Molecular cellular biology > SNAREs > Flashcards

SNAREs Flashcards

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1
Q

in what roles is membrane fusion important?

A
  • synaptic vesicle fusion - communication between neurons and tissues
  • secretory granule fusion - endocrine and exocrine pancreas
  • secretion of serum proteins e.g. antibodies from plasma cells
  • mucus secretion from epithelial mucosal cells
  • intracellular transport of proteins between organelles
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2
Q

how were secretory vesicles discovered?

A

via electron microscopy in 1938
- synaptic zymogen granules disappeared when synapses were stimulated

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3
Q

what were the 3 ways in which the molecular machinery of vesicle fusion was discovered?

A
  1. biochemical reconstitution
  2. yeast genetics
  3. cloning

interaction of these 3 methods allowed identification of SNAREs which drive membrane fusion

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4
Q

what is biochemical reconstitution? what inhibitor was identified?

A
  • apply radioactive label to cells to target Golgi membranes
  • as budding events occur and more golgi vesicles are formed, radioactivity is increased
  • add inhibitors, fractionate the cytosol and identify factors which are necessary for this process to occur
  • N-ethylmaleimide was shown to inhibit the Golgi budding process, as radioactivity decreased
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5
Q

how was NSF identified? what are SNAPs?

A
  • the target of N-ethylmaleimide was purified and named the N-ethylmaleimide sensitive factor (NSF)
  • NSF is an ATPase - key in driving membrane fusion
  • when membranes are salt-washed, NSF can no longer bind to membranes - the protein on the membrane was purified and named SNAP soluble NSF attachment protein
  • SNAPs bind to membranes via hypothetical proteins called SNAP receptors (SNAREs)
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6
Q

how do NSF and SNAP interact?

A

NSF binds to SNAP molecules and cycles on and off in an ATP-dependent manner
- cycle of ATP hydrolysis
- addition of ATP inhibitors would kill this reaction

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7
Q

what is the 20S complex?

A

a purified complex of NSF, SNAP and a receptor
- it was hypothesised that this complex was key for membrane fusion

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8
Q

what secretory genes did Scheckman identify using yeast genetics?

A

he isolated sec mutants by inhibiting membrane trafficking:
- sec1 - SNARE binding protein
- sec17 - encodes alpha-SNAP
- sec18 - encodes NSF (it has sequence homology to NSF)

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9
Q

How were VAMP and syntaxin discovered?

A

by cloning synaptic vesicle proteins:
- antbodies were raised against synaptic vesicles purified from pacific electric rays
- the antibobies were then used to expression clone VAMP and syntaxin

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10
Q

how was VAMP discovered to be involved in membrane trafficking?

A

clostridial neurotoxin botox was known to inhibit vesicle fusion:
- botulinum toxin is a protease which targeted and cleaved VAMP and disrupted vesicle fusion
- therefore clear that VAMP has a role in membrane trafficking

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11
Q

how were SNAREs biochemically purified by Rothman?

A
  • scientists found that they could purify a large complex that disassembles when ATP is hydrolysed
  • NSF is the ATPase
  • NSF was added to rat brains with a tag
  • NSF was prevented from hydrolysing ATP
  • ATP is swapped out so it can be hydrolysed, and the complex was released for electrophoresis
  • produced bands which showed syntaxin B, syntaxin A, SNAP-25 and VAMP
  • they found that these were key machinery in membrane fusion
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12
Q

which key molecules were in Rothman’s SNARE hypothesis?

A
  • syntaxin = target-SNARE
  • VAMP = v-SNARE
  • syntaxin and VAMP interact with NSF and alpha-SNAP to form a large 20S complex
  • through ATP hydrolysis this complex brings the vesicle in close proximity to drive vesicle fusion
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13
Q

what was Rothman’s SNARE hypothesis?

A
  1. SNAREs for each transport step within the cell
  2. SNAREs should provide specificity for transport
  3. SNAREs should be sufficient to drive lipid bilayer fusion
  4. proposed that NSF and ATP hydrolysis catalyses membrane fusion (this is wrong)
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14
Q

How many SNAREs are there?

A
  • 38 SNAREs encoded in human genome
  • involved in various transport steps and fusion reactions
  • main prototypic SNARE: VAMP, syntaxin, SNAP25
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15
Q

what is the crystal structure of the SNARE complex and how does it funciton?

A

VAMP, syntaxin, SNAP25
- the 3 molecules have coiled-coil domains (alpha-helices wrap around each other)
- the molecules zipper up in a parallel fashion to drive vesicle and target membranes in close proximity
- this disrupts lipid bilayers and allow them to fuse
- lots of energy is needed which is provided by the zipping of molecules

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16
Q

how are SNAREs regulated?

