structural studies of heterogeneous amyloid species of...
TRANSCRIPT
Structural studies of heterogeneous
amyloid species of lysozymes and de novo protein albebetin and their
cytotoxicity
Vladimir Zamotin
Department of Medical Biochemistry and Biophysics Umeå University
Umeå, Sweden 2007
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Моей маме, жене и доче
To my family
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Abstract A number of diseases are linked to protein folding problems which lead to
the deposition of insoluble protein plaques in the brain or other organs. These diseases include prion diseases such as Creutzfeld-Jakob disease, Alzheimer's disease, Parkinson's disease and type II (non-insulin dependent) diabetes. The protein plaques are found to consist of amyloid fibrils - cross-beta-sheet polymers with the beta-strands arranged perpendicular to the long axis of the fibre. Studies of ex vivo fibrils and fibrils produced in vitro showed that amyloid structures possess similar tinctorial and morphological properties. These suggest that the ability to form amyloid fibrils is an inherent property of polypeptide chains.
The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the involved mechanisms. The model proteins used in the studies were the equine and hen lysozymes and de novo designed protein albebetin.
Lysozymes are naturally ubiquitous proteins. Equine lysozyme belongs to an extended family of structurally related lysozymes and α-lactalbumins and can be considered as an evolutional bridge between them. Hen lysozyme is one of the most characterized protein and its amyloidogenic properties were described earlier. De novo protein albebetin and its constructs are designed to perform the function of grafted polypeptide sequence. Fibrils of equine lysozyme are formed at acidic pH and elevated temperatures where a partially folded molten globule state is populated. We have shown that lysozyme assembles into annular and linear protofilaments in a calcium-dependent manner. We showed that albebetin and its constructs are inherently highly amyloidogenic under physiological conditions. Fibrillation proceeds via multiple pathways and includes a hierarchy of amyloid structures ranging from oligomers to protofilaments and fibrils, among which two distinct types of oligomeric intermediates were characterized. Pivotal oligomers comprise of 10-12 monomers and on-pathway amyloid-prone oligomers constitute of 26-30 molecules. We suggest that transformation of the pivotal oligomers into the amyloid-prone ones is a limiting stage in albebetin fibrillation. Cytotoxic studies of albebetin amyloid species have revealed that initial, pivotal oligomers do not effect on cell viability while amyloid-prone ones induce cell death. We suggest that oligomeric size is important for the stabilizing cross-beta-sheet core which is crucial for cell toxicity. Cytotoxic studies of both oligomers and fibrils of hen lysozyme have revealed that both species induce cell death. The amyloid sample containing cross-β-sheet oligomers induces an apoptosis-like cell death. The oligomers without cross-β-sheet appeared to be non-toxic, indicating that the stabilization of this structural pattern is critical for the induced toxicity. In contrast, the fibrils induce more rapid, necrosis-like death.
These studies gained insights into a structure–function relationship of different forms of amyloid and general pathways of cell death. This is an important step in understanding the mechanisms of amyloid-associated degeneration and defining specific therapeutic targets.
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Contents
ABSTRACT 5
CONTENTS 6
LIST OF PUBLICATIONS 8
INTRODUCTION 10
Amyloid diseases and related proteins 10
MISFOLDING AND AGGREGATION 12
Generic properties of amyloids 12
Amyloid precursor state 16
Conditions of amyloid formation 18
Effect of mutation on amyloidogenic properties 21
Models of amyloid formation 23
CYTOTOXICITY OF AMYLOIDS 26
Amyloid toxic species 26
Cellular targets for amyloids 27
General pathways of cell death 30
STUDIED PROTEINS 33
Lysozymes 33
De novo designed protein albebetin and its constructs 37
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APPLICATION OF ATOMIC FORCE MICROSCOPY IN AMYLOID STUDIES 39
AFM volume measurements 41
INTRODUCTION TO THE ARTICLES 44
CONCLUSIONS 50
ACKNOWLEDGEMENTS 52
REFERENCES 53
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List of publications
The thesis is based upon the following papers:
I. Malisauskas, M., Zamotin, V., Jass, J., Noppe, W., Dobson, C.M.,
Morozova-Roche, L.A. (2003) Amyloid protofilaments from the
calcium-binding protein equine lysozyme: formation of ring and linear
structures depends on pH and metal ion concentration. J. Mol. Biol.
330, 879-890.
II. Morozova-Roche,* L.A., Zamotin,* V., Malisauskas, M., Ohman, A.,
Chertkova, R., Lavrikova, M.A., Kostanyan, I.A., Dolgikh, D.A.,
Kirpichnikov, M.P. (2004) Fibrillation of carrier protein albebetin and
its biologically active constructs. Multiple oligomeric intermediates and
pathways. Biochemistry, 43, 9610-9619. (*- with equal contributions)
III. Zamotin,* V., Gharibyan,* A., Gibanova, N.V., Lavrikova, M.A.,
Dolgikh, D.A., Kirpichnikov, M.P., Kostanyan, I.A., Morozova-Roche,
L.A. (2006) Cytotoxicity of albebetin oligomers depends on cross-β-
sheet formation. FEBS Letters, 580, 2451-2457. (*- with equal
contributions)
IV. Gharibyan, A.L., Zamotin, V., Yanamandra, K., Moskaleva, O.S.,
Margulis, B.A., Kostanyan, I.A., Morozova-Roche, L.A. (2007)
Lysozyme Amyloid Oligomers and Fibrils Induce Cellular Death via
Different Apoptotic/Necrotic Pathways. J. Mol. Biol. 365, 1337-1349.
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The articles not included in the thesis:
1. Gruden, M.A., Davudova, T.B., Malisauskas, M., Zamotin, V.V.,
Sewell, R.D., Voskresenskaya, N.I., Kostanyan, I.A., Sherstnev, V.V.,
Morozova-Roche, L.A. (2004) Autoimmune responses to amyloid
structures of Aβ(25-35) peptide and human lysozyme in the serum of
patients with progressive Alzheimer's disease. Dement. Geriatr. Cogn.
Disord. 18, 165-171.
2. Malisauskas, M., Ostman, J., Darinskas, A., Zamotin, V., Liutkevicius,
E., Lundgren, E., Morozova-Roche, L.A. (2005) Does the cytotoxic
effect of transient amyloid oligomers from common equine lysozyme in
vitro imply innate amyloid toxicity? J. Biol. Chem. 280, 6269-6275.
3. Lavrikova, M.A., Zamotin, V.V., Malisauskas, M., Chertkova, R.V.,
Kostanyan, I.A., Dolgikh, D.A., Kirpichnikov, M.P., Morozova-Roche,
L.A. (2006) Amyloidogenic properties of the artificial protein albebetin
and its biologically active derivatives. The role of electrostatic
interactions in fibril formation. Biochemistry (Mosc). 71, 306-314.
4. Malisauskas, M., Darinskas, A., Zamotin, V.V., Gharibyan, A.,
Kostanyan, I.A., Morozova-Roche, L.A. (2006) Intermediate amyloid
oligomers of lysozyme: is their cytotoxicity a particular case or general
rule for amyloid? Biochemistry (Mosc), 71, 505-512.
5. Wilhelm, K.R., Yanamandra, K., Gruden, M.A., Zamotin, V.,
Malisauskas, M., Casaitec, V., Darinskasd, A., Forsgrene, L.,
Morozova-Roche L.A. (2007) Immune reactivity towards insulin, its
amyloid and protein S100B in blood sera of Parkinson’s disease
patients. European J. Neurology, 14, 327-334.
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Introduction
Amyloid diseases and related proteins
Proteins, term originated from the Greek πρώτα ("prota")- "of primary
importance", were first described and named by Jöns Jakob Berzelius in 1838.
They are most multi-purposed macromolecules in living organisms,
participating in essentially all biological processes. They function as catalysts
and transporters; they store other molecules, provide mechanical support and
immune protection, transmit nerve impulses, control growth and differentiation
of cells and perform other functions. The structure of proteins and their ability
to carry out their correct functions are very tightly controlled, as even small
structural defects can lead to protein folding diseases. A number of human
diseases are associated with the deposition of protein aggregates. These include
prion diseases such as bovine spongiform encephalopathy and its human
equivalent Creutzfeld-Jakob disease, Alzheimer's and Parkinson’s diseases,
Skelefteå disease, type II (non-insulin dependent) diabetes and others. In most
of amyloid disorders, the protein deposits are accumulated extracellular. In
Parkinson's disease, however, amyloid deposits of α-synuclein were found
intracellularly.
The major constituent of the proteinaceous deposits are amyloid fibrils.
They can be constituted of different proteins or peptides, depending on their
location in the body. To date more than 30 proteins are known to be involved
in the human amyloid diseases (Table 1). In spite of their different primary
sequences and secondary and tertiary structures, when the individual
polypeptides are assembled into fibrils, the latter exhibit very similar structural
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characteristics such as cross-β-sheet core with the β-strands arranged
perpendicular to the long axis of the fibre [Glenner, 1980; Blake & Serpell,
1996]. The common structural properties of the fibrils imply that the general
structural mechanism govern their formation and therefore the principles found
for one particular protein have a general application for understanding this
phenomenon as a whole.
Table 1. Human amyloid disease and associated proteins.
Disease Aggregating polypeptide Structure of polypeptide
Neurodegenerative diseases Alzheimer's disease Amyloid β peptide Natively unfolded Spongiform encephalopathies
Prion protein or fragments Natively unfolded (residues 1–120) and α-helical (residues 121–230)
Parkinson's disease α-Synuclein Natively unfolded Dementia with Lewy bodies
α-Synuclein Natively unfolded
Amyotrophic lateral sclerosis
Superoxide dismutase-1 All-β, Ig like
Huntington's disease Huntingtin with polyQ expansion
Largely natively unfolded
Spinocerebellar ataxia 17 TATA box-binding protein with polyQ expansion
α+β, TBP like (residues 159–339); unknown (residues 1–158)
Nonneuropathic systemic amyloidoses AL amyloidosis Immunoglobulin light chains
or fragments All-β, Ig like
AA amyloidosis Fragments of serum amyloid A protein
All-α, unknown fold
Senile systemic amyloidosis
Wild-type transthyretin All-β, prealbumin like
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Disease Aggregating polypeptide Structure of polypeptide
Familial amyloidotic polyneuropathy
Mutants of transthyretin All-β, prealbumin like
Hemodialysis-related amyloidosis
β2-microglobulin All-β, Ig like
Apo amyloidosis N-terminal fragments of apolipoproteins
Natively unfolded
Lysozyme amyloidosis Mutants of lysozyme α+β, lysozyme fold Fibrinogen amyloidosis Variants of fibrinogen α-
chain Unknown
Nonneuropathic localized diseases Type II diabetes Amylin, also called islet
amyloid polypeptide (IAPP) Natively unfolded
Injection-localized amyloidosis
Insulin All-α
Corneal amylodosis associated with trichiasis
Lactoferrin α+β
Cataract γ-Crystallins All-β
Misfolding and aggregation
Generic properties of amyloids
Amyloid was discovered in 1854 by German physician Rudolph
Virchow, who named it in the belief that the iodine-staining component was
starch-like [Cohen & Jones, 1991; Sipe, 2000]. Positive response by iodine
staining led Virchow to the conclusion that the substance underlying the
evident macroscopic abnormality was cellulose and he gave it the name of
amyloid, derived from the Latin amylum and the Greek amylon [Cohen &
Jones, 1991]. In 1859 Friedreich and Kekule demonstrated that the amyloids
contain the depositions of proteinaceous mass. [Kyle, 2001].
The amyloid depositions were most commonly identified by various
types of microscopy and immunohistochemical dyes such as Congo red and
thioflavin T. (Figure 1 A, B)
A B C
Figure 1. Tinctorial properties of amyloid. Congo red staining of transthyretin (A) and thioflavin T staining of human lysozyme fibrils (B) and X-ray diffraction pattern of Ile56Thr (C) [Morozova-Roche et al., 2000; Saraiva, 2002].
Congo red stained deposits of amyloid were observed in a variety of
tissues using polarization light microscopy, as they demonstrate a characteristic
apple green birefringence under polarised light. Congophilia with apple green
birefringence was used as the first criterion of amyloid. [Missmahl & Hartwig,
1953] The secondary diazo dye Congo red is schematically presented in Figure
2. It has been suggested that it bindings specifically to amyloids via insertion
between the individual strands of cross-β-sheet [Carter & Chou, 1998]
Figure 2. Chemical structure of secondary diazo dye Congo red.
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Benzothiazole dye thioflavin T is extensively applied in the histological
analysis of ex vivo amyloids as well as in in vitro amyloid studies. The
fluorescence of thioflavin T increases significantly upon binding to amyloid
fibrils. Krebs and co-workers suggested that thioflavin T molecules bind to
fibrils in the grooves on the face of the β-sheets that form the fibrillar backbone
[Krebs et al., 2005]. (Figure 3)
A B
Figure 3. Chemical structure of thioflavin T (A) and model of binding with amyloid structure (B) [Krebs et al., 2005].
