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Botulinum neurotoxin type A (BoNT/A) is a biopharmaceutic widely used for the treatment of neurological diseases and in aesthetic medicine to achieve months-long paralysis of target muscles and glands. Large numbers of mice are used in the mouse bioassay (MBA) for various botulinum-related applications including batch release potency testing, antitoxin testing, countermeasure development, and basic research. BoNT/A intoxication causes severe suffering to the mice used for testing, and application-specific, non-animal alternatives are urgently needed. Cell-based assays (CBA) need to replicate all the physiological steps of botulinum intoxication, comprising neuronal binding, internalization, endosomal escape, and cleavage of synaptosomal-associated protein of 25 kDa (SNAP25). However, the CBA currently in use have limitations. In this study we show that LAN5 cells, a human neuroblastoma-derived cell line, are sensitive to BoNT/A and can be engineered to express a recombinant NanoLuciferase (NanoLuc)-tagged SNAP25 reporter molecule. On exposure, the reporter molecule is cleaved and releases a NanoLuc-SNAP25 fragment that can be captured on a 96-well plate for quantitative luminometry using a cleavage-specific SNAP25 antibody. We demonstrate, using purified BoNT/A and a commercial BoNT/A product, that the sensitivity of this new cell-based assay is in the fM range, comparable to that of the MBA. The new assay could replace the MBA in research and commercial testing of BoNT/A, benefiting both scientific progress and animal welfare. [ABSTRACT FROM AUTHOR]

This review discusses the expanding application of botulinum neurotoxin in treating neurological conditions. The article specifically explores novel approaches to using non-paralytic botulinum molecules. These new molecules, such as BiTox or el-iBoNT, offer an alternative for patients who face limitations in using paralytic forms of botulinum neurotoxin due to concerns about muscle function loss. We highlight the research findings that confirm not only the effectiveness of these molecules but also their reduced paralytic effect. We also discuss a potential cause for the diminished paralytic action of these molecules, specifically changes in the spatial parameters of the new botulinum molecules. In summary, this article reviews the current research that enhances our understanding of the application of new botulinum neurotoxins in the context of common conditions and suggests new avenues for developing more efficient molecules. [ABSTRACT FROM AUTHOR]

The interactions between [alpha]-synuclein and docasahexaenoic acid (DHA) are investigated by using photophysical, biochemical and morphological methods. The results have identified a potential biological function for [alpha]-synuclein and have defined an omega-3-linked pathway leading to [alpha]-synuclein aggregation.

Author(s): Frederic Darios [1]; Bazbek Davletov (corresponding author) [1] Growth of neurite processes from the cell body is the critical step in neuronal development and involves a large increase in [...]

Botulinum neurotoxins are the most potent toxins designed by nature. They are produced by Clostridium bacteria to cause long-lasting paralysis and death. However, in the past century one member of the botulinum family--botulinum neurotoxin type A--has been put to good use, and is now widely employed in clinical neurology and, even more often, in beauty clinics. This article will critically assess the value of individual members of the botulinum family for experimental neuroscience and medicine. The seven botulinum neurotoxins all act by targeting the same vesicle fusion machinery, and yet they cause paralysis of different durations. The distinct behaviors of botulinum neurotoxins will be discussed, with the aim of highlighting advantages and limitations for their future exploitation.

Author(s): Kuang Hu; Joe Carroll; Sergei Fedorovich; Colin Rickman; Andrei Sukhodub; Bazbek Davletov (corresponding author) Release of neurotransmitter occurs when synaptic vesicles fuse with the plasma membrane. This neuronal exocytosis [...]

The C[sub.2] domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs., Author(s): Ming Zhao [1]; Daniel A. Beauregard [1]; Louiza Loizou [1]; Bazbek Davletov [2]; Kevin M. Brindle (corresponding author) [1] Apoptosis is an active process of cellular self-destruction that plays [...]

[C.sub.2] domains are found in many proteins involved in membrane traffic or signal transduction. Although [C.sub.2] domains are thought to bind calcium ions, the structural basis for calcium binding is unclear. Analysis of calcium binding to [C.sub.2] domains of synaptotagmin I and protein kinase C-β by nuclear magnetic resonance spectroscopy revealed a bipartite calcium-binding motif that involves the coordination of two calcium ions by five aspartate residues located on two separate loops. Sequence comparisons indicated that this may be a widely used calcium-binding motif, designated here as the [C.sub.2] motif., Calcium ions regulate a wide variety of biological functions through binding to proteins. Most [Ca.sup.2+]-binding proteins can be grouped into families with common structural motifs such as the EF-hand motif [...]

The three-dimensional structure of the first C2 domain of synaptotagmin I at 1.9 angstrom resolution in both Ca2+-bound and Ca2+-free forms has an eight-stranded beta-sandwich constructed around a conserved four-stranded motif called the C2 key. Synaptotagmin I is a synaptic vesicle membrane protein that functions as the Ca2+ sensor in exocytosis. It contains two C2 domains, regulatory sequence motifs.

Synaptotagmin I, an intrinsic protein of the synaptic vesicles, possesses the properties of AP-2 receptors and participates in endocytosis and exocytosis. Synaptic vesicles possess a high affinity AP-2 site, which is identical to the sites found in fibroblasts. In nerve terminals endocytosis occurs immediately after the Ca2+-induced synaptic vesicle exocytosis, identical to the receptor-mediated endocytosis in fibroblasts. The endocytosis requires clathrin coated pits. The assemblage of clathrin coated pits requires the binding of clathrin AP-2 adaptor complex to high affinity membrane receptors, the synaptotagmin I.

Abstract Background Many techniques in molecular biology, clinical diagnostics and biotechnology rely on binary affinity tags. The existing tags are based on either small molecules (e.g., biotin/streptavidin or glutathione/GST) or peptide tags (FLAG, Myc, HA, Strep-tag and His-tag). Among these, the biotin-streptavidin system is most popular due to the nearly irreversible interaction of biotin with the tetrameric protein, streptavidin. The major drawback of the stable biotin-streptavidin system, however, is that neither of the two tags can be added to a protein of interest via recombinant means (except for the Strep-tag case) leading to the requirement for chemical coupling. Results Here we report a new immobilization system which utilizes two monomeric polypeptides which self-assemble to produce non-covalent yet nearly irreversible complex which is stable in strong detergents, chaotropic agents, as well as in acids and alkali. Our system is based on the core region of the tetra-helical bundle known as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. This irreversible protein attachment system (IPAS) uses either a shortened syntaxin helix and fused SNAP25-synaptobrevin or a fused syntaxin-synaptobrevin and SNAP25 allowing a two-component system suitable for recombinant protein tagging, capture and immobilization. We also show that IPAS is suitable for use with traditional beads and chromatography, planar surfaces and Biacore, gold nanoparticles and for protein-protein interaction in solution. Conclusions IPAS offers an alternative to chemical cross-linking, streptavidin-biotin system and to traditional peptide affinity tags and can be used for a wide range of applications in nanotechnology and molecular sciences.