Computational%20Method%20for%20%20Predicting%20Amyloidogenic%20Sequences%20%20%20Bill%20Welsh%20UMDNJ-%20Robert%20Wood%20Johnson%20Medical%20School

1
Computational Method for Predicting
Amyloidogenic Sequences Bill WelshUMDNJ-
Robert Wood Johnson Medical School
welshwj_at_umdnj.edu
2
Amyloid Fibril Formation A Common Mechanism
for Protein Misfolding Diseases

• Numerous amyloid misfolding diseases
• All of them are incurable at present
• Short list of more familiar examples
• Alzheimers disease
• Parkinsons disease
• Huntingtons disease
• Crutzfeld-Jakob disease (Mad Cow)
• Familial Amyloidosis
• Type II Diabetes
• Triggered by short sequences that convert from
  native a-helix or coil to b-strand
• We call this trait hidden b-strand propensity

3
Problems

• No sequence specificities
• Absence of detailed structural information on
• misfolded proteins (amyloid fibrils)

4
Intriguing Relationship Between Tertiary
Contacts and Secondary Structure
Tertiary Contact (TC)
Two non-H atoms ?4Å apart separated by more than
4 residues in sequence
Based on SCOP20v1.57
Striking Conclusion
a-helix dominates in low-TC regions b-sheet
dominates in high-TC regions
5
TC Influence on Secondary Structure Propensity
Average Tertiary Contacts (TCs) in SCOP20
L S A Q E K D N R I F T M P G W C V Y H
9.9 8.3 6.4 10.2 8.6 8.0 9.1 10.6 15.9 11.0 18.5 9.3 11.9 7.0 6.5 25.9 12.7 10.1 21.7 16.2
6
The CSSP Algorithm Locating Sequences Exhibiting
HbP
Similar Sequences
Database of gt450,000 7-residue sequences with
secondary structure TCs
7
Sensitivity of the CSSP Method Cameleon Sequences
1AMP
1GKY
Aminopeptidase
Guanylate kinase
ASVKQVS in a-helix
ASVKQVS in b-sheet
8
Hidden ß-propensity in Alzheimers Disease
KLVFF are key residues in amyloid fibril
polymerization (Tjernberg et al., JBC 1996)

• Amyloidogenic wild type Aß fragment

Helix Beta Coil
Propensity
Strong
Moderate

• Non-amyloidogenic mutant Aß fragment

Weak
Very weak
Yoon and Welsh, Protein Science (2004) ibid.,
Proteins (2005)
9
hIAPP sequence (Type 2 Diabetes)
-NFLVH- -FLVHS- Mazor et al., JMB (2002)
-NFGAIL- Zanuy, Nussinov, et al. Biophysical
Journal (2003)

• hIAPP sequence (4-34) associated with type II
  diabetes

NAC sequence (Parkinsons disease)
Bodles et al., J Neurochem (2001)

• NAC sequence of a-synuclein associated with
  Parkinsons disease

10
Beta propensity of acetylcholinesterase (AChE)
and its homolog butyrylcholinesterase (BuChE)
Cottingham et al., Biochemistry (2002) ibid.,
(2003) AChE586-599 and BuChE573-586

• Amyloidogenic AChE586-599 fragment

• Nonamyloidogenic BuChE573-596 fragment

11
Amyloid Formation by G334VMutant p53 Associated
with Lung Cancer

• Higashimoto et al, Biochemistry 45, 1608-1619
  (2006)

12
Amyloidogenic Sequence Knowledge Base (ASKB)

• CSSP Algorithm that predicts
• Hidden b-Strand Propensity
• in Proteins Polypeptides
• Searchable peptide database

http//askb.umdnj.edu/askb/welcome.html
13
Estimating Free Energies
Unfolded
Partially Folded
a-helix
b-strand
b-rich amyloid
Random coil
14
Predicted vs. Exptl b-Sheet Structure of Prion
Protein Peptide

• Decatur and coworkers employed FTIR spectroscopy
  to determine b-sheet structure for peptides
  based on residues 109-122 of the Syrian hamster
  prion protein (H1) substituted at position 117.
• We plotted our calculated HbP metrics for the
  sequences H1, A117G, A117V, A117L, and A117I vs.
  Decaturs exptl values.
• Strong correlation (R20.96) suggests that
• calculated HbP profiles are excellent
  predictors
• of b-sheet nature.

SA Petty, T Thorsteinn, SM Decatur,
Biochemistry 444720-4726 (2005)
15
(No Transcript)
16
Thank You!welshwj_at_umdnj.edu
General Observations and Implications

• The CSSP algorithm successfully pinpoints
  amyloidogenic sequences in numerous examples
  where exptl data are available
• These sequences possess hidden b-strand
  propensity
• generally short sequences (4-7 residues) that
  serve as core nucleation motifs to trigger
  amyloid fibril formation
• adopt a-helix in low contact regions (low TC) and
  b-strand in high contact regions (high TC)
• These sequences are conformationally ambivalent
• interconvertible between a-helix and b-strand
• highly sensitive to tertiary environment
• generally contain hydrophobic, aromatic residues
  (Phe, Trp, Tyr)
• consistent with recent findings Rojas Quijano et
  al Biochemistry (2006)
• Ability to form amyloid is a generic trait of all
  proteins