Electron and atomic force microscopic studies of amyloids
[Chamberlain et al., 2000] showed that they exhibit a fibrillar morphology; the
fibrils range in width from 60 to 130 Å (average 75 to 100 Å) and in length up
to microns.
Figure 4. The structure of fibrils assembled from human lysozyme [Chamberlain et al., 2000]. Transmission electron microscopy (A); Atomic force microscopy (B). 14
X-ray diffraction analyses of isolated amyloid protein fibrils [Bonar et
al., 1969; Glenner et al., 1974; Sunde and Blake, 1998] showed specific
diffraction pattern, which was assigned to cross-β-sheet packing (Figure 1C).
The β-sheet formation accompanied the amyloid formation is also
monitored by using circular dichroism and Fourier-transformed infrared
spectroscopy (Figure 5). Up to date, all these methods are widely used in the
amyloid research.
A B
Figure 5. Fourier-transformed infrared spectrum of fibrillar amylin (A) and the far-UV circular dichorism spectrum (B) of fibrillar human lysozymes, demonstrating the β-sheet formation [Nilsson & Raleigh, 1999; Morozova-Roche et al., 2000].
During the last decade, a number of proteins not associated with
diseases have been found to convert into amyloid fibrils in vitro, showing the
physicochemical characteristics of those related to diseases (Table 2). The
similarity in dimensions and morphology, as well as in tinctorial properties
between ex-vivo and in vitro protein fibrils has been demonstrated. All fibrils
independent of their origin are stabilised by an array of inter- and
intramolecular hydrogen bonds between the main-chain amide and carbonyl
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groups that are common to all peptides and proteins. Based on the accumulated
data, it has been suggested that the ability to form amyloid fibrils is an inherent
property of polypeptide chains and most if not all polypeptides have ability to
form amyloids, though their propensity to amyloid self-assembly can be
different (Dobson et al., 1999, 2001).
Table 2. Non-disease related amyloidogenic proteins
Protein References
Curlin Chapman et al., 2002
Hydrophobin EAS Wosten & Vocht, 2000
Myoglobin Fandrich et al., 2001
Proteins of the chorion of the eggshell Iconomidou et al., 2000
Spidroin Kenney et al., 2002
Ure2p (prion) Taylor et al., 1999
Sup35p (prion) King et al., 1997
HET-s (prion) Dos Reis et al., 2002
SH3 Guijarro et al., 1998
Cytochrome C Pertinhez et al., 2001
Equine lysozyme Malisauskas et al., 2003
α-lactalbumin Goers et al., 2002
Hen lysozyme Goda et al., 2000
Albebetin Morozova-Roche et al., 2004
Amyloid precursor state
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In the amyloid field, particular attention was focused on amyloid
precursor state, which can be converted into fibrils. The multiple
conformational states of proteins are presented schematically in Figure 2 using
the energy folding landscape (Figure 6). Equilibrium partially folded states are
considered as stable counterparts of kinetic intermediates transiently populated
during protein folding kinetics [Heegaard et al., 2001]. Among equilibrium
partially folded states molten globule ones are the most extensively studied.
They are characterised by the native-like secondary structure, but labile and
large destabilised tertiary structure. Many globular proteins have been shown
to adopt several stable conformations, e.g. the native, molten globule, pre-
molten globule and unfolded states [Dunker et al., 2001; Kayed et al., 1999;
Lashuel et al., 2002; Merlini et al., 2003; Smith et al., 2003; Teplow et al.,
1998; Uversky et al., 2002]. A common observation is that fibrillization starts
from an intermediate state either partially unfolded or partially folded [Rochet
& Lansbury, 2000]. Many globular proteins such as human and hen lysozymes
[Morozova-Roche et al., 2000, Krebs et al., 2000], phosphoglycerate kinase
[Damaschun et al., 1999], cystatin C [Ekiel & Abrahamson, 1996],
acylphosphatase [Chiti et al., 1999] and transthyretin [Lai et al., 1996] and
other need to be partial unfolded in order to undergo fibril formation. In the
case of unfolded polypeptides such as α-synuclein [Uversky et al., 2000; 2001]
and islet amyloid polypeptide, the amyloid precursor intermediates have to be
partially folded. The free-energy barriers of the protein folding landscape
(Figure 3) can be rather high [Damaschun et al., 1999; Ferreira & De Felice,
2001; Kelly, 1999], leading to existence of parallel conformational states [Soto
& Saborio, 2001], including oligomeric, fibrillar and aggregated forms.
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Conditions of amyloid formation
The amyloid-precursor state can be accumulated and the subsequent
aggregation can occur under variety of conditions, which have been studied
scrupulously [Ahmad et al., 2003; Canet et al., 2002; Hoyer et al., 2004; Lai, et
al., 1999; McParland et al., 2002; Smith et al., 2003]. The incubation at low
pH and elevated temperatures is widely used as a common condition to
produced fibrils from a number of proteins, including lysozymes (equine,
human and hen) [Malisauskas et al., 2003; Mishra et al., 2007; Morozova-
Roche et al., 2000], β2 microglobulin [Sasahara et al., 2007], insulin [Range et
al., 1997], transthyretin [Pasquato et al., 2007], apomyoglobin [Picotti et al.,
2007] and others.
These conditions cause the population of partially folded states in the
cases of lysozymes and β2 microglobulin. The low pH results in dissociation of
transthyretin tetramers and insulin hexamers into monomers or dimers,
respectively, which is critical for the amyloid assembly of these proteins
[Colon & Kelly, 1992; Ahmad et al., 2003].
Acylphosphatase and wild type hen lysozyme can be converted into
amyloid fibrils in the presence of moderate concentration of 2,2,2-
trifluoroethanol [Chiti et al., 1999; Krebs et al., 2000]. Under these conditions
hydrophobic residues and main-chain hydrogen bond donors and acceptors
become partially exposed, thus non-covalent intermolecular interactions and
particular hydrogen bonding within the polypeptide chain become favourable.
High hydrostatic pressure modulates protein – protein and protein –
solvent interactions through volume changes. Thereby it affects the equilibrium
between native and denatured conformational states as well as between the
monomeric, oligomeric and aggregated forms without the addition of
chemicals. High hydrostatic pressure has provided to be a useful tool to
populate and stabilize folding intermediates, which are characterised by more
perturbed and solvated structure as well as by smaller volume than the native
state [Randolph et al., 2002; Silva & Weber, 1993].
Figure 6. The energy folding landscape. The surface shows the multitude of conformations 'funneling' towards the native state via intramolecular contact formation, or towards the formation of amyloid fibrils via intermolecular contacts [Jahn & Radford, 2005].
Partially unfolded conformations with high propensity to form amyloid
have been populated by using this method for a range of proteins, including
insulin [Jansen et al., 2005], human lysozyme [De Feliche et al., 2004],
interferon-γ [Webb et al., 2001], interleukin-1 receptor antagonist [Seefeldt et
al., 2005], and β-lactoglobulin [Panick et al., 1999].
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Amyloid fibrils can be induced by adding transient metal ions.
Miranker and co-workers demonstrated that Cu2+ significantly destabilizes β2
microglobulin and enhances fibrils formation of β2 microglobulin at neutral pH
[Morgan et al., 2001]. The addition of copper, iron, manganese or zinc ions
induced fibrillation of synthetic Aβ peptide [Huang et al., 1997; Atwood et al.,
1998]. Similar role of transient metal has been shown in the conversion of PrPC
to PrPSc [Jones et al., 2004]. In most of the cases the metal binding site of the
proteins involves histidine residues: in the case of Aβ peptide these are His6,
His13 and His14, while in PrP the binding site includes HGGGW segment of
the N-terminal domain. For β2 microglobulin different binding sites were
demonstrated for the native and amyloid precursor states; in the native state the
binding site involves His31, while in the non-native state the coordination of
Cu2+ is mediated by residues His13, His51 and His84, but not His31. [Eakin et
al., 2002].
The net charge of the proteins is critical for amyloid formation. The
kinetics and the quantity of the amyloid of albebetin, which carries a net charge
-12 under the neutral pH, was significantly enhanced by increasing ionic
strength, shielding the overall electrostatic repulsions between protein
molecules [Morozova-Roche et al., 2004]. Similarly, the fibrillation of
pharmaceutical peptides glucagon and insulin is also affected by both cations
and anions, changing the kinetics and morphology of amyloids by minimizing
the electrostatic interactions [Nielsen et al., 2001; Pedersen et al., 2006].
Chemical denaturants such as urea and guanidine-HCl at moderate
concentrations have been used extensively in protein-folding studies to trap
folding intermediates [Cymes et al., 1996; Ayed & Duckworth, 1999]. They
affect protein stability by disrupting hydrogen-bonding of the peptide
backbone, thus destabilizing secondary structure, and by allowing greater
solvation of hydrophobic side chains. For example, moderate urea
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concentrations accelerates fibril formation of insulin [Ahmad et al., 2004] and
β-lactoglobulin [Hamada & Dobson, 2002; Rasmussen, 2007]. In prion protein
increasing urea concentration led at first to an increase in fibril formation
kinetics, which followed by a decrease in a rate of assembly, with the
maximum rate at 2.4 M urea, corresponding to the midpoint of protein
unfolding [Baskakov et al., 2004]. A bell-shaped dependence of the speed of
the fibrillation of β-lactoglobulin has been observed also by Hamada and co-
workers [Hamada & Dobson, 2002]. The authors showed that the rate of β-
lactoglobulin fibril formation decreased at the urea concentrations more than 5
M, although the population of unfolded protein molecules increased under this
condition. In order to aggregate both high population of unfolded proteins and
strong interactions between them are required. At the increasing concentration
of denaturant such as urea, the intermolecular interactions decline substantially
leading to decrease in amyloid assembly [Chiti et al., 1999; Bellotti et al.,
2000].
Finally for natively unfolded proteins such as α-synuclein, partial
folding has been shown to be essential first step in self-assembly [Der-
Sarkissian et al., 2003], underlining the importance of partially folded species
as amyloid precursors.
Effect of mutation on amyloidogenic properties
Most mutations associated with accelerated fibrillation in vitro and with
protein deposition in the body have been shown to destabilize the protein
native structure, increasing the concentration of partially folded conformers
[Rochet et al., 2000; Nielsen et al., 2001; Canet et al., 1999; Heegaard et al.,
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2001]. In the case of human lysozyme, single point mutations in the gene cause
the fibril deposition in several tissues. Two amyloidogenic variants, Ile56Thr
and Asp67His, have been studied in detail and shown to be significantly less
stable than the wild-type protein [Morozova-Roche et al., 2000]. They showed
also a lack of cooperativity of the native structure, leading to an increased
concentration of partially folded states at equilibrium [Booth et al., 1997].
Transthyretin amyloidogenic variants are characterised by decreased stability
and increased dissociation rate constant of tetramer [Hammarstrom et al.,
2002]. Chiti and co-authors systematically analysed more than 50 mutants of
acylphosphatase. They demonstrated that destabilization of the native
conformation of acylphosphatase renders it more prone to amyloid formation.
The correlation drawn between the conformational stability of native
acylphosphatase and the tendency to form aggregates indicates that amyloid
fibrils originate from an ensemble of denatured conformations of the protein
under conditions in which the formation of non-covalent interactions is still
favourable [Chiti et al., 2000].
Nielsen and co-workers [Nielsen et al., 2001] have performed thorough
studies of amyloid properties of insulin mutations. They have demonstrated the
significant increase in the lag time of fibril formation of insulin upon amino
acid substitutions to more polar residues, as well as mutations to charged
residues. The authors suggested that mutations may affect the amount of
oligomeric intermediates formed under the conditions of experiments or may
perturb their structure, thus affecting the propensity to form fibrils. These
results demonstrate the importance of both hydrophobic and electrostatic
interactions in the initial stages of fibrillation [Nielsen et al., 2001].
Models of amyloid formation
The kinetics of fibril formation can be described as a nucleation
dependent process involving three major steps: nucleation, fibril growth and
stationary phases [Jarrett & Lansbury, 1993]. Nucleation involves the assembly
of several protein monomers into an organized structure – nucleus, serving the
role of precursor for the formation of fibrils. A lag phase of fibrillation kinetics
is related to the time required for the formation of nuclei. Subsequent addition
of monomers to the nuclei elongates them into fibrils [Teplow, 1998]. Seeding
or addition of preformed nuclei diminishes the lag-phase (Figure 7) [Han et al.,
1995; Wood et al., 1999; Morozova-Roche et al., 2000].
Figure 7. Aggregation of hen lysozyme. Data are shown for the aggregation of 1 mM hen lysozyme at pH 2.0 and 65°C monitored by thioflavin-T fluorescence in the absence of added fibrils (down triangles) and following addition of 5% (v/v) (black squares), 2.5% (v/v) (circles), 1% (v/v) (triangles), and 0.5% (v/v) fibrils preformed from hen lysozyme at pH 2.0 (diamonds). Seeding with a 5% (v/v) aliquot of a solution of amyloid fibrils containing purified full-length hen lysozyme gave essentially identical kinetics (gray squares) to aliquots formed simply by incubation at pH 2.0 and 65°C [Krebs et al., 2004].
