SNARES

Which chemical agent was used by scientists to inhibit transport reactions, leading to the identification of the fusion protein NSF?

N-ethylmaleimide (NEM)

What does the acronym NSF stand for in the context of membrane fusion?

N-ethylmaleimide-sensitive factor

In the Rothman intra-Golgi transport assay, what is the defining characteristic of the 'donor' compartment?

It contains the cargo but lacks the enzyme required to modify it.

In the Rothman intra-Golgi transport assay, how is successful vesicle transport biochemically confirmed?

By measuring the addition of a labeled sugar to the G protein in the acceptor compartment.

Which protein acts as a bridge to allow NSF to bind to Golgi membranes?

Soluble NSF attachment protein (SNAP)

What term is used for the hypothetical membrane-bound proteins that act as receptors for SNAPs?

SNAREs (SNAP receptors)

The yeast protein Sec18 is the evolutionary homologue of which human fusion protein?

NSF

The yeast protein Sec17 is the evolutionary homologue of which human fusion protein?

alpha-SNAP

Why was the Pacific electric ray chosen as a model for cloning synaptic vesicle proteins?

Its neurons are densely packed with synaptic vesicles.

Which specific SNARE protein is located on the membrane of the synaptic vesicle?

VAMP (also known as a v-SNARE)

Which specific SNARE protein is located on the target plasma membrane (presynaptic membrane)?

Syntaxin (also known as a t-SNARE)

Which two neurotoxins were used to prove the importance of VAMP by specifically cleaving it?

Tetanus and Botulinum B neurotoxins

According to Rothman's SNARE hypothesis, what provides the specificity for vesicle transport to correct destinations?

Specific SNAREs for each transport step

What aspect of Rothman's original SNARE hypothesis was later proven incorrect?

The proposal that NSF and ATP hydrolysis directly catalyze membrane fusion.

How many SNAREs are encoded in the human genome?

38

The SNARE complex is composed of a bundle of how many helices?

4

Which SNARE protein contributes two helical domains to the 4-helix bundle?

SNAP-25

In what direction does the zippering of the SNARE complex occur?

From the N-terminal toward the C-terminal.

What physical barrier must the mechanical force of SNARE zippering overcome to allow fusion?

The electrostatic/electrical repulsion between two negatively charged lipid membranes.

What is the status of the SNARE proteins when they are in a 'trans' configuration?

The proteins reside on two different membranes.

Describe the 'hemi-fusion' stage of membrane fusion.

The outer leaflets of the two membranes merge to create a stalk, while contents remain separated.

At what stage of the fusion pathway is the cargo first allowed to leak into the destination space?

Fusion-pore opening

What is the 'cis-SNARE complex'?

A stable complex where all SNARE proteins are located on the same integrated membrane after fusion.

Functional SNARE complexes must follow a strict ratio of ____ helices to 1 R helix.

3 Q

Under the Q/R classification system, which type of SNARE is Syntaxin typically categorized as?

Qa-SNARE

The N-terminal part of SNAP-25 is classified as which type of SNARE?

Qb-SNARE

The C-terminal part of SNAP-25 is classified as which type of SNARE?

Qc-SNARE

Where are SNARE proteins generally anchored within the cell?

They are usually C-terminally anchored in the membrane.

What is the primary role of NSF after the membrane fusion step is complete?

Recycling SNAREs by unzipping the stable cis-SNARE complex.

In the Drosophila 'comatose' mutant, which protein's function is temperature-sensitive?

NSF

Why do 'comatose' flies collapse at 38 degrees

NSF stops working, preventing SNARE recycling and causing synaptic vesicles to run out.

The Drosophila 'shibire' mutation affects which protein responsible for vesicle budding?

Dynamin

The Drosophila 'para' mutation affects which component of the neuron?

Voltage-gated channels (the electrical trigger for fusion)

Which Drosophila mutant recovers most rapidly when the temperature drops from 38 degrees to 23 degrees?

para (voltage-gated channel)

What is the phenotype of VAMP2 or SNAP25 knockout mice?

They die at birth due to a loss of synaptic transmission.

Heterozygous mutations in VAMP2 in humans lead to which group of symptoms?

Global developmental delay, hypotonia, and autistic/Rett-like features.

What is the structural effect of the S75P mutation in VAMP2?

Serine is replaced by proline, disrupting hydrogen bonds and forming kinks that impair the SNARE zipper.

Which stage of the SNARE cycle is specifically impaired by the S75P VAMP2 mutation?

Fusion-pore opening

Familial haemophagocytic lymphohistiocytosis (FHL) is characterized by the over-proliferation of which cells?

T cells, natural killer (NK) cells, B cells, and macrophages.

A mutation in which SNARE protein is the primary cause of FHL4?

Syntaxin-11

Why does a lack of Syntaxin-11 lead to the symptoms of FHL?

Killer cells cannot release toxic granules to eliminate targets, causing a compensatory cytokine storm and inflammation.

What is a common clinical sign of FHL involving the internal organs?

Enlarged liver (hepatomegaly) or spleen (splenomegaly).

What is the curative treatment for Familial haemophagocytic lymphohistiocytosis?

Stem cell transplant

What is the estimated LD50 of clostridial neurotoxins?

1-2ng/kg

Tetanus is characterized by permanent muscle contraction because the toxin blocks _ neurotransmitters.

Inhibitory

Botulinum toxin causes flaccid paralysis by blocking _ neurotransmitters at the neuromuscular junction.

Excitatory

What triggers the clostridial toxin light chain to be released from the endosome into the cytosol?

The acidic environment of the endosome.

How does Tetanus toxin (TeNT) reach inhibitory interneurons after entering a motor neuron?

Via retrograde transport in signalling endosomes to the spinal cord, then jumping across the synapse.

Most cosmetic Botulinum products (like Botox) are based on Botulinum A and target which protein?

SNAP-25

Why is it not possible to create medications based on the tetanus toxin?

Because the general population is vaccinated against the toxin.

Which SNARE protein is located on hepatocytes to facilitate the secretion of albumin?

The material lists 'secretion of serum proteins' as a fusion requirement but does not specify the SNARE name for albumin.

What is the specific ratio of Arginine (R) to Glutamine (Q) in a functional SNARE complex?

1 R-SNARE to 3 Q-SNAREs

In the SNARE cycle, what is the role of ATP hydrolysis?

It provides the energy for NSF to pry the stable cis-SNARE complex apart.

Which Drosophila mutant accumulates docked vesicles at the restrictive temperature 38 degrees?

comatose (NSF)

Which genetic condition is associated with 'floppy baby syndrome'?

Infant botulism (or hypotonia in VAMP2 disorders)

The mutation in the Muc18-2 binding site of Syntaxin leads to which specific disease?

FHL5

Identify the annual worldwide death toll attributed to tetanus.

~50,000