Flavio Seno

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Biophys 07 Biology and beyond September 3-5,
2007 Arcidosso (Grossetto)
Insight into the structure of amyloid fibrils
from the analysis of globular proteins

• Flavio Seno
• Dipartimento di Fisica G. GalileiUniversita
  di Padova
• CNISM and INFN Sezione di Padova

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Amyloid fibrils The name comes from the early
mistaken identification of the substance as
starch (amylum in Latin)
One of the most intriguing issues in biology is
the occasional conversion of proteins into stable
fibrillar aggregates.
Such structures, known as amyloid fibrils are
responsabile for over 20 neurodegenerative human
diseases.
An electron microscope image of amyloid fibrils
in vitro
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Such structures are responsabile for over 20
human diseases.

• Alzheimers disease
• Parkinsons disease
• Atrial Amyloidosis
• Hereditary Renal Amyloidosis
• Secondary Systematic Amyloidosis
• Injection-Localized Amyloidosis
• Type II diabetes
• Chronic Wasting Disease (CWD)
• Scrapie
• BSE- Mad Cow Disease
• Kuru
• Creutzfeldt-Jakob Disease

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AMYLOID FORMATION IS NOT LIMITED TO THE FEW
PROTEINS ASSOCIATED WITH DISEASES.
Fandrich, Fletcher, and Dobson, Nature 410,
165-166 (2001)
Myglobin is a compact and highly soluble protein
without any native state properties to suggest
that it has a predisposition to form amyloid
fibrils.
pH 9.0 at T65 C
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This generic ability has been exploited by living
systems
Sup35p adopts a fibrillar confromation that
enables the expression of silent genes and
creates new phenotypes.
( True and Lindquist,
Nature 407, 477 (2000))
Pmel17 amyloid play a role in mitigating the
toxicity associated with melanin formation by
minimizing diffusion of toxic precursors.
(Fowler et al., PLos Biology, 4, 100, (2006))
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HOMOPOLYMER MODEL WHICH CAN FORM HYDROGEN BONDS
BENDING RIGIDITY (SECONDARY STRUCTURE PROPENSITY)
HYDROPHOBIC EFFECT (Hoang,Trovato, F.S,
Banavar,Maritan, PNAS 101, 7960-7964, (2004).
PREDETERMINED FOLDS
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Hoang,Trovato,Marsella, FS,Banavar,Maritan, PNAS
103, 6883-6887 (2006)
PREDETERMINED FOLDS FOR MULTIPLE CHAINS
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Hoang,Trovato,Marsella, FS,Banavar,Maritan, PNAS
103, 6883-6887 (2006)
PREDETERMINED FOLDS FOR MULTIPLE CHAINS
amyloid structure and the native folds of
globular proteins are stabilised by the same
physicochemical determinants
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Experimental techniques

• Solid state nuclear magnetic resonance
• X-ray micro and nano-crystallography
• Systematic protein engineering coupled to site
  direct spin or fluorescence labelling

In many (but not all!) cases filaments have
parallel, in register alignament and are
perfectly stacked along the fiber axis
It is possible to identify the regions of a
sequence forming and stabilising the fibril
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In register parallel aligment Ferguson et
al., PNAS 103, 16248 (2006)
Solid state NMR atomic level structure of amiloyd
fibrils of WW domain in human CA150 (a
transcriptional activator involved in
Huntingtons disease)
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ARE WE ABLE TO IDENTIFY THE REGIONS OF A
PROTEIN THAT ARE INVOLVED IN AMYLOID FORMATION?
ARE WE ABLE TO UNDERSTAND IN REGISTER
ALIGNEMENT?
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Phenomenological models based on physicochemical
propertiesOne-dimensional search!
Prediction of sequence-dependent and mutational
effect on the aggregation of peptides and
proteins (TANGO) A.M. Fernandez-Escamilla, F.
Rousseau, J. Schymkowitz and L. Serrano Nature
Biotechnology, 22, 1302 (2005)
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Energy function for aggregation propensity
SPECIFIC PAIRING OF TWO SEQUENCE STRETCHES OF
THE SAME LENGTH
CHAIN 1
CHAIN 2
IS THERE A PART OF CHAIN 1 WHICH PREFER TO FORM
BETA-STRAND WITH ANOTHER PART OF CHAIN 2?
Do they like to form hydrogen bonds?
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DATA SET OF GLOBULAR PROTEIN NATIVE STRUCTURES
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EMPIRICAL PAIRWISE POTENTIALS
Energies can be associated to the occurence of
parallel or anti parallel beta pairing of two
amino acids of type a and b by assuming that the
database is a system in thermodynamic equilibrium
Probability (propensities) are defined as the
ratio of the observed frequency over the expected
one in the reference state
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Energy function for aggregation propensity
Propensity of two residue types to be found
paired in neighbouring strands within
beta-sheets in globular proteins. (Samudrala and
Moult, 1998)
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Histograms of energies
PHE-PHE
CYS-CYS
PRO-PRO
VAL-VAL
ILE-ILE
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j
i
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Prediction of specific pairings and sequence
aggregation propensities
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Prediction of specific pairings and sequence
aggregation propensities
PROBABILITY THAT A GIVEN AMINO-ACID K BELONGS TO
AN AGGREGATED SEGMENT OF LENGTH L (EITHER P OR AP)
PROBABILITY THAT A.A. K IN FIRST CHAIN FORMS AN
HYDROGEN BOND WITH J IN THE SECOND CHAIN.
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Human amyloid beta-peptide 1-40 (Alzheimer
disease)
12 24 30
40