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There are several models of fibril formation based on the kinetic studies
such as monomer-derived conversion [Prusiner, 1982], template assisted [Soto,
2001] and nucleated polymerization model [Lomakin et al., 1996]. The
monomer derived conversion model suggests that a monomeric peptide can
adopt a conformation called the A state that is analogous to the conformation
adopted in the fibril. The rate-determining step is the A and S monomer
binding and converting S state monomer, which results in the A state dimer
(Figure 8 A). The dimer then dissociates and the A structured monomers
rapidly add to the end of the growing fibril [Prusiner, 1982]. In the template-
assisted model, a preassembled nucleus binds to a soluble state peptide present
in a random coil conformation. This step is followed by a rate-determining
structural change to add the peptide to the growing end of the fibril or filament,
presumably as part of the β-sheet-rich quaternary structure (Figure 8B) [Soto,
2001]. The nucleation polymerisation is characterized by the rate-limiting
formation of a nucleus arising from equilibrium between monomers. Once a
nucleus is established, assembly occurs by the addition of assembly competent
monomers to the growing end of the fibril (the nucleus). [Lomakin et al., 1996]
Serio and co-workers proposed the nucleation conformational conversion
model, which incorporated features of nucleated polymerization and template-
assisted ones. [Serio et al., 2000].
Figure 8. The models of protein conversion into amyloid fibrils. A, Templated assembly; B, monomer-directed conversion; C, nucleated polymerisation; D, nucleated conformational conversion. A-state denoted by smooth circles, rough circles represent S state [Kelly, 2000].
This model suggests that native oligomers accumulate and associate into a
“nucleus”, where conformational conversion takes place as a rate-determining
step. Oligomeric complexes are crucial intermediates forming the amyloid
nucleus. When these complexes undergo a conformational change on
association with the nuclei, rapid fibrillar assembly follows [Serio et al., 2000].
Each particular amyloid scenario need to be carefully investigated to conclude
which specific mechanisms is involved.
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Cytotoxicity of amyloids
Amyloid toxic species
In spite of the large amount of experimental data on the role of protein
self-assembly in amyloid related diseases, the nature of the toxic amyloid
structures remains elusive. A number of studies have supported the view that
the prefibrillar aggregates are the most cytotoxic species. By means of electron
and atomic force microscopy, size exclusion chromatography and other
techniques it has been demonstrated that the broad range of structures
characterized by a different degree of assembly of misfolded proteins and
variable morphology are toxic in vitro. These include ADDLs – β-amyloid-
derived diffusible ligands [Walsh et al., 2002, Gong, et al., 2003], 8-mers to
20-mers of equine lysozyme [Malisauskas et al., 2005], 20-mer cross-β-sheet
containing oligomers of albebetin [Zamotin et al., 2006], misfolded monomers
and hexamers of transthyretin [Reixach et al., 2004], 8–9 nm globular
structures of transthyretin mutants [Andersson et al., 2002], 4– 200 nm
diameter granules of the PI3-SH3 domain, 4 – 8 nm width aggregates of HypF
[Bucciantini et al., 2002], 3 – 5 nm spherical aggregates of α-synuclein [Ding
et al., 2002; Volles et al., 2001] and others. Kayed and co-authors reported that
intermediate-size soluble oligomers of Aβ(1–40) and five other proteins form
structures sharing a similar conformational epitope, suggesting that they exert
cytotoxicity via similar mechanisms [ Kayed et al., 2003]. Even the process of
nucleation-dependent polymerization of Aβ(1–40) itself has been suggested as
a primary cause of cytotoxicity [Wogulis et al., 2005].
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A greater controversy exists on the cellular toxicity of the amyloid
fibrils. Initially, they have been considered as an inert material substantially
accumulated in the tissues and organs [Andersson et al., 2002; Anderluh et al.,
2005; Bhatia et al., 2000; Chromy et al., 2003; Sirangelo et al., 2004]. There is
also a body of experimental evidence clearly demonstrating the neurotoxicicity
and cytotoxicity of amyloid fibrils.[ Forloni 1996; Forloni et al., 1996; Howlett
et al., 1995; Lorenzo& Yankner, 1994; Novitskaya et al., 2006; Weldon et al.,
1998; ] Amongst the recent findings, Petkova and co-authors showed that
mature fibrils from Aβ(1–40) produced under two different conditions, and
consequently characterized by different morphologies, exert significantly
different toxicity on neuronal cells [Petkova et al., 2005]. The origin of the
toxicity of amyloid species, especially in the body, require further
investigation.
Cellular targets for amyloids
The similarity and inherent toxicity of the amyloid structures suggests
that the toxic agents may share specific cellular targets [Kayed et al., 2003].
Cellular membranes, separating the ordered living cells from the entropic chaos
outside, are considered to be such common target [Hartley, 1999; Janson et al.,
1999]. Prefibrillar oligomers of α-synuclein and Aβ peptide were shown to lead
to neuron dysfunction and degeneration by forming the structures with pore-
like morphologies, which can be inserted into the cellular membranes, altering
their permeability [Lashuel & Lansbury, 2006]. The formation of amyloid
pores in mitochondria results in the loss of mitochondria membrane potential,
overloading with Ca2+ and subsequently to oxidative stress [De Giorgi et al.,
28
2002]. Some studies suggest that lipids can be absorbed by amyloids during
their formation on the membranes, leading to the membrane rupture due to
gradual lipid depletion [Sparr et al., 2004; Knight & Miranker, 2004].
Another main target involved in amyloid cytotoxicity is mitochondria
which can be affected by cytotoxic species in direct or indirect manner [Eckert,
2003; Brookes et al., 2004; Swerdlow et al., 2004; Takuma et al., 2005;
Nunomura et al., 2006]. Mitochondria are essential for the regulation of
intracellular Ca2+ homeostasis and as the principal generators of intracellular
reactive oxygen species. Therefore, the defects of mitochondrial functions can
result in excessive production of reactive oxygen species [Butterfield et al.,
2001; Wyttenbach et al., 2002]. The formation of the transmembrane pores are
linked to release of small proteins, such as cytochrome C and apoptosis-
inducing factor [Crompton, 1999; Rodriguez-Enriquez et al., 2004]. The
mitochondrial dysfunctions were proposed as potential mechanisms in the
development and pathogenesis of Alzheimer’s disease and most of the studies
was focused on the effect of Aβ peptide on mitochondrial activity [Ojaimi &
Byrne, 2001]. Growing evidence indicates that cell death can be initiated by the
Aβ peptide induced generation of free radicals [Butterfield et al., 1994],
leading to lipid peroxidation and oxidative stress. Oxidative stress,
subsequently, induces intracellular accumulation of Aβ [Misonou et al., 2000].
The experiments performed on isolated mitochondria demonstrated the ability
of Aβ to be directly toxic to the organelle [Casley et al., 2002]. It has been
shown that Aβ inhibits the mitochondrial electron transport chain complexes,
cytochrome C oxidase and promotes Ca2+-induced assembly of the
transmembrane pores. It has been also demonstrated that Aβ is present in
mitochondria, presumably in association with Aβ-binding alcohol
dehydrogenase, and promotes mitochondrial dysfunction and reactive oxygen
29
species production. This provides a direct link to Aβ-induced mitochondrial
toxicity [Lustbader, et al., 2004]. Recently it has been shown that Aβ induces
neuronal apoptosis via mechanisms involving induction of Fas ligand and the
release of Smac via AP-1/Bim activation in mitochondria [Morishima et al.,
2001; Yin et al., 2002]. In the case of Parkinson’s disease, it was shown, that
fibrillar deposits of α-synuclein also cause oxidative stress and mitochondria
dysfunction [Cooper et al., 2006].
Another abnormality found in neurodegenerative diseases is linked to
the damage of endoplasmatic reticulum. Endoplasmatic reticulum stress results
in the disruption of Ca2+ homeostasis, inhibition of protein N-linked
glycosylation, expression of mutant proteins and other types of cellular stresses
caused by the accumulation of misfolded proteins in the lumen of
endoplasmatic reticulum [Kaufman, 1999; Katayama et al., 2004]. It has been
suggested that the endoplasmatic reticulum stress-mediated apoptotic signal
pathways, specifically activation of caspase-4, may also contribute to the
pathogenesis of Alzheimer’s disease [Katayama et al., 2004]. As Aβ is
synthesized and accumulated in the endoplasmatic reticulum, Aβ has been
suggested as a direct mediator of the endoplasmatic reticulum stress responses
and apoptosis. Aβ(1–42) activates caspase-12 in primary neurons through
calpain activation, therefore, the caspase-12 knocked-out neurons are partially
resistant to Aβ-induced cell death [Nakagawa et al., 2000].
In familial amyloid polyneuropathies, the interaction of transthyretin
fibrils with RAGE receptors (receptor for advanced glycation end products)
were suggested as contributing to cellular stress and toxicity [Sousa et al.,
2001]. These indicate that fibrils can act via specific mechanisms, rather than
inducing only accidental cell damage.
30
General pathways of cell death
The general pathways of cell death induced by amyloids are also a
subject of intense debates. In the studies described above it has been shown
that amyloids activate caspases and receptor-mediated signaling pathways,
associated with apoptosis. There are, however, findings that HypF-N amyloid
exerts necrotic rather than apoptotic death of NIH-3T3 fibroblasts [Bucciantini
et al., 2002]. Here we give the definitions of apoptotic and necrotic cell deaths,
which can be useful for our further analysis. Apoptosis, or programmed cell
death, is a highly regulated process that allows a cell to self-degrade in order to
eliminate unwanted or dysfunctional cells in the body. During apoptosis, the
genome of the cell fractures, the cell shrinks and part of it disintegrates into
smaller apoptotic bodies (Figure 9). Unlike necrosis, where the cell dies by
swelling and bursting its content in the surrounding area, which causes an
inflammatory response, apoptosis is a very clean and controlled process.
During apoptosis the content of the cell is kept strictly within the cell
membrane during its degradation. The apoptotic cell is phagocytosed by
macrophages before the cell’s contents have a chance to leak into the
neighborhood [Van Cruchten & Van Den Broeck W, 2002]. Therefore,
apoptosis can prevent unnecessary inflammatory response.
Apoptosis can be triggered in a cell through either the extrinsic or
intrinsic pathways. The extrinsic pathway is initiated through the stimulation of
the transmembrane death receptors, such as the Fas or TNF receptors, located
on the cell membrane. In contrast, the intrinsic pathway is initiated through the
release of signal factors by mitochondria within the cell (Figure 10).
Figure 9. Hallmarks of the apoptotic and necrotic cell death process [Van Cruchten & Van Den Broeck W, 2002].
The caspase pathway leading to classical apoptosis [Mattson, 2000;
Strasser et al., 2000] is initiated by the release of cytochrome C from the
mitochondrial intermembrane space. Together with other essential factors, such
as ATP, it triggers assembly of the apotosome complex, which forms the
template for efficient caspase processing [Hengartner, 2000; Strasser et al.,
2000;]. The second mitochondrial death pathway leads to necrosis, without
necessarily activating caspases. Intracellular control of this pathway can be
attenuated by antioxidants [Schulze-Osthoff et al., 1992; Vercammen et al.,
1998]. The third distinct mitochondria pathway involves the release of the
apoptosis-inducing factor from the intermembrane space [Susin et al., 1999]. It
induces caspase-independent formation of large (50 kb) chromatin fragments
[Strasser et al., 2000; Susin et al., 1999]. The biochemical difference between
various cell death pathways is reflected in morphology of the condensed
chromatin (Figure 10). Often, a few pathways are activated simultaneously 31
[Jäättelä et al., 1998; Mattson, 2000; Susin et al., 1999]. The cell fate is then
determined by the relative speed of each process in a given model. The
amyloids can also activate the extrinsic apoptotic pathways, which are
followed by intrinsic pathways switching between apoptosis and necrosis,
depending on the timing and severity of mitochondria derangement.
Figure 10. Mitochondrial regulated pathways of cell death. A cell may switch back and forth between different death pathways [Leist & Jaattela, 2001].
32
33
Studied proteins
Lysozymes
Lysozyme is a protective enzyme which lyses bacteria by catalyzing the
hydrolysis of mucopolysaccharides of bacterial cell walls. Human lysozyme is
found in a variety of organs, including spleen, lung and kidney as well as in
extracellular fluids such as plasma, milk, saliva and tears. Five pathogenic
mutants of human lysozyme (I56T, F57I, W64R, D67H, W112R) are found to
be associated with non-neuropathic systemic amyloidosis, where mutant
lysozymes are deposited in kidney, lung, liver and intestines [Pepys et al.,
1993; Röcken et al., 2006; Valleix et al., 2002; Yazaki et al., 2003]. The
effects of several amyloidogenic mutations on the folding properties of
lysozyme have been investigated [Booth et al., 1997; Canet et al., 1999;
Merlini and Bellotti, 2005]. All mutants show the lower stability of the native
fold, which enables them to enter partially folded molten globule states under
physiological conditions. The molten globule state is also populated for the
wild-type lysozyme, but under more severe denaturing conditions, which may
explain why wild-type lysozyme is not found in amyloid deposits.
