1. Protein conformational disorders
Alice Skoumalová

2. Protein structure
Primary structure is determined by the amino-acid sequence
Secundary structure is given by the spatial arrangement of the peptide strand
α-helix where the hydrogen bonds are within the same strand
β-sheets that are formed of alternating peptide pleated strands linked by hydrogen bonding between one strand and another
Tertiary structure represents the final arrangement of the protein

3. 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

4. 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

5. Loss of biological function
α-helix
β-sheet
Conformational change
the starting point is the natural protein folded in the native and active conformation
normal protein is rich in α-helix conformations (folded structure)
the end-point is the same protein adopting prevalent β-sheets structure
it is disease-associated protein (misfolded structure)
Aggregation
Gain of toxic activity
Loss of biological function

6. Protein misfolding causes disease!
the hallmark event in PCD is a formation of β-sheet conformations
the production of β-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

7. Diseases Protein involved Alzheimer‘s disease Amyloid-β
Parkinson disease
α-Synuclein
Diabetes type 2
Amylin
Amyotrophic lateral sclerosis
Superoxide dismutase
Haemodialysis-related amyloidosis
β2-microglobulin
Cystic fibrosis
Cystic fibrosis transmembrane regulator
Sickle cell anemia
Hemoglobin
Hungtington disease
Huntingtin
Creutzfeldt-Jakob disease
Prion protein
Amyloidosis
Ten other proteins

8. Polymerization hypothesis Conformational hypothesis
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)

9. 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 β-sheet oligomers
These oligomers by further growth produce amyloid fibrils
Conformational changes trigger oligomerization that is essential for the stabilization of protein misfolding

10. Proteins that are not able to achieve the native state:
Neurodegenerative diseases
Recognition
Prion diseases
Degradation (protein quality control system)
1.Chaperones
2. Ubiquitin proteasome system

11. Accumulation (Amyloidoses)
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)

12. Protein quality control in the cell
Interplay of molecular chaperones and proteases in the cell. Substrate proteins are shown in red; an ATP-dependent chaperone, such as GroEl, is shown in bluse; a Clp chaperone is purple and is associated with a compartmentalized protease shown in green.
Molecular chaperones play a critical role in protein quality control during the course of cell growth as well as during stress conditions. Normal protein synthesis produces nascent unfolded proteins. Although some nascent polypeptides are able to fold spontaneously (1), others require the action of molecular chaperones, icluding members of the DnaK/Hsp70 and GroEL/Hsp60 families, to facilitate folding (2). Unfolded and misfolded proteins also arise in cells as a result of environmental stresses, such as heat shock, or pathologic conditions, such as inflammation, tissue damage, infection, and genetic diseases involving mutant proteins. Molecular chaperones are able to refold and reactive some misfolded proteins (2). Ther irreversibly misfolded proteins are recognized by the proteasome. These multicomponent proteases use associated chaperones to unfold and deliver damaged proteins to the proteases for degradation (3). Finally, proteins that are neither refolded nor degraded form insoluble aggregates in the cell (4). Aggregates are not always an end product, but can be dissolved by molecular chaperones.

13. 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.
Chemical chaperones influence the protein folding environment inside the cell, stabilize proteins against thermal and chemical denaturation (glycerol).
Pharmacological chaperones bind to specific conformations and stabilize them. They are effective in rescuing proteins from proteasomal degradation.

14. 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)

15. Alzheimer disease a progressive degenerative disease of the brain
the extracellular deposition of β amyloid (A)
the neuropathological feature
the key molecule in the pathology of AD
resulting from the proteolytic processing of a membrane-bound -amyloid precursor protein (APP)
in the brain of dementing patients, A-amyloidosis is found in senile plaques and in the blood vessels
the misfolded protein is rich in -sheet conformation - formation of -sheets is usually stabilized by protein oligomerization or aggregation - the misfolded protein becomes deposited in amyloid-like aggregates

16. Amyloid β (Aβ) is formed after sequential cleavage of the amyloid
Amyloid β (Aβ) is formed after sequential cleavage of the amyloid precursor protein ; an aggregation and accumulation of Aβ (amyloid plaques in Alzheimer disease); pathogenesis of AD

17. 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 β-sheet sructure, designed peptides prevent and reverse β-sheet formation (β-sheet breakers)
Molecular chaperones play an important role in protein folding,
chemical and pharmacological chaperones are experimentally studied

18. Questions The hallmark event in PCD and consequences
Some examples of PCD
The fate of a misfolded protein in the cell; the role of chaperons
The pathogenesis of Alzheimer disease

19. Summary Protein misfolding leads to the conformational changes
Misfolded protein causes disease
Protein misfolding is the primary cause of Alzheimer disease, Parkinson disease, diabetes mellitus, cystic fibrosis, amyloidoses
Therapy aims to reverse conformational changes