Protein misfolding

Protein misfolding

1. Overview

   • When does it go wrong?
     • Protein synthesis
     • Protein degradation
   • Why does it go wrong?
     • Mutation in protein
     • Lack of enzyme needed for correct folding
     • Accumulated damage to protein
     • Conformational change
       • Conformational diseases
         • If a protein misfolds it should be removed by degradation
   • What happens in disease as a result?
     • Protein is non-functional
     • Protein is lacking or in short supply
     • Protein is in the wrong place
     • Aggregates accumulate causing disruption
       • Aggregation
         • Protein self-assembles and forms aggregates

2. Protein misfolding and disease: Aggregation and degradation

   • Protein is no longer in the right place to be functional
   • Misfolding occurs during protein synthesis and folding causing loss of function
   • Cystic fibrosis
     • 1/2000 live births
     • Autosomal recessive
     • 200 different mutations
     • CFTR
       • A plasma membrane chloride channel in epithelial cells
       • An ABC transporter
       • An enzyme that utilises ATP
       • Many components needed to fold for the protein to be functional
         • Hence lots of mutations
         • Folding pathway
           • Complicated due to many subunits
           • Different points of folding
           • Co-translational folding where folding occurs during synthesis
           • Post-translational folding where all components are put together
       • Deletion of F508
         • CFTR folds poorly
           • Degraded in endoplasmic reticulum
         • F508 usually forms a hydrophobic pocket and is important in protein folding
         • Mutant F508 CFTR accumulates in endoplasmic reticulum
           • Does not attach to plasma membrane
       • Molecular consequences of CFTR mutations
         • No CFTR synthesis
           • Non sense mutation
           • Deletion of TT
           • Change in splice junction
         • Block in processing
           • Missense mutation
           • AA deletion
         • Block in regulation
           • Missense mutation
         • Altered conductance
           • Missense mutation
         • Reduced synthesis
           • Missense mutation
           • Alternative splicing
   • Serpins and disease
     • Serpins
       • Serine protease inhibitors
       • Alpha-1-antitrypsin
       • Antithrombin
       • Cell conserved structure
         • 3 beta sheets
         • 9 alpha helices
       • Reactive centre loop
         • Negatively unfolded
     • Serpin mechanism
       • Protease will cleave reactive centre loop
         • Inhibitor changes molecule conformation
           • Reactive centre loop becomes beta strand
             • Protease is denatured
               • Conformational change opens up the protein for misfolding
     • Serpin polymerisation
       • Occurs in conformational diseases
       • Alpha-1 ant-trypsin is a protease inhibitor in the lungs
         • Without protease the lungs are susceptible to emphysema
       • Serpin polymers
         • alpha1-antitrypsin forms filaments which have beads on a string appearance
         • Hyperthermostable
         • Polymer formation is irreversible
         • Alpha1-antitrypsin is made in the liver
           • Transported to lungs
             • Forms aggregates
               • Causes liver disease
               • Loss of function in lungs causes emphysema
     • Alpha-1 anti-trypsin deficiency
       • Acute phase protein
         • Overexpressed following inflammation
       • 1 in 20 hetereozygous
       • Homozygotes suffer from emphysema and liver disease
       • Z alpha1-anti-trypsin
         • Replaced glutamate for lysine
           • Results in loss of salt bridge
           • Affects beta sheet where reactive loop inserts
             • Reactive centre loop of another molecule is able to insert into this gap
       • Circular dichroism shows structures of alpha1 and zalpha1
         • Very similar
         • Tertiary structure has aromatic residues in different environments