Hen egg white and equine lysozymes belong to the same family of c-
type lysozymes and were extensively studied during the last decades [Krishna
& Englander, 2007; Matagne & Dobson, 1998; Permyakov et al., 2006;
Mizuguchi et al., 1998; Morozova-Roche et al., 1997; Morozova et al., 1995].
In contrast to most c-type lysozymes, which do not bind calcium, equine
lysozyme is a calcium-binding protein. The calcium-binding properties
affected its structural organisation, stability and subsequently folding
34
properties. Equine lysozyme displays about 50 % sequence homology to
human and hen lysozymes, similar degree of the amino acid sequence
homology exists between the members of lysozyme family and other group of
proteins – α-lactalbumins. Lysozymes and α-lactalbumins show remarkable
homology in their tertiary and secondary structures. Equine lysozyme occupies
a special position within this family, combining the structural and folding
properties of non-calcium-binding lysozymes and calcium-binding α-
lactalbumins. Equine lysozyme was suggested to serve a role of an
evolutionary link between these groups of proteins [Nitta et al., 1987; Acharya
et al., 1994]. Specifically, it contains the active-site residues Glu35 and Asp52,
involved in lysozyme enzymatic activity [Tsuge et al., 1991; 1992], and the
conserved, high-affinity calcium-binding site of α-lactalbumins (Figure 11)
[Permyakov and Berliner, 2000].
As shown schematically in Figure 11, lysozymes and α-lactalbumins consist of
two sub-domains: the α-domain is comprised by four major α-helices, while the
β-domain contains a triple-stranded β-sheet and several long loops. All proteins
of this super-family possess also highly conserved four disulfide bridges. The
equine lysozyme calcium-binding property was initially predicted by Stuart
and co-workers [Stuart et al., 1986], since in the domain interface it has the
same calcium-binding ligands as α-lactalbumins (Figure 11). Then the binding
constant of 2×106 M-1 was determined experimentally [Nitta et al., 1987] and
the structure of the binding site confirmed by crystallographic and NMR
structural analysis [Acharya et al., 1994; Tsuge et al., 1991; 1992]. The
presence of the calcium-binding site most likely contributes to equine
lysozyme’s lower stability and cooperativity compared to other lysozymes
[Morozova et al., 1991, 1995; Morozova-Roche et al., 1999; Van Dael et al.,
1993]. If hen and human lysozymes undergo highly cooperative two-state
35
denaturation above 70 °C, and even the amyloidogenic variants of human
lysozyme are destabilised by only ca. 12 °C [ Privalov, 1979; Booth et al.,
1997], then by contrast, apo-equine lysozyme displays a three-state transition
in a wide temperature range from room temperature to 80 °C (Figure 12)
[Griko et al., 1995; Permyakov et al., 2006]. Calcium-binding significantly
stabilises the protein structure, shifting the first transition, which depend on the
calcium concentration, towards higher temperatures. However, even in the
presence of high calcium concentration the thermal denaturation of equine
lysozyme cannot be described by two-state, but three-state model.
Figure 11. Equine lysozyme – an evolutional link between lysozymes and calcium-binding α-lactalbumins [Blanch et al., 2000].
Equine lysozyme enters into various molten globule states during the
thermal, chemical or pH denaturation [Morozova et al., 1991; 1995;
Morozova-Roche et al., 1999; Van Dael et al., 1993]. Amide-proton hydrogen
exchange analysis provided residue-specific information on the nature of
equine lysozyme molten globule stability [Morozova et al., 1995]. The acidic
A-state of equine lysozyme shows high protection of the A, B and D α-helices
and particularly the amides of hydrophobic residues facing the helical interface
and making contacts with each other. The model of molten globule was
proposed, which possesses an extended hydrophobic core based on the native-
like tertiary interactions of the three out of four major α-helices. This was the
most stable molten globule within lysozyme-α-lactalbumin family [Morozova
et al., 1995, Morozova-Roche et al., 1997]. Thus, the first thermodynamic
transition of equine lysozyme corresponds to the conversion of the native state
to molten globule, while the unfolding of the core, rather than the whole α-
domain [Griko et al., 1995], gives an enthalpic contribution to the second
transition from molten globule to thermally-unfolded state [Kobashigawa et al.,
2000; Koshiba et al., 2000; Morozova et al., 1995; Van Dael et al., 1993].
Figure 12. Specific heat capacity of equine lysozyme at various calcium concentrations [Permyakov et al., 2006].
Hen lysozyme also populates an equilibrium partially folded state under
strongly acidic conditions as well as a kinetic intermediate [Kuwajima et al.,
1985; Sasahara et al., 2000]. In our research we have explored the partially
folded states of equine lysozyme and destabilised conformation of hen
lysozyme as amyloid precursors to produce various forms of amyloids
described below. 36
De novo designed protein albebetin and its constructs
De novo designed albebetin (ABB) is a 7.4 kDa protein, containing 73
amino acid residues in its sequence. ABB is characterised by a double repeat of
αββ-motif [Dolgikh et al., 1991; Fedorov et al., 1992], arranged in such a way
that four β-strands form an anti-parallel β-sheet covered by α-helices (Figure
13).
The compactness of ABB was optimised by the short loops connecting
the elements of the secondary structure and limiting the number of
conformations which can be adopted. While ABB possesses a well defined
secondary structure, it is characterised by a labile tertiary structure, which
correlates with its low immunogenicity [Bocharova et al., 2002].
Figure 13. Schematic representation of the ABB molecule [Koradi et al., 1996]. In order to increase the solubility of ABB, 22 charged amino acid residues
were introduced throughout the protein primary structure, including 17 Glu and
37
Asp residues and 5 Arg and Lys residues, creating a high net charge of -12 at
the neutral pH (Figure 14).
The large electrostatic repulsion contributes to an overall instability of
ABB. As a result, albebetin is characterized by the conformational mobility of
molten globule type at neutral pH and room temperature. While the molten
globules are most commonly induced by the additional perturbations or
destabilising conditions, neither of them are required in the case of albebetin. It
exists in the molten globule state under physiological conditions by definition
of its design.
N-tgEnhR-
N-lKEKKysp- spED lggN- mDpgDpDc tgg lEqllRR
α helix
tvhvEv β strand
vEvEv β strand Fused
Albebetin
lggs tgg spEDR-C EclEqllRR α helix
pgDp vEvEv β strand
tvhvEv β strand
Figure 14. Primary structure of ABB. Peptides of interferon-α2 or HLDF-6 fused to ABB are shown as arrows. Positively charged amino acids are denoted by italic capital letters. Negatively charged amino acids are denoted with bold capital letters [Morozova-Roche et al., 2004]
Due to the low immunogenicity of albebetin, it was planned to be used
as a carrier protein in drug delivery. The octapeptide LKEKKYSP of human
interferon-α2 or the hexapeptide TGENHR of human leukemia differentiation
factor were fused to the N-terminus of albebetin [Aphasizheva et al., 1998],
which resulted in two biologically active constructs possessing the
functionality of the fused peptides. The first construct, ABB-I, activates the
thymocyte blast transformation similarly to interferon-α2 [Chertkova et al.,
2003], while the second, ABB-DF, induces the differentiation and inhibits
proliferation of human leukemia cells similarly to molecules of differentiation
38
39
factor [Dolgikh et al., 1996; Kostanyan et al., 1994]. These peptides do not
perturb the structure of albebetin; both constructs retain the molten globule
state. Due to importance of the molten globule in amyloid scenario as an
amyloid precursor state, the amyloidogenic propensities of albebetin and its
two constructs were analysed in our research.
Application of atomic force microscopy in amyloid studies
Atomic force microscopy (AFM) allows two and three-dimensional
imaging and measurements of unstained and uncoated biological samples in air
or fluid. AFM belongs to an extended family of scanning probe microscopy
methods. AFM was first described by Binnig, Quate and Gerber in 1986 and
since then the method is rapidly developing.
AFM measurements are based on the interaction of the very sharp tip-
probe with the atomically flat surfaces on which the object of interest is
located. Images are formed by recording the interaction forces between the
probe and the surface as the cantilever carrying the probe is scanned over the
sample row by row. An optical system is used to measure the changes of the
reflection of the laser beam from the gold-coated back of the cantilever onto a
position-sensitive photodiode. This allows registering the changes in the
position of the incident laser at sub-nanometer scale. AFM operates in several
modes that can be chosen depending on the sample, environment and
measurements required. The contact mode is the original AFM imaging mode,
which can be implemented in both air and fluid. In this mode, the AFM probe
is brought into contact with the sample surface and raster-scanned across the
surface. Changes in the cantilever deflection during scanning are monitored
40
using an electronic feedback circuit. The soft biological samples, though, can
be drugged and damaged applying this mode.
The tapping mode is most commonly used in biological sample
imaging, In this mode the imaging probe is vertically oscillated at or near the
resonant frequency of the integrated cantilever. The probe-sample interactions
reflected in the changes of the amplitude and phase of oscillation. These
changes are transformed into the height or topographical image.
Simultaneously, amplitude and phase images can be produced. The advantage
of the tapping imaging that all these images can be obtained in a single scan.
Because the interactions between the tip and the surface depend not only on the
topography of the sample, but also on other characteristics, such as hardness,
elasticity, adhesion or friction, the movement of the cantilever depends also on
these properties, which are reflected in the phase shift during the tapping mode
scan. Therefore, the phase imaging can detect, for example, different
components in polymers related to their stiffness or areas of different
hydrophobicity in hydrogels immersed in saline solutions.
There are two major approaches to produce the cantilever oscillations in
the tapping mode. This can be achieved by acoustic vibrations induced by a
piezoelectric transducer attached to the cantilever holder. This method is called
an acoustic mode. Recently another approach becomes increasingly popular, in
which the cantilever is caused to oscillate directly without involving the
cantilever housing. The cantilever coated with a magnetic material is driven
into oscillation by magnetic field generated by a solenoid positioned close to
the cantilever housing. This mode is called magnetic mode. The application of
the magnetic mode has particular advantages during imaging in liquid, due to
minimising the noise produced by the system. The resolution of AFM in height
measurements both in air and in liquid environment can be of ± 0.1 nm.
Schematic diagram of AFM is presented in Figure 15.
41
AFM volume measurements
AFM can be used to determine the molecular volumes of individual particles
including single protein molecules as it was suggested by Schneider and co-
workers [Schneider et al., 1998]. We used this method to identify the volumes
of amyloid oligomers and consequently their stoichiometries. For this purpose
we measured the parameters of individual particles.
Figure 15. Schematic diagram of AFM [www.molec.com].
AFM usually overestimates the diameter of particles due to the
geometry of the tip, which introduces the broadening effect in the image. In
order to compensate this we measured the height and the diameter at half-
maximal height of the individual particles. Their volumes were calculated by
using the model of sphere segment which is presented in the Figure 16 and
equation 1.
Figure 16. Spherical cup model.
The equitation 1:
VAFM = (πh/6)×(3r2 + h2) (1)
Here, h is the particle height and r is the radius at half-height.
The molecular volume of monomeric protein was estimated by equation 2:
Vm = (M0/N0)×(V1 + dV2), (2) where M0 is the protein molecular weight, N0 is Avogadro’s number, d is the
extent of protein hydration (0.4 mol of H2O/mol of protein), V1 and V2 are the 42
43
partial specific volumes of individual protein (0.74 cm3 g -1) and water (1 cm3
g-1), respectively.
The number of molecules in oligomeric species was determined by the
equation 3.
n= VAFM / Vm, (3) where VAFM is the volume calculated from AFM measurements, Vm is volume of the single molecule.
44
Introduction to the articles
The results of this thesis are important for general understating the
mechanisms of amyloid formation and amyloid induced cytotoxicity. In this
section the main results and conclusions of the four articles, constituting the
thesis, are reviewed. The figures are referred to their numbers as given in the
original articles.
Paper I. Amyloid protofilaments from the calcium-binding protein equine
lysozyme: formation of ring and linear structures depends on pH and
metal ion concentration.
The destabilising mutations in human lysozyme are associated with the
hereditary systemic amyloidoses. Equine lysozyme is a naturally destabilised
protein, which is characterised by the formation of a range of molten globule
states. Equine lysozyme is considered as an evolutionary link between c-type
lysozymes and α-lactalbumins, as it performs the bacterial lyses similar to
lysozymes but binds calcium like α-lactalbumins. Here we have explored the
conditions, under which equine lysozyme populates molten globule state, in
order to produce amyloids. We have found that it self-assembled into
protofilaments of amyloid type under both pH 2.0 and pH 4.5 and elevated
temperatures. The morphology of its amyloid structures strongly depends on
environmental conditions such as calcium concentration, pH and temperature.
