Protein Folding

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Created by
Beth

Cards (39)

  • ATP synthase
    • made of protein
    • molecular motor found in the mitochondria
    • responsible for making ATP
    • rotation of a turbine synthesises ATP
    • Two motors
  • Actin forms filaments for muscle contraction.
  • Immunoglobulins fight infection
  • Insulin hexamer controls glucose levels.
  • Disease can result from absent, inactive, overactive, mis-located or malfunctioning proteins. All stages of the process need to be error free
    • DNA - needs to be without mutations
    • RNA - needs to be properly transcribed without error
    • Protein - needs to be translated, folded and located in the correct part of the cell or outside it
  • Central dogma of biology
    • DNA transcripted to RNA (via RNA polymerase)
    • RNA translated to protein (via ribosome)
  • Protein synthesis at the ribosome
    • Ribosomes read mRNA (messenger) and assemble a chain of amino acids, joined by peptide bonds
    • A specific tRNA (transfer) binds each triplet of RNA bases, leaving behind the amino acids for which they code
  • Primary, secondary and tertiary structure of proteins
    1.      String of amino acids
    2.      Alpha helices and beta sheets
    3.      Functional domains
  • Quaternary structures
    • Potassium channel
    • Has four identical chains each 97 amino acids long
    • Antibody
    • Has four amino acid chains, two short and two long
    • G-protein
    • Has three non-identical amino acid chains
  • Lipid bilayer forms the basic structure of cell membrane
    • Most membrane functions are carried out by membrane proteins
    Membrane proteins include:
    • Enzymes (adenylyl cyclase)
    • Receptors (EGFR/PDGFR)
    • Anchors (integrins)
    • Transporters
    • CFTR (a chloride transporter is a membrane protein).
    • The translocon in endoplasmic reticulum is essential for membrane proteins
  • CFTR
    • CFTR has 1480 amino acids and 12 alpha helices that cross the membrane
    • It also has a globular domain on the inside face of the membrane
  • Protein trafficking through the cell
    • Inserted into the endoplasmic reticulum via the translocon where it is folded and modified then transported to the cell membrane via the Golgi apparatus
    • The translocon is a protein pore in the ER membrane through which proteins are synthesised
    • The ribosome sits on top of it and the new protein chain is inserted
  • Membrane proteins enter membrane at the endoplasmic reticulum
    • Facilitated by Signal recognition peptide
    • SRP binds to signal sequence of newly synthesised peptide as it emerges from ribosome
    • Pauses protein synthesis until complex find SRP receptor on endoplasmic reticulum membrane and docks.
    • New peptide chain inserted into translocon channel where it enters endoplasmic reticulum.
    • Protein synthesis will then resume once the SRP has been released from the ribosome
  • Five main steps in CFTR synthesis
    • Transcription
    • Translation and protein folding
    • Post-translational modification
    • Protein trafficking
    • Surface expression
    • Cells have an inbuilt quality control mechanism to deal with misfolded and aggregated proteins
    • For membrane proteins like CFTR this control process happens in the ER (where they are made)
    • If a protein is misfolded but salvageable then it will be refolded by chaperones in the ER
    • If a protein is terminally misfolded, it is passed back through the ER membrane (retrotranslocated) into the cytoplasm where it is degraded by protease enzymes
    • Proteins that won’t function properly are NOT inserted into the cell membrane
  • Endocytosis is a key mechanism in regulating the density of membrane proteins and anything that interferes with these pathway as well as the function of CFTR when its at the cell surface can cause cystic fibrosis. After endocytosis there are two options for the CFTR protein
    o   Either recycled back to cell membrane
    o   Targeted for degradation
  • CFTR Class 1
    • No functional CFTR protein
    • Nonsense, frameshift, canonical splice
  • CFTR Class 2
    o   CFTR trafficking deficit
    o   Missense, amino acid deletion
  • CFTR Class 3
    o   Defective channel regulation
    o   Missense, amino acid change
  • CFTR Class 4
    o   Decreased channel conductance
    o   Missense, amino acid change
  • CFTR Class 5
    o   Reduced synthesis of CFTR
    o   Splicing defect, missense
  • CFTR Class 6
    o   Decreased CFTR stability
    o   Missense, amino acid change
  • the most common cause of CF is the p.Phe508del mutation
    • This is a deletion of three DNA bases which results in a missing F Phenylalanine (F) amino acid at position 508 in the protein
