L14-15 SNARES

Question 1

Membrane fusion is happening in our bodies all the time!

Answer 1

• 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

Question 2

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

Answer 2

Biochemical reconstitution.
Yeast Genetics.
Cloning

Question 3

Biochemical reconstitution: Intra-Golgi transport assay

Answer 3

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

Question 4

The identification of NSF

Answer 4

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).

Question 5

The isolation of sec mutants (NSF and a-SNAP)

Answer 5

SEC1 (SNARE binding protein), SEC17 encodes -SNAP,

SEC18 encodes NSF

Question 6

The cloning of synaptic vesicle proteins (VAMP and Syntaxin)

Answer 6

Antibodies were raised against synaptic vesicles purified from electric rays. The antibodies were then used to expression clone VAMP and Syntaxin.

Question 7

Clostridial neurotoxins tetanus and boutlinum B cleave VAMP / (synaptobrevin)

Answer 7

SDS-PAGE gel of SSV treated with tetanus toxin

Question 8

The biochemical purification of SNAREs

Answer 8

• Synaptosomal associated protein of 25 kD (SNAP25)

- Found they could purify a large complex that dissembles when ATP is hydrolyzed.

Question 9

Rothman’s SNARE hypothesis

Answer 9

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!)

Question 10

Are there SNAREs for every transport step?

Answer 10

38 SNAREs encoded in the human genome. Involved in various transport steps and fusion reactions

Question 11

Crystal structure of the neuronal SNARE complex

Answer 11

SNAREs zipper in a parallel coiled coil

Question 12

SNARE zippering is thought to provide energy to drive membrane fusion

Answer 12

Bring two membranes together and fusing them is energetically unfavorable

Question 13

Ratio?

Answer 13

• 3Q:1R ratio conserved in all complexes

- Mutation of a Q/R inhibits SNARE activity.

Question 14

Do SNAREs provide the specificity of membrane fusion?

Answer 14

• 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)

Question 15

Common features of SNARE proteins

Answer 15

Generally small 14-40kDa

All have at least 1 coiled-coil or SNARE motif Generally C-terminally anchored

Question 16

A large number of knock out mice have been generated to study SNARE function

Answer 16

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

Question 17

Several rare human diseases are caused mutations in SNARE proteins

Answer 17

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)

Question 18

Familial hemophagocytic lymphohistiocytosis

Answer 18

• 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

Question 19

Mutation in syntaxin 11 cause FHL4

Answer 19

• 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

Question 20

Clostridial neurotoxins

Answer 20

• 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

Question 21

Infant botulism is the most common form of the disease

Answer 21

• Babies < 6 months most susceptible

- Floppy baby syndrome

Question 22

Botulinum neurotoxins and there clinical uses

Answer 22

• 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.