Fibrillar structures were characterised by using atomic force microscopy,
biochemical and spectroscopic methods. We demonstrated that the calcium-
bound equine lysozyme assembles into linear protofilaments, while the apo-
protein forms ring-shaped amyloids (Figure1, 2). The amyloid-rings are
45
characterised by diameters 45 - 50 nm at pH 4.5 and 70 - 80 nm at pH 2.0,
respectively. They bind amyloid dyes Congo red and thioflavin T similar to the
disease-related ring structures found for Aβ peptide and α-synuclein, which
were considered as a major pathogenic cause in Alzheimer’s and Parkinson’s
diseases. We suggested that the formation of annular amyloids is not limited to
disease-related polypeptides, but the amyloid-rings represent a second generic
type of amyloids in addition to the more common linear filaments.
We found that during the exposure to the low pH and elevated
temperatures equine lysozyme undergoes partial acidic hydrolysis, We have
shown that under appropriate conditions and limited exposure to the acidic pH
the full length equine lysozyme is preserved and gives rise to amyloid. This
indicates that a full length protein is able to assemble into amyloid structures
and partially hydrolysis is not required for this process. Upon further exposure
to acid pH two major peptides of 1 - 80 and 54 - 125 amino acid residues from
the primary structure of equine lysozyme were accumulated as shown by using
mass-spectrometry and gel electrophoresis. They are both prone to amyloid
formation, which indicates that the acidic hydrolysis can contribute to
amyloidogenic process on a longer time scale.
We have shown that equine lysozyme amyloid protofilaments do not
undergo maturation and significant growth. This was attributed to the presence
of the very stable core in its structure. Although the amino acid sequences most
likely forming the fibrillar cross-β-sheet correspond to the equine lysozyme β-
domain, the stable α-helical core forms a non-adhesive fibrillar interface,
preventing further fibrillar growth. These results suggest that it is not only the
propensity to form β-sheet structure governs the amyloid formation, but also
the conformational plasticity of the sequences involved in the fibrillar
interface. Therefore, the design of the regions with a high local stability and
46
cooperatively, in addition to the overall stabilisation of protein molecules, can
be explored in amyloid-preventing strategies.
Paper II. Fibrillation of carrier protein albebetin and its biologically active
constructs. Multiple oligomeric intermediates and pathways.
Albebetin was designed to adopt a molten globule conformation
under neutral pH. It is comprised of two αββ repeats, the compactness of
albebetin is achieved by the introducing short linking loops between the
elements of its secondary structure. Albebetin is characterised by a
conformational mobility of molten globule and rather low immunogenicity.
Albebetin was intended to be used as a drug carrier protein. For these purposes
the constructs of ABB were created by grafting to the N-terminus octapeptide
peptide from human intereferon-α2 and hexapeptide from human leukemia
differentiation factor. Both constructs possess the activity of the grafted
peptides, the first one, ABB-I, efficiently activates the thymocyte blast
transformation, while the second one, ABB-DF induces differentiation and
inhibits proliferation of human leukaemia cells.
We have demonstrated that albebetin and its constructs are highly
amyloidogenic at physiological conditions and form a wide range of amyloids
after hours and days of incubation under physiological pH. The combination of
methods including atomic force microscopy, Congo red and thioflavin T
binding and circular dichroism were used to follow development the amyloid
fibrils and their morphology. We have shown that the fibrillation proceeds via
multiple pathways and includes a hierarchy of amyloid structures ranging from
oligomers to protofilaments and fibrils. Comparative height and volume
microscopic measurements allowed us to identify two distinct types of
oligomeric intermediates: pivotal oligomers 1.2 nm in height comprised of 10 -
47
12 monomers and on-pathway amyloid-competent oligomers ca. 2 nm in height
constituted of 26 - 30 molecules. The pivotal oligomers can assemble into
chains and rings with “bead-on-string morphology”, in which a “bead”
corresponds to an individual oligomer. The rings and chain assemblies of
oligomers are very stable and, once formed, they remain in solution
simultaneously with fibrils. The amyloid-competent oligomers give rise to
protofilaments and fibrils, and their formation is concomitant with an
increasing level of thioflavin T binding. The amyloid nature of filamentous
structures was also confirmed by a pronounced thioflavine T and Congo red
binding as well as characteristic β-sheet-rich far-UV circular dichroism
spectrum. We suggest that transformation of the pivotal oligomers into the
amyloid-prone ones is a limiting stage in amyloid assembly. Peptides, either
fused to albebetin or added into solution, and an increased ionic strength
promote fibrillation of albebetin, carrying a net charge of -12 at the neutral pH,
by counterbalancing critical electrostatic repulsions. This finding demonstrates
that the fibrillation of newly designed polypeptide-based products can produce
multimeric amyloid species with a potentially “new” functionality, raising
questions about their potential safety in biomedical applications.
Paper III. Cytotoxicity of albebetin oligomers depends on cross-β-sheet
formation.
The cytotoxicity of amyloid prefibrillar and fibrillar species is a focus
of much current research. It is viewed as a common amyloid characteristic,
which the polypeptides involved in amyloid formation do not possess prior
their assembly into amyloids. The advantage of using albebetin in the
cytotoxicity studies is related to the fact that it forms amyloid under
physiological conditions and therefore its amyloid structures will not be
48
perturbed, when subjected to the conditions of cytotoxicity experiments.
Albebetin populated also well-defined and distinct amyloid oligomers of two
types, as described above, and protofilaments. Therefore we were able to
explore the cytotoxicity of two types of amyloid oligomeric assemblies, with
and without cross-β-sheet. We have shown that the initial or pivotal oligomers
consisted of 10–15 molecules as determined by atomic force microscopy, do
not bind thioflavin-T and do not affect viability of granular neurons and SH-
SY5Y cells. When these oligomers were converted into the larger ones
possessing ca. 30 - 40 albebetin molecules and developing cross-β-sheet
structure, they induce the reduction of the viability of both types of neuronal
cells. Neither monomers nor protofilaments of albebetin displayed cellular
toxicity on both neuronal cells. We have suggested that oligomeric size is
important for stabilizing cross-β-sheet core. The stable core may prevent
amyloid oligomers from the dissociation upon the penetration through cellular
membranes as well as inside of the cellular environment. The cytotoxicity of de
novo protein albebetin accentuates the universality of this phenomenon. As
albebetin was designed and intended to use as a carrier-protein for drug
delivery, its cytotoxicity once more necessitates the examination of
amyloidogenic properties prior subjecting the proteinaceous compounds to the
large scale applications.
Paper IV. Lysozyme amyloid oligomers and fibrils induce cellular death
via different apoptotic/necrotic pathways.
Amyloid cytotoxicity was further examined using hen lysozyme as a
model protein. Among the newly gained amyloid properties, its cytotoxicity
plays a key role. Lysozyme is a ubiquitous protein which has been shown to be
49
involved in systemic amyloidoses in vivo and able to form amyloid under
destabilizing conditions in vitro. We characterized both oligomers and fibrils of
hen lysozyme by atomic force microscopy and spectroscopic technique. The
well-defined amyloid species were subjected to the cellular toxicity assays after
different periods of aging. We demonstrated that both oligomers and fibrils of
hen lysozyme induce a dose (5 - 50 μM) and time-dependent (6 - 48 h) effect
on the viability of neuroblastoma SH-SY5Y cells. Using a wide range of cell
toxicity assays to probe the apoptotic and necrotic cell death pathways, we
have demonstrated that the oligomers and fibrils act on a different time scale.
We revealed that fibrils induce a decrease of cell viability after 6 h due to
membrane damage shown by inhibition of tetrazole reduction (WST-1 assay),
lactate dehydrogenase release and propidium iodide intake. By contrast, the
amyloid oligomers activate caspases after 6 h, but cause the cell viability
decline only after 48 h, as shown by fluorescent-labelled annexin V binding to
externalized phosphatidylserine, propidium iodide DNA staining, lactate
dehydrogenase release, and by typical apoptotic shrinking of cells. We
concluded that amyloid oligomers induce apoptosis-like cell death, while the
fibrils lead to necrosis-like death. As polymorphism is a common property of
an amyloid, we demonstrated that it is not a single uniform species, but rather a
continuum of cross-β-sheet-containing amyloids that are cytotoxic. An
abundance of lysozyme once more illuminates a universality of the amyloid-
associated toxicity, and accentuates that amyloid toxicity should be assessed in
all clinical applications involving proteinaceous materials.
50
Conclusions
• We have demonstrated that equine lysozyme forms amyloids under
conditions in which its molten globule states are populated. The
morphology of equine lysozyme amyloids is strongly dependent on
environmental conditions such as calcium concentration, pH and
temperature.
• We suggested that the ring-shaped amyloids represent an alternative
generic form of amyloid assembly in addition to most commonly
populated linear protofilaments. The ring formation is not limited to
Alzheimer’s and Parkinson’s disease-related amyloids, but they can be
formed even by such an abundant protein as lysozyme.
• The presence of very stable hydrophobic core in equine lysozyme
structure prevents amyloid protofilaments from significant elongation
and lateral assembly. The strategy of designing of the specific highly
stable and cooperative areas within protein molecule can be explored as
amyloid preventing approach in additional to the overall stabilisation of
the protein fold.
• We have shown that full-length equine lysozyme forms amyloid,
though on the longer time-scale acidic hydrolysis can also contribute to
the fibril formation.
• We have shown that de novo protein albebetin and its constructs with
biologically active peptides are inherently highly amyloidogenic,
forming a range of amyloid species under physiological conditions.
• We demonstrated the multiple pathways of amyloid assembly of
albebetin, including formation of a hierarchy of on- and off-pathway
oligomeric intermediates. The pivotal oligomers comprised of 12
51
protein monomers are the key-point, in which the amyloid pathways
split apart. The conversion of the pivotal oligomers into amyloid-prone
ones is considered as a rate limiting step in amyloid assembly.
Amyloid-prone oligomers are characterized by the cross-β-sheet
formation, giving rise to amyloid protofilaments and fibrils.
• Electrostatic interactions have been shown to be critical in amyloid
assembly of albebetin. The fusion of the short polar peptides to the N-
terminus of albebetin as well as increasing ionic strength significantly
enhances its amyloidogenicity.
• We demonstrated that the amyloid toxicity of albebetin is clearly
correlates with the development the cross-β-sheet in their oligomeric
assemblies. The pivotal oligomers, which do not contain cross-β-sheet
core, are apparently non-toxic, while the larger amyloid-prone
oligomers, comprised of ca. 30 and more molecules and possessing
well-developed cross-β-sheet pattern, induce the cell death. The initial
monomeric albebetin and its protofilaments do not display toxicity on
the neuronal cells.
• We demonstrated that amyloid oligomers and fibrils of hen lysozyme
induce death of SH-SY5Y cells via different mechanisms.
• We conclude that oligomers induce apoptosis-like cell death, while the
fibrils lead to necrosis-like death.
52
Acknowledgements I would like to express my sincere gratitude to:
Entire department of Medical Biochemistry and Biophysics for creating the
nice atmosphere.
My supervisor Ludmilla Morozova-Roche, for everything you have done for
me both in science and in life in general. I will never get better boss than you!
Gerhard Gröbner, for the support, infinite optimism and for the introducing the
phrase “gut, gut” in my lexicon.
Mantas, for the friendship and for the stories beginning “once upon a time we
visited Systembolaget”.
Anna, Kristina, Kiran – we are the very group.
Our administrator, Ingrid Råberg, for the guidance through the peculiarities of
life in Sweden.
Gena, for the hockey and the brain storm in chess.
Maribel, for your shine. No pasaran!
Katya, Mattias, Linus, Peter - tack att ni finns.
And all russian-speaking community in Umeå.
Моим друзьям и тем, кто был в Умео визитом на короткое время, я вас
всех помню.
Кате, за твой созидательно-подстригательный креатив, способный
потрясти окружающих, включая мою жену.
Моей маме за ее безграничную любовь и веру!
Моим любимым, жене и дочери, за приобретение смысла в жизни! Я
люблю вас, мои родные!
Thank you!!!
53
References
Acharya K.R., Stuart D.I., Phillips D.C., McKenzie H.A. and Teahan C.G. (1994) Models of the three-dimensional structures of echidna, horse, and pigeon lysozymes: calcium-binding lysozymes and their relationship with α-lactalbumins. J. Prot. Chem. 6, 569-584.
Ahmad A., Millett I. S., Doniach S., Uversky V.N. Fink A L. (2003) Partially folded intermediates in insulin fibrillation. Biochemistry 42, 11404–11416.
Ahmad A., Millett I.S., Doniach S., Uversky V.N., Fink A.L. (2004) Stimulation of insulin fibrillation by urea-induced intermediates. J. Biol. Chem. 279, 14999-5013.
Anderluh G., Gutierrez-Aguirre I., Rabzelj S., Ceru S., Kopitar-Jerala N., Macek P., Turk V, Zerovnik E. (2005) Interaction of human stefin B in the prefibrillar oligomeric form with membranes. Correlation with cellular toxicity. FEBS J. 272, 3042–3051.
Andersson K., Olofsson A., Nielsen E. H., Svehag S. E., Lundgren E. (2002) Only amyloidogenic intermediates of transthyretin induce apoptosis. Biochem. Biophys. Res. Commun. 294, 309–314.