    • Just one missing F results in a protein that cannot fold correctly
    • This misfolded CFTR is recognised by the cell’s quality control machinery and held in the endoplasmic reticulum (ER) and then is degraded
    • P.Phe508del cannot perform its function (as it doesn’t reach the plasma membrane of the cell) and this results in the symptoms of CF
  • Membrane protein quality control
    • Chaperone proteins like BiP and calnexin are in the ER to hold back misfolded proteins
    • There is also much slower turnover of correctly folded membrane proteins
  • Until recently treatment for CF has been limited to targeting the secondary effects of the defective CTFR
    o   Bronchodilators, antibiotics, corticosteroids
    o   Pulmozyme, insulin, bisphosphonates
    o   Vaccinations and flu jabs
    o   Enzymes, diet and nutrition
    o   Physiotherapy, airway clearance
    o   Lung transplantation
    These treatments are for symptoms of CF they do not address the cause of CF, a defective CFTR protein
  • Fixing the defective CFTR protein depends on the effect of the CFR mutation of the CFTR protein
    • Reduction in protein function
    • Class 3 and 4 mutations
    • Potentiators
    • Reduction in amount
    • Class 1,2,5,6 mutations
    • Correctors
    • Production correctors
  • Potentiators
    • Increase the activity of defective CFTR at the cell surface. Potentiators can either act on gating or conductance defects
    • Open and close the channel
  • Production correctors Instruct ribosomes to read-through premature termination codons (PTCs) during mRNA translation
  • VX-770
    • Also called Ivacaftor = Kalydeco
    • Identified by high throughput screening
    • a potentiator
    • binds to CFTR channel, increases opening time and restores chloride transport
  • Clinical effects of ivacaftor on patients with at least one class 3 CFTR mutation
    • Increase (~10%) in FEV1 (lung function)
    • Weight gain (~2.8kg)
    • Decreased pulmonary exacerbations (~50%)
    • Decreased sweat chloride concentration
    • Decreased endobronchial colonisation with P. aeruginosa
  • Channel regulation/conductance mutations are very rare so limited patient impact – class 2 more common.
    -        Most common CFTR mutation = p.Phe508del mutation – a class 2 mutation
  • Targeting the p.Phe508del mutation
    • VX-809 – lumicaftor
    • Identified in same screening as VX-770
    • A corrector
    • Targets p.Phe508del CFTR proteins
    • Increases the number of CFTR proteins that are trafficked to the cell surface
    Lumicaftor did not show the outstanding success of Ivacaftor
    • Excellent results in vitro
    • Improved function of sweat duct but
    • No improvement in lung function
    Results led to a new trial of a lumicaftor AND ivacaftor
  • Ivacaftor + Lumicaftor = Orkambi
    • Hypothesised that p.Phe508del CFTR need to a combination of lumicaftor and ivacaftor to get the CFTR to the cell surface and the facilitate channel opening and closing
    • The combination was clinically effective
    o   Reduced pulmonary exacerbations
    o   Increased BMI
    o   Increased FEV1
  • Problem with Orkambi
    -        Lumicaftor (corrector) appears to induce the CYP3A4 enzyme which is involved in metabolising Ivacaftor (reducing the amount available)
  • Two correctors better than one
    -        Shown in vitro two correctors
    -        Distinct binding sites on CFTR
    -        Complementary mechanism of action
    -        Increases the amount of Phe508del CFTR protein at the cell surface to a greater extent than either corrector alone
    -        Tezacaftor, VX-445 and VX-659 = correctors
  • Alzheimer’s disease
    o   Acetylcholinesterase inhibitors
    o   Prevent acetylcholinesterase from breaking down acetylcholine in the brain
    o   Increased concentration of acetylcholine leads to increased communication between nerve cells
    o   Temporarily alleviate or stabilise some symptoms of Alzheimer’s disease
    o   Amyloid plaques and neurofibrillary tangles
  • Protein folding diseases – therapeutic strategies
    • Amyloid plaques
    o   Amyloid beta peptides
    o   Proteolytic processing of APP protein
    o   BACE1 and gamma secretase enzymes.
    • Alzheimer’s specific therapeutic targets
    o   Inhibiting BACE1
    o   Inhibiting secretase
    • Other targets:
    o   Inhibit protein production, inhibit aggregation of misfolded proteins, increased clearance of misfolded proteins, pharmacopherone drugs
    • Understanding more about how particular proteins fold can help us develop novel treatments
    • Computer simulations can help with this – although some information is needed from lab experiments, predictions can be made about how proteins fold