Protein misfolding

Protein misfolding

1. Overview

   • When?
     • Protein synthesis
     • Protein degradation
       • Sometimes small fragments will associate with one another instead of being fully degraded
   • Why?
     • Mutation in protein
     • Lack of enzyme needed for correct folding
     • Accumulated damage to protein
       • Become less efficient
     • Conformational changes
   • Impact
     • Protein is non-functional
     • Protein is lacking or in short supply
     • Protein is in the wrong place
     • Aggregates can accumulate and cause disruption

2. Aggregation and degradation

   • Type 1 procollagen
     • Osteogenesis imperfecta
   • alpha1-antitrypsin
     • Deficiency
   • CFTR
     • Cystic fibrosis
       • 1/2000 live births
       • Autosomal recessive
       • Epithelial plasma membrane chloride channel
         • ABC transporter
       • 200 different mutations
         • May account for commonness of condition
     • We make significantly more than we need because misfolding is so common
       • Co-translational folding
         • During synthesis, on the ribosome
       • Post-translational folding
       • F508 CFTR doesn't accumulate but is degraded in the ER
         • The change is so minimal, but it affects the key S7-H6 loop
     • Types of mutation
       • Type I: No synthesis
         • G542X
       • Type II: Block in processing
         • delta-F508
       • Type III: Block in regulation
         • Missense G551D
       • Type IV: Altered conductance
         • MIssense R117H
       • Type V: Reduced synthesis
         • MIssense A455E
   • LDL receptor
     • Familial hypocholesterolaemia

3. Serpins

   • SERine Protease INhibitors
     • alpha1-antitrypsin
       • Mutation
         • Emphysema
         • Liver disease
       • Acute-phase protein
         • Overexpressed following mutation
       • 1 in 20 northern Europeans heterozygous for Z-allele of alpha1-antitrypsin
         • Loss-of-function, 60% inhibitory activity retained
           • Replacement of acidic glutamine for basic lysine
             • Loss of salt bridge
             • Where the reactive centre loop opens up
         • Homozygosity
           • Emphysema
           • Liver disease
             • EM
               • Filaments in "beads-on-a-string" formation
                 • Consistent with assembly into polymers
             • PAGE
               • Native runs as a ladder
                 • Because all different length chains have assembled
         • Circular dichroism analysis
           • Z form has less pronounced peaks and troughs
             • Central aromatic residues in different environments
     • alpha1-antichymotrypsin
     • Antithrombin
     • Large family
       • Look similar
         • Quite different roles
   • Structure well conserved
     • 3 beta-sheets
     • 9 alpha-helices
     • Reactive centre loop
       • Important for function
     • Natively unfolded
   • Mutations
     • Dementia
       • Neuroserpin
   • Mechanism
     • Cleaved by protease. Brings one side down and inserts into reactive beta sheet. Opens protein for misfolding whilst denaturing the protease
   • Polymer formation is irreversible
     • Serpin polymers are hyperthermostable

4. Aggregation and accumulation

   • Amyloidoses
     • Amyloid
       • Insoluble fibrils that are formed by polymerisation of normally soluble proteins and are deposited in the tissues.
         • Intra- or extracellular
       • Smooth and not recognisable as original proteins
       • Fibrils formed from different proteins have very similar structures
         • Stain with Congo Red, green birefringence under cross-polarised light
           • There is a particular ordering of the dye molecules
       • EM
         • Long
         • Straight
         • Unbranching
       • X-ray
         • Cross-beta diffraction pattern
           • Repeating structure
             • A ladder of beta-sheets running perpendicular to the vertical axis
     • There are many different proteins found
       • Extracellular
         • Transthyretin
         • Gelsolin
         • Lysozyme
       • Intracellular
         • alpha-synuclein
         • Tau
       • Beta-sheet
       • Alpha-helical
       • Alpha/beta
       • Peptides
       • It's such an organised structure that it works for any size of protein
     • Causes
       • Mutations
         • Transthyretin
       • High concentration
         • Immunoglobulin
       • Infectious
         • Prion
           • CJD
     • 1. Monomer
       • 2. Aggregate
         • 3. Oligomer
           • 4. Protofibril
             • 5. Amyloid fibril
               • 6. Amyloid plaque
             • Protofilaments are individual fibres that wrap around one another
   • Huntingtin
     • Huntington's disease
   • Alpha1-antitrypsin
     • Deficiency
   • Alpha-synuclein
     • Parkinson's
   • Cu/Zn superoxide dismutase
     • ALS
   • Disease relates to accumulation of deposited material and disruption of tissues
     • Cell death