Aphasizheva, I. Y., Dolgikh, D. A., Abdullaev, Z. K., Uversky, V. N., Kirpichnikov, M. P., and Ptitsyn, O. B. (1998) Can grafting of an octapeptide improve the structure of a de novo protein? FEBS Lett. 425, 101-104.
Atwood C.S., Moir R.D., Huang X., Scarpa R.C., Bacarra N.M., Romano D.M., Hartshorn M.A., Tanzi R.E., Bush A.I. (1998) Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J. Biol. Chem. 273, 12817-12826.
Ayed A., Duckworth H.W. (1999) A stable intermediate in the equilibrium unfolding of Escherichia coli citrate synthase. Protein Sci. 8, 1116-1126.
Baskakov I.V., Legname G., Gryczynski Z., Prusiner S.B. (2004) The peculiar nature of unfolding of the human prion protein. Protein Sci. 13, 586-595.
Bellotti V., Mangione P. and Merlini G. (2000) Review: Immunoglobulin light chain amyloidosis - the archetype of structural and pathogenic variability. J. Struct. Biol. 130, 280–289
Blake C.C.F. and Serpell L.C. (1996) Synchrotron X-ray studies suggest that the core of the transthyretin amyloid fibril is a continuous beta-sheet helix. Structure 4, 989-998.
54
Bhatia R., Lin H., Lal R. (2000) Fresh and globular amyloid β protein (1–42) induces rapid cellular degeneration: evidence for AβP channel-mediated cellular toxicity. FASEB J. 14, 1233–1243.
Bocharova O.V., Moshkovskii S.A., Chertikova R.V., Abdullaev Z.Kh., Kolesanova E.F., Dolgikh D.A. and Kirpichnikov M.P. (2002) Introduction of biologically active fragments of interferon-R2 and insulin into the artificial protein albebetin affects immunogenicity of the final construct. J. Mol. Biol. 6, 84-90.
Bonar L., Cohen A.S., Skinner M.M. (1969) Characterization of the amyloid fibril as a cross-beta protein. Proc. Soc. Exp. Biol. Med. 131, 1373–1375.
Booth D.R., Sunde M., Bellotti V., Robinson C.V., Hutchinson W.L., Fraser P.E., Hawkins P.N., Dobson C.M., Radford S.E., Blake C.C., Pepys M.B. (1997) Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 385, 787-793.
Blanch E.W., Morozova-Roche L.A., Hecht L., Noppe W., Barron L.D. (2000) Raman optical activity characterization of native and molten globule states of equine lysozyme: comparison with hen lysozyme and bovine alpha-lactalbumin. Biopolymers 57, 235-248.
Brange J., Dodson G.G., Edwards D.J., Holden P.H., Whittingham J.L. (1997) A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin). Proteins 27, 507-516.
Brookes P.S., Yoon Y., Robotham.JL., Anders M.W., Sheu.SS. (2004) Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol. Cell. Physiol. 287, C817-C833
Bucciantini M., Giannoni E., Chiti F., Baroni F., Formigli L., Zurdo J., Taddei N., Ramponi G., Dobson C.M., Stefani M (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507–511.
Butterfield D.A., Hensley K., Harris M., Mattson M., Carney J. (1994) Betaamyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence-specific fashion: implications to Alzheimer’s disease. Biochem. Biophys. Res. Commun. 200, 710–715.
Butterfield D.A., Drake J., Pocernich C., Castegna A. (2001) Evidence of oxidative damage in Alzheimer's disease brain: central role for amyloid beta-peptide. Trends Mol. Med. 7, 548-554.
Canet D., Sunde M., Last A.M., Miranker A., Spencer A., Robinson C.V., Dobson C.M. (1999) Mechanistic studies of the folding of human
55
lysozyme and the origin of amyloidogenic behavior in its disease-related variants. Biochemistry 38, 6419–6427.
Canet D., Last A.M., Tito P., Sunde M., Spencer A., Archer D.B., Redfield C., Robinson C.V., Dobson C.M. (2002) Local cooperativity in the unfolding of an amyloidogenic variant of human lysozyme. Nat. Struct. Biol. 9, 308–315.
Casley C.S., Canevari L., Land J.M., Clark J.B., Sharpe M.A. (2002) Beta-amyloid inhibits integrated mitochondrial respiration and key enzyme activities. J. Neurochem. 80, 91–100.
Carter D.B., Chou K.C. (1998) A model for structure-dependent binding of Congo red to Alzheimer beta-amyloid fibrils. Neurobiol. Aging. 19, 37-40.
Chamberlain A.K., MacPhee C.E., Zurdo J., Morozova-Roche L.A., Hill H.A., Dobson C.M., Davis JJ. (2000) Ultrastructural organization of amyloid fibrils by atomic force microscopy. Biophys. J. 79, 3282-3293.
Chapman M.R., Robinson L.S., Pinkner J.S., Roth R., Heuser J., Hammar M., Normark S., Hultgren S.J. (2002) Role of Escherichia coli curli operons in directing amyloid fiber formation. Science 295, 851-855
Chertkova R.V., Kostanian I.A., Astapova M.V., Surina E.A., Dolgikh D.A. and Kirpichnikov M.P. (2003) An artificial protein, possessing biological activity of HL-60 human promyelocytic leukemia differentiation factor. Bioorg. Khim. 29, 30-37.
Chiti F., Webster P., Taddei N., Clark A., Stefani M., Ramponi G. & Dobson C.M. (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc. Natl Acad. Sci. USA 96, 3590–3594.
Chiti F, Taddei N, Bucciantini M, White P, Ramponi G, Dobson CM. 2000 Mutational analysis of the propensity for amyloid formation by a globular protein. EMBO J. 19, 1441-1449.
Chiti F., Taddei N., Baroni F., Capanni C., Stefani M., Ramponi G. and Dobson C.M. (2002) Kinetic partitioning of protein folding and aggregation. Nature Struct. Biol. 9, 137–143.
Chromy B.A., Nowak R.J., Lambert M.P., Viola K.L., Chang L., Velasco P.T., Jones B.W., Fernandez S.J., Lacor P.N., Horowitz P., Finch C.E., Krafft G.A., Klein W.L. (2003) Selfassembly of Aβ(1-42) into globular neurotoxins. Biochemistry 42, 12749–12760.
Cohen A.S., Jones L.A. (1991) Amyloidosis. Curr. Opin. Rheumatol. 3, 125-38.
Cooper A.A., Gitler A.D., Cashikar A., Haynes C.M., Hill K.J., Bhullar B., Liu K., Xu K., Strathearn K.E., Liu F., Cao S., Caldwell K.A.,
56
Caldwell G.A., Marsischky G., Kolodner R.D., Labaer J., Rochet J.C., Bonini N.M., Lindquist S. (2006) Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson's models. Science 313, 24–328.
Crompton M. (2004) Mitochondria and aging: a role for the permeability transition? Aging Cell. 3, 3-6.
Cymes G.D., Grosman C., Delfino J.M., Wolfenstein-Todel C. (1996) Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process. Protein Sci. 5, 2074-2079.
Damaschun G., Damaschun H., Fabian H., Gast K., Krober R., Wieske M. & Zirwer D. (1999) Conversion of yeast phosphoglycerate kinase into amyloid-like structure. Proteins 39, 204–211.
Damaschun G., Damaschun H., Gast K. and Zirwer D. (1999) Proteins can adopt totally different folded conformations. J. Mol. Biol. 291, 715–725.
De Felice F.G., Vieira M.N., Meirelles M.N., Morozova-Roche L.A., Dobson C.M., Ferreira S.T. (2004) Formation of amyloid aggregates from human lysozyme and its disease-associated variants using hydrostatic pressure. FASEB J. 10, 1099-1101.
De Giorgi F., Lartigue L., Bauer M.K., Schubert A., Grimm S., Hanson G.T., Remington S.J., Youle R.J., Ichas F. (2002) The permeability transition pore signals apoptosis by directing Bax translocation and multimerization. FASEB J. 16, 607-609.
Der-Sarkissian A, Jao CC, Chen J, Langen R. (2003) Structural organization of alpha-synuclein fibrils studied by site-directed spin labeling. J. Biol. Chem. 278, 37530-37535.
Ding T.T., Lee S.J., Rochet J C. & Lansbury, P.T., Jr (2002). Annular alpha-synuclein protofibrils are produced when spherical protofibrils are incubated in solution or bound to brain-derived membranes. Biochemistry 41, 10209–10217.
Dobson C.M. (1999) Protein misfolding, evolution and disease. Trends Biochem. Sci.24, 329-332.
Dobson C.M. (2001) The structural basis of protein folding and its links with human disease. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356, 133-145.
Dolgikh D.A., Fedorov A.N., Chermeris V.V., Chernov B.K., Finkel’shtein A.V., Shul’ga A.A., Alakhov I.B., Kirpichnikov M.P., and Ptitsyn O.B. (1991) Preparation and study of albebetin, an artificial protein with a given spatial structure. Dokl. Akad. Nauk SSSR 320, 1266-1269.
57
Dolgikh D. A., Uversky V. N., Gabrielian A. E., Chemeris V. V., Fedorov A. N., Navolotskaya E. V., Zav’yalov V. P. and Kirpichnikov M. P. (1996) The de novo protein with grafted biological function: transferring of interferon blast- transforming activity to albebetin. Protein Eng. 9, 195-201.
Dos Reis S, Coulary-Salin B, Forge V, Lascu I, Begueret J, Saupe S.J. (2002) The HET-s prion protein of the filamentous fungus Podospora anserina aggregates in vitro into amyloid-like fibrils. J. Biol. Chem. 277, 5703-5706.
Dunker A.K., Lawson J.D., Brown C.J., Williams R.M., Romero P., Oh J.S., Oldfield C.J., Campen A.M., Ratliff C.M., Hipps K.W., Ausio J., Nissen M.S., Reeves R., Kang C., Kissinger C.R., Bailey R.W., Griswold M.D., Chiu W., Garner E.C. Obradovic Z. (2001) Intrinsically disordered protein. J. Mol. Graph. Model. 19, 26–59.
Eakin C.M., Knight J.D., Morgan C.J., Gelfand M.A., Miranker A.D. (2002) Formation of a copper specific binding site in non-native states of beta-2-microglobulin. Biochemistry 41, 10646-10656.
Eckert A., Keil U., Marques C.A., Bonert A., Frey C., Schussel K., Muller W.E. (2003) Mitochondrial dysfunction, apoptotic cell death, and Alzheimer's disease. Biochem. Pharmacol. 66, 1627-1634.
Ekiel I. & Abrahamson M. (1996) Folding-related dimerization of human cystatin C. J. Biol. Chem. 271, 1314–1321.
Fandrich M., Fletcher M.A., Dobson C.M. (2001) Amyloid fibrils from muscle myoglobin. Nature 410, 165-166.
Ferreira S.T. & De Felice F.G. (2001) Protein dynamics, folding and misfolding: from basic physical chemistry to human conformational diseases. FEBS Lett. 498, 129–134.
Fedorov A.N., Dolgikh D.A., Chemeris V.V., Chernov B.K., Finkelstein A.V., Schulga A.A., Alakhov Y.B., Kirpichnikov M.P. and Ptitsyn O.B. (1992) De novo design, synthesis and study of albebetin, a polypeptide with a predetermined threedimensional structure. Probing the structure at the nanogram level. J. Mol. Biol. 225, 927-931.
Forloni G., Bugiani O., Tagliavini F. & Salmona M. (1996) Apoptosis-mediated neurotoxicity induced by β-amyloid and PrP fragments. Mol. Chem. Neuropathol. 28, 163–171.
Forloni G. (1996) Neurotoxicity of β-amyloid and prion peptides. Curr. Opin. Neurol. 9, 492–500.
Guijarro JI, Sunde M, Jones JA, Campbell ID, Dobson CM. 1998 Amyloid fibril formation by an SH3 domain. Proc Natl Acad Sci U S A. 95, 4224-4228.
58
Glenner G.G., Eanes E.D., Bladen, H.A. (1974) Beta-pleated sheet fibrils. A comparison of native amyloid with synthetic protein fibrils, J. Histochem. Cytochem. 22, 1141–1158.
Glenner G. (1980) Amyloid deposits and amyloidosis. N. Engl. J. Med. 302, 1283-1292.
Goda S., Takano K., Yamagata Y., Nagata R., Akutsu H., Maki S., Namba K., Yutani K. (2000) Amyloid protofilament formation of hen egg lysozyme in highly concentrated ethanol solution. Protein Sci. 9, 369-375.
Goers J., Permyakov S.E., Permyakov E.A., Uversky V.N., Fink A.L. (2002) Conformational prerequisites for alpha-lactalbumin fibrillation. Biochemistry 41, 12546-12551.
Gong Y., Chang L., Viola K.L., Lacor P.N., Lambert M.P., Finch C.E. Grant A. Krafft G.A. and Klein W.L. (2003) Alzheimer's diseaseaffected brain: presence of oligomeric Aβ ligands (ADDLs) suggests a molecular basis for reversible memoryloss. Proc. Natl. Acad. Sci.USA 100, 10417–10422.