A

SNAREs have regulatory domains to prevent overactivity
- VAMP and syntaxin have transmembrane domains
- SNAP25 has 2 coiled-coil domains
- NSF unscrews the SNARE molecules after fusion

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17
Q

how do SNAREs provide energy to drive membrane fusion?

A
  1. trans-SNARE complex: SNARE coiled-coil domains zipper up
  2. Hemi-fusion where lipid bilayers fuse together
  3. cis-SNARE complex
    - fusion pore between vesicle and target membrane to release contents
    - fusion pore enlarges for contents to enter the target membranes
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18
Q

is membrane fusion energetically favourable?

A
  • bringing 2 membranes together and fusing them is energetically unfavourable
  • NSF has to hydrolyse and recycle the SNARE complex using ATP
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19
Q

how do SNAREs interact?

A
  • interactions between alpha-helices are hydrophobic in nature
  • in the 0-layer position, there is a salt-bridge interaction between 3 glutamines and an arginine
  • R-SNARE (VAMP) on the vesicle contains arginine
  • Q-SNAREs (syntaxin and SNAP25) on the target membrane contain glutamine
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20
Q

what is the R-SNARE?

A

VAMP - has arginine at the 0-layer position

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21
Q

what are Q-SNAREs?

A

syntaxin and SNAP25: have glutamine at the 0-layer position

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22
Q

what is the glutamine to arginine ratio of SNAREs?

Study These Flashcards
A

3 glutamine: 1 arginine
(3Q:1R)

3Q-coiled domains to 1R SNARE

23
Q

why is the 3Q:1R complex important in SNAREs?

Study These Flashcards
A

this ratio is conserved in all SNARE complexes:
- mutation of glutamine or arginine inhibits SNARE activity
- if the glutamine in syntaxin is changed to arginine, it causes disruption to membrane fusion

24
Q

do SNAREs provide specificity of membrane fusion?

