Lecture 20 Snares II

Question 1

How do snares drive membrane fusion

Answer 1

• SNAREs form a coiled-coil
• Vamp on vesicle, Snap 25/syntaxin on target membrane are brought in close proximity
• These zipper up to drive membrane fusion of bilayers
• Release of contents e.g. NT/insulin
• NSF recycles the SNAREs (unzippering)

Question 2

What happens when heat shibera mutant to 38 degrees

Answer 2

Become paralysed

Question 3

3 types of mutants identifies when inhibit membrane fusion

Answer 3

• Shibire – dynamin (endocytosis disrupted)
• Comatose – NSF
• Paralytic – α-subunit of VG Na+ channel

Question 4

Shibire mutant due to

Answer 4

dynamin

Question 5

Comatose mutant due to

Answer 5

NSF

Question 6

Paralytic mutant due to

Answer 6

α-subunit of VG Na+ channel

Question 7

What accumulates at the restrictive temperature in comatose flies + what was concluded

Answer 7

Docked vesicles accumulate so concluded NSF recycles Snare molecules

Question 8

How was snare function studied

Answer 8

KO mice

Question 9

KO VAMP2 phenotype

Answer 9

die at birth

loss of synaptic transmission so cant breath

Question 10

Synatxin1a KO phenotype

Answer 10

no gross abnormalities

subtle defects in synaptic transmission

Question 11

Synatxin1b KO phenotype

Answer 11

die after birth

reduced synaptic transmission

Question 12

SNAP25 KO phenotype

Answer 12

die at birth

loss of synaptic transmission so cant breath

Question 13

How many different Snares are there

Answer 13

38

Question 14

What still occurs in KO mice

What occurs in the KO of both isoforms of syntaxin

Answer 14

There is some spontaneous fusion events due to redundancy shown through the 2 isoforms of syntaxin in the brain. Yet, KO both isoforms leads to a phenotype like VAMP2 KO.

Question 15

What occurs when neurons are stimulated electrically

Answer 15

When neurons are stimulated electrically, there is no fusion of synaptic vesicles. i.e. a redundant system is working but the electrically activated neurons do not

Question 16

Can disease from SNAREs be genetic? How was this found?

Answer 16

Yes
Some can be inherited
Through sequencing of the genome

Question 17

Disease from mutation in VAMP2

Answer 17

Neurodevelopmental disorder with hypotonia (floppy baby syndrome) and autistic features with or without hyperkinetic movements

Question 18

Disease from mutation in SNAP25b

Answer 18

Neurodevelopmental disorder with seizures, intellectual diability, severe speech delay, cerebellar taxia

Question 19

Disease from mutation in SNAP29

Answer 19

Cerebral dysgenesis
Neuropathy
Ichthyosis (scaly skin)
Palmoplantar keratoderma syndrome - thick skin

Question 20

What is the difference between SNAP 25 and 29

Answer 20

• Snap 29 is similar to Snap 25 but is expressed in the rest of the body as well as the brain so there are developmental AND skin issues

Question 21

Disease from mutation in Syntaxin 11

Answer 21

Familial hemophagocytic lymohohistiocytosis type 4 (FHL4)

SO expressed in the immune system

Question 22

What causes serious neurodevelopmental disorders

Answer 22

Heterozygous mutations in VAMP2 (+/m)

Question 23

Heterozygous mutations in VAMP2 (+/m):

1. What did all have from birth
2. type of mutation
3. Need to check what

Answer 23

1. All had hypotonia from birth.
2. These are spontaneous mutations so not inherited.
3. Botulinim cleaves Vamp and also leads to hypotonia (floppy baby syndrome). Checked changes were deleterious in VAMP and not just a polymorphism.

Question 24

Where could the heterozygous mutations be mapped to

Answer 24

The SNARE domain of VAMP2
• In coiled-coil domain which is important for the zippering up of SNAREs
• These are R SNAREs

Question 25

What mutation slows the rate of liposome fusion and how

Answer 25

S75P mutation Proline disrupts the ability to form alphas helices and so coiled-oil structures so having serine to proline disrupts coiled coil so rate of fusion is significantly inhibited

Question 26

Is Vamp 2 mutation inherited

Answer 26

No

Question 27

How common are human diseases from snare proteins

Answer 27

very rare

Question 28

What type of mutation is S75P

Answer 28

Dominant negative mutation | In assay used 50% of WT and 50% mutant – interferes with WT copy to fuse i.e. dominant negative

Question 29

What disease is caused by mutation in syn11/munc 18-2

Answer 29

Familial hemophagocytic lymphohistiocytosis

Question 30

How common is Familial hemophagocytic lymphohistiocytosis

Answer 30

very rare disease of immune system

Question 31

Familial hemophagocytic lymphohistiocytosis affects predominantly

Answer 31

infants

Question 32

Familial hemophagocytic lymphohistiocytosis is from the overproliferation of...