17
A Common Mechanism for Protein Misfolding
Diseases
Amyloid Fibril Formation

• Alzheimers disease b-amyloid
• Parkinsons disease a-Synuclein
• Huntingtons disease Huntinton
• Human version of
• mad cow disease Prion protein
• Familial British dementia BriL
• Hereditary cerebral
• amyloid angiopathy Cystatin C

• Familial amyloidosis Gelsolin
• Familial subepithelial corneal amyloid
• 
  Lactotransferrin
• Cataracts TGF-BI

Brain
Eyes
Pituary gland amyloidosis
Prolactin

• Cystic fibrosis CFTR
• Chronic obstructive pulmonary
• disease a1-antichymotrypsin

Thyroid Medullary carcinoma
Calcitonin
Lung

• Heart
• Atrial amyloidosis Atrial natriuretic factor

• Familial amyloid polynueropathy III
• Apolipoprotein A1

Viscera

• Type 2 diabetes hIAPP
• Injection-localized
• amyloidosis Insulin

Pancrease
Peripheral nerves

• Familial amyloid
• polyneuropathy I Transthyrectin variants

Kidney Herediary renal amyloidosis
Fibrinogen
Skeletal muscle

• Hemodialysis-related
• amyloidosis b2-microglobulin

Mutiple targets

• Primary systemic amyloidosis Ig
  light chain
• Secondary systemic amyloidosis Serum amyloid A
• Senile systemic amyloidosis
  Transthyrectin
• Familial non-neuropathic amiloidosis
  Lysozyme

Blood system

• numbness blood proteins
• Familial amyotrophic lateral
• sclerosis Superoxide dismutase

18
Detecting Amyloid Fibril-Forming Sequence
Amyloid fibrils from myoglobin
Fandrich et al., 2001 Nature
Predicting Myoglobins Secondary Structure
19
CSSP Method Contact-dependent Secondary
Structure Propensities
Non-local effect (Tertiary interactions)
Local effect (Sequence) (side-chain
interaction) (i,i1) (i,i2) (i,i3)
Low
Structure 1 Structure 2 Structure 3
Similar Sequences
Tertiary Contacts
High
Protein Data Bank 3D protein structures PDB
Non-homologous protein database SCOP20
P(a low) P(b high)
7 residue length
Tertiary Contact
 A distance of ?4 Å between two heavy atoms that
are more than 4-residues apart in sequence
Average Tertiary contacts in SCOP20
L S A Q E K D N R I F T M P G W C V Y H
9.9 8.3 6.4 10.2 8.6 8.0 9.1 10.6 15.9 11.0 18.5 9.3 11.9 7.0 6.5 25.9 12.7 10.1 21.7 16.2
20
CSSP Algorithm
Query
GEAVELA
Retrieve similar sequences (templates)
DB of SCOP20 453,787 fragments
Intermediate TCs
Low TCs
High TCs
Discarded
GSSVELA b GDTVELT b GEAVGLP a GEGVEPA
coil VDSVELA b GDIVELT b GAGVDLA coil AAAVEMA
a SEGVELV a LEAVELP coil PERVELA b GEVVEVG b
GQAVALA a GEDVELD coil SEAVKLA a PEEVELA
a GNAVNLA a KEAVELG a GETVDLD a GEVVEEA a NDDVELA
a GKAVHLA a
f
g
21
Alzheimer's Disease b-amyloid N-terminal
sequence (4-25)
KLVFF are key residues in amyloid fibril
polymerization (Tjernberg et al., JBC 1996)
Wild type sequence
Low TC
High TC
Mutant sequence
Color index
Low TC
High TC
Type II Diabetes Human Islet Amyloid Precursor
Proteins (hIAPP)
-NFLVH- -FLVHS- Mazor et al., JMB 2002
Low TC
High TC
AA NTATCATQRLANFLVHSSNNFGAILSSTNVG PHD
HHHHHHHHHHHH EEE PrH
0012558999999887410000000000000 PrE
0011100000000000000011156530001
22
Parkinson's Disease Amyloidogenic NAC fragments
of a-synuclein
Bodles et al., J Neurochem (2001)
Low TC
High TC
Cottingham et al., Biochemistry (2002) ibid.,
(2003) AChE586-599 and BuChE573-586
Amyloidogenic Acetylcholinesterase fragment
(586-599)
AA QWKAEFHRWSSYMVHWKNQF PHD
HHHHHHHHHHH PrH 02334413578899999740 PrE
01121111000000000000
Low TC
High TC
Non-amyloidogenic Butyrylcholinesterase fragment
(573-596)
AA EWKAGFHRWNNYMMDWKNQF PHD HHHH
HHHHHHHHHH PrH 03455512588899988530 PrE
00121100000000000000
Low TC
High TC
23
?-amyloid peptide (Ab) and Alzheimers Disease
a-synuclein / NAC and Parkinsons / Alzheimers
Diseases
Yoon and Welsh, Protein Science (2004) ibid.,
Proteins (2005)
24
Exptl vs HbP-predicted propensities for b-strand
formation of four repeat fragments in tau
The hIAPP(4-34) sequence associated with amyloid
fibril formation in type II diabetes
25
CSSP-predicted b-strand propensity
profilesAChE586-599 (amyloidogenic) versus
BuChE573-596 (non-amyloidogenic)