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SNAREs Flashcards

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AP® Biology flashcards Biology 101 flashcards
1
Q

What were the 3 main approaches taken to identify the machinery of vesicle transport?

A
  1. Biochemical reconstitution
  2. Yeast genetics
  3. Cloning
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2
Q

What is biochemical reconstitution?

A

Break down a cell and rebuild it with the parts we want, to test the function of parts of the cell.

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3
Q

What did Jim Rothman’s intra-Golgi transport assay (biochemical reconstitution) allow us to measure?

A

Allows us to measure budding and fusing of transport vesicles by measuring radioactive sugar transported from one Golgi to another via vesicles

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4
Q

What did Jim Rothman’s intra-Golgi transport assay (biochemical reconstitution) show us?

A

The presence of an ATPase called NSF which is required for fusion of membranes.

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5
Q

What is the 20 S complex?

A

Syntaxin (Q SNARE), VAMP (R SNARE), and SNAP25 (Q SNARE) interact with NSF and αSNAP to form 20 s complex

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6
Q

What gene encodes for NSF in yeast and humans?

A

SEC 18

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7
Q

What is the function of the 20 s complex?

A

Via ATP hydrolysis this complex allows for the SNARE complexes to dissociate.

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8
Q

What was the experiment that brough together all previous experiments vesicle proteins and what was the conclusion?

A

Rothman’s biochemical purification of SNAREs showed what made up the 20s complex. They concluded that SNAREs are used for every transport step and provide specificity to vesicle transport. They were incorrect in thinking ATP hydrolysis drives this however.

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9
Q

What is the prototypic neuronal SNARE complex comprised of?

A

VAMP (on vesicle), Synatxin and SNAP25 (both on target membrane)

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10
Q

How many SNAREs are there?

A

38 SNAREs working on different membranes.

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11
Q

How do vesicles fuse with membranes through the neuronal SNARE complex?

A
  1. VAMP (on vesicle), Syntaxin and SNAP25 (both on target membrane) can form coil domains (SNARE domains) together (alpha helices which wrap around each other), at this stage is called Trans-SNARE complex (fusion has just begun)
  2. This provides the energy to drive the two membranes together, disrupting the lipid bilayer so they can fuse. At this stage is called Cis-SNARE complex as fusion pore is enlarging.
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12
Q

Why is it energetically unfavourable for membranes to fuse, and what provides the energy allowing for them to fuse?

A

> Energetically unfavourable process, as lipids repel each other, so requires energy to drive membranes together (as membranes have hydrophilic heads which repel)

> The zipping of VAMP, Synatxin and SNAP25 provides this energy.

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13
Q

What is a) Trans-SNARE complex b) CIs-SNARE complex?

A

a) Occurs first, just when lipid bilayers start fusing so the fusion pore starts opening

b) Occurs second, fusion pore enlarges.

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14
Q

What is the function of NSF in vesicular transport?

A

NSF uncoils the SNAREs so they can be recycled, ATP is required to take the SNARE complex apart (NSF is an ATPase).

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15
Q

What SNAREs are a) R SNAREs b) Q SNAREs, where are these found and why are they in this groups?

A

a) VAMP molecule (as has arginine on 0 layer), R SNAREs on vesicle

b) Syntaxin and SNAP25 (as has glutamine on 0 layer), Q SNAREs on target membrane

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16
Q

What forms between R SNAREs and Q SNAREs to allow them to coil?

A

> The Arginine (R SNAREs) and glutamines (Q SNAREs) between these molecules form salt bridges when forming coils.

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17
Q

What is the structure of salt bridge formation in the nueronal SNARE complex and what is conserved between all SNARE complexes?

A

> In the neuronal SNARE complex, there are 4 salt bridges, two from glutamine (Q) on the two coil domains of SNAP25, one glutamine (Q) from Syntaxin, and one Arginine (R) from VAMP.

