1

Protein folding and diseases of
misfolding

Helen Saibil
Crystallography and
Ìnstitute of Structural and Molecular Biology
Birkbeck College London
Textbook: Biochemistry, Berg, Tymoczko & Stryer
7
th
edition, WH Freeman
How do our proteins get assembled into the right
shape so that they can carry out the functions we
need for life?
What happens if protein assembly goes wrong?
What are diseases of protein misfolding, and what
is amyloid?
Central Dogma
DNA RNA Protein Protein
sequence fold
Proline cis-trans isomerisation
Disulfide bond formation
Glycosylation
Proteolysis
FOLDÌNG
Globular soluble protein
Stable and soluble
Functional
Unfolded or misfolded proteins
Aggregation-prone
Non functional
Protein folding is necessary for function
Protein misfolding causes disease
Hydrophilic amino acid
Hydrophobic amino acid
Unfolded protein chains are prone to proteolysis
Protein folding

A linear chain of amino acids goes from a random set of
dynamic conformations in solution to its native, folded and
functional structure.
What drives this process?
The folded state should have a lower free energy:
G=H-TS
where G is change in Gibbs free energy, H is change in
enthalpy, T is temperature, and S is change in entropy. A
reaction will go spontaneously in the direction of lower free
energy. Enthalpy reflects the contributions from chemical
bonds. Making bonds lowers the enthalpy. Entropy reflects the
degree of disorder. Loss of disorder is energetically
unfavourable.

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A folded protein is only slightly more stable than its unfolded form
The amino acid sequence of a protein
determines its native, 3-dimensional structure

Under suitable conditions, a protein will fold spontaneously to
its native state, which normally has the lowest free energy.

However, at physiological concentrations and temperature,
many proteins would become trapped in misfolded states or in
aggregates. The cell has an elaborate system of quaIity
controI machinery that helps proteins to fold, not by providing
steric information, but by preventing or rescuing them from
misfolded and aggregated states.

Protein quality control is carried out by moIecuIar chaperones
(folding helpers) and by reguIated proteoIysis.
Bovine pancreatic ribonuclease A
The native fold has 4 disulfide bonds
Figure: DW Brooks, Univ of Nebraska-Lincoln
The Anfinsen experiment on RnaseA:
Ribonuclease sequence determines its fold
Aggresomes: Cellular rubbish dumps
Kolodziesjka et al, PNAS 2005 Kopito, TÌCB, 2000
When it all goes wrong
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Folding intermediate Folded Reversible aggregate
Ìrreversible aggregate
(amyloid)
Protein misfolding causes disease
Loss of function disease: cystic fibrosis
Gain of function disease: (toxicity) Alzheimers, Parkinsons
Misfolding of cystic fibrosis transmembrane conductance regulator (CFTR)
vacuole
Nucleus
An aggregate of amyloid fibres: Yeast prions
Cell membrane
Ribosomes/
cytoplasm
Prion
aggregate
The stress response

When cells are exposed to elevated temperatures or other
stresses, e.g. osmotic stress, toxic chemical such as alcohols, an
emergency programme of gene expression is initiated ÷ the heat
shock response or stress response. Normal protein synthesis is
replaced by elevated expression of a small group of proteins known
as heat shock proteins or molecular chaperones. Chaperones bind
to unfolded or misfolded proteins, prevent their aggregation and
help them to refold to the native state.

Regulated proteolysis

Proteins that are irreversibly misfolded, damaged or mutated are
targetted to the proteolysis machinery (the proteasome) for
degradation.
Protein Quality Control
Folding and degradation pathways
Proteasome
Ubiquitin-
labelled
protein
Amyloid Diseases