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A
  • only SNARE complexes that fit 3Q:1R ratio can fuse
  • SNAREs show some promiscuity but on the whole they predominantly interact with SNAREs from the appropriate membranes
  • SNAREs from ER will not interact with SNAREs at PM due to spatial separation and specificity
  • rabs, coat proteins and tethers also contribute to SNARE specificity
25
what are the common features of SNARE proteins?
- small molecules: 14-40kDa - all have at least one coiled-coil SNARE domain (alpha helix) - 4 coils are needed to drive membrane fusion - generally C-terminus is membrane-anchored (N-terminus is in cytoplasm) - R-SNARE (VAMP) is located on the vesicle - Q-SNAREs (syntaxin and SNAP25) are located on target membrane - N-terminal extension on syntaxin is a regulatory domain that can fold back and cause dissociation of SNARE complex
26
are SNAREs the minimal fusion machinery needed? can they drive membrane fusion alone?
TIRF microscopy: fluorescent dye added to lipids with SNAREs on glass slide - vesicles could fuse to the membranes with just the SNAREs HOWEVER: - SNAREs are not enough to drive FAST or SPECIFIC membrane fusion when alone
27
how is calcium important in membrane fusion?
calcium triggers fusion events: - it neutralises the charge between the vesicle and target membranes - helps to buffer electrostatic charges between membranes - synaptotagmin is a calcium sensor which manipulates the SNARE complex formation - calcium influx triggers a switch from trans- to cis-SNARE complex
28
what is the role of NSF in SNARE fusion?
NSF recycles the SNAREs after fusion: - NSF isn't required for fusion itself - NSF, VAMP, SNAP25 and syntaxin form the 20S complex, leading to ATP hydrolysis and SNARE recycling
29
how does NSF/20S complex function?
- NSF unwinds the cis-SNARE coil by binding to it, hydrolysing ATP and pulling them apart - SNAREs can then be recycled
30
what happens when you inhibit NSF in animals?
mutagenesis screens in flies showed that NSF mutation caused flies to become paralysed when in high temperatures - comatose is the mutation in NSF - shibire is the mutation of dynamin (dynamin pinches off vesicles from plasma membranes to recycle membranes) - mutations in these proteins mean that the SNAREs are maintained in the cis-SNARE complex, leading to paralysis
31
what happens at restrictive temperatures in comatose NSF mutated flies?
docked vesicles accumulate: - comatose NSF mutation at 33C causes accumulation of lots of cis-SNARE vesicles and less SNARE recycling - causes paralysis
32
what happens if VAMP2 is knocked out in mice?
If VAMP2 is knocked out, it is not compatible with life as it impairs all forms of synaptic transmission - mouse dies
33
what happens if syntaxin1a is knocked out in mice?
Syntaxin 1a mutation has some defects in transmission, but there are no gross abnormalities (subtle defects in synaptic transmission)
34
what happens if syntaxin 1b is knocked out in mice?
Syntaxin 1b mutation impairs synaptic transmission and is not compatible with life - mouse dies
35
what happens if SNAP25 is knocked out in mice?
SNAP25 mutation impairs synaptic transmission and is not compatible with life - mouse dies
36
what heterozygous diseases can be caused by mutations in SNARE proteins?
1. VAMP2: neurodevelopmental disorder with hypotonia and autistic features with or without hyperkinetic movements 2. SNAP25b: neurodevelopmental disorder with seizures, intellectual disability, severe speech delay and cerebellar ataxia 3. SNAP29: cerebral dysgenesis, neuropathy, ichthyosis and palmoplantar keratoderma syndrome (CEDNIK syndrome) - SNAP29 mainly found in other tissues like skin 4. Syntaxin 11 (Q-SNARE): familial hemophagocytic lymphohistiocytosis type 4
37
what do heterozygous mutations in VAMP2 cause?
serious neurodevelopmental disorder characterised by hypotonia present since birth, intellectual disability and autistic features - little power in muscular movements as VAMP2 cannot pass proper signals
38
why are mutations in VAMP2 so detrimental?
- coiled-coil domain have hydrophobic repeats to allow SNAREs to interact - residues in the coiled-coil domains are highly conserved, so if these are mutated in VAMP2, the SNARE complex can no longer function
39
what does the S75P mutation in VAMP2 cause? how is this observed?
liposome fusion assay: fusion of VAMP2 with syntaxin shown by fluorescent markers on liposome - wild-type VAMP2 has high fluorescence - S75P VAMP2 has lower fluorescence, indicating decreased VAMP activity in liposome fusion
40
what kind of mutation is S75P VAMP2?
it is a dominant negative mutation as it causes LOF of VAMP, where it reduces VAMPs ability to fuse
41
what does a mutation in syntaxin 11 cause?
Familial hemophagocytic lymphohistiocytosis type 4 (FHL4) - rare disease of immune system - effects infants - overproliferation of T cells, NK cells, B cells and macrophages - causes cytokine storm - patients can die from infection due to defective T cells which cannot secrete cytotoxic granules
42
what is syntaxin 11? what are its implications in FHL4?
it is a Q-SNARE without a transmembrane domain - patients with FHL4 have reduced levels of syntaxin 11 - loss of syntaxin 11 causes defective degranulation from cytotoxic T cells by an unclear mechanism
43
what mutation in syntaxin causes FHL5?
- mutation in Munc18 which is a regulatory protein of syntaxin 11 - mutation in Munc18 causes less munc18 to be available, and therefore less syntaxin 11 - therefore munc18 must be important for the stability of syntaxin 11 - mutation causes disruption to T cell activity
44
what are the 2 main clostridial neurotoxins?
Clostridium tetani - tetanus (lockjaw) Clostridium botulinum - botulism most potent biological toxins known to man
45
what is tetanus (lockjaw)?
- 50,000 people die from tetanus each year - causes intense muscle spasms - inhibits the inhibitory neurons, so there is neuronal overexcitation - vaccination is effective against tetanus
46
what is infant botulism?
- floppy baby syndrome - babies less than 6 months are most susceptible - caused by ingestion of bacteria, leading to hypotonia - respirator is needed to keep alive until the action of botulinum wears off - around 100-200 people get botulism each year
47
how do clostridial neurotoxins affect membrane fusion?
- target SNARE molecules - targeting domain binds to neurons - translocation domain enables entry into endosomes - protease domain cleaves the SNARE molecules
48
what is the mechanism of botulinum toxin?
- botulinum binds to neuron surface - endocytosis occurs to take up the toxin - translocation domain of the toxin pokes holes into the endosome, so it is released into the cytoplasm - protease domain cleaves a SNARE protein - they therefore inhibit release of neurotransmitter BontC cleaves syntaxin BontA,E,C cleaves SNAP25 BontB,D,F,G cleaves VAMP
49
which neurons do tetanus and botulinum toxins affect?
tetanus inhibits inhibitory neurons, leading to muscle spasms and overstimulation by excitatory neurons botulinum inhibits excitatory neurons, leading to low neuronal activity and paralysis
50
what clinical uses does botulinum toxin have?
- Cosmetic uses - Strabismus, blepharospasm, hemifacial spasm, cervical dystonia, axillary hyperhidrosis, over active bladder, GI tract disorders, sialorrhea, temporomandibular disorder and limb spasticity - Most products are based around Botulinum A and target SNAP25. - Treatments last for several months - Due to their high efficacy, tolerance, longevity and satisfactory safety profile, BoNTs are now the most widely used therapeutic proteins.
51
why can't tetanus be used clinicaly?
everyone is vaccinated against tetanus toxin
52
what is botulinum A used for?
cosmetic purposes - it inhibits SNAP25 it can also be used to non-surgically treat Strabismus
53
what can botulinum B be used for?
treat cervical dystonia
Molecular cellular biology
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