Molecular Mechanisms of Protein Folding and Misfolding Molecules and Cells

A. Importance of Proteins as the Machines of the Cell 1. The Inner Life of the Cell (proteins are the stars!) http://multimedia.mcb.harvard.edu/anim_innerlife_hi.html

2. The cell is a crowded protein-rich environment

Component cell membranes & associated proteins cytoplasm nuclear material blood serum proteins

Color green blue & purple red & orange yellow & brown

Terry Oas Department of Biochemistry Rm. 230C Nanaline Duke [email protected] 684-4363

3. Definition of Protein Folding The process by which unfolded polypeptides discover their lowest energy native conformation, either in or out of the cell.

4. Anfinsen experiment a) Used chemical denaturants (urea and guanidinium chloride) to unfold purified ribonuclease and reducing agent to break disulfide bonds. b) Removed denaturant and allowed disulfides to re-form. c) Obtained high yields of active ribonuclease with correct disulfide bonds. d) Proteins contain all of the information necessary to determine their three dimensional structure within their amino acid sequence. e) Native structure is the lowest energy state under native conditions. Science 181: 223 (1973)

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B. Why Protein Folding is Difficult and Complex 1. Structural Complexity of the Amino Acids

a) Individual amino acid residues can be very important. (1) Patients who are homozygous for the deletion of phenylalanine 508 in the CFTR protein account for 70% of all cystic fibrosis cases.

http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/cftr.shtml

(2) Many genetic diseases arise from single site (missense) amino acid substitutions that preclude proper folding (e.g. sickle cell anemia: glutamic acid 6 →valine in Hemoglobin β chain) 3

b) Levinthalʼs Paradox (1) 150 residue protein has 10300 possible conformations! In a typical folding time can only sample 108 conformations. 10300 >> 1077 (number of atoms in universe) (2) Most proteins fold in seconds, giving time to sample only 108 conformations. (3) Solution: sequence-determined pathways (4) Perhaps Anfinsen was wrong- if proteins canʼt sample all of their possible conformations, how do they know that the native state is the lowest energy state?

2. Chemical Complexity of the Amino Acids a) Hydrophobicity (1) Tendency of apolar compounds (some amino acid side chains) to avoid solvation by water. (2) Burial of hydrophobic surface area generally thought to be the primary driving force of protein folding. (3) Hydrophobic surface area can be burying by multimolecular association. b) Hydrogen bonding (1) H-bonds formed by backbone groups (secondary structure) and between polar side chains. (2) Provide some stability and specificity (3) Almost always form between buried polar residues. (4) Form at intermolecular interfaces.

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3. Multiple folding pathways via intermediates a) Folding Funnels

Ann. Rev. Biochem. 59: 631-60 (1990) Nat. Struct. Biol. 4: 10-19 (1997)

(1) Intermediate-free protein folding funnel: • each conformation maps to a different x-y coordinate • z coordinate (vertical height) corresponds to the energy of the conformation • bottleneck: transition state of folding reaction

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(2) Levinthalʼs funnel: • no downhill slope to guide folding chain to N • like trying to hit a golf ball to the hole on a flat green blindfolded

(3) A funnel with partially folded intermediates: •

some (but not all) protein molecules will fold to local minima and may be kinetically trapped there

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lifetime of the intermediate determined by the height of the barrier out of the local minimum

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energy landscape of an unevolved protein (“frustrated”)

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many kinetic traps, no way to find the global minimum 6

b) Dead-end Pathways (1) Molten globule intermediates: secondary structure without fixed tertiary interactions. (2) Partially disulfide bonded intermediates (3) Cis/trans proline isomerization (4) Intermolecular association of partially folded intermediates

C. When proteins get into trouble: Misfolding of partially folded intermediates 1. Efficient folding of most proteins is hard and often fails in vivo a) Variety of pathogenic states caused by misfolding b) Crowded environment of the cell encourages misfolding and aggregation Proc. Natl. Acad. Sci. 18: 1107 (1997)

2. Properties of partially folded intermediates a) b) c) d) e) f)

some regions of backbone highly disordered solvent accessible surface area greater than native conformation radius smaller than fully denatured conformation (collapsed) exposed hydrophobic surface area incomplete tertiary structure sometimes non-native interactions

3. Reactions involving partially folded intermediates a) b) c) d) e)

folding to the native conformation unfolding to denatured conformation(s) folding to a long-lived (kinetically trapped) non-native conformation aggregation proteolytic degradation (ubiquination →proteosome, ERAD)

4. Role of cellular chaperones a) Provide an dynamic polarity environment for monomeric folding b) Prevent association of partially folded intermediates c) Some chaperones (e.g., Hsp104) can dissagregate 7

D. Aggregation: Intermolecular Association 1. Symmetric self-association a) dimerization

b) oligomerization c) aggregation

d) fibrillation

2. Amorphous Precipitation/Aggregation a) Inclusion body formation b) Refractory to cellular proteases

"Aggresome" formation promoted by cellular machinery

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E. Two pathogenic outcomes of protein misfolding a) Gain of toxicity b) Loss of function

FEBS Letters 498:204-207 (2002)

Although aggregation is associated with many protein misfolding events, it may not always be the cause of disease

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F. Molecular Basis of Protein Misfolding Diseases Clinical Syndrome

Protein

Parkinsonʼs disease

α-Synuclein

Spongiform encephalopathies (kuru, CJD)

Prion protein

Huntingtonʼs disease

Huntingtin

Familial amyotrophic lateral sclerosis (ALS)

Superoxide dismutase

Chronic obstructive pulmonary disease

α1-Antichymotrypsin

Cystic fibrosis

CFTR protein

Sickle cell anemia

Hemoglobin

Amyloidosis (see below)

numerous

1. Two forms of protein misfolding disease a) spontaneous b) genetic: earlier onset & more severe

Mutations that destabilize the native conformation more than a partially folded intermediate can lead to pathogenic conformations.