A family of degenerative, and so far, incurable diseases
Amyloid diseases of the brain cause loss of memory and
cognitive ability, movement disorders and emotional
disturbances, depending on the particular brain regions
affected.
They include Alzheimer's, Parkinson's, Huntingdon's and
the prion diseases.
The prion diseases are the only ones known to be
transmissible.
Amyloid diseases in other organs are also incurable and
lethal.
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What is amyloid?
Characteristic fibrillar protein deposits contain carbohydrate whose
staining properties led to the name amyloid (starch-like) in the mid 19th
century.
A normally innocuous soluble protein or peptide polymerizes into
insoluble fibrils whose accumulation is associated with cell death.
Polymerization occurs if high concentrations of a suitable, partially
folded intermediate accumulate, so these diseases tend to involve
abundant proteins.
Known amyloid diseases involve around 20 unrelated proteins or
peptides in a conformational change that leads to a very similar fibril
assembly.
The conformational change thought to take place in the acidic
environment of the lysosome or endosome.
Clinical syndrome Fibril subunit Precursor
structure
Monoclonal protein systemic (AL) amyloidosis All or part oI V
L
domain oI Ig light chain All
Reactive systemic (AA) amyloidosis 76-residue N-term. Iragment oI amyloid A /
Familial amyloidotic polyneuropathy All or part oI transthyretin variants All
Hereditary apoA1 amyloidosis N-terminal Iragments oI apoA1 variants Predicted /
Hereditary lysozyme amyloidosis Full-length lysozyme variants ¹
Type II diabetes mellitus 37-residue Iragment oI islet-associated
polypeptide
Unknown
Alzheimer`s disease A protein residues 1-39 to 43 , or coil
Insulin-related amyloid Full-length wild-type insulin ¹
Transmissible spongiIorm encephalopathies Full-length or Iragments oI prion protein ¹
Medullary carcinoma oI the thyroid Fragments oI calcitonin Unknown
Senile systemic amyloidosis Full-length or Iragments oI WT transthyretin All
Hemodialysis-related amyloidosis Full-length wild-type
2
-microglobulin All
Isolated atrial amyloidosis Atrial natriuretic Iactor Unknown
Hereditary cerebral amyloid angiopathy 110-residue Iragment oI variant cystatin-C ¹
Finnish hereditary amyloidosis 71-residue Iragment oI gelsolin variants /
Hereditary Iibrinogen -chain amyloidosis Fragments oI Iibrinogen -chain variants Unknown
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Diagnostic appearance in tissue sections
Amyloid fibril structure
Amyloid fibrils are rope-like structures with varying numbers of
protofilaments wound together.
There is a common core structure of the protofilament ÷ the cross-
structure.
-strands are arranged in long ribbons,
with the strand direction perpendicular
to the fibril axis, at a spacing of 4.7 Å.
The ribbons are packed against each
other with an inter-sheet spacing of
about 10 Å.
The 4.7 Å spacing along the fibre axis
and the 10 Å spacing perpendicular to
the fibre axis are observed by X-ray
fibre diffraction.
Example of an antiparallel
cross- structure
500 Å
Negative stain EM images and
model of insulin amyloid fibrils
What causes cell death in amyloid disease?
The mechanism of toxicity is not understood
The fibrils are often found in the extracellular space, but they
may form inside the cell, which then dies.
The toxic species may be fibril precursors or intermediates in
fibril formation and breakdown, rather than the fibrils themselves.
Certain aggregates are cytotoxic, possibly by damaging
membranes or stimulating apoptotic pathways.
The amyloidogenic protein may be a variant whose sequence
predisposes it to undergo the conformational change, probably by
destabilizing the native structure.
Ìn some cases, the normal wild-type protein slowly forms
amyloid and disease only manifests itself in the very elderly.
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Lysozyme
Two variants of lysozyme cause systemic amyloidosis and lead to
death by age 40.
The effects of the mutations on the 2 domain lysozyme structure is
mainly to destabilize the interface between the domains, facilitating
partial unfolding.
The mutant proteins are less stable
than the wild type, and the unfolding
transition is uncooperative, unlike the
wild type.
This results in a significant population
of the partially folded state, which is
thought to be the precursor to amyloid
formation.
domain domain
Lysozyme amyloid fibre
Transthyretin
Transthyretin (TTR) is a tetrameric -sheet protein that transports
thyroxine and also binds and transports the retinol binding protein.
Dissociation of the tetramer and partial unfolding at low pH can
induce amyloid fibril formation in vitro. The wild-type protein can
slowly convert into amyloid, causing senile systemic amyloidosis,
with heavy amyloid deposits in the heart leading to congestive heart
failure. Ìn addition to this slow conversion of the wild-type protein,
there are many variant TTRs that can cause severe amyloid disease
