular, botulinum toxin, the most poisonous biological substance known, is used for treatment of a myriad of human neuromuscular disorders characterized by involuntary muscle contractions. Since approval of type-A botulinum toxin by the US Food and Drug Administration in December 1989 for three disorders (strabismus, blepharospasm, and hemifacial spasm), the number of indications being treated has increased greatly to include numerous focal dystonias, spasticity, tremors, cosmetic applications, migraine and tension headaches, and other maladies. Many of these diseases were previously refractory to pharmacological and surgical treatments. The remarkable therapeutic utility of botulinum toxin lies in its ability to specifically and potently inhibit involuntary muscle activity for an extended duration. The clostridia produce more protein toxins than any other bacterial genus and are a rich reservoir of toxins for research and medicinal uses. Research is underway to use clostridial toxins or toxin domains for drug delivery, prevention of food poisoning, and the treatment of cancer and other diseases. The remarkable success of botulinum toxin as a therapeutic agent has created a new field of investigation in microbiology.
192 citations
Patent•
14 Jul 2000TL;DR: In this article, a system for the rapid characterization of multi-analyte fluids, in one embodiment, including a light source, a sensor array, and a detector, is presented, where the sensor array is formed from a supporting member into which a plurality of cavities may be formed.
Abstract: A system for the rapid characterization of multi-analyte fluids, in one embodiment, includes a light source, a sensor array, and a detector. The sensor array is formed from a supporting member into which a plurality of cavities may be formed. A series of chemically sensitive particles are, in one embodiment positioned within the cavities. The particles may be configured to produce a signal when a receptor coupled to the particle interacts with the analyte. Using pattern recognition techniques, the analytes within a multi-analyte fluid may be characterized.
161 citations
TL;DR: This Review provides a broad overview of several drug discovery efforts focused on metalloenzymes and attempts to map out the current landscape of high-value metaloenzyme targets.
Abstract: Metalloenzymes are central to a wide range of essential biological activities, including nucleic acid modification, protein degradation, and many others The role of metalloenzymes in these processes also makes them central for the progression of many diseases and, as such, makes metalloenzymes attractive targets for therapeutic intervention Increasing awareness of the role metalloenzymes play in disease and their importance as a class of targets has amplified interest in the development of new strategies to develop inhibitors and ultimately useful drugs In this Review, we provide a broad overview of several drug discovery efforts focused on metalloenzymes and attempt to map out the current landscape of high-value metalloenzyme targets
154 citations
TL;DR: Recent progress in developing therapeutic countermeasures against bioterrorism and the potential use of biological weapons against both military and civilian populations is covered.
Abstract: The threat of bioterrorism and the potential use of biological weapons against both military and civilian populations has become a major concern for governments around the world. For example, in 2001 anthrax-tainted letters resulted in several deaths, caused widespread public panic and exerted a heavy economic toll. If such a small-scale act of bioterrorism could have such a huge impact, then the effects of a large-scale attack would be catastrophic. This review covers recent progress in developing therapeutic countermeasures against, and diagnostics for, such agents.
149 citations
References
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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
TL;DR: Botulinum toxin type A, one of the most lethal biologic toxins, has been found to be of therapeutic value in the treatment of a variety of neurologic and ophthalmologic disorders.
Abstract: BOTULISM had been recognized by the 18th century, but the observation that a toxin produced by an anaerobic organism might be responsible for food poisoning was not made until 1897.1 Although seven immunologically distinct toxins have since been identified, only types A, B, and E have been linked to cases of botulism in humans.2 , 3 Botulinum toxin type A (hereafter referred to as botulinum toxin), one of the most lethal biologic toxins, has been found to be of therapeutic value in the treatment of a variety of neurologic and ophthalmologic disorders.4 The Food and Drug Administration recently approved botulinum toxin (Oculinum) . . .
854 citations
TL;DR: Light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus, suggesting that Snapchat may play an important role in the synaptic function of specific neuronal systems.
Abstract: cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.
830 citations
TL;DR: Recent findings suggest that research is moving quickly toward a full determination of the cellular and subcellular actions of the clostridial toxins, and this is an encouraging tum of events.
Abstract: Botulinum toxin is a term that has been used to describe eight different substances designated types A, B, Cb C2, D, E, F, and G. For many years it was assumed that these eight substances acted at the neuromuscular junction to block acetylcholine release. It is now known that this assumption is not entirely correct. Seven Of the substances do act at cholinergic junctions (types A, B, C b D, E, F, and G), and they are properly referred to as botulinum neurotoxins. The eighth substance (type C2) is unique in its structure and pharmacological actions. It is called the botulinum binary toxin. Tetanus toxin is a term that has been used to describe a single substance that acts mainly in the central nervous system to block inhibitory transmission. There are many similarities between tetanus toxin and botulinum neurotoxin, including a common origin, a closely related structure, and perhaps the same subcellular mechanism of action. The similarities between tetanus toxin and the botulinum binary toxin are less clear. The purpose of the present review is to discuss the proposed mechanism( s) of action of the three groups of clostridial toxins. Recent findings suggest that research is moving quickly toward a full determination of the cellular and subcellular actions of these substances. This is an encouraging tum of events, because the clostridial toxins are generally regarded as the most poisonous substances known to mankind.
464 citations
TL;DR: It is shown that a larger signature comprising this sequence is common to most of the known zinc‐dependent endopeptidases, and that the presence of the signature can be indicative of membership in the family.
438 citations