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Advanced Molecular > SNARES > Flashcards

SNARES Flashcards

SNARE complex, formation and associated diseases

1
Q

Name some examples of membrane fusion (3)

A
  • Synaptic vesicles fusion
  • Secretory granule fusion (endocrine and exocrine)
  • Secretion of serum proteins i.e. antibodies from plasma cells
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2
Q

How are secretory vesicles visualised?

A

Electron microscopy
- You can see the vesicles transfer when certain things are stimulated under a microscope

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3
Q

What were the 3 main approaches to identify the machinery of vesicle transport and what do they mean?

A
  1. Biochemical reconstituion
    - Taking cells apart and putting them back together to try and identify the key bits
  2. Yeast genetics
    - Genetic screens of simple organisms to identify key components
  3. Cloning
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4
Q

What did Jim Rothman do?

A
  • Reconstituting DNA replication working on DNA polymerases
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5
Q

What was the system Jim Rothman used?

A
  • He took a cell and grinded it up
  • Purified the intracellular organelles, the golgi
  • He took cells infected with a virus where the VSV G protein involved in membrane fusion normally gets glycosylated involving lots of different enzymes
  • He had a mutant cell which was missing one of these key enzymes
  • If you take Golgi membranes from each of these, wild type and mutant, and mix them together if you have VSV G budding off a vesicle and fusing it gets incorporated
  • This can be seen using radioactivity
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6
Q

What does NEM do and what did the scientists discover?

A
  • Inhibits budding off vesicle reaction
  • The vesicles are docked on to the golgi membrane which shows the NEM is inhibiting the protein needed to ensure they dock off
  • They identified the protein as NSF which is an ATPase which showed ATP is needed for this reaction
  • Also identifies SNAP protein which helps bind NSF to membrane
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7
Q

What did Schekman do?

A
  • Understand intracellular trafficking through yeast genetics
  • Discovered SEC1 (SNARE binding protein but didn’t know at time) , SEC17 which encodes SNAP and SEC18 which encodes NSF
  • It all links!
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8
Q

What did Scheller and Kelly do?

A
  • Wanted to understand how the brain works
  • Pacific electric ray has big neurons and components can be purified
  • USed antibody based cloning
  • Took synaptic vesicles from electric ray and injected it into animals to get antibodies for them which they used to clone and see the components of the synaptic vesicle
  • Identified a protein called VAMP (on the vesicle) and syntaxin (on the synapse)
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9
Q

What did Montecucco do?

A
  • Took purified synaptic vesicles
  • Found VAMP as well idk
  • If u muck about with VAMP you get paralysed so there’s something going on with membrane fusion
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10
Q

What did Jim Rothman do regarding ATP hydrolysis?

A
  • Thought hydrolysis was required for membrane fusion as NSF is a ATPase
  • Tagged NSF to broken up cells with bits of membrane
  • Added a version of ATP which cant be hydrolysed so it locks the NSF
  • Switched ATP to a normal one and the complex was released
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11
Q

What was Rothmans Snare hypothesis?

A

You have a vesicle, a molecule which is the VAMP, plasma membrane, Syntaxin molecule and SNAP 25 molecule
- VAMP and syntaxin zipper together and through the action of NSF you get membrane fusion
Hypothesis:
1. Snares for each transport step in the cell
2. SNAREs should provide specificity to vesicle transport
3. SNAREs should be sufficient to drive lipid bilayer fusion
4. Proposed that NSF and ATP hydrolysis catalyses membrane fusion

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12
Q

How many SNAREs are encoded in the human genome?

A

38

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13
Q

How do SNAREs work?

A
  • Coiled coil domains
  • VAMP and Syntaxin coil up and bring the vesicle and the target domain closer and closer together driving bilayer fusion
  • Fusion pore enlargement so the component inside the vesicle can be released into the membrane
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14
Q

What are Q and R SNAREs?

A

Q SNAREs = Syntaxin molecules and SNAP
R SNAREs = Vamp molecules

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15
Q

How many strands of the coil does each molecule provide?

A

Syntax provides one strand
Vamp provides one strand
Snap 25 provides 2 strands

3Qs to 1R ratio, only get fusion with SNAREs that fit this

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16
Q

What other machinery contributes to SNAREs specificity?

A

Coat proteins
Tethers

Ensure specific vesicle trafficking which is the lead up to SNAREs

17
Q

What are some common features of SNARE proteins?

