Lecture 11: SNAREs II

Role of SNAREs in Membrane Fusion

• Function of SNAREs: SNARE proteins are the primary drivers of membrane fusion, facilitating the merging of vesicles with target membranes.

• Stages of SNARE-Driven Fusion:

  • Nucleation: The initial association of vesicle SNAREs (v-SNAREs) and target SNAREs (t-SNAREs).

  • Zippering: The progressive wrapping of SNARE domains from the N-terminus toward the C-terminus, which brings membranes into close proximity.

  • Fusion-Pore Opening: The process where a physical opening allows the contents of the vesicle to be released. This step is often triggered by Calcium ions ($Ca^{2+}$).

• Recycling Machinery:

  • After fusion is complete, the SNARE complex must be disassembled to allow for subsequent rounds of fusion.

  • NSF (N-ethylmaleimide-sensitive factor) is an enzyme that uses energy from $ATP$ hydrolysis ($ATP \rightarrow ADP + P$) to disassemble the complexes.

  • SNAPs (Soluble NSF Attachment Proteins) act as adapters that assist NSF in binding to the SNARE complex.

Animal Models of SNARE Dysfunction

• Drosophila melanogaster (Fruit Fly) Mutants: Research by David Suzuki’s lab in the 1970s (specifically published in PNAS 1976 73 (9) 3253-3257) utilized temperature-sensitive mutants to study membrane fusion.

  • Permissive Temperature: The temperature at which the protein functions normally, allowing the flies to survive and behave typically.

  • Restrictive Temperature: The temperature at which the protein function is inhibited. At this temperature, the flies "keel over" or become paralyzed but can recover if returned to the permissive temperature.

• Specific Drosophila Mutations:

  • shibire: Affects dynamin, which is involved in endocytosis and vesicle budding.

  • comatose: Affects NSF, leading to a failure in SNARE recycling. In these flies, docked vesicles accumulate under the plasma membrane because they cannot be recycled after fusion.

  • paralytic: Affects the $\alpha$-subunit of voltage-gated sodium channels ($Na^+$ channels).

Phenotypes of SNARE Knockout Mice

• Nearly 30 different SNAREs exist, and almost all have been studied via gene knockouts in mammals. While many are expressed mainly in the brain, their absence affects various physiological processes.

• Gene-Specific Phenotypes:

  • VAMP2 (Synaptobrevin): Mice die at birth due to a total loss of synaptic transmission. They are unable to breathe because neuromuscular junction activity is inhibited.

  • Syntaxin 1A: No gross structural abnormalities; however, there are subtle defects in synaptic transmission.

  • Syntaxin 1B: Mice die shortly after birth, exhibiting significantly reduced synaptic transmission. Syntaxin 1A may partially compensate for the loss of 1B, but not sufficiently for survival.

  • SNAP25: Mice die at birth with a total loss of synaptic transmission.

  • VAMP3: Mice appear nearly normal despite the knockout.

  • VAMP8: Mice are unable to absorb food and die.

Human Diseases and SNARE Mutations

• VAMP2 Mutations: Associated with neurodevelopmental disorders characterized by hypotonia ("floppy baby syndrome"), autistic features, and occasionally hyperkinetic movements. These are typically heterozygous mutations.

  • Mapping studies show mutations occur in the v-SNARE domain, which is highly conserved across species (Chimpanzee, Marmoset, Rat, Rabbit, Cow, Dog, Zebrafish).

  • S75P Mutation: A substitution of Serine for Proline at position 75. In liposome fusion assays (measuring NBD Fluorescence), this mutation significantly slows the rate of fusion. It acts as a dominant negative mutation, meaning the presence of the mutant protein interferes with the function of the wild-type protein, and Munc18-1 cannot compensate for it.

  • E78A Mutation: A substitution of Glutamic acid for Alanine at position 78.

• SNAP25b Mutations: Lead to neurodevelopmental disorders involving seizures, intellectual disability, severe speech delay, and cerebellar ataxia.

• SNAP29 Mutations: Cause Cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar keratoderma syndrome (CEDNIK syndrome).

• Syntaxin 11 Mutations: Lead to Familial hemophagocytic lymphohistiocytosis type 4 (FHL4).

Familial Hemophagocytic Lymphohistiocytosis (FHL)

• Disease Overview: A rare, life-threatening immune system disease primarily affecting infants. It is characterized by a "cytokine storm" caused by the over-proliferation of T cells, natural killer (NK) cells, B cells, and macrophages.

