1. Protein-Misfolding Diseases PHY6940 April 8 th 2009 Jessica Nasica

2. 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

3. General facts on protein folding

4. 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 : 1 Lowest energy state only 1 amino acid sequence

5. General facts on protein folding The Protein Quality Control (PQC) system found in ER and cytosol

6. 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

7. 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

8. 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 )

9. 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)

10. 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.

11. Causes of misfolding and protein aggregation States accessible to a protein molecule Free-energy folding landscape for chaperone-mediated protein folding

12. 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.

13. 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.

14. Cellular consequences of protein aggregation the slowing down of polypeptides translation +

15. Protein-misfolding diseases include 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)

16. Protein-misfolding diseases

17. 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)

18. 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 )

19. 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

20. Causes of the formation of amyloid fibrils

21. 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

22. 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

23. 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

24. 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

25. Consequences of amyloid fibril formation

26. Neurodegenerative diseases They are localized amyloidoses affecting the brain & the most-occurring type of age-related PMDs

27. Neurodegenerative diseases Aggregation happens in neurons in the brain & spinal cord deterioration of neurons or their myelin sheath dysfunction movement coordination memory loss impaired Schematic view of a neuron

28. Alzheimer’s disease  most common progressive neurodegenerative disorder  massive 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

29. Alzheimer’s disease Effects on the brain Brain shrinkage

30. Alzheimer’s disease Resulting symptoms Memory impairment Inability to think by themselves Inability to function independently

31. Parkinson’s disease  2 nd 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 affected Α-synuclein accumulating neurons

32. Parkinson’s disease Resulting symptoms Muscular rigidity  extreme pain Postural instability Resting tremor

33. 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 inclusion In neocortex

34. 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

35. Therapeutic solutions 1. Inhibition of amyloid ß (Aß) fibril neurotoxicity by laminin.

36. 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)

37. Therapeutic solutions 3. Clonidine and Minoxidil enhance the clearance of aggregate prone proteins, including mutant Huntingtin and mutants of α-synuclein

38. 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

39. Curing Alzheimer’s disease

40. Preventing Alzheimer’s disease

41. Thank you !