SOD1ALS link:

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Molecular Mechanisms of CuZnSOD-Linked ALS
Lecture 1 SOD1-ALS link Gain of
function mechanism Protein aggregation
vs. oxidative damage Chemistry of superoxide
and other ROS Lecture 2 ALS Clinical
aspects SODs and SOD mechanisms Lecture 3
Model studies in cell culture and ALS Tg mice
and rats WT and mutant SOD1
structures-CONTINUED Lecture 4 Protein
aggregation and disease Oxidative stress in
ALS? Lecture 5 Biophysical properties of WT
and mutant SOD1s
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Three FALS SOD1 Metal-Binding Region Mutant
Structures (Green or Blue) Superimposed on
Wild-Type SOD1 (Gray)
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S134N Subunit A mixture of Cu and Zn ions in
the Cu-binding site and zinc alone in the
Zn-site. Subunit B Zn in the Cu- site and no
metal in the Zn-site. (1.3 Å, Hart lab)
Zn-H46R Zinc alone in the Zn-site. (2.15 Å,
Hasnain lab)
apo-H46R (2.5 Å, Hart lab)
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(No Transcript)
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Metal-bound Native SOD1 Cannot Form Linear
Filaments (Negative Design)
Zn Loop residues
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S134N and apo H46R SOD1 Linear Filaments
Elam, J. S., et al., Amyloid-like filaments and
water-filled nanotubes formed by SOD1 mutant
proteins linked to familial ALS, Nat Struct Biol
10, 461-7 (2003).
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S134N FALS SOD1 Gain-of-Interaction
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Molecular Mechanisms of CuZnSOD-Linked ALS
Lecture 1 SOD1-ALS link Gain of
function mechanism Protein aggregation
vs. oxidative damage Chemistry of superoxide
and other ROS Lecture 2 ALS Clinical
aspects SODs and SOD mechanisms Lecture 3
Model studies in cell culture and ALS Tg mice
and rats WT and mutant SOD1
structures Lecture 4 Protein aggregation and
disease Oxidative stress in ALS? Lecture 5
Biophysical properties of WT and mutant SOD1s
8
Aggregation of Proteins in Neurodegenerative
Diseases
Taylor JP, Hardy J, Fischbeck KH. (2002) Science
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Aggregation of misfolded proteins in
microscopically visible inclusions or plaques in
various neurodegenerative diseases. (A)
Alzheimer's disease. Arrowhead, intracellular
neurofibrillary tangles arrow, extracellular
amyloid plaque. (B) Fibrillar tau inclusions in
Pick's disease. (C) PrPSc amyloid deposition in
prion disease. (D) Multiple Lewy bodies in a
nigral neuron in Parkinson's disease. (E)
Neuronal intranuclear inclusions of mutant
ataxin-3 in Machado-Joseph's disease. (F) Higher
power micrograph of nuclear inclusion of mutant
ataxin-3, demonstrating that it is distinct from
the nucleolus. Magnification, 40.
Toxic Proteins in Neurodegenerative Disease, JP
Taylor et al., Science, 296, 1991-1995, 2002
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M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
11

• Protein Misfolding Diseases
• A specific protein may be unable to carry out its
  normal function because it is improperly folded
  or because it is not sufficiently stable due to
  misfolding.
• A protein may be unable to carry out its normal
  function because it is not trafficked to the
  proper location due to misfolding.
• A protein may fail to fold or to remain folded
  correctly and consequently aggregate (often with
  other components) leading to amyloid diseases.
  (Amyloidosis refers strictly to diseases in
  which extracellular deposits are formed, but the
  terms amyloid diseases or protein aggregation
  diseases are now being used to refer to diseases
  in which protein deposits are intra- or
  extracellular.)
• Some of the clinical symptoms of the
  non-neurological amyloidoses seem to be due to
  the accumulation of large deposits of aggregated
  proteins in vital organs
• In neurodegenerative diseases, cellular function
  appears to be impaired by the interaction of
  aggregated proteins with cellular components.
  This impairment is associate with evidence of
  elevated oxidative stress, but the mechanism is
  unknown.

