SNAREs Flashcards
What membrane fusion processes happen in our bodies?
Synapatic vesicle fusion
Secretory granule fusion
Secretion of serum proteins
Mucus secretion
What are the three approaches to identifying vesicle machinery?
Biochemical reconstitution
Yeast genetics
Cloning
How is biochemical reconstitution done to find SNAREs?
Cells are broken open and put back together, reconstituting vesicles
Inhibitors and fractionation are used to identify pathways and molecules being inhibited
How is glycosylation in CHO cells followed?
CHO cells missing glycosylation had radioactive sugars added.
Enzymes catalysing glycosylation bud off golgi of another cell. The vesicles can bud and fuse to the other golgi, leading to transfer of the radioactive sugar.
How does NSF stimulate vesicle fusion?
Binds to SNAP receptors catalysed by ATP
When membranes are salt washed, NSF no longer binds to membranes
What do SEC1, 17 and 18 encode?
1- SNARE binding protein
17- α-snap
18- NSF
What does tetanus toxin cleave in protein?
VAMP
Outline the SNARE hypothesis
- Involved in each transport step within the cell
- Should provide specificity for vesicle transport
- Should be sufficient to drive lipid bilayer fusion
- NSF and ATP hydrolysis catalyse vesicle fusion
What sort of structure do SNAREs form?
A zip in a coiled coil
What is syntaxin’s function
Drives proteins closer together and allows membrane fusion
What is in the zero layer of SNAREs
Hydrophilic electrostatic interaction between 3 Gly and 1 Arg. This 3Q:1R ratio is conserved
What is the different between Q and R SNAREs?
R- Arg at 0
Q- Syntaxin 1a at 0
Describe SNAREs
Generally 14-40kDa
At least 1 coiled-coil SNARE motif
Generally C-terminally anchored
Recombinant SNAREs can drive fusion of purified liposomes
What recycles SNAREs after fusion?
NSF
Outline the steps leading to budding
Nucleation
Zippering
Fusion-pore opening
20s complex formation with SNAPs and NSF
Budding
What make up the trans SNARE?
SNAP25 and syntaxin
Why is VAMP2 expressed in high levels in the endocrine pancreas?
Absorb nutrients
What happens when VAMP2 is knocked out?
Death at birth due to loss of synaptic transmission
What happens when SNAP25 is knocked out?
Death at birth due to loss of synaptic transmission
What disease is VAMP2 associated with?
Neurodevelopment disorder. Floppy baby syndrome (Hypertonia), autistic features and can have hyperkinetic movements
What disease is SNAP25b associated with?
Neurodevelopment disorder. Seizures, speech delay and cerebellar ataxia
What disease is SNAP29 associated with?
Cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma syndrome (CEDNIK syndrome)
What disease is syntaxin 11 associated with?
Familial hemophagocytic lymphohystiocytosis
What does the S75P mutation in VAMP2 do and why?
Slows rate of liposome fusion as proline disrupts coiled coils
This was measured with a fluorescent dye on a synthetic membrane
What is lymphohistiocytosis (FHL4)?
Immune disease mainly affecting infants.
Over proliferation of T-cells, B-cells, NK cells and macrophages
Cytokine storms make this life threatening
Syntaxin 11 is mutated as well as other genes.
Why doesn’t phagocytosis take place without syntaxin11?
It makes up SNAREs which regulate phagocytosis
Why does FHL4 cause degranulation from cytotoxic T-cells?
STX11 levels are lower, preventing phagocytosis
STX11 is a Q-SNARE lacking a TM domain
What is FHL5?
Reduced STX11 levels and a mutation in Munc18-2 (a regulating protein)
How do Clostridial neurotoxins work?
Cause muscle to tense
These toxins are only up by neurons and cleave different SNAREs.
How do the different domains in clostradial neurotoxins work?
Targeting domain
Translocation domain- to get to endosome
Protease domain- cleave SNAREs
What are botulinum neurotoxins used to treat have?
Strabismus (botulinum A)
Blepharospasm
hemifacial spasm
cervical dystonia (Botulinum B)
overactive bladder
GI tract disorders
What does botulinum target?
SNAP25
