MEDCHEM E CHEMBIO Lecture8 Proteins as toxins

• Botulism


  Rare but potentially life-threatening bacterial illness
• Clostridium botulinum bacteria


  • Grows on food and produces toxins that, when ingested, cause paralysis
• Botulism neurotoxins


  1. Prevent neurotransmitters from functioning properly by inhibiting motor control

  2. Toxin binds specifically to nerves which use the neurotransmitter acetylcholine

  
• Botulism progression


  1. Patient experiences paralysis from top to bottom, starting with the eyes and face and moving to the throat, chest, and extremities

  2. Paralysis reaches the chest, resulting in death from inability to breathe
• Botulinum toxins


  Proteins as toxins
• Botulinum toxin production


  Before their release via bacterial autolysis, botulism toxins are produced as progenitor toxins (variously sized complexes composed of inactive single polypeptide toxin chains and other non-toxic accessory proteins)
• Active form of botulism toxins


  • Consists of three distinct domains endowed with different biological properties: the C-terminal domain is responsible for the specific presynaptic binding of the toxins; the N-terminal domain (the L-chain) is a Zn2+-dependent endopeptidase; and the middle domain mediates translocation of the L-chain into the nerve terminal cytosol

  
• Structure of the toxin
• Mechanism of action of botulinum toxins


  1. Intoxication includes cell surface recognition, vesicle internalization, translocation of the catalytic domain (light chain) into the cytosol, and proteolytic cleavage of one of the proteins of the SNARE complex

  2. These steps lead to inhibition of neurotransmitter-containing vesicle release

  3. Botulinum toxins B, D, F, and G cleave proteins of the VAMP family, and Botulinum toxins A, C, and E cleave SNAP-25

  4. Botulinum toxins C can also cleave syntaxin

  
• Botulinum toxins anchor the vesicles that bind to the cell wall and release acetylcholine
• Botulinum Antitoxin


  • Antibodies or antibody antigen-binding fragments that block the neurotoxin
  • The antibody fragments bind free botulinum toxin, which then prevents the toxin from being internalized at the post-synaptic cholinergic receptor
  • Antitoxin only binds free botulinum toxin, so it prevents progression of symptoms but does not reverse any paralysis already present

  
• Antigen


  • Substance that is capable of stimulating an immune response
  • An antigen interacts with an antibody that matches the antigen's molecular structure
• Botulinum toxin A


  • Highly specific for peripheral cholinergic nerve terminals and it does not spread significantly from the site of injection
  • The action of Botulinum toxin is reversible, but can last for up to six months, thus frequent applications are not needed
• Limitations of Botulinum neurotoxins: could elicit neutralizing antibodies against the corresponding toxin, thus reducing the beneficial effects or rendering the patient completely unresponsive to further treatment.
• Applications of Botulinum toxin


  • Blepharospasm
  • Hemifacial spasm
  • Laryngeal dysphonia
  • Head and neck dystonias
• Cholera Toxin is a protein enterotoxin, secreted by toxic species of the bacterium Vibrio cholera, and is spread mostly from dirty water
• Cholera affects an estimated 3–5 million people worldwide and causes 58,000–130,000 deaths a year
• Cholera


  Potentially epidemic and life-threatening secretory diarrhea characterized by numerous, voluminous watery stools, often accompanied by vomiting, resulting in hypovolemic shock and acidosis
• Mechanism of action of cholera toxin


  1. The toxin causes over activation of the signalling pathway that controls the activity of chloride channel proteins

  2. The toxin activates the chloride channel proteins to open and allow the movement of chloride ions out of the cell in the absence of the signalling molecule
• Structure of the cholera toxin


  • Cholera toxin has two types of subunits: subunit A and subunit B
  • A subunit contains A1 domain, which includes the enzymatic active site, and A2 domain, which has a a–helix tail
  • The B subunit contains 5 chains that form a pentameric ring around the central pore in structure
  • Subunit A and subunit B are assembled by the α–helix tail of A2 domain, which inserts into the central pore
• Mechanism of action of the cholera toxin


  1. Cholera toxin binds to ganglioside receptors on the surface of intestinal epithelium cells and is internalised by endocytosis, triggering the production of cyclic AMP (a second messenger) within the cell

  2. Cyclic AMP (cAMP) activates specific ion channels within the cell membrane, causing an efflux of ions from the cell

  3. The build up of ions in the intestinal lumen draws water from cells and tissues via osmosis – causing acute diarrhea