3. Protein misfolding and disease: Aggregation and acculumulation

   • Disease related to accumulation of deposited material and disruption of tissues
   • Amyloid
     • Insoluble fibrils formed by polymerisation of normally soluble proteins
       • Deposited in tissue
     • Large number of proteins can form amyloid fibrils
       • Proteins are all different to each other and cause different diseases
     • Amyloid formation involves a conformational change in the native protein
     • What makes a native protein aggregate form amyloid in disease?
       • Mutations
         • Destabilises structure making them more likely to form amyloid fibrils
       • High concentration
       • Infections
     • Amyloid fibrils formed from different proteins have similar structures
       • Congo red staining
       • EM
         • Shows long, straight, unbranching fibrils
       • X-ray fibre diffraction
         • Shows charcteristic cross beta diffraction pattern
     • Amyloid fibrils can be defined in different ways
       • Structure of proteins
         • Grow a crystal and use diffraction pattern to tell you arrangement of atoms
       • Fibre diffraction and cross beta structure
         • Diffraction patterns form specific fingerprint
         • Repeating structure revealed where beta sheets are joined by hydrogen bonds
       • Structure of amyloid
         • Fibrils are made of several protofilaments that are formed by many beta sheet structures
       • Negative design of natural beta proteins
         • Proteins avoid potential beta assembly
           • Edge of beta sheets are protected with beta bulges
         • Beta sandwich proteins
           • Beta bulge causes twist in edge strand preventing hydrogen bonding upon elongation
           • Inward pointing charged side chains
             • Aggregation leads to burial of charge which is energetically unfavourable
           • Helix at the end of beta sheet preventing it from associating with other beta sheets
     • Amyloid fibres consist of a mixture of fibrils
       • Lots of short peptides form amyloid fibrils
       • Fibre diffraction analysis used to see how peptides pack together
       • Crystal structure of amyloid
         • Must be soluble and hetereogenous, in solution and correct environmental conditions
           • To find if the crystal structure is representative of the amyloid fibre oberve under electron microscope
         • Compare 2 crystals structures by comparing fibre diffraction and electron micrographs
   • Alzhiemers and brain degeneration
     • Memory loss and personality changes
     • Most common form of dementia
     • Sporadic AD: patients over 70
     • Familial AD: 30-40 years
     • Large vacuoles appear in brain tissue
     • Accumulation of Alzheimers peptide AB
       • AB cleaved from amyloid precursor protein (APP)
         • APP is a membrane protein
         • Cleaved by secretases
       • Everyone has AB
       • Amount of AB affects the chance of a person developing Alzheimers
       • Monomeric protein
         • When aggregates it results in a toxic oligomer
           • Forms toxic protofibrils
             • Results in amyloid fibres
               • Form amyloid plaques
     • Amyloid plaques made of amyloid fibrils accumulate in the brain
       • Composed of cross beta structure
         • Forms gradually twisting structure
           • Neurofibrillary tangles
             • In cell bodies of neurons
             • Composed of paired helical filaments and tau
             • Associated with neuronal degradation and cell death
       • Extracellular in the neutrophil
     • Less efficient at protein folding with age
     • Genetic predispositions
       • Presenlin 1/2
         • Secretases involved in the production of AB
       • ApoE4
         • More likely to get Alzhiemers than someone with ApoE2
     • Environmental factors
       • Trauma
       • Inflammation

4. Infectious proteins-prions

   • Prions form amyloid fibres
   • Mad cow disease and Creutzfelt Jakob disease (CJD)
   • Normal cellular prion is not a beta sheet structure
     • Normal form can be transformed into infectious by toxic infection
       • Mechanism requires a host protein to be infected
         • Build up leads to increase in aggregation
   • Prion hypothesis
     • A toxic oligomer in one cell can spread to others
   • How is the amyloid toxic and transmitted?
     • Cellular membranes
       • AB has retained the ability to work within the membrane from its precursors
     • Seeding/template aggregation
       • AB needs to move from cell to cell

5. Test for the effects of AB

   • AB affects lipid membranes
     • Use biometric lipid vesicles
       • Vesicles have fluorescent dye inside them which only fluoresces when released
         • AB comes along and disrupts the membrane
           • Fluorescent dye leaks out and this can be measured
             • As AB assembles it becomes less able to permeate membranes
   • Atomic force microscopy of planar membranes
     • Shows permeation by AB
   • AB42 oligomers are toxic to neuronal cells when added and incubated for 24 hours
   • Antibody labelling
     • Show accumulation
   • Electron microscopy
     • AB accumulates on surface of neuroblastoma cells and is internalised