with early onset. The native structure is not
altered, but is destabilized in the variants,
so that they more readily unfold in mildly
acidic conditions.
Thyroxine binding stabilizes the native
tetramer, and similar small molecule ligands
provide potential therapeutics, by stabilizing
the native form.
Transthyretin dimer
Alzheimer's Disease (AD)
AD is the most common cause of dementia, the progressive decline of cognitive
processes that affects about 20% of people over 75.
Extracellular plaques of the Amyloid- peptide and intraneuronal tangles of
hyperphosphorylated tau protein (a microtubule associated protein) in the brains of
AD patients are associated with neuronal death and loss of cholinergic pathways.
AD is not usually inherited, but some genetic factors predispose individuals to the
disease and influence the age of onset. Most of the genes known to be involved
play a role in the metabolism of APP (-amyloid precursor protein), a
transmembrane protein which is cleaved by proteases termed secretases to
release the A- peptide.
A- is normally 40 residues long, but abnormal cleavage produces A- 1-42, which
has a strong tendency to form amyloid.
APP is on chromosome 21, which is disrupted in Down's syndrome. Down's
patients develop the symptoms of AD by their 40s.
Other genes known to be involved in early onset of AD code for 2 closely related
transmembrane proteins, the presenilins, possibly involved in the secretory
pathway. Ìn addition, the allele of the lipid transporter apolipoprotein E influences
the risk and age of onset of AD.
Brain pathology and peptide processing in Alzheimer's Disease
Neurofibrillar tangles are
another type of deposit found in
AD brains. These aggregates
consist of an aggregated,
hyper-phosphorylated form of
the microtubule-associated
protein Tau.
Prions
The prion diseases (transmissible spongiform encephalopathies) appear to
be unique among the amyloid diseases in that they are transmissible. Ritual
cannibalism practiced by the Fore people of New Guinea spread the fatal
disease kuru through the population.
Creutzfeld Jacob disease (CJD) has been transmitted to patients receiving
human growth hormone, and about 200 people died of variant CJD, derived
from mad cow disease, itself propagated in cows via contaminated feed in
the 1980s.
The prion protein Prp is a brain glycoprotein on the cell
surface (GPÌ-anchored) that may play a role (so far
unknown) in synaptic function. Ìn its normal (cellular)
form, Prp
C
, it has a mainly -helical structure. The -
sheet containing Prp
SC
(the scrapie form, named after the
original animal disease) from diseased brain can induce
Prp
C
in previously healthy individuals to convert to the
scrapie form, by an unknown mechanism.
Prp knockout mice are not susceptible to infection.
The structure of Prp
C
,
residues 97-231
Huntingdon's Disease (HD)
A group of severe neurodegenerative diseases are caused by expansion
of glutamine repeats (CAG codon).
The protein huntingtin (>3000 residues, with long stretches of glutamine
and also proline repeats near the N terminus) is found in intranuclear
deposits of degenerating neurons. Ìt is essential for embryonic neural
development.
Ìndividuals with less than 38 repeated glutamines do not develop the
disease, but those with over 40 glutamines do, in precise correlation with
its in vitro aggregation into amyloid-like fibrils and ribbons.
Ubiquitin is usually associated with the aggregates. Misfolded proteins are
targeted for degradation by ubiquitination, which targets them to the
proteasome, but the aggregates are resistant to degradation.
Glutamine repeats engineered onto an unrelated protein produce
neurotoxic aggregates when expressed in Drosophila. Neurodegeneration
is reduced by overexpression of the chaperone Hsp70, or by deletion of a
pro-apoptotic gene, but increased by defects in the ubiquitin-proteasome
system.
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Parkinson's Disease (PD)
PD, and the related Lewy body disease cause movement abnormalities
and dementia. Lewy body disease accounts for about 20% of dementia
cases.
Lewy bodies are diffuse intracellular deposits of the protein -synuclein, a
highly conserved synaptic protein (named for both synaptic and nuclear
localization) related to apolipoproteins.
-synuclein appears to be involved in synaptic plasticity, and is abundant in
the brains of songbirds during song development. The most conserved
region of the protein is predicted to form an amphipathic helix, consistent
with a role in binding lipids or vesicles. This region of the sequence has
only 2 residues different between canary and man.
The distribution of the protein in brain is similar to that of -amyloid in AD,
and peptides derived from -synuclein are also found in A- plaques. The
mechanism of this association is unknown, since the plaques are
extracellular and the synuclein is intracellular. As with the other
neurodegenerative diseases, 80% of the neurons in the affected brain
areas can be lost before symptoms appear.

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