Protein conformational disorders Alice Skoumalová

Hypothetical protein folding pathway: (hierarchical) local segments of secondary structure tertiary structure (subdomains, domains) stable conformation

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

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

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

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

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

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)

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

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

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)

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)

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.

Molecular chaperones Hsp 70 - prevent folding of nascent chain Chaperonins – reverse misfolded structures

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

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

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

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

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