Protein misfolding

Protein misfolding

Overview
1. When does it go wrong?
  1. Protein synthesis
  2. Protein degradation
2. Why does it go wrong?
  1. Mutation in protein
  2. Lack of enzyme needed for correct folding
  3. Accumulated damage to protein
  4. Conformational change
    1. Conformational diseases
      1. If a protein misfolds it should be removed by degradation
3. What happens in disease as a result?
  1. Protein is non-functional
  2. Protein is lacking or in short supply
  3. Protein is in the wrong place
  4. Aggregates accumulate causing disruption
    1. Aggregation
      1. Protein self-assembles and forms aggregates
Protein misfolding and disease: Aggregation and degradation
1. Protein is no longer in the right place to be functional
2. Misfolding occurs during protein synthesis and folding causing loss of function
3. Cystic fibrosis
  1. 1/2000 live births
  2. Autosomal recessive
  3. 200 different mutations
  4. CFTR
    1. A plasma membrane chloride channel in epithelial cells
    2. An ABC transporter
    3. An enzyme that utilises ATP
    4. Many components needed to fold for the protein to be functional
      1. Hence lots of mutations
      2. 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
    5. Deletion of F508
      1. CFTR folds poorly
        Degraded in endoplasmic reticulum
      2. F508 usually forms a hydrophobic pocket and is important in protein folding
      3. Mutant F508 CFTR accumulates in endoplasmic reticulum
        Does not attach to plasma membrane
    6. Molecular consequences of CFTR mutations
      1. No CFTR synthesis
        Non sense mutation
        Deletion of TT
        Change in splice junction
      2. Block in processing
        Missense mutation
        AA deletion
      3. Block in regulation
        Missense mutation
      4. Altered conductance
        Missense mutation
      5. Reduced synthesis
        Missense mutation
        Alternative splicing
4. Serpins and disease
  1. Serpins
    1. Serine protease inhibitors
    2. Alpha-1-antitrypsin
    3. Antithrombin
    4. Cell conserved structure
      1. 3 beta sheets
      2. 9 alpha helices
    5. Reactive centre loop
      1. Negatively unfolded
  2. Serpin mechanism
    1. Protease will cleave reactive centre loop
      1. Inhibitor changes molecule conformation
        Reactive centre loop becomes beta strand
        Protease is denatured
        Conformational change opens up the protein for misfolding
  3. Serpin polymerisation
    1. Occurs in conformational diseases
    2. Alpha-1 ant-trypsin is a protease inhibitor in the lungs
      1. Without protease the lungs are susceptible to emphysema
    3. Serpin polymers
      1. alpha1-antitrypsin forms filaments which have beads on a string appearance
      2. Hyperthermostable
      3. Polymer formation is irreversible
      4. Alpha1-antitrypsin is made in the liver
        Transported to lungs
        Forms aggregates
        Causes liver disease
        Loss of function in lungs causes emphysema
  4. Alpha-1 anti-trypsin deficiency
    1. Acute phase protein
      1. Overexpressed following inflammation
    2. 1 in 20 hetereozygous
    3. Homozygotes suffer from emphysema and liver disease
    4. Z alpha1-anti-trypsin
      1. 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
    5. Circular dichroism shows structures of alpha1 and zalpha1
      1. Very similar
      2. Tertiary structure has aromatic residues in different environments
Protein misfolding and disease: Aggregation and acculumulation
1. Disease related to accumulation of deposited material and disruption of tissues
2. Amyloid
  1. Insoluble fibrils formed by polymerisation of normally soluble proteins
    1. Deposited in tissue
  2. Large number of proteins can form amyloid fibrils
    1. Proteins are all different to each other and cause different diseases
  3. Amyloid formation involves a conformational change in the native protein
  4. What makes a native protein aggregate form amyloid in disease?
    1. Mutations
      1. Destabilises structure making them more likely to form amyloid fibrils
    2. High concentration
    3. Infections
  5. Amyloid fibrils formed from different proteins have similar structures
    1. Congo red staining
    2. EM
      1. Shows long, straight, unbranching fibrils
    3. X-ray fibre diffraction
      1. Shows charcteristic cross beta diffraction pattern
  6. Amyloid fibrils can be defined in different ways
    1. Structure of proteins
      1. Grow a crystal and use diffraction pattern to tell you arrangement of atoms
    2. Fibre diffraction and cross beta structure
      1. Diffraction patterns form specific fingerprint
      2. Repeating structure revealed where beta sheets are joined by hydrogen bonds
    3. Structure of amyloid
      1. Fibrils are made of several protofilaments that are formed by many beta sheet structures
    4. Negative design of natural beta proteins
      1. Proteins avoid potential beta assembly
        Edge of beta sheets are protected with beta bulges
      2. 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
  7. Amyloid fibres consist of a mixture of fibrils
    1. Lots of short peptides form amyloid fibrils
    2. Fibre diffraction analysis used to see how peptides pack together
    3. Crystal structure of amyloid
      1. 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
      2. Compare 2 crystals structures by comparing fibre diffraction and electron micrographs
3. Alzhiemers and brain degeneration
  1. Memory loss and personality changes
  2. Most common form of dementia
  3. Sporadic AD: patients over 70
  4. Familial AD: 30-40 years
  5. Large vacuoles appear in brain tissue
  6. Accumulation of Alzheimers peptide AB
    1. AB cleaved from amyloid precursor protein (APP)
      1. APP is a membrane protein
      2. Cleaved by secretases
    2. Everyone has AB
    3. Amount of AB affects the chance of a person developing Alzheimers
    4. Monomeric protein
      1. When aggregates it results in a toxic oligomer
        Forms toxic protofibrils
        Results in amyloid fibres
        Form amyloid plaques
  7. Amyloid plaques made of amyloid fibrils accumulate in the brain
    1. Composed of cross beta structure
      1. 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
    2. Extracellular in the neutrophil
  8. Less efficient at protein folding with age
  9. Genetic predispositions
    1. Presenlin 1/2
      1. Secretases involved in the production of AB
    2. ApoE4
      1. More likely to get Alzhiemers than someone with ApoE2
  10. Environmental factors
    1. Trauma
    2. Inflammation
Infectious proteins-prions
1. Prions form amyloid fibres
2. Mad cow disease and Creutzfelt Jakob disease (CJD)
3. Normal cellular prion is not a beta sheet structure
  1. Normal form can be transformed into infectious by toxic infection
    1. Mechanism requires a host protein to be infected
      1. Build up leads to increase in aggregation
4. Prion hypothesis
  1. A toxic oligomer in one cell can spread to others
5. How is the amyloid toxic and transmitted?
  1. Cellular membranes
    1. AB has retained the ability to work within the membrane from its precursors
  2. Seeding/template aggregation
    1. AB needs to move from cell to cell
Test for the effects of AB
1. AB affects lipid membranes
  1. Use biometric lipid vesicles
    1. Vesicles have fluorescent dye inside them which only fluoresces when released
      1. 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
2. Atomic force microscopy of planar membranes
  1. Shows permeation by AB
3. AB42 oligomers are toxic to neuronal cells when added and incubated for 24 hours
4. Antibody labelling
  1. Show accumulation
5. Electron microscopy
  1. AB accumulates on surface of neuroblastoma cells and is internalised

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	Criado por sophie_connor mais de 11 anos atrás