> The 3Q:1R ratio (3Q coil domains and 1R SNARE) is conserved between all SNARE complexes

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18
Q

What 3 other machineries contribute to membrane fusion specificity?

A

rabs, coat proteins and tethers

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19
Q

Why are SNAREs specific in their interactions?

A

SNAREs usually interact with their same type, e.g. endoplasmic SNAREs interact with each other.

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20
Q

What are 3 typical features of all SNARE proteins?

A
  1. Generally small 14-40kDa
  2. All have at least 1 coiled-coil or SNARE motif (alpha helices)
  3. Generally C-terminally anchored (C terminus is in membrane)
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21
Q

Can SNAREs carry out fusion on their own?

A

SNAREs on their own can’t do fast membrane fusion or specific, needs extra machinery.

22
Q

What fusion pathways are SNAREs required for?

A

Required for all fusion steps in the endocytic/ biosynthetic pathways.

23
Q

What is the role of Ca2+ influx on vesicle and membrane fusion?

A

CA2+ helps neutralise the charge between vesicle and cell membranes (buffers electrostatic charges between membranes) making it more energetically favourable to fuse (calcium influx triggers a switch from trans- to cis-SNARE complex)

24
Q

Describe membrane fusion via the neuronal SNARE complex in 6 steps

A
  1. VAMP SNARE (R SNARE) on vesicle
  2. SNAP 25 (2 Q coil domain), Synatixn (1 Q SNARE) on target membrane, form alpha helices coils with VAMP (3Q:1R salt bridge ratio)
  3. Influx of Ca2+ at synapse, buffering electrostatic charges between membranes (making more energetically favorable)
  4. Trans to Cis SNARE complex
  5. NSF comes in with αSNAP to form 20s complex (NSF, αSNAP, SNAP25, Syntaxin, VAMP)
  6. ATP hydrolysis, NSF unwinds SNAREs so they are free to be recycled
25
Q

Why do SNAREs need to be recycled after fusion?

A

As the R and Q SNAREs end up both on the target membrane after fusion, so the R SNAREs (VAMP) need to be recycled back to a vesicle

26
Q

What are mutations in 2 drosophila proteins that cause paralysis due to incorrect SNARE function, and what are the human version of these proteins?

A
  1. shibire: dynamin
    > Dynamin: Important for pinching off vesicles from plasma membrane (reclining membranes for synaptic transmission)
  2. comatose: encodes NSF
    >Mutation in NSF (recycling SNAREs)
27
Q

What is dynamin important for?

A

Important for pinching off vesicles from plasma membrane (reclining membranes for synaptic transmission)

28
Q

What does a mutation in NSF cause?

A

Accumulation of SNAREs as NSF is used to uncoil SNAREs so vesicles can be recycled (VAMP R SNARE stays on the target membrane with SNAP25/ Syntaxin Q SNAREs)

29
Q

What is observed when the phenotype for the genes a) VAMP2 b) Syntaxin 1A c) Syntaxin 1B d) SNAP25 are knocked out?

A

a)Without VAMP2 die at birth due to paralysis and can’t breathe. Loss of synaptic transmission

b) No gross abnormalities. Subtitle defects in synaptic transmission

c) Die after birth. Reduced synaptic transmission

d) Die at birth. Loss of synaptic transmission

30
Q

What happens to most people with SNARE diseases?

A

Most people with SNARE diseases die at birth.
Most alive cases are heterozygous diseases

31
Q

What are the diseases caused by mutations in the genes a) VAMp2 b) SNAP25b c) SNAP29 d) Syntaxin11

A
  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
    >(expressed in brain) Cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma syndrome (CEDNIK syndrome)
  4. Syntaxin 11
    >Familial hemophagocytic lymphohistiocytosis type 4 (FHL4)
32
Q

What domain of SNAREs is conserved across all species?

A

SNARE (coil) domain

33
Q

How do the SNARE (coil) domain allow SNAREs to interact?