Griko Y.V., Freire E., Privalov G., van Dael H. and Privalov P.L. (1995) The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme. J Mol Biol. 252, 447-459.
Han H., Weinreb P.H. and Lansbury P.T.Jr (1995) The core Alzheimer’s peptide NAC forms amyloid fibrils which seed and are seeded by beta-amyloid: is NAC a common trigger or target in neurodegenerative disease? Chem. Biol. 2, 163–169.
Hamada D., Dobson C.M. (2002) A kinetic study of beta-lactoglobulin amyloid fibril formation promoted by urea. Protein Sci. 10, 2417-2426.
Hammarstrom P, Jiang X, Hurshman A.R., Powers E.T., Kelly J.W. (2002) Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity. Proc Natl Acad Sci USA 99, 16427-16432.
Hartley D.M., Walsh D.M., Ye C.P., Diehl T., Vasquez S., Vassilev P.M., Teplow D.B., Selkoe D.J. (1999) Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J. Neurosci. 19, 8876-8884.
Heegaard N.H., Sen J.W., Kaarsholm N.C., Nissen, M.H. (2001) Conformational intermediate of the amyloidogenic protein beta 2-microglobulin at neutral pH. J. Biol. Chem. 276, 32657–32662.
Hengartner M.O. (2000)The biochemistry of apoptosis. Nature 407, 770–776.
59
Howlett D.R., Jennings K.H., Lee D.C., Clark M.S., Brown F., Wetzel R. Wood S.J., Camilleri P., Roberts G.W. (1995) Aggregation state and neurotoxic properties of Alzheimer β-amyloid peptide. Neurodegeneration 4, 23–32.
Hoyer W., Cherny D., Subramaniam V. and Jovin T. M. (2004) Impact of the acidic C-terminal region comprising amino acids 109–140 on alpha-synuclein aggregation in vitro. Biochemistry 43, 16233–16242.
Huang X., Atwood C.S., Moir R.D., Hartshorn M.A., Vonsattel J.P., Tanzi R.E., Bush A.I. (1997) Zinc-induced Alzheimer's Abeta(1-40) aggregation is mediated by conformational factors. J. Biol. Chem. 272, 26464-26470.
Iconomidou V.A, Vriend G., Hamodrakas S.J. (2000) Amyloids protect the silkmoth oocyte and embryo. FEBS Lett. 479, 141-145.
Jäättelä M., Wissing D., Kokholm, K, Kallunki T. & Egeblad M. (1998) Hsp70 exerts its anti-apoptotic function downstream of caspase-3-like proteases. EMBO J. 17, 6124–6134.
Jahn T.R., Radford S.E. (2005) The Yin and Yang of protein folding. FEBS J. 272, 5962-5970.
Jansen R., Dzwolak W., Winter R. (2005) Amyloidogenic self-assembly of insulin aggregates probed by high resolution atomic force microscopy. Biophys. J. 88, 1344-1353.
Janson J., Ashley R.H., Harrison D., McIntyre S., Butler P.C. (1999) The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles. Diabetes 48, 491-498.
Jarrett J.T. and Lansbury P.T. (1993) Seeding "one-dimensional crystallization" of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73, 1055-1058.
Jones C.E., Abdelraheim S.R., Brown D.R., Viles J.H. (2004) Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein. J. Biol. Chem. 279, 32018-32027.
Katayama T., Imaizumi K., Manabe T., Hitomi J., Kudo T., Tohyama M. (2004) Induction of neuronal death by ER stress in Alzheimer’s disease. J. Chem. Neuroanat. 28, 67–78.
Kaufman R.J. (1999) Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes. Dev. 13, 1211–1233.
Kayed R., Bernhagen J., Greenfield N., Sweimeh K., Brunner H., Voelter W. and Kapurniotu A. (1999) Conformational transitions of Islet
60
Amyloid Polypeptide (IAPP) in amyloid formation in vitro. J. Mol. Biol. 287 781–796.
Kayed R., Head E., Thompson J.L., McIntire T.M., Milton S.C., Cotman C.W. & Glabe C.G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489.
Kelly J.W. (1998) The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 8, 101–106.
Kenney J.M., Knight D., Wise M.J., Vollrath F. (2002) Amyloidogenic nature of spider silk. Eur J Biochem. 269, 4159-4163.
King C.Y., Tittmann P., Gross H., Gebert R., Aebi M., Wuthrich K. (1997) Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments. Proc. Natl. Acad. Sci. USA 94, 6618-6622.
Knight J.D., Miranker A.D. (2004) Phospholipid catalysis of diabetic amyloid assembly. J. Mol. Biol. 341, 1175-1187.
Kobashigawa Y., Demura M., Koshiba T., Kumaki Y., Kuwajima K. and Nitta K. (2000) Hydrogen exchange study of canine milk lysozyme: stabilization mechanism of the molten globule. Proteins 40, 579-589.
Koradi R., Billeter M., and Wuthrich K. (1996) MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graphics 14, 51-55.
Koshiba T., Yao M., Kobashigawa Y., Demura M., Nakagawa A., Tanaka I., Kuwajima K. and Nitta K. (2000) Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme. Biochemistry 39, 3248-3257.
Kostanyan I. A., Astapova M. V., Starovoytova E. V., Dranitsina S. M. and Lipkin V. M. (1994) A new human leukemia cell 8.2 kDa differentiation factor: isolation and primary structure. FEBS Lett. 356, 327-329.
Krebs M.R., Wilkins D.K., Chung E.W., Pitkeathly M.C., Chamberlain A.K., Zurdo J., Robinson C.V., Dobson C.M. (2000) Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain. J. Mol. Biol. 300, 541-549.
Krebs M.R., Morozova-Roche L.A., Daniel K., Robinson C.V., Dobson C.M. (2004) Observation of sequence specificity in the seeding of protein amyloid fibrils. Protein Sci. 13, 1933-1938.
Krebs M.R., Bromley E.H., Donald A.M. (2005) The binding of thioflavin T to amyloid fibrils: localisation and implications. J. Struct. Biol. 149, 30-37.
Krishna M.M., Englander S.W. (2007) A unified mechanism for protein folding: predetermined pathways with optional errors. Protein Sci. 16, 449-464.
Kuwajima K., Hiraoka Y., Ikeguchi M., Sugai S. (1985) Comparison of the transient folding intermediates in lysozyme and -lactalbumin. Biochemistry 24, 874-881
Kyle R.A. (2001) Amyloidosis: a convoluted story. Brit. J. Haem. 114, 529-538.
Lai Z., Colon W. & Kelly J.W. (1996) The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid. Biochemistry 35, 6470–6482.
Lashuel H.A., Hartley D., Petre B.M., Walz T., Lansbury P.T. Jr. (2002) Neurodegenerative disease: amyloid pores from pathogenic mutations. Nature 418, 291.
Lashuel H.A., Lansbury P.T. Jr. (2006) Are amyloid diseases caused by protein aggregates that mimic bacterial pore-forming toxins? Q Rev Biophys. 39, 167-201.
Leist M., Jaattela M. (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol. 2, 589-598.
Lomakin A., Chung D.S., Benedek G.B., Kirschner D.A. and Teplow D.B. (1996) On the nucleation and growth of amyloid beta-protein fibrils: detection of nuclei and quantitation of rate constants. Proc. Natl. Acad. Sci. USA 93, 1125–1129.
Lorenzo A. & Yankner B. A. (1994) β-amyloid neurotoxicity requires fibril formation and is inhibited by congo red. Proc. Natl Acad. Sci. USA 91, 12243–12247.
Lustbader J.W., Cirilli M., Lin C., Xu H.W., Takuma K., Wang N., Caspersen C., Chen X., Pollak S., Chaney M., Trinchese F., Liu S., Gunn-Moore F., Lue L.F., Walker D.G., Kuppusamy P., Zewier Z.L., Arancio O., Stern D., Yan S.S., Wu H. (2004) ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science 304, 448-452.
Malisauskas M., Zamotin V., Jass J., Noppe W., Dobson C.M., Morozova-Roche L.A. (2003) Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration. J. Mol. Biol. 330, 879-890.
Malisauskas, M., Ostman, J., Darinskas, A., Zamotin, V., Liutkevicius, E., Lundgren, E. & Morozova-Roche, L. A. (2005). Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity? J. Biol. Chem. 280, 6269–6275.
61
62
Matagne A., Dobson C.M. (1998) The folding process of hen lysozyme: a perspective from the 'new view'. Cell Mol. Life. Sci. 54, 363-371.
Mattson M.P. (2000) Apoptosis in neurodegenerative disorders. Nature Rev. Mol. Cell Biol. 1, 120–129
McParland V.J., Kalverda A.P., Homans S.W., and Radford S.E. (2002) Structural properties of an amyloid precursor of beta(2)-microglobulin. Nat. Struct. Biol. 9, 326–331.
Merlini G. and Bellotti V. (2003) Molecular mechanisms of amyloidosis. N. Engl. J. Med. 349, 583–596.
Merlini G. and Bellotti V. (2005) Lysozyme: a paradigmatic molecule for the investigation of protein structure, function and misfolding. Clin. Chim. Acta 357, 168–172.
Mishra R., Sorgjerd K., Nystrom S., Nordigarden A., Yu Y.C., Hammarstrom P. (2007) Lysozyme amyloidogenesis is accelerated by specific nicking and fragmentation but decelerated by intact protein binding and conversion. J. Mol. Biol. 366, 1029-1044.
Misonou H., Morishima-Kawashima M., Ihara Y. (2000) Oxidative stress induces intracellular accumulation of amyloid beta-protein (Abeta) in human neuroblastoma cells. Biochemistry 39, 6951–6959.
Missmahl H.P. and Hartwig M. (1953) Polarisationsoptischeuntersuchungen an der amyloidsubstance, Virchows. Arch. Pathol. Anat. 324, 489–508.
Mizuguchi M., Arai M., Ke Y., Nitta K., Kuwajima K. (1998) Equilibrium and kinetics of the folding of equine lysozyme studied by circular dichroism spectroscopy. J. Mol. Biol. 283, 265-277.
Morgan C.J., Gelfand M., Atreya C., Miranker A.D. (2001) Kidney dialysis-associated amyloidosis: a molecular role for copper in fiber formation. J. Mol. Biol. 309, 339-345.
Morishima Y., Gotoh Y., Zieg J., Barrett T., Takano H., Flavell R., Davis R.J., Shirasaki Y, Greenberg M.E. (2001) Beta-amyloid induces neuronal apoptosis via a mechanism that involves the c-Jun N-terminal kinase pathway and the induction of Fas ligand. J. Neurosci. 21, 7551–7560.
Morozova L., Haezebrouck P. and Van Cauwelert F. (1991) Stability of equine lysozyme. Thermal unfolding behaviour. Biophys. Chem. 41, 185–191.
Morozova L.A., Haynie D.T., Arico-Muendel C., Van Dael H., Dobson C.M. (1995) Structural basis of the stability of a lysozyme molten globule. Nat. Struct. Biol. 10, 871-875.
Morozova-Roche L.A., Arico-Muendel C.C., Haynie D.T., Emelyanenko V.I., Van Dael H., Dobson C.M. (1997) Structural characterisation and
63
comparison of the native and A-states of equine lysozyme. J. Mol. Biol. 268, 903-921.
Morozova-Roche L.A., Jones J.A., Noppe W. & Dobson C.M. (1999) Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme. J. Mol. Biol. 289, 1055-1073.
Morozova-Roche L.A., Zurdo J., Spencer A., Noppe W., Receveur V., Archer D.B., Joniau M., Dobson C.M. (2000) Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants. J. Struct. Biol. 130, 339-351.
Morozova-Roche L.A., Zamotin V., Malisauskas M., Ohman A., Chertkova R., Lavrikova M.A., Kostanyan I.A., Dolgikh D.A, Kirpichnikov M.P. (2004) Fibrillation of carrier protein albebetin and its biologically active constructs. Multiple oligomeric intermediates and pathways. Biochemistry 43, 9610-9619.
Nakagawa T., Yuan J. (2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol. 150, 887–894.
Nielsen L., Khurana R., Coats A., Frokjaer S., Brange J., Vyas S., Uversky V.N., Fink A.L. (2001) Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. Biochemistry 40, 6036-6046.
Nielsen L., Frokjaer S., Brange J., Uversky V.N. and Fink A.L. (2001) Probing the mechanism of insulin fibril formation with insulin mutants. Biochemistry 40, 8397–8409.
Nilsson M.R., Raleigh D.P. (1999) Analysis of amylin cleavage products provides new insights into the amyloidogenic region of human amylin. J. Mol. Biol. 294, 1375-13785.
Nitta K., Tsuge H., Sugai S. and Shimazaki K. (1987) The calcium-binding property of equine lysozyme. FEBS Lett. 223, 405-408.
Novitskaya V., Bocharova O.V., Bronstein I. & Baskakov I.V. (2006) Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J. Biol. Chem. 281, 13828–13836.
