Local Protein Unfolding and Pathogenesis of PolyglutamineExpansion Disease

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Local Protein Unfolding and Pathogenesis of
Polyglutamine-Expansion Disease

• Yu Wai Chen
• Centre for Protein Engineering and Cambridge
  University Chemical Laboratory, Cambridge, United
  Kingdom
• Proteins 20035168-73
• speaker S.C.Fu
  24/04/03

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What is Polyglutamine-Expansion Disease?

• Polyglutamine-Expansion
• Abnormal lengthening of polyglutamine (polyQ)
  repeat sequence inside the protein.
• Inherited neurodegenerative disease such as
  Huntingtons disease and spinocerebellar ataxia
  (SCA) have been shown to be caused by polyQ
  repeat.

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Elongation of CAG repeat

• Petruska et. al. 1998

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These Inherited neurodegenerative diseases

• Causing misfolding and aggregation of disease
  proteins.
• Expansion of Glutamine repeat gt 3540 residues
  confers on disease proteins a toxic gain of
  function that cause tissue-specific neuronal
  death.
• Progress via a similar pathogenic mechanism.
• Apart from polyQ repeat, these proteins and their
  genes have nothing in common.

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Hypothesis

• PolyQ disease proteins may share a common feature
  of being natively unfolded and are thus
  susceptible to misfolding and aggregation.

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Unfolded
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• Proteins that are natively unfolded are
  characterized by high net charge and low
  hydrophobicity.
• They found that for a given protein with a mean
  net charge of ltRgt, a boundary value exists for
  its mean hydrophobicity, ltHgt, below which the
  protein is likely to be natively unfolded. This
  empirical relationship is expressed as

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Materials and Methods

• Dataset (from the SWISS-PROT database)
• Huntingtons disease protein (or huntingtin, Htt,
  P42858)
• androgen receptor (AR, P10275)
• atrophin-1 (Atf1,also known as dentatorubral-palli
  doluysian atrophy protein,P54259)
• spinocerebellar ataxin-1 (Atx1, P54253),
• ataxin-2 (Atx2, Q99700)
• ataxin-3 (Atx3, also known as Machado-Joseph
  disease protein, MJD, P54252)
• ataxin-6(Atx6, O00555)
• ataxin-7 (Atx7, O15265)
• sperm whale myoglobin (Mb, P02185)
• The TATA-box-binding protein (TBP or TF2D,
  referred to as Atx17 here, P20226)

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Materials and Methods

• Using ProtParam program from ExPASy for net
  calculation.
• ProtScale is used to calculate hydrophobicity.

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ProtParam
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Global protein folding prediction

• blue dots (excluding polyQ)
• red dots (including the polyQ)

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Result of global and local protein folding
prediction
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Local protein folding prediction

• blue dots (excluding polyQ)
• red dots (including the polyQ)

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Compared with experimental observation

• An artificial system to study the structural
  consequences of introducing repeats of 12, 28,
  35, and 50 glutamines into sperm whale myoglobin
  (Mb).
• Mb-12
• Mb-28
• Mb-35
• Mb-50

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Compared with experimental observation
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Compared with experimental observation

• The studies of secondary structures of several
  fusion constructs of Atx3 using circular
  dichorism (CD) spectroscopy
• Atx3 and maltose-binding protein fusions
  (MBP-Atx3-Q27 and MBPAtx3-Q78)
• hexahistidine-tagged ataxin-3 (H6-Atx3-Q27)

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Circular dichorism

• Circular dichroism is the difference in
  absorption between left and right handed circular
  polarized light.
• is used to gain information about the secondary
  structure of proteins and polypeptides in
  solution.
• very sensitive to the secondary structure of
  polypeptides and proteins.

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Standard Curves
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Example of CD
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Compared with experimental observation
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Discussion

• Contribution
• Limitation of this method
• Need more information about domain boundary.
• Ligands or ions that affect local folding should
  be considered.
• Limited in applying to proteins w/ multiple
  domain.

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Simulation of Unfolding
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Thanks for your attention