Download Daggers of Death

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)