Nunomura A., Castellani R.J., Zhu X., Moreira P.I., Perry G., Smith M.A. (2006) Involvement of oxidative stress in Alzheimer disease J. Neuropathol. Exp. Neurol. 65, 631-641.
Ojaimi J., Byrne E. (2001) Mitochondrial function and alzheimer's disease. Biol. Signals Recept. 10, 254-262.
Panick G., Malessa R. and Winter R. (1999) Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering, Biochemistry 38, 6512–6519.
64
Pasquato N., Berni R., Folli C., Alfieri B., Cendron L., Zanotti G. (2007) Acidic pH-induced conformational changes in amyloidogenic mutant transthyretin. J. Mol. Biol. 366, 711-719.
Pedersen J.S., Dikov D., Flink J.L., Hjuler H.A. Christiansen G., Otzen D.E. (2006) The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting. J. Mol. Biol. 355, 501-523.
Pepys M.B., Hawkins P.N., Booth D.R., Vigushin D.M., Tennent G.A. and Soutar A.K. (1993) Human lysozyme gene mutations cause hereditary systemic amyloidosis, Nature 362, 553–557.
Permyakov E.A. and Berliner L.J. (2000) α-Lactalbumin: structure and function. FEBS Lett. 473, 269-274.
Permyakov E.A., Permyakov S.E., Deikus G.Y., Morozova-Roche L.A., Grishchenko V.M., Kalinichenko L.P., Uversky V.N. (2003) Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges. Proteins 51, 498-503.
Permyakov S.E., Khokhlova T.I., Nazipova A.A., Zhadan A.P., Morozova-Roche L.A. and Permyakov E.A. (2006) Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature "phase diagrams". Proteins 65, 984-998.
Pertinhez T.A., Bouchard M., Tomlinson E.J., Wain R., Ferguson S.J., Dobson C.M., Smith L.J. (2001) Amyloid fibril formation by a helical cytochrome. FEBS Lett. 495, 184-186.
Petkova A.T., Leapman R.D., Guo Z., Ya, W.M., Mattson M.P. & Tycko R. (2005) Self-propagating, molecular-level polymorphism in Alzheimer's β-amyloid fibrils. Science 307, 262–265.
Picotti P., De Franceschi G., Frare E., Spolaore B., Zambonin M., Chiti F., de Laureto P.P., Fontana A. (2007) Amyloid fibril formation and disaggregation of fragment 1-29 of apomyoglobin: insights into the effect of ph on protein fibrillogenesis. J. Mol. Biol. 367, 1237-1245.
Privalov P.L. (1979) Stability of proteins: small globular proteins. Adv. Protein Chem. 33, 167-241.
Prusiner S.B. (1982) Novel proteinaceous infectious particles cause scrapie. Science 216, 136–144
Randolph T.W., Seefeldt M., Carpenter J.F. (2002) High hydrostatic pressure as a tool to study protein aggregation and amyloidosis. Biochim. Biophys. Acta 1595, 224–234.
Rasmussen P., Barbiroli A., Bonomi F., Faoro F., Ferranti P., Iriti M., Picariello G., Iametti S. (2007) Formation of structured polymers upon controlled denaturation of beta-lactoglobulin with different chaotropes. Biopolymers 86, 57-72.
65
Reixach N., Deechongkit S., Jiang X., Kelly J.W. & Buxbaum J.N. (2004) Tissue damage in the amyloidoses: transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture. Proc. Natl. Acad. Sci. USA, 101, 2817–2822.
Rochet J.C. and Lansbury P.T. Jr (2000) Amyloid fibrillogenesis: themes and variations. Curr. Opin. Struct. Biol. 10, 60–68.
Rodriguez-Enriquez S., He L., Lemasters J.J (2004) Role of mitochondrial permeability transition pores in mitochondrial autophagy. Int. J. Biochem. Cell Biol. 36, 2463-24672.
Röcken C., Becker K., Fändrich M., Schroeckh V., Stix B., Rath T. Kahne T., Dierkes J., Roessner A., Albert F.W. (2006) ALys amyloidosis caused by compound heterozygosity in exon 2 (Thr70Asn) and exon 4 (Trp112Arg) of the lysozyme gene. Hum. Mutat. 27, 119–120.
Saraiva M.J.M. (2002) Hereditary transthyretin amyloidosis: molecular basis and therapeutical strategies Exp. Rev. Mol. Med. 14 May
Sasahara K., Demura M., Nitta K. (2000) Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy. Biochemistry 39, 6475-6482.
Sasahara K., Yagi H., Naiki H., Goto Y. (2007) Heat-triggered conversion of protofibrils into mature amyloid fibrils of beta(2)-microglobulin. Biochemistry 46, 3286-3293.
Schneider S.W., Larmer J., Henderson R.M., and Oberleithner H. (1998) Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pfluegers Arch. 435, 362-367.
Seefeldt M.B., Ouyang J., Froland W.A., Carpenter J.F. and Randolph T.W. (2004) High-pressure refolding of bikunin: Efficacy and thermodynamics, Protein Sci. 13, 2639–2650.
Serio T. R., Cashikar A. G., Kowal A. S., Sawicki G. J., Moslehi J. J., Serpell L. et al. (2000) Nucleated conformational conversion and the replication of conformational information by a prion determinant. Science 289, 1317–1321
Schulze-Osthoff K., Bakker A.C., Vanhaesebroeck B., Beyaert R., Jacob W.A., Fiers W. (1992) Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J. Biol.Chem. 267, 5317-5323.
Silva J.L. and Weber G. (1993) Pressure stability of proteins, Annu. Rev. Phys. Chem. 44, 89–113.
Sipe, J.D. & Cohen, A.S. (2000) History of the amyloid fibril. J. Struct. Biol. 130, 88–98.
66
Sirangelo I., Malmo C., Iannuzzi C., Mezzogiorno A., Bianco M. R., Papa M. & Irace G. (2004) Fibrillogenesis and cytotoxic activity of the amyloid forming apomyoglobin mutant W7F and W14F. J. Biol. Chem. 279, 13183–13189.
Smith D.P., Jones S., Serpell L.C., Sunde M., Radford S.E. (2003) A systematic investigation into the effect of protein destabilisation on beta-2-microglobulin amyloid formation. J. Mol. Biol. 330, 943–954.
Soto C. & Saborio G.P. (2001) Prions: disease propagation and disease therapy by conformational transmission. Trends Mol. Med. 7, 109–114.
Soto C. (2001) Protein misfolding and disease; protein refoldingand therapy. FEBS Lett. 498, 204–207.
Sparr E., Engel M.F., Sakharov D.V., Sprong M., Jacobs J., de Kruijff B., Hoppener J.W., Killian J.A. (2004) Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers. FEBS Lett. 577, 117-120.
Strasser A., O'Connor L., Dixit V.M. (2000) Apoptosis signaling. Annu. Rev. Biochem. 69, 217-45.
Stuart D.I., Acharya K.R., Walker N..P, Smith S.G., Lewis M. and Phillips D.C. (1986) α-Lactalbumin possesses a novel calcium binding loop. Nature 324, 84-87.
Sousa M.M., Du Yan S., Fernandes R., Guimaraes A., Stern D. & Saraiva, M.J. (2001) Familial amyloid polyneuropathy: receptor for advanced glycation end products-dependent triggering of neuronal inflammatory and apoptotic pathways. J. Neurosci. 21, 7576–7586.
Sunde M., and Blake C.C.F. (1998) From the globular to the fibrous state: protein structure and structural conversion in amyloid formation, Q. Rev. Biophys. 31, 1–39.
Susin S.A., Lorenzo H.K., Zamzami N., Marzo I., Snow B.E., Brothers G.M., Mangion J., Jacotot E., Costantini P., Loeffler M., Larochette N., Goodlett D.R., Aebersold R., Siderovski D.P, Penninger J.M., Kroemer G. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441-446.
Swerdlow R.H., Khan S.M. (2004) A "mitochondrial cascade hypothesis" for sporadic Alzheimer's disease. Med. Hypotheses. 63, 8-20.
Takuma K., Yan S.S., Stern D.M, Yamada K. (2005) Mitochondrial dysfunction, endoplasmic reticulum stress, and apoptosis in Alzheimer's disease. J. Pharmacol. Sci. 97, 312-316
Taylor K.L., Cheng N., Williams R.W., Steven A.C., Wickner R.B. (1999) Prion domain initiation of amyloid formation in vitro from native Ure2p. Science 283, 1339-1343.
67
Teplow B., (1998) Structural and kinetic features of amyloid beta-protein fibrillogenesis. Amyloid-Int. J. Exp. Clin. Invest. 5, 121–142
Tsuge H., Koseki K., Miyano M., Shimazaki K., Chuman T., Matsumoto T., Noma M., Nitta K. and Sugai S. (1991) A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR. Biochim. Biophys. Acta 1078, 77-84.
Tsuge H., Ago H., Noma M., Nitta K., Sugai S. and Miyano M. (1992) Crystallographic studies of a calcium binding lysozyme from equine milk at 2.5 Å resolution. J. Biochem. 111, 141-143.
Uversky V.N. (2002) What does it mean to be natively unfolded? Eur. J. Biochem. 269, 2–12
Uversky V.N., Gillespie J.R. & Fink A.L. (2000) Why are 'natively unfolded' proteins unstructured under physiologic conditions? Proteins 41, 415–427.
Uversky, V.N., Li, J. & Fink, A.L. (2001) Evidence for a partially folded intermediate in α-synuclein fibril formation. J. Biol. Chem. 276, 10737–10744.
Valleix S., Drunat S., Philit J.B., Adoue D., Piette J.C., Droz D., MacGregor B, Canet D., Delpech M., Grateau G. (2002) Hereditary renal amyloidosis caused by a new variant lysozyme W64R in a French family, Kidney Int. 61, 907–912.
Van Cruchten S., Van Den Broeck W. (2002) Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat Histol Embryol. 31, 214-223.
Van Dael H., Haezebrouck P., Morozova L., Arico-Muendel C. and Dobson C.M. (1993) Partially folded states of equine lysozyme. Structural characterization and significance for protein folding. Biochemistry 32, 11886-11894.
Vercammen D., Brouckaert G., Denecker G., Van de Craen M., Declercq W., Fiers W, Vandenabeele P. (1998) Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways. J. Exp. Med. 188, 919–930.
Volles M.J., Lee S.J., Rochet J.C., Shtilerman M.D., Ding T.T., Kessler J.C. & Lansbury P. T., Jr (2001) Vesicle permeabilization by protofibrillar alpha-synuclein: implications for the pathogenesis and treatment of Parkinson's disease. Biochemistry 40, 7812–7819.
Walsh D.M., Klyubin I., Fadeeva J.V., Cullen W.K., Anwyl R., Wolfe M.S., Rowan M.J., Selkoe D.J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535-539
68
Webb J.N., Webb S.D., Cleland J.L., Carpenter J.F. and Randolph T.W. (2001) Partial molar volume, surface area, and hydration changes for equilibrium unfolding and formation of aggregation transition state: High-pressure and cosolute studies on recombinant human IFN-gamma. Proc. Natl. Acad. Sci. USA 98, 7259–7264.
Weldon D.T., Rogers S.D., Ghilardi J.R., Finke M.P., Cleary J.P., O'Hare E., Esler W.P., Maggio J.E., Mantyh P.W. (1998) Fibrillar β-amyloid induces microglial phagocytosis, expression of inducible nitric oxide synthase, and loss of a select population of neurons in the rat CNS in vivo. J. Neurosci. 18, 2161–2173.
Wogulis M., Wright S., Cunningham D., Chilcote T.,Powell K. & Rydel, R.E. (2005) Nucleation-dependent dependent polymerization is an essential component of amyloid-mediated neuronal cell death. J. Neurosci. 25, 1071–1080.
Wood S.J., Wypych J., Steavenson S., Louis J. C., Citron, M. and Biere A.L. (1999) alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson’s disease. J. Biol. Chem. 274, 19509–19512.
Wosten H.A., de Vocht M.L. (2000) Hydrophobins, the fungal coat unravelled. Biochim. Biophys. Acta. 1469, 79-86.
Wyttenbach A., Sauvageot O., Carmichael J., Diaz-Latoud C., Arrigo A.P., Rubinsztein D.C. (2002) Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum. Mol. Genet. 11, 1137-1151
Yazaki M., Farrell S.A. and Benson M.D. (2003) A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis, Kidney Int. 63, 1652–1657.
Yin K.J., Lee J.M., Chen S.D., Xu J., Hsu C.Y. (2002) Amyloid-beta induces Smac release via AP-1/Bim activation in cerebral endothelial cells. J Neurosci 22, 9764–9770.
Zhao H., Tuominen E.K., Kinnunen P.K. (2004) Formation of amyloid fibers triggered by phosphatidylserine-containing membranes. Biochemistry 43, 10302-10307.
Zamotin V., Gharibyan A., Gibanova N.V., Lavrikova M.A., Dolgikh D.A., Kirpichnikov M.P. and Morozova-Roche L.A. (2006) Cytotoxicity of albebetin oligomers depends on cross-β-sheet formation. FEBS Letters, 580, 2451–2457.