A
  • VAMP, Syntaxin and snap 25
  • Very small
  • Each SNARE has at least one coil domain
  • Regulatory domain of SNARE which opens and closes
18
Q

What did Suzuki do in his lab?

A
  • Looked at genes involved in neurotransmission
  • Chemical mutagenesis in flies
  • Looked for flies which were temperature sensitive and then put them in a restrictive temp much hotter than normal
  • Flies become paralysed due to effects to dynamin and its role in recycling SNAREs, vesicles accumulate under the plasma membrane when looked under electron microscope
19
Q

What happens if you get rid of VAMP in a knockout experiment with a mouse?

A
  • Mouse dies at birth as it can’t breathe due to a loss of synaptic transmission
20
Q

What happens if you get rid of Syntaxin in a knockout experiment with a mouse?

A

In 1A there’s no gross abnormailites
1B Die after birth due to reduced synaptic transmission

21
Q

What happens if you get rid of SNAP25 in a knockout experiment with a mouse?

A

Die at birth because they cant breathe due to a loss of synaptic transmission

22
Q

What happens when there’s mutations in VAMP2?

A
  • Hypotonia ( floppy baby syndrome weak muscles)
  • Autism
23
Q

What happens when there’s mutations in SNAP25?

A
  • Seizures, severe speech delay, intellectual disability
24
Q

What happens when theres mutations in SNAP29?

A
  • SNAP29 is a SNARE protein
  • Lots of it in skin
  • Neurodevelopmental defects but also ‘scaly’ and ‘fish’ skin
25
Q

What happens when there is a mutation in Syntaxin 11 and what is it?

A
  • Immune specific SNARE so mainly in immune cells
  • FHl4 (name is toooo long)
26
Q

What are purified liposomes and what are they used for?

A
  • Artificially made vesicles that have purified lipids
  • SNAREs can be inserted into them
  • I.e. Vamp2 on one, Syntaxin on another and SNAP25 on another
  • Florescent dye added to liposomes
  • Dye only works when its diluted so its visualised when the membranes between liposomes join together and fuse due to dye spreading across membranes
  • If there’s less dye visualisedthat shows the mutation disables the ability to form SNARE complexes
27
Q

What does a negative dominant mutation mean?

A

the presence of the mutated allele is sufficient to exert its effect, even in the presence of a functional copy of the gene.

  • Happened in experiments with liposomes becuase the mutations are often spontaneous, if the mutated protein reacts with a normal protein it has negative effects
28
Q

What is FHL4?

A
  • Familial hemophagocytic lymphohistiocytosis
  • Rare immune disease due to mutation in Syntaxin 11
  • Multiple gene mutations can cause this disease
  • Mainly effects children
  • Overproliferation of T, B cells and macrophages
  • When you get an infection a cytokine storm happens (overactivation)
  • Patients will die from an infection due to defective killing of T cells
29
Q

What are Clostridial neurotoxins?

A

Interfere with the release of neurotransmitters at neuromuscular junctions e.g.
Clostridium tetani : causes tetanus
Clostridium botulinum : botulism
1-2ng can kill

30
Q

What is the most common form of botulism?

A
  • Infant botulism
  • Effects babies less than 6 months old
  • Floppy baby syndrome
31
Q

What is the structure of a clostridial neurotoxin?

A
  • Targeting domain which binds to the neuron
  • Translocation domain which allows it to get inside
  • Protease domain (light chain) which cleaves the SNARE molecule, very specific e.g. botox only cleaves SNAP25
32
Q

How does the toxin get in?

A
  • Bind to lipid/protein receptors on surface
  • Endocytosed
  • Translocation domain punches holes so it can get into cytoplasm
  • Protease is released and cleaves SNARE molecules
33
Q

Why do tetanus and Botulin toxins have similar mechanisms but different effects?

A
  • Botulin acts on SNAREs at neuromuscular junction
  • Tetanus acts in inhibitory neurons and inhibits them
34
Q

What are the uses of neurotoxins in industry?

A
  • Clinical (Botox, to control sweating etc.)
  • Anaesthetic
35
Q

What restores normal neurotransmission after betulinum?

A
  • Peripheral sprouts (nerve endings) are formed which restores neurotransmission
  • Light chains are degraded ( the bits that cleaved VAMP off)
  • Peripheral sprouting occurs within 28 days and normal transmission reoccurs within 3 months
36
Q
A

Advanced Molecular (13 decks)

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