Protein conformational disorders Amyloid

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Protein conformational disordersAmyloid
Alice Skoumalová
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• Hypothetical protein folding pathway
• (hierarchical)
• local segments of secondary structure
• tertiary structure (subdomains, domains)
• stable conformation

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Local minimum (alternative conformation)
Global minimum (native state)

• the protein folding proceeds from a disordered
  state to progressively more ordered
  conformations corresponding to lower energy
  levels
• there are more ways of folding (the same protein
  can aquire more conformations alternative
  conformations are represented by local energy
  minima)

Alternative conformations various function of
the protein disease-associated protein
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a-helix
ß-sheet
Conformational change

• the starting point is the natural protein folded
  in the native and active conformation
• normal protein is rich in a-helix conformations
  (folded structure)

• the end-point is the same protein adopting
  prevalent ß-sheets structure
• it is disease-associated protein (misfolded
  structure)

Aggregation
Gain of toxic activity
Loss of biological function
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• The conformational change
• a change in the secundary or tertiary structure
  of a normal protein without alteration of the
  primary structure
• the biological function of a protein depends on
  its tridimensional structure
• Protein conformatinal disorders (PCD)
• diverse diseases arise from protein misfolding
• the conformational change may promote the disease
  by either gain of a toxic activity or by the
  lack of biological function of the natively
  folded protein

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• Protein misfolding causes disease!
• the hallmark event in PCD is a formation of
  ß-sheet conformations
• the production of ß-sheets is usually stabilized
  by protein oligomerization and aggregation
• the misfolded protein self-associates and becomes
  deposited in amyloid-like aggregates in diverse
  organs, inducing tissue damage and organ
  dysfunction

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Three different hypotheses have been proposed to
describe the relationship between conformational
changes and aggregation
Polymerization hypothesis Aggregation induces the
protein conformational changes
Conformational hypothesis Protein misfolding is
independent of aggregation, which is a
non-necessary end point of conformational
changes (the factors inducing the protein
structural changes are e.g. mutations, oxidative
stress)
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Conformation-oligomerization hypothesis Slight
conformational changes result in the formation of
an unstable intermediate which is stabilized by
intermolecular interactions with other molecules
forming small ß-sheet oligomers
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Proteins that are not able to achieve the native
state
Recognition
Degradation (protein quality control
system) 1.Chaperones 2. Ubiquitin proteasome
system
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Protein quality control in the cell
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DNA
Ubiquitin
Ribosome
RNA
ATP
Chaperones
Native protein
Misfolded protein
Aggregate/fibrillar amyloid
Chaperones
Proteasome
Accumulation (Amyloidoses)
Degraded protein
Gain of toxicity (Alzheimer disease)
Loss of protein function (Cystic fibrosis)
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• Implication of protein misfolding
• 1. Gain of toxicity
• The harmfull effect of the misfolded protein may
  be due to deleterious gain of function as seen in
  many neurodegenerative disorders (Alzheimer
  disease, Parkinson disease, Hungtington disease),
  in which protein misfolding results in the
  formation of harmfull amyloid. Neurodegenerative
  diseases are characterized by the accumulation of
  misfolded proteins and formation of aggregates
• 2. Loss of function
• Other effect of the misfolded protein may be due
  to loss of function, as observed in cystic
  fibrosis. There is a mutation in the CFTR
  sequence
• 3. Accumulation
• Protein aggregates are sometimes converted to a
  fibrillar structure. Fibrils themselves are not
  toxic but insoluble. Their accumulation cause
  tissue damage (amyloidoses)

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• Chaperones
• assist other proteins to achieve a functionally
  active 3D structure
• prevent the formation of a misfolded or
  aggregated structure
• Molecular chaperones recognise misfolded protein,
  bind to the hydrophobic surfaces and inhibit
  aggregation. Most of these molecules are heat
  shock proteins (formed during thermal
  damage)-protect against denaturation.
• Pharmacological chaperones bind to specific
  conformations and stabilize them. They are
  effective in rescuing proteins from proteasomal
  degradation.

