Lecture 20 Snares II Flashcards
How do snares drive membrane fusion
- SNAREs form a coiled-coil
- Vamp on vesicle, Snap 25/syntaxin on target membrane are brought in close proximity
- These zipper up to drive membrane fusion of bilayers
- Release of contents e.g. NT/insulin
- NSF recycles the SNAREs (unzippering)
What happens when heat shibera mutant to 38 degrees
Become paralysed
3 types of mutants identifies when inhibit membrane fusion
- Shibire – dynamin (endocytosis disrupted)
- Comatose – NSF
- Paralytic – α-subunit of VG Na+ channel
Shibire mutant due to
dynamin
Comatose mutant due to
NSF
Paralytic mutant due to
α-subunit of VG Na+ channel
What accumulates at the restrictive temperature in comatose flies + what was concluded
Docked vesicles accumulate so concluded NSF recycles Snare molecules
How was snare function studied
KO mice
KO VAMP2 phenotype
die at birth
loss of synaptic transmission so cant breath
Synatxin1a KO phenotype
no gross abnormalities
subtle defects in synaptic transmission
Synatxin1b KO phenotype
die after birth
reduced synaptic transmission
SNAP25 KO phenotype
die at birth
loss of synaptic transmission so cant breath
How many different Snares are there
38
What still occurs in KO mice
What occurs in the KO of both isoforms of syntaxin
There is some spontaneous fusion events due to redundancy shown through the 2 isoforms of syntaxin in the brain. Yet, KO both isoforms leads to a phenotype like VAMP2 KO.
What occurs when neurons are stimulated electrically
When neurons are stimulated electrically, there is no fusion of synaptic vesicles. i.e. a redundant system is working but the electrically activated neurons do not
Can disease from SNAREs be genetic? How was this found?
Yes
Some can be inherited
Through sequencing of the genome
Disease from mutation in VAMP2
Neurodevelopmental disorder with hypotonia (floppy baby syndrome) and autistic features with or without hyperkinetic movements
Disease from mutation in SNAP25b
Neurodevelopmental disorder with seizures, intellectual diability, severe speech delay, cerebellar taxia
Disease from mutation in SNAP29
Cerebral dysgenesis
Neuropathy
Ichthyosis (scaly skin)
Palmoplantar keratoderma syndrome - thick skin
What is the difference between SNAP 25 and 29
• Snap 29 is similar to Snap 25 but is expressed in the rest of the body as well as the brain so there are developmental AND skin issues
Disease from mutation in Syntaxin 11
Familial hemophagocytic lymohohistiocytosis type 4 (FHL4)
SO expressed in the immune system
What causes serious neurodevelopmental disorders
Heterozygous mutations in VAMP2 (+/m)
Heterozygous mutations in VAMP2 (+/m):
- What did all have from birth
- type of mutation
- Need to check what
- All had hypotonia from birth.
- These are spontaneous mutations so not inherited.
- Botulinim cleaves Vamp and also leads to hypotonia (floppy baby syndrome). Checked changes were deleterious in VAMP and not just a polymorphism.
