Protein-Misfolding DiseasesPHY6940April 8th 2009

Jessica Nasica

Overview of the presentation

• General facts on protein folding• Causes of misfolding and protein aggregation• Cellular consequences of protein aggregation• Protein-misfolding diseases• Amyloidoses• Amyloid fibril formation• Neurodegenerative diseases and Alzheimer’s disease• Therapy solutions• Curing Alzheimer’s disease

General facts on protein folding

General facts on protein folding

The “new view”

• A stochastic search of the many conformations accessible to one particular sequence

• A polypeptide chain searches for the lowest-energy state by a process of trial and error

• Native-like interactions between residues are more stable than non-native ones

• To undergo correct folding, the native structure must go through a transition state

Possible starting configurations : 10^16

Compact configurations : 10^10

Transition states : 10^3

Native conformation : 1Lowest energy state

only 1 amino acid sequence

General facts on protein folding

The Protein Quality Control (PQC) system

found in ER and cytosol

General facts on protein folding

Role of chaperones• To help proteins in their folding process• To unfold misfolded proteins before their degradation by

the proteasome unit• To protect proteins from interfering interactions during

folding

General facts on protein folding

Chaperones

The concentration of chaperones is genetically self-regulated and increases with the presence of misfolded proteins.

They possess an ATPase domain that reversibly binds with the hydrophobic parts of partially folded proteins

General facts on protein folding

• Important elements for protein folding:

– The amino acid sequence

– The right cellular environment

– The perfect balance between the various folding states

– A fully functional Protein Quality Control (PQC) system

(T ,P ,pH , etc …)

(Chaperones, proteasome unit )

Causes of misfolding and protein aggregation

3 main causes leading to aggregation :• Genetic mutations resulting in a substitution, deletion or

addition of amino acids

• Changes in environmental conditions of the cell leading to dysfunction of the PQC system or the mitochondria and/or the creation of unwanted interactions with folding proteins

• Post-translational accidental changes to the polypeptide after RNA-translation into amino-acids

α-synuclein gene mutations (Parkinson’s)

Causes of misfolding and protein aggregation

How protein aggregates form

• Change in cellular conditions more misfolded proteins the PQC system is overwhelmed -> aggregation is favored

• Aggregation is thought to be set in by protein segments containing hydrophobic amino acids residues, β-sheet predisposition and low net charge.

Causes of misfolding and protein aggregation

States accessible to a protein molecule Free-energy folding landscape for chaperone-mediated protein folding

Causes of misfolding and protein aggregation

Protein aggregation is a 2-stage event

1. The nucleation proteins start attaching reversibly to a growing nucleus

2. Proteins attach irreversibly to the nucleus until it becomes a larger aggregate.

Cellular consequences of protein aggregation

• Loss-of-function pathogenesis: if misfolded proteins are prematurely degraded by PQC system

protein deficiency disease

• Gain-of-function pathogenesis: if misfolded proteins are not eliminated but accumulated instead

disease pathology

toxicity

Some diseases display both pathogenic mechanisms.

Cellular consequences of protein aggregation

the slowing down of polypeptides translation+

Protein-misfolding diseasesinclude conditions where a protein:

• fails to fold correctly (cystic fibrosis, Marfan syndrome, amyotonic lateral sclerosis)

• is not stable enough to perform its normal function (many forms of cancer)

• fails to be correctly trafficked (familial hypercholesterolemia, α1-antitrypsin deficiency)

• forms insoluble aggregates that deposit toxically (neurodegenerative diseases: Alzheimer’s, type II diabetes, Parkinson’s and many more)

Protein-misfolding diseases

Protein-misfolding diseases

Conditions may be :

• Familial the disorder is genetically inherited and symptoms appear during childhood ( e.g., Huntington )

• Sporadic patternless and characterized by a late onset. Primarily due to aging or to an incorrect lifestyle.

Not associated with gene mutations. (e.g., most of Alzheimer’s and Parkinson’s cases and many more)

• Transmissible (e.g., prion disease, spongiform encephalopathies and fatal familial insomnia)

A closer look at amyloidoses

Main family of protein-misfolding diseases most clinically relevant due to the high occurrence of neurodegenerative diseases and type II diabetes

2 types:

• Systemic amyloidoses large amounts of fibrils accumulate everywhere.

