L14-15 SNARES Flashcards
Membrane fusion is happening in our bodies all the time!
- Synaptic vesicles fusion (communication between neurons and muscles)
- Secretory granule fusion (endocrine and exocrine pancreas)
- Secretion of serum proteins (albumin from hepatocytes and antibodies from plasma cells)
- Mucus secretion (epithelial mucosal cells)
- Intracellular transport of proteins between organelles in all of your cells
3 main approaches were taken to identify the machinery of vesicle transport
Biochemical reconstitution.
Yeast Genetics.
Cloning
Biochemical reconstitution: Intra-Golgi transport assay
Traffic from different departments and then lets them go to the golgi and then transfects them. Takes golgi from different cells. Protein trafficking studing by using radioactive sugars
The identification of NSF
N-ethylmaleimide inhibits reaction (alkalyting reagent).
Target purified and named N-ethylmaleimide Sensitive Factor (NSF 1988). NSF is an ATPase
When membranes are salt washed NSF can no longer bind to membranes. Target purified and named SNAP Soluble NSF Attachment Protein (1990).
The isolation of sec mutants (NSF and a-SNAP)
SEC1 (SNARE binding protein), SEC17 encodes -SNAP,
SEC18 encodes NSF
The cloning of synaptic vesicle proteins (VAMP and Syntaxin)
Antibodies were raised against synaptic vesicles purified from electric rays. The antibodies were then used to expression clone VAMP and Syntaxin.
Clostridial neurotoxins tetanus and boutlinum B cleave VAMP / (synaptobrevin)
SDS-PAGE gel of SSV treated with tetanus toxin
The biochemical purification of SNAREs
- Synaptosomal associated protein of 25 kD (SNAP25)
- Found they could purify a large complex that dissembles when ATP is hydrolyzed.
Rothman’s SNARE hypothesis
SNAREs for each transport step within the cell.
SNAREs should provide specificity to vesicle transport.
SNAREs should be sufficient to drive lipid bilayer fusion.
Proposed that NSF and ATP hydrolysis catalyses membrane fusion (this bit is wrong!)
Are there SNAREs for every transport step?
38 SNAREs encoded in the human genome. Involved in various transport steps and fusion reactions
Crystal structure of the neuronal SNARE complex
SNAREs zipper in a parallel coiled coil
SNARE zippering is thought to provide energy to drive membrane fusion
Bring two membranes together and fusing them is energetically unfavorable
Ratio?
- 3Q:1R ratio conserved in all complexes
- Mutation of a Q/R inhibits SNARE activity.
Do SNAREs provide the specificity of membrane fusion?
- Only get fusion with SNARE complexes which fit 3Q:1R ratio.
- SNAREs show some promiscuity but on the whole they predominantly interact with SNAREs from
the appropriate membranes. - Lots of additional machinery contribute to specificty (i.e. rabs/ coat proteins and tethers)
Common features of SNARE proteins
Generally small 14-40kDa
All have at least 1 coiled-coil or SNARE motif Generally C-terminally anchored
A large number of knock out mice have been generated to study SNARE function
Gene/ Phenotype
VAMP2
Die at birth. Loss of synaptic transmission
Syntaxin1A
No gross abnormalities. Subtitle defects in synaptic transmission
Syntaxin1B
Die after birth. Reduced synaptic transmission
SNAP25
Die at birth. Loss of synaptic transmission
Several rare human diseases are caused mutations in SNARE proteins
Gene/Disease
VAMP2
Neurodevelopmental disorder with hypotonia and autistic features with or without hyperkinetic movements
SNAP25b
Neurodevelopmental disorder with seizures, intellectual disability, severe speech delay, and cerebellar ataxia
SNAP29
Cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma syndrome (CEDNIK syndrome)
Syntaxin 11
Familial hemophagocytic lymphohistiocytosis type 4 (FHL4)
Familial hemophagocytic lymphohistiocytosis
- Rare disease of the immune system
- Predominantly effects infants
- Over proliferation of T cells, natural killer cells, B cells and macrophages
- Life threatening condition (cytokine storm)
- Caused by mutation in several different genes
- Patients can die from infection due to defective killing in T-cells
Mutation in syntaxin 11 cause FHL4
- STX11 is an unusual Q-SNARE as it does not have a trans membrane domain
- Patients with FHL4 have significantly reduced levels of STX11
- Loss of STX11 causes defective degranulation from cytotoxic T-cells by an unclear mechanism
Clostridial neurotoxins
- Clostridium tetani: tetanus (lock jaw)
- Approximately 50,000 people die from tetanus each year
- Clostridium botulinum: botulism
- Around 100-200 people get botulism each year
- Most potent biological toxins known to man: LD50 1-2ng/k
Infant botulism is the most common form of the disease
- Babies < 6 months most susceptible
- Floppy baby syndrome
Botulinum neurotoxins and there clinical uses
- Cosmetic uses
- Strabismus, blepharospasm, hemifacial spasm, cervical dystonia, axillary hyperhidrosis, over active bladder, GI tract disorders, sialorrhea, temperomandibular disorder and limb spasticity
- Most products are based around Botulinum A and target SNAP25. Treatment last for several months
- Can’t make medicines based around tetanus as everybody is vaccinated against the toxin.