• Mechanism of Killing: T-cells kill infected cells by secreting cytotoxic granules. This process requires precise degranulation and membrane fusion.

• FHL4 (Syntaxin 11): Syntaxin 11 (STX11) is an unusual Q-SNARE because it lacks a transmembrane domain. Mutations in STX11, which are expressed in immune cells, lead to significantly reduced levels of the protein and defective degranulation. This condition is incredibly rare, with as few as one known case in the UK.

• FHL5 (Munc18-2): Mutations in Munc18-2 reduce the stability and levels of STX11, drastically reducing the killing capacity of T-cells compared to healthy donors.

Clostridial Neurotoxins: Tetanus and Botulism

• Potency: These are the most potent biological toxins known. The median lethal dose ($LD_{50}$) is estimated at $1-2\,ng/kg$.

• Tetanus (Clostridium tetani): Causes tetanus, commonly known as "lockjaw."

  • Characterized by rigid paralysis and intense muscle spasms that can be strong enough to break bones.

  • Approximately 50,000 people die from tetanus annually world-wide.

  • Historical depictions include the Charles Bell painting of a dying soldier (1808).

• Botulism (Clostridium botulinum): Causes flaccid paralysis.

  • Approximately 100-200 cases occur annually, with infant botulism (children < 6 months) being the most common form.

  • Infant Botulism: Often linked to honey consumption. Initial signs include droopy eyelids and "floppy baby syndrome."

  • Sources include bacteria in soil and food poisoning.

Mechanism of Toxin Action

• Structural Domains: The toxins consist of three primary domains:

  1. Targeting Domain: Binds specifically to receptors on the surface of neurons.

  2. Translocation Domain: Allows the toxin to enter the cell cytoplasm through the membrane after being endocytosed.

  3. Protease Domain: A zinc-dependent protease ($Zn^{2+}$) that cleaves specific SNARE proteins.

• Specific SNARE Cleavage:

  • BoNT/A and BoNT/E: Cleave SNAP25.

  • BoNT/B, BoNT/D, BoNT/F, BoNT/G, and TeNT: Cleave VAMP (Synaptobrevin).

  • BoNT/C: Cleaves both Syntaxin and SNAP25.

• Cellular Pathway Differences:

  • Botulinum Neurotoxins (BoNT): Remain within the motor neuron at the neuromuscular junction, inhibiting synaptic transmission and causing flaccid paralysis.

  • Tetanus Neurotoxin (TeNT): Retrotranslocates up the motor neuron into the spinal cord, where it enters inhibitory neurons. By cleaving VAMP2 in these inhibitory neurons, it prevents the release of inhibitory neurotransmitters, causing the motor neurons to fire excessively, leading to rigid paralysis.

Clinical and Cosmetic Applications of Botulinum Toxins

• Duration: Treatments typically last for several months.

• Cosmetic Uses: Targeting facial lines such as forehead lines, frown lines, eyebrow lifts, crow's feet, lower eyelid wrinkles, bunny lines, gummy smiles, lip lines, marionette lines, and chin dimpling.

• Medical/Neurological Uses: Specifically for conditions associated with neuronal hyperactivity:

  • Strabismus (crossed eyes) and Blepharospasm (eyelid spasms).

  • Hemifacial spasm and Cervical dystonia (treated with Myobloc/Botulinum B).

  • Axillary hyperhidrosis (excessive sweating) and sialorrhea (excessive drooling, common in Parkinson's).

  • Overactive bladder and GI tract disorders.

  • Temperomandibular disorder and limb spasticity.

• Note on Tetanus Toxin: TeNT cannot be used for medicine because the general population is vaccinated against it.

• Product Examples:

  • BOTOX® (OnabotulinumtoxinA): Produced by Allergan; 50 Units/vial. Targets SNAP25.

  • MYOBLOC® (RimabotulinumtoxinB): Produced by US WorldMeds; 5,000 Units/mL. Targets VAMP.

Global Market and Industry

• Market Value: The botulinum toxin market was valued at nearly 5 billion dollars as of 2018/2019.

• Projected Growth: Compound Annual Growth Rate (CAGR) of $8.1\%$ from 2017 to 2026.

• Geographic Focus: North America, Europe, Asia-Pacific, and the rest of the world.

• Key Industry Players: Allergan, Merz, US WorldMeds, Revance, Medytox, and Ipsen.