Protein conformational disordersAmyloid

Alice Skoumalová

Hypothetical protein folding pathway:

(hierarchical)

local segments of secondary structure

tertiary structure (subdomains, domains)

stable conformation

Global minimum(native state)

Local minimum(alternative conformation)

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 proteindisease-associated protein

α-helixβ-sheet

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)

Conformational change

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

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)

Polymerization hypothesis

Aggregation induces the protein conformational changes

Three different hypotheses have been proposed to describe the relationship between conformational changes and aggregation

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

Misfolded protein

Mutation(familial forms)

Error in the folding process(sporadic)

Degradation (protein quality control system)

1.Chaperones

2. Ubiquitin proteasome system

Recognition

Proteins that are not able to achieve the native state:

Protein quality control in the cell

DNA

RNA Ribosome

Native protein Misfolded protein

Chaperones

Ubiquitin

ATP

Degraded protein

Loss of protein function (Cystic fibrosis)

Proteasome

Aggregate/fibrillar amyloidChaperones

Gain of toxicity (Alzheimer disease)

Accumulation (Amyloidoses)

Implication of protein misfolding

1. Gain of toxicityThe 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 functionOther 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. AccumulationProtein 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.

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

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

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- β“ 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

Polypeptide Major disease states

Transthyretin Heart, kidney, peripheral neuropathy

Serum amyloid A Kidney, peripheral neuropathy

Immunoglobulin light chain Kidney, heart

Immunoglobulin heavy chain Spleen

β2-Microglobulin Carpal tunnel syndrome,

osteoarthropathies

Islet amyloid polypeptide Diabetic pancreatic islet cells

Fibrinogen α-chain Kidney

Apolipoprotein A1 Peripheral neuropathy, liver

Atrial natriuretic peptide Heart

Amyloid β-protein (Aβ) Brain (Alzheimer‘s disease,

cerebral amyloid angiopathy)

α-Synuclein Brain (Parkinson‘s disease)

Huntingtin polyglutamine Brain (Huntington‘s disease)

sequence

Prion protein (PrP) Brain (Creutzfeldt-Jakob disease,

mad cow disease)

Cystatin C, Gelsolin Brain (cerebral amyloid angiopathy)

ABri Brain (familial British dementia)

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)

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 β-sheet structure (the β-sheets are perpendicular to the fibril axis and bind together by the hydrogen bonds)

β2-microglobulin amyloid fibrils

Overview of amyloid diseases (amyloidosis)

Systemic amyloidosis1. PrimaryThe 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. SecondaryCaused by infection, inflammatory diseases, and sometimes cancer3.FamilialMutations that make the proteins more prone to aggregation and amyloid

deposition (e.g. transthyretin)

Organ-specific amyloidosisDiabetes mellitus type 2 (amylin)Alzheimer‘s disease (Aβ)Parkinson‘s disease (α-synuclein)Huntington‘s disease (huntingtin)Transmissible spongioform encephalopathies (prion protein)Cardiac amyloidosis (PrP or transthyretin)

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 deposits2. Membrane depolarization resulting from channels created by amyloid fibril

assembly intermediates inserted into membranes3. 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

Questions

1. Describe the protein folding funnel

2. The hallmark event in PCD and consequences

3. The fate of a misfolded protein in the cell

4. The role of chaperons

5. Amyloid - formation, toxicity

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)