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Molecular chaperones
Hsp 70 - prevent folding of nascent chain
Chaperonins reverse misfolded structures
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• Therapy
• Considering that protein misfolding and
  aggregation are central in the pathogenesis of
  PCD, a therapy directed to the cause of the
  disease should aim to inhibit and reverse the
  conformational changes
• Development of novel peptides which can
  destabilize the abnormal conformation might be
  useful to correct protein misfolding. Misfolded
  protein is rich in ß-sheet sructure, designed
  peptides prevent and reverse ß-sheet formation
  (ß-sheet breakers)
• Molecular chaperones play an important role in
  protein folding,
• chemical and pharmacological chaperones are
  experimentally studied

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• Amyloid
• Amyloid is an aggregated protein sructure
  consisting of unbranched microscopic fibrils
  often found in dense tissue deposits and
  associated with a variety of human diseases
• The term amyloid does not pertain to a specific
  protein molecule or sequence, but rather to a
  general folding motif that appears in various
  proteins
• The amyloid structure exhibit a characteristic
  folding pattern, called a cross- ß structure
• Amyloid is a pathogenic structure, formed by
  accident under conditions of molecular, cellular,
  or organismic stress, from proteins that evolved
  to fold and function in different structural
  states

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Polypeptide Major disease states Transthyretin
Heart, kidney, peripheral neuropathy Serum
amyloid A Kidney, peripheral neuropathy Immunogl
obulin light chain Kidney, heart Immunoglobulin
heavy chain Spleen ß2-Microglobulin Carpal
tunnel syndrome, osteoarthropathies Islet
amyloid polypeptide Diabetic pancreatic islet
cells Fibrinogen a-chain Kidney Apolipoprotein
A1 Peripheral neuropathy, liver Atrial
natriuretic peptide Heart Amyloid ß-protein
(Aß) Brain (Alzheimers disease, cerebral
amyloid angiopathy) a-Synuclein Brain
(Parkinsons disease) Huntingtin polyglutamine
Brain (Huntingtons disease) sequence Prion
protein (PrP) Brain (Creutzfeldt-Jakob
disease, mad cow disease) Cystatin C,
Gelsolin Brain (cerebral amyloid
angiopathy) ABri Brain (familial British
dementia)
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• Molecular factors in amyloid formation
• Protein misfolding is central to amyloid
  formation
• Protein stability- the resistance of the folded
  conformation to misfolding- is an important
  factor in determining susceptibility to amyloid
  formation
• Destabilizing factors
• 1. Extreme environments in the body, such as
  acidic cell compartments
• 2. Proteolytic removal of a portion of a protein
  by an endogenous protease
• 3. Mutations that alter the primary structure
  (many of the amyloid diseases involve amino acid
  substitutions in an amyloid precursor protein)

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• Amyloid fibril structure
• Straight, unbranched, diameters in the range of
  80-160A
• Composed of two to six protofilaments of diameter
  30-40A
• Rich in a type of ß-sheet structure (the ß-sheets
  are perpendicular to the fibril axis and bind
  together by the hydrogen bonds)

ß2-microglobulin amyloid fibrils
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• Overview of amyloid diseases (amyloidosis)
• Systemic amyloidosis
• 1. Primary
• The cause is unknown abnormal production of
  immunoglobulins insoluble protein fibers are
  deposited in tissues and organs, impairing their
  function The organs affected tongue, intestines,
  skeletal and smooth muscles, nerves, skin,
  ligaments, heart, liver, spleen, and kidneys
• 2. Secondary
• Caused by infection, inflammatory diseases, and
  sometimes cancer
• 3.Familial
• Mutations that make the proteins more prone to
  aggregation and amyloid deposition (e.g.
  transthyretin)
• Organ-specific amyloidosis
• Diabetes mellitus type 2 (amylin)
• Alzheimers disease (Aß)
• Parkinsons disease (a-synuclein)
• Huntingtons disease (huntingtin)
• Transmissible spongioform encephalopathies (prion
  protein)
• Cardiac amyloidosis (PrP or transthyretin)

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• Toxicity of amyloid fibrils
• 1. Amyloid can cause life-threatening disease by
  accumulating in such high mass that normal tissue
  structure and function are disrupted (systemic
  amyloidosis)
• 2. The accumulated mass of amyloid is very low
  compared to the surrounding cell mass
  (neurodegenerative diseases)
• 1. Collateral damage caused by immune responses
  to an amyloid deposits
• 2. Membrane depolarization resulting from
  channels created by amyloid fibril assembly
  intermediates inserted into membranes
• 3. Recruitment of other proteins into growing
  aggregates, which has the effect of denying the
  cell activity of the recruited proteins
• 4. Disruption of the normal cellular apparatus
  for breakdown and elimination of misfolded
  proteins, such as the ubiquitin-proteasome system
  and the molecular chaperones

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Questions

• Describe the protein folding funnel
• The hallmark event in PCD and consequences
• The fate of a misfolded protein in the cell
• The role of chaperons
• Amyloid - formation, toxicity

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Pictures used in the presentation Marks Basic
Medical Biochemistry, A Clinical Approach, third
edition, 2009 (M. Lieberman, A.D.
Marks) Principles of Biochemistry, Third Edition,
2008 (D.J. Voet, J.G. Voet, C.W. Pratt)