M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
12
A schematic energy landscape for protein folding.
The surface funnels the multitude of denatured
conformations to the unique native structure. The
critical region on a simple surface such as this
one is the saddle point corresponding to the
transition state, the barrier that all molecules
must cross if they are to fold to the native
state. The yellow spheres in this ensemble
represent the three key residues in the
structure when these residues have formed their
native-like contacts the overall topology of the
native fold is established.
CM Dobson, Protein folding and misfolding,
Nature, 426, 884-890 (2003)
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The natural equilibrium of proteins
Schematic representation of the equilibria
existing between different conformational states
of a protein in a cell
Calloni, G. et al. J. Biol. Chem.
200528010607-10613
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CM Dobson, Protein folding and misfolding,
Nature, 426, 884-890 (2003)
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Many protein folds, one amyloid core structure
M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
16
Misfolded proteins are normally detected and
cleared from cell (or stored in aggresomes)
Regulation of protein folding in the ER. Many
newly synthesized proteins are translocated into
the ER, where they fold into their
three-dimensional structures with the help of a
series of molecular chaperones and folding
catalysts (not shown). Correctly folded proteins
are then transported to the Golgi complex and
then delivered to the extracellular environment.
However, incorrectly folded proteins are detected
by a quality-control mechanism and sent along
another pathway (the unfolded protein response)
in which they are ubiquitinated and then degraded
in the cytoplasm by proteasomes.
CM Dobson, Protein folding and misfolding,
Nature, 426, 884-890 (2003)
17
M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
18
MY Sherman and AL Goldberg, Cellular Defenses
against Unfolded Proteins A Cell Biologist
Thinks about Neurodegenerative Diseases, Neuron,
Vol. 29, 1532, January, 2001,
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MY Sherman and AL Goldberg, Cellular Defenses
against Unfolded Proteins A Cell Biologist
Thinks about Neurodegenerative Diseases, Neuron,
Vol. 29, 1532, January, 2001,
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MY Sherman and AL Goldberg, Cellular Defenses
against Unfolded Proteins A Cell Biologist
Thinks about Neurodegenerative Diseases, Neuron,
Vol. 29, 1532, January, 2001,
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General mechanism of aggregation to form amyloid
fibrils
Unfolded or partially unfolded proteins associate
with each other to form small, soluble aggregates
that undergo further assembly into protofibrils
or protofilaments (a) and then mature fibrils
(b). The fibrils often accumulate in plaques or
other structures such as the Lewy bodies
associated with Parkinsons disease (c). Some of
the early aggregates seem to be amorphous or
micellar in nature, although others form
ring-shaped species with diameters of
approximately 10 nm (d).
CM Dobson, Protein folding and misfolding,
Nature, 426, 884-890 (2003)
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Some amyloid-related peptides/proteins form early
aggregates of globular appearance that further
organise into beaded chains, globular annular
'doughnut' shaped assemblies eventually giving
mature protofilaments and fibrils. Pre-fibrilar
aggregates may interact with reconstituted
phospholipid membranes and with cell membranes
where they form aspecific channels (pores)
disrupting cellular homeostasis. The latter
possible mechanism of toxicity is similar to that
displayed by antimicrobial peptides, pore-forming
eukaryotic proteins and bacterial toxins and
newly synthesised cyclic peptide antibiotics.
M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
23
Precursors of amyloid fibrils can be toxic to
cells
Amyloid aggregates of the N-terminal domain of
the bacterial hydrogenase maturation factor HypF,
a protein unrelated to any amyloid disease, are
cytotoxic in their pre-fibrillar organisation.
Protein samples at differing times and stages of
aggregation were added to the culture medium of
NIH-3T3 or PC12 cells. Only globular,
pre-fibrillar aggregates displayed cytotoxicity,
whereas mature fibrils were substantially
harmless.
M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
24
M Stefani and CM Dobson, Protein aggregation and
aggregate toxicity new insights into protein
folding, misfolding diseases and biological
evolution, J. Mol. Med 81678-699 (2003)
25
M Stefani, Protein misfolding and aggregation
new examples in medicine and biology of the dark
side of the protein world, BBA 1739 (2004) 5-25.