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Daggers of Death Kettle Moraine SMART Team: Dams, Greg; Drachenberg, Disa; Goelz, Michael; Laux, Jacob; Kasper, David; Krause, Kris; Mayes, Melanie; Murray, Nathan; Swanson, Kaitlin; Tessman, Marsha Teachers: DeBoer, Karen; Nielsen, Peter; Plum, Stephen Mentor: Joseph Barbieri, Ph.D., Medical College of Wisconsin Abstract Clostridium botulinum, which causes Botulism, is a bacterium found in soil and in improperly processed foods. Botulism causes neuroparalytic diseases, where paralysis results in a part of the body because the nerves that supply it are diseased. Common symptoms of Botulism usually appear 12 to 36 hours after consumption and include a dry mouth, difficulty swallowing, slurred speech or difficulty speaking, muscle weakness, blurred or double vision, and drooping eyelids. Botulism works by blocking the release of neurotransmitters – chemicals which trigger neighboring nerves or activate muscle cells – through the action of Clostridal Neurotoxins (CNTs) that break down soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are essential for fusion of the vesicle carrying the neurotransmitters with the cell membrane, thus releasing the neurotransmitters. If the neurotransmitters are not released, communication between nerve and muscle cells is halted, thus leading to paralysis. Botulinum Toxins (BoNTs) are composed of three domains: receptor, translocation, and catalytic. The receptor domain of BoNTs binds to receptors in the surface of neurons and enters the neuron by receptor-mediated endocytosis. Once inside the neuron, the catalytic domain is translocated across the membrane of the vesicle by the translocation domain, into the cytosol, where the catalytic domain cleaves SNARE proteins. This blocks the release of neurotransmitters and leads to paralysis. Molecular Structure of Botulism Serotype-A Catalytic Botulinum Neurotoxin serotype A Receptor Binding Catalytic PBD:3BTA SNAP25’s Role in Vesicle Fusion In a normal neuron, SNAP25 fuses with the v-SNARE protein to allow the release of a neurotransmitter into the synapse. The muscle cell can then respond to stimuli. Introduction (Rossetto O, Montecucco, 2003) Translocation (Daggers of Death) How BOTOX® cleaves SNAP25 SNAP25 is… •One of three SNARE proteins •Cleaved by BoNT/A at specific sites called exosites •Defined as SyNaptosome-Associated Protein-25 kiloDaltons BoNT/A Avian Botulism: SNAP25 Ducks with flaccid paralysis in the neck (Leighton, 2000) Blepharospasm: (uncontrolled blinking) Before and After Botox Treatment (Burns, 1986) •Clostridium botulinum, a bacteria found in the soil, produces a toxin which targets neurons, causing paralysis. •This toxin cleaves SNARE proteins, resulting in inhibited vesicle fusion (the muscle can no longer communicate with the nerve). •Botulism is the most toxic protein to humans! •However, Botulism is also the most commonly used protein in medicine. •The Botulinum Neurotoxin has three domains: Receptor Binding, Catalytic, and Translocation. •There are seven serotypes of Botulism Neurotoxin (A-G). Serotype A is used in BOTOX®. (Rossetto O, Montecucco,2003) When the botulinum toxin is present, the light chain (the catalytic domain) cleaves SNAP25 and SNAP 25 can no longer fuse with the v-SNARE protien; therefore, the neurotransmitters cannot be released, and the muscle cell cannot respond. In our model, there are mutations at two amino acid residues on the light chain: E224Q and Y366F (orange). Scientists did this in order to visualize the interactions between BoNT/A and Snap25. This mutated light chain bound, but did not cleave, SNAP25. Physical model of catalytic domain of BoNT/A complexed with SNAP25 a) SNAP25 is attached to a presynaptic membrane b) Binding of BoNT/A with SNAP25 Conclusion Botulism causes (limp) paralysis by blocking the release of neurotransmitters. •It breaks down SNARE proteins responsible for transmittance between nerve and muscle cells. •There are seven serotypes of the botulism neurotoxin: A-G •BoNT/A (the serotype used in BOTOX®) can be used to treat muscle spasms. This treatment results in normal control. •The SNAP25 complex in the neurotoxin breaks the SNARE protein •When BoNT/A is mutated at two specific places it becomes ineffective as a cleaver of the SNARE protein, even though it binds Because the protein binds at exosites, the mutated version could help scientists design specific inhibitors. c) Complex leading to cleavage of SNAP25 Therapeutic Uses Botulinum Endotoxin (BOTOX®) has been approved by the Food and Drug Administration to treat: Strabismus (lazy eye), blepharospasm (uncontrolled blinking), cervical dystonia (involuntary muscle contractions in the neck), focal dystonia (the misfiring of neurons resulting in an undesirable muscle contractions), and severe primary axillary hyperhidrosis (excessive underarm sweating). BOTOX® is used for cosmetic treatments of wrinkles and frown lines. In the future, BOTOX® may be useful for treating tinnitus (ringing in the ears), urinary incontinence, and excessive scarring. References Breidenbach, M, and Brunger, A. Substrate recognition strategy for botulinum neurotoxin serotype A. Nature 432(2004): 925-929. SNAP25-LC Schematic: Physical model of catalytic domain complexed with SNAP25 coordinates from PDB 1XTG Burns, CL, Gammon, JA, and Gemmill, MC. 1986. Ptosis associated with botulinum toxin treatment of strabismus and blepharospasm. Ophthalmology. 1986. 93(12):16217. Early description of the therapeutic use of BoNT Gomersall, Gomersall. (2008). Syringes. Retrieved February 5, 2008, from http://www.aic.cuhk.edu.hk/web8/Syringes.html Leighton, F.A. 2000. Signs of Disease in Affected Birds. In Type C Botulism in Birds. Retrieved February 5, 2008, from http://wildlife1.usask.ca/wildlife_health_topics/botulism/botulismc.php Rossetto O, Montecucco C. Chapter 2. How botulinum toxins work. In: Moore P, Naumann M, editors. Handbook of Botulinum Toxin Treatment. 2nd Ed. Blackwell Science 2003. (Gomersall, 2008) A SMART Team project supported by the National Institutes of Health (NIH) – National Center for Research Resources Science Education Partnership Award (NCRR-SEPA)