• Organ-limited amyloidoses fibrils accumulate locally in one organ ( e.g., brain )

A closer look at amyloidoses

Characterized by the formation, accumulation and deposition of similar highly-organized insoluble fibrillar aggregates amyloid fibrils

AFM image of β2-microglobulin amyloid fibrils

Causes of the formation of amyloid fibrils

Amyloid fibril formation

Structural properties• Insoluble fibrous aggregates• Specific optical behavior (binds to dye “Congo red”) • Highly organized macrostructure a few nm in diameter• Characterized by a cross-β quaternary structure• Cross-β composed of 2-β sheets facing each other,

closely interacting• β-sheets have their β-strands perpendicular to fibril axis

Laser scanning confocal microscopy

Amyloid fibril formation

Mechanism of formation 3 steps:1. Alignment of the molecules to form β-sheets fastest

stage involves H-bonds

2. Formation of the cross-β structure slower than step 1 involves Van-der-Waals forces interdigitation of residues side chains “steric zipper” structure

3. Fibril formation involves non-covalent bonds

Amyloid fibril formation

Amyloid fibril formation is a nucleated-growth process presence of a pre-formed nucleus

rapid growth: additional proteins added more easily once the large entropy barrier is

overcome

Amyloid fibrils are stabilized by the protein concentration and by the formation of steric zippers

Aggregation rates depend on the charge, secondary structure propensities, hydrophobicity and length of the proteins

The efficiency of the PQC system is also very important

Toxicity of amyloid fibrils

annular pores

Schematic representation of structural species during amyloid formation.Protofibrils may form spherical, chain-like or annular pore-like structures to go through the cell membranes

Consequences of amyloid fibril formation

Neurodegenerative diseases

They are localized amyloidoses affecting the brain

& the most-occurring type of age-related PMDs

Neurodegenerative diseases

Aggregation happens in neurons in the brain & spinal cord

deterioration of neurons or their myelin sheath

dysfunction

movement coordination memory loss

impairedSchematic view of a neuron

Alzheimer’s disease most common progressive neurodegenerative disordermassive loss of neurons

Accumulation of Aβ-amyloid protein (extracellular)Accumulation of tau protein (intracellular)

Formation of amyloid plaques (Aβ )Formation of neurofibrillar tangles or NFTs ( tau protein )

Plaques

NFTs

Alzheimer’s disease

Effects on the brain

Brain shrinkage

Alzheimer’s disease

Resulting symptoms

• Memory impairment• Inability to think by themselves• Inability to function independently

Parkinson’s disease

2nd most common neurodegenerative disease due to aging

Degeneration of specific dopaminergic neurons in the substantia nigra

Accumulation of α-synuclein protein in presence of dopamine

Other neurons are not affectedΑ-synuclein accumulating neurons

Parkinson’s disease

Resulting symptoms

• Muscular rigidity extreme pain• Postural instability• Resting tremor

Huntington’s disease

• Polyglutamine disease mutation encoding for an addition of Q amino-acids

• Accumulation of the misfolded protein Huntingtin• Formation of toxic inclusions in brain cells• Degeneration of glutamatergic striatal neurons

polyQ inclusionIn neocortex

Therapeutic solutions

3 main approaches:

1. Inhibition of protein aggregation

2. Interference with post-translational peptide changes before the misfolding/aggregation step

3. Upregulation of molecular chaperones or aggregate-clearance mechanisms

Therapeutic solutions

1.

Inhibition of amyloid ß (Aß) fibril neurotoxicity by laminin.

Therapeutic solutions

2.

• Targeting Aβ-formation by inhibiting β- and γ- secretase proteins responsible for the formation of amyloid plaques

• Inhibition of tau protein phosphorylation (hyperphosphorylation of tau protein is responsible for its aggregation)

Therapeutic solutions

3.

Clonidine and Minoxidil enhance the clearance of aggregate prone proteins, including mutant Huntingtin and mutants of α-synuclein

Curing Alzheimer’s disease

Disease affecting 37 million people worldwide

In 40 years, 1 in 85 people will develop AD

Current drug research aims at preventing Aβ-formation and blocking the formation of amyloid plaques to slow the progression of the disease

A few programs targeting the tau protein

Curing Alzheimer’s disease

Preventing Alzheimer’s disease

Thank you !