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2. Amyloid diseases

a) Identification of amyloid (1) First identified in the form of plaques in brains of Alzheimer's patients (2) Histology: Binding of small molecule dyes: congo red, thioflavins and maromolecules such as amyloid P component b) Forms of amyloid (1) amorphous aggregates (2) profibrils (3) fibrils (4) filaments 11

c) Is amyloid causative or an epiphenomenon? (1) Amyloid hypothesis: Amyloid deposits are cytotoxic and are the direct cause of neuronal cell death and other pathologies. Evidence: Factors that increase the amyloidgenecity of a protein also produce more pathology. (2) Not yet clear how fibril formation induces cell death and extent of observed amyloid deposition doesnʼt always correlate with the severity of the disease. d) Structure of fibrils

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e) Models of fibril formation

FEBS Letters 498:204-207 (2001) 13

3. Prion Diseases Transmissible spongiform encephalopathies (Creutzfeldt-Jakob Disease) a) Correlated with prion protein (PrP), which can be transmitted from one individual to another b) "Prion Hypothesis", Stanley Prusiner:

c) Crosses species barriers: BSE ("Mad Cow") → Spontaneous CJD d) Also found in familial forms. PrPC (non-scrapie form)

PrPSC (scrapie form)

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4. Polyglutamine expansion diseases a) Subset of trinucleotide expansion diseases b) Caused by expansion of (CAG)n repeats c) Neurodegenerative Diseases Disease

Protein Affected

Huntington's

Huntingtin

Spinobulbar muscular atrophy (Kennedy's)

Androgen Receptor

Number of Repeats Normal Disease 6 -35 37 - 100 9 - 36

38 - 62

Symptoms dementia, involuntary movements, abnormal posture progressive muscle weakness and atrophy, male infertility

d) polyQ tracts are found at the N-terminus of huntingtin e) Polar zipper hypothesis (Perutz)

PNAS 99: 5596 (2002)

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f) The greater the number of repeats, the earlier the onset of disease

Aggregation of KKQnKK repeat peptides at high and low concentrations. (a) Experimental results (symbols) and simulations of KKQ36KK aggregation at three high concentrations. (b) Simulated aggregation progress of KKQ28KK, KKQ36KK, and KKQ47KK at cellular concentration. There is good correlation with age-of-onset for patients with similar polyQ repeats in their huntingtin protein. PNAS 99: 11884 (2002) g) Huntingtin aggregates are sequestered in nucleus and have profound transcriptional effects. h) Aggregates stimulate apoptosis (programmed cell death) via caspase-1.

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G. Early Therapeutic Approaches to Protein Misfolding 1. General Strategies:

NEUROLOGY 66:S118 (2006)

a) Limiting the expression or availability of the protein substrate molecules that stabilize the amyloidogenic protein precursor. b) Stabilizing the native protein conformation to inhibit conversion. c) Destabilizing the misfolded protein conformation to enhance clearance. d) Stimulating the existing cellular clearance mechanism to reduce concentrations of the misfolded product

2. Mechanisms to interfere with protein aggregation/ amyloid formation a) β-sheet breaker peptides b) small molecule inhibitors that disrupt β structure

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3. Favoring non-amyloidogenic forms over amyloid precursors in TTR

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H. Further Reading 1. Excellent current summary of the most pertinent questions (and some answers) in the field of protein misfolding: http://www.nature.com/horizon/proteinfolding/keyquestions.html 2. Reviews and papers on the role of chaperones and protein degradation pathways in the formation of amyloid a) b) c) d) e) f) g) h) i) j) k)

Trends in Molecular Medicine 8: 411(2002) EMBO Journal 22: 355 (2003) Science 295: 1852 (2002) Nature 416: 507 (2002) Nature 426: 826 – 910 (2003): Excellent series of minireviews on protein misfolding diseases Structure 11: 243 (2003) Interesting proposal for the mechanism by which monomeric proteins might aggregate. FASEB J. 18: 617 (2004) Comparison of AD and Parkinson's. Trends Mol Med 8: 370-4 (2002) Connection between atherosclerosis and misfolding. NEUROLOGY 66: S118 (2006) A review of pharmaceutical strategies against misfolding. Proc Natl Acad Sci 102: 10427-32 (2005). A proposal that amyloids cause ionic channel formation in cell membranes that disrupts ionic homeostasis leading to cell death. Current Opinion in Structural Biology 20: 54-62 (2010) A description of the structure based design of TTR amyloidosis inhibitors. Some prospective drugs are in Stage II & III human clinical trials. The most promising therapeutic approach to any amyloid disease to date.

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