Answer 32

T cells, natural killer cells, B cells and macrophages

Question 33

Symptoms of Familial hemophagocytic lymphohistiocytosis

Answer 33

death due to cytokine storm | death due to defective killing in T cells

Question 34

How do T cells kill infected cells

Answer 34

secreting cytotoxic granules | that contain perforin to perforate cancer or infected cancer cells

Question 35

What structure is formed between cancer cells and T cells

Answer 35

Immunological synapse so granules will polarise and fuse driven by Snares and syntaxin 11

Question 36

What causes FHL4 and what do patients with this display

Answer 36

mutation in syntaxin 11 | display reduced levels of syntaxin 11

Question 37

Why is syntaxin 11 unusual

Answer 37

• STX11 is an unusual Q-SNARE as it does not have a TM domain (but instead a lipid anchor)

Question 38

What does loss of syntaxin 11 lead to

Answer 38

• Loss of STX11 causes defective degranulation from cytotoxic T-cells by an unclear mechanism and so leads to cancer

Question 39

What does a mutation in Munc18-2 cause

Answer 39

FHL5 and reduces the levels of STX11

Question 40

What occurs as the ratio of cancer cells to T cells is increased

Answer 40

• As the ratio of cancer cells to T cells is increased, the ability of T cells to kill is compromised

Question 41

Give 2 examples of Clostridial neurotoxins:

Answer 41

* Clostridium tetani: Tetanus | * Clostridium botulinum: Botulism

Question 42

Clostridium tetani: Tetanus causes and number of deaths

Answer 42

- Causes paralysis | - Vaccinations have reduced deaths globally from 300,000 to 50,000 die a year

Question 43

Clostridium botulinum: Botulism is due to, most common in, leads to, number infected

Answer 43

- Anaerobic bacteria – spores – canned food - if don’t heat for long enough - Infant botulism is the most common form of this disease, specifically babies under the age of 6 months - Leads to floppy baby syndrome i.e. weak muscles - 100-200 people a year get this (but is treatable)

Question 44

Clostridium tetani: Tetanus, Clostridium botulinum: Botulism are described as

Answer 44

Most potent biological toxins known to man

Question 45

Median lethal dose of tetanus and botulinum

Answer 45

1-2ng/kg

Question 46

How many types of clostridial neurotoxins are there

Answer 46

several different types

Question 47

What do clostridial neurotoxins have in common

Answer 47

conserved protein structure

Question 48

clostridial neurotoxins protein structure

Answer 48

* Targeting domain – binds to neurons * Protease – cleaves snare ONLY * Translocation domain – allows escape

Question 49

clostridial neurotoxins are what type of proteases

Answer 49

Toxins are zinc dependent proteases

Question 50

What are clostridial neurotoxins only taken up by

Answer 50

Neurons This is because the targeting domains bind to specific lipids on the surface of neurons. Also bind to neuronal R’s and so there is not paralysis in all cell types. Taken up by neurons by endocytosis. Escape out of endosome by translocation domain, toxin is reduced and it clips the SNARE molecules.

Question 51

Clostridial neurotoxins cleave what

Answer 51

Different snares

Question 52

Tetanus and Botulinum toxins have a similar ... but ... different neurons

Answer 52

mode of action | intoxicate

Question 53

Tetanus and Botulinum toxins are both

Answer 53

potent inhibitors of SNARE function

Question 54

Explain symptoms of Tetanus and Botulinum

Answer 54

* Botulinum toxin goes into MN and acts at NMJ so don’t get Ach release  floppy paralysis as inhibiting release of ACh * Tetanus cleaves VAMP like BoNTs/B/D/F and G - goes into endosomes, tracts up axon into cell body and transferred into inhibitory interneuron  spasms as inhibiting regulation

Question 55

What is botulinum used to treat

Answer 55

• Used to treat neurological conditions associated with neuronal hyperactivity

Question 56

Uses of botulinum toxins

Answer 56

* Cosmetic uses - safe in small quantities and injected locally so only inhibit neurons in close proximity * Strabismus – is eye is wrong direction, paralyses the eye to straighten it up , Blepharospasm – eye spasm, over active bladder, axillary hyperhidrosis (sweat)

Question 57

Why do you have to have botox repetitively

Answer 57

• SNARES turn over and synapses reforms so need to get treatment periodically

Question 58

What are most products based around

Answer 58

• Most products are based around Botulinum A and target SNAP 25

Question 59

Why cant you make medicine against tetanus

Answer 59

• Can’t make medicine based around tetanus as everyone is vaccinated against the toxin

Question 60

Value of botulinum market

Answer 60

$5 billion

Question 61

What us botulinum A used for

Answer 61

• Botulinum A is used for cosmetic purposes and other indications e.g. non-surgical Strabismus – cleaves SNAP 25 proteolytically which is then degraded so you can no longer form complex so get inhibition of ability to fuse vesicles at NMJ so get paralysis of these muscles – has longer lasting action

Question 62

What is botulinum B used for

Answer 62

• Botulinum B can be used to treat cervical dystonia – cleaves VAMP (synaptobrevin) – botulinum B heavy chain binds to synaptotagmin and light chain that is endocytosed into vesicle. The light chain translocates to the vesicular membrane to cleave synaptobrevin. This means Ach vesicles cannot fuse so muscle contraction ceases.