A

Hydrophobic repeats and ionic layers, made up of conserved residues, interact between SNAREs allowing coiling

34
Q

Where would loss of function mutations occur in SNAREs?

A

In the highly conserved SNARE (coil) domain.

35
Q

What is the effect of mutated coil domains in VAMP SNAREs in a Liposome fusion assay?

A

Liposomes with mutated VAMP2 cannot fuse or dissociate properly with Liposomes with Syntaxin

36
Q

How is Syntaxin11 important for regulating the immune response?

A

Loss of STX11 causes defective degranulation from cytotoxic T-cells by an unclear mechanism

37
Q

What is the effect of Familial hemophagocytic lymphophistiocytosis and mutations in which SNARE causes this?

A

> Rare immune disease were over proliferation of T cells, natural killer cells, B cells and macrophages causes a cytokine storm leading to death.

> Syntaxin11

38
Q

How is Syntaxin11 effected in patients with Familial hemophagocytic lymphophistiocytosis forms a) FHL4 b) FHL5

A

a) FHL4 patients lose STX11

b) FHL5 patients have mutated Munc18-2 which binds and regulates STX11, so causes defects in STX11 machinery too.

39
Q

What is the overall mechanism of Tetanus and Botulism toxins?

A

Cleave SNAREs

40
Q

What bacteria secrete the toxins a) Botulism b) Tetanus?

A

a) Clostridium botulinum

b) Clostridium tetani

41
Q

What is a disease caused infant botulism contact?

A

Infant botulism which can also cause Floppy baby syndrome (hypertonia)

42
Q

What are the 3 steps for Clostridial toxins to cleave SNAREs and which domains of the toxins do each part?

A
  1. Toxin binds to neuron
    >By targeting domain of toxin
  2. Toxin pokes holes in endosome to enter cytoplasm
    >By translocation domain of toxin
  3. Protease cleaves SNAREs
    >By protease domain of toxin
    >Cleaves specific SNAREs, and nothing else, inhibiting neuro transmission.
43
Q

What proteases in Clostridial neurotoxins cleaves the SNAREs of the neuronal SNARE complex?

A

1) SNAP-25 cleaved by BoNT A

2) Syntaxin cleaved by BoNT C

3) VAMP cleaved by BoNT D,F,G

44
Q

Why do Tetanus and Botulinum toxins have a similar mode of action but have different symptoms?

A

The toxins have different effects due to cleaving SNAREs in different neurons.

45
Q

What are the symptoms of a) Tetanus b) Botulinum toxins?

A

a) Rigid and spasm

b) Paralysis

46
Q

How does Tetanus toxin cause seizures?

A

Enters through neuromuscular junction but Tetanus goes back and effects inhibitory neurons, cleaving their SNAREs, causing seizures due to lack of inhibitory regulation of Aps.

47
Q

How does Botulinum toxin cause paralysis?

A

Enters through neuromuscular junction and effects this region (unlike Tetanus toxin), cleaving SNAREs causing paralysis due to decreased AP firing at neuromusuclar junctions.

48
Q

Why are Botulinum neurotoxins useful clinically?

A

Useful clinically as only need small amounts of the toxins to act for a long time 3-4 months.

49
Q

What are 3 examples of symptoms Botulinum toxins can treat?

A

Stops over salivating in people with Parkinson’s, treating chronic pain, or stopping muscle spams

50
Q

What do most Botulinum toxins that are used for cosmetic purposes target?

A

SNAP25 by BoNT A protease domain

51
Q

Why can’t we make medicines based around Tetanus toxins?

A

Can’t make medicines based around tetanus as everybody is vaccinated against the toxin.

52
Q

What types of conditions can Botulinum toxins treat and 2 examples

A

> Botulinum toxins can be used to treat neurological conditions associated with neuronal hyperactivity (as cleaving SNAREs decrease AP firing)

> E.g. Strabismus by Botulinum A, Cervical dystonia by Botulinum B

Advanced Molecular Biology (9 decks)

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