12-20 with 12-20 parallel
score -6.12
31-40 with 31-40 parallel
score -6.11
12-21 with 12-21 parallel
score -5.49
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Human amyloid beta-peptide 1-40 (Alzheimer
disease)
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Human amyloid beta-peptide 1-40 (Alzheimer
disease)
TANGO
PAWAR ET AL.
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Human amyloid beta-peptide 1-40 (Alzheimer
disease)
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PHF43 tau fragment 1-40 (Alzheimer disease)
Gamblin, Berry, Binder, Biochemistry 42, 15009
(2003)
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Human alpha synuclein (Parkinson and Dementia
with Lewy Bodies)
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101
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Human alpha synuclein (Parkinson and Dementia
with Lewy Bodies)
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Myglobin
TANGO
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Comparison with 179 peptides studied by Tango
TANGO
US
150 OUT of 179 predicted
correctly 8 false positive 21
false negatives
155 OUT of 179 predicted
correctly 21 false positive 3
false negatives
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IN REGISTER ALLIGNMENT IS SO FAVOURED?
IN REGISTER
CHAIN 1
CHAIN 2
Search only among 20 diagonal elements with many
favourable entries
NOT IN REGISTER
CHAIN 1
CHAIN 2
Search among 210 elements
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NNQQNY
PARALLEL 1.65
AP3.45
KFFEAAAKKFFE
AP-2.23
P 3.82
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Strand 2 parallel to strand 3
Strand 1 parallel to strand 4
3
4
2
2
4
3
1
1
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CONCLUSIONS

• THEORETICAL EVIDENCE THAT IN REGISTER ALLIGNMENT
  IS HIGHLY FAVOURED
• PROPENSITY OF RESIDUE PAIRS TO FORM BETA-SHEET
  STRUCTURE IN NATIVE FOLDED PROTEINS IS SIMILAR IN
  AMYLOIDS
• ENERGIES ARE OBTAINED IN A SIMPLE WAY THERE
  SHOULD BE SOMETHING ROBUST BEHIND IT
• IT IS POSSIBLE TO USE THESE PROPENSITIES TO
  DRIVE SIMULATIONS AND TO PREDICIT AMYLOID
  STRUCTURES
• PROTEIN-PROTEIN INTERACTION BY ß-ADDITION

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COLLABORATORS

• Fabrizio Chiti Dipartimento di
  Biochimica, Firenze (Italy)
• Amos Maritan Dipartimento di Fisica,
  Padova (Italy)
• Antonio Trovato Dipartimento di Fisica,
  Padova (Italy)
• Silvio C.E. Tosatto Dipartimento di Biologia,
  Padova (Italy)

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A. Trovato, F. Chiti, A. Maritan and F.
Seno INSIGHT INTO THE STRUCTURE OF AMYLOID
FIBRILSFROM THE ANALYSIS OF GLOBULAR PROTEINS
Plos Computational Biology , vol. 2(12), e170 ,
(2006)
A. Trovato, A. Maritan and F. Seno
AGGREGATION OF NATIVELY FOLDED PROTEINS A
THEORETICAL APPROACH Journal of Physics C, vol
19, p. 285221(2007)
A.Trovato, F. Seno, S.C.E. Tosatto THE PASTA
SERVER FOR PROTEIN AGGREGATION PREDICTION Protein
Engineering , Design and Selection, in press
(2007)
http//protein.cribi.unipd.it/pasta/
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Statistical analysis (I)
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i-j0 in register
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Statistical analysis (II)
In other words, in register parallel pairing is
favoured, with respect to other parallel
alignments, because it includes some of the most
favourable entries, and cause them to be found
more likely
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Statistical analysis (III)
2
3
4
5 6
1
Twice 2-5 ,3-4 and 1-6
4
3
2 1
6 5
3
4
5
6 7
2
Twice 3-5 ,2-6 and once 4-4 and 1-7
4
3
2 1
6 5