Where could the heterozygous mutations be mapped to
The SNARE domain of VAMP2
• In coiled-coil domain which is important for the zippering up of SNAREs
• These are R SNAREs
What mutation slows the rate of liposome fusion and how
S75P mutation
Proline disrupts the ability to form alphas helices and so coiled-oil structures so having serine to proline disrupts coiled coil so rate of fusion is significantly inhibited
Is Vamp 2 mutation inherited
No
How common are human diseases from snare proteins
very rare
What type of mutation is S75P
Dominant negative mutation
In assay used 50% of WT and 50% mutant – interferes with WT copy to fuse i.e. dominant negative
What disease is caused by mutation in syn11/munc 18-2
Familial hemophagocytic lymphohistiocytosis
How common is Familial hemophagocytic lymphohistiocytosis
very rare disease of immune system
Familial hemophagocytic lymphohistiocytosis affects predominantly
infants
Familial hemophagocytic lymphohistiocytosis is from the overproliferation of…
T cells, natural killer cells, B cells and macrophages
Symptoms of Familial hemophagocytic lymphohistiocytosis
death due to cytokine storm
death due to defective killing in T cells
How do T cells kill infected cells
secreting cytotoxic granules
that contain perforin to perforate cancer or infected cancer cells
What structure is formed between cancer cells and T cells
Immunological synapse so granules will polarise and fuse driven by Snares and syntaxin 11
What causes FHL4 and what do patients with this display
mutation in syntaxin 11
display reduced levels of syntaxin 11
Why is syntaxin 11 unusual
• STX11 is an unusual Q-SNARE as it does not have a TM domain (but instead a lipid anchor)
What does loss of syntaxin 11 lead to
• Loss of STX11 causes defective degranulation from cytotoxic T-cells by an unclear mechanism and so leads to cancer
What does a mutation in Munc18-2 cause
FHL5 and reduces the levels of STX11
What occurs as the ratio of cancer cells to T cells is increased
• As the ratio of cancer cells to T cells is increased, the ability of T cells to kill is compromised
Give 2 examples of Clostridial neurotoxins:
- Clostridium tetani: Tetanus
* Clostridium botulinum: Botulism
Clostridium tetani: Tetanus causes and number of deaths
- Causes paralysis
- Vaccinations have reduced deaths globally from 300,000 to 50,000 die a year
Clostridium botulinum: Botulism is due to, most common in, leads to, number infected
- Anaerobic bacteria – spores – canned food - if don’t heat for long enough
- Infant botulism is the most common form of this disease, specifically babies under the age of 6 months
- Leads to floppy baby syndrome i.e. weak muscles
- 100-200 people a year get this (but is treatable)
Clostridium tetani: Tetanus, Clostridium botulinum: Botulism are described as
Most potent biological toxins known to man
Median lethal dose of tetanus and botulinum
1-2ng/kg
How many types of clostridial neurotoxins are there
several different types
What do clostridial neurotoxins have in common
conserved protein structure
clostridial neurotoxins protein structure
- Targeting domain – binds to neurons
- Protease – cleaves snare ONLY
- Translocation domain – allows escape
clostridial neurotoxins are what type of proteases
Toxins are zinc dependent proteases
What are clostridial neurotoxins only taken up by
Neurons
This is because the targeting domains bind to specific lipids on the surface of neurons. Also bind to neuronal R’s and so there is not paralysis in all cell types. Taken up by neurons by endocytosis. Escape out of endosome by translocation domain, toxin is reduced and it clips the SNARE molecules.
Clostridial neurotoxins cleave what
Different snares
Tetanus and Botulinum toxins have a similar … but … different neurons
mode of action
intoxicate
Tetanus and Botulinum toxins are both
potent inhibitors of SNARE function
Explain symptoms of Tetanus and Botulinum
- Botulinum toxin goes into MN and acts at NMJ so don’t get Ach release floppy paralysis as inhibiting release of ACh
- Tetanus cleaves VAMP like BoNTs/B/D/F and G - goes into endosomes, tracts up axon into cell body and transferred into inhibitory interneuron spasms as inhibiting regulation
What is botulinum used to treat
• Used to treat neurological conditions associated with neuronal hyperactivity
Uses of botulinum toxins
- Cosmetic uses - safe in small quantities and injected locally so only inhibit neurons in close proximity
- Strabismus – is eye is wrong direction, paralyses the eye to straighten it up , Blepharospasm – eye spasm, over active bladder, axillary hyperhidrosis (sweat)
Why do you have to have botox repetitively
• SNARES turn over and synapses reforms so need to get treatment periodically
What are most products based around
• Most products are based around Botulinum A and target SNAP 25
Why cant you make medicine against tetanus
• Can’t make medicine based around tetanus as everyone is vaccinated against the toxin
Value of botulinum market
$5 billion
What us botulinum A used for
• Botulinum A is used for cosmetic purposes and other indications e.g. non-surgical Strabismus – cleaves SNAP 25 proteolytically which is then degraded so you can no longer form complex so get inhibition of ability to fuse vesicles at NMJ so get paralysis of these muscles – has longer lasting action
What is botulinum B used for
• Botulinum B can be used to treat cervical dystonia – cleaves VAMP (synaptobrevin) – botulinum B heavy chain binds to synaptotagmin and light chain that is endocytosed into vesicle. The light chain translocates to the vesicular membrane to cleave synaptobrevin. This means Ach vesicles cannot fuse so muscle contraction ceases.
