The amyloid: structure, properties and application

Mantas Mališauskas

Department of Medical Biochemistry and Biophysics Umeå University

Umeå, Sweden 2007

Copyright © 2007 by Mantas Mališauskas

Printed in Sweden by VMC, KBC, Umeå University,

Umeå 2007

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Mano tėvams

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Contents ABSTRACT............................................................................................................ 6

ABBREVIATIONS ................................................................................................ 7

LIST OF PUBLICATIONS .................................................................................. 8

INTRODUCTION................................................................................................ 11 PROTEINS AND AMYLOIDOSES ..................................................................................................... 11 LYSOZYME AMYLOIDOSIS............................................................................................................ 13 GENERAL STRUCTURE OF AMYLOIDS ........................................................................................... 14 HIERARCHY OF AMYLOID STRUCTURES ....................................................................................... 18 AMYLOID PRECURSOR STATE....................................................................................................... 20 AMYLOID PROPERTIES ................................................................................................................. 21 APPLICATIONS OF AMYLOID IN NANOTECHNOLOGY..................................................................... 25 EQUINE LYSOZYME PROPERTIES .................................................................................................. 26

METHODS AND TECHNIQUES USED IN THE STUDY............................. 29 AMYLOID SPECIFIC DYE BINDING................................................................................................. 29 ATOMIC FORCE MICROSCOPY....................................................................................................... 30

Spherical cap model............................................................................................................... 32 AIMS OF THE THESIS...................................................................................... 34

SUMMARY OF THE PRESENT STUDY ........................................................ 35 PAPER I. AMYLOID PROTOFILAMENTS FROM THE CALCIUM-BINDING PROTEIN EQUINE LYSOZYME: FORMATION OF RING AND LINEAR STRUCTURES DEPENDS ON PH AND METAL ION CONCENTRATION..................................................................................................................................................... 35 PAPER II. DOES THE CYTOTOXIC EFFECT OF TRANSIENT AMYLOID OLIGOMERS FROM COMMON EQUINE LYSOZYME IN VITRO IMPLY INNATE AMYLOID TOXICITY? ............................................... 36 PAPER III. ULTRATHIN SILVER NANOWIRES PRODUCED BY AMYLOID BIOTEMPLATING. .............. 37

ACKNOWLEDGMENTS ................................................................................... 39

CITED LITERATURE ....................................................................................... 40

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Abstract

Protein aggregation, leading to the formation and depositions of amyloids, is a cause for a number of diseases such as Alzheimer’s and Creutzfeld-Jacob’s disease, systemic amyloidoses, type II diabetes and others . More than 20 proteins are associated with protein misfolding diseases and even a larger number of proteins can self-assemble into amyloid in vitro. Relating structural and functional properties of amyloid is of particular interest, as this will lead to the identification of the main factors and mechanisms involved in the process of protein misfolding and aggregation; consequently, this will provide a basis for developing new strategies to treat protein misfolding diseases. The aim of the thesis is to investigate structural aspects of amyloid formation and relate that to the functional properties of amyloid. The first paper describes the amyloid formation of equine lysozyme (EL). We have demonstrated that EL enters an amyloid forming pathways under conditions where the molten globule state is populated. We have found that the morphology of the amyloids depend on the calcium-binding to lysozyme, specifically the holo-protein assembles into short, linear protofilaments, while the apo-EL forms ring-shaped structures. The morphology of EL amyloid significantly differs from the amyloid fibrils of human and hen lysozymes. We have suggested that the stable alpha-helical core of EL, which remains structured in the molten globule intermediate, may obstruct the formation of fibrilar interface and therefore leads to assembly of short, curly fibrils and rings. In the second paper, we describe the cytotoxicity of EL amyloids. We have analysed the amyloid intermediates on the pathway towards amyloid fibrils. The sizes of amyloid oligomers were determined by atomic force microscopy (AFM) and the formation of cross-beta sheet was shown by thioflavin T (ThT) binding. The toxicity studies show that the oligomers formed during amyloid growth phase are toxic to a range of cell lines and cultures and the toxicity is size-dependant. The last manuscript describes a novel method for manufacturing of silver nanowires by the biotemplating using amyloid fibrils. The amyloid assembled from an abundant and cheap hen egg white lysozyme was used as a scaffold for casting ultrathin silver nanowires. We have manufactured nanowires with a diameter of 1.0-2.5 nm and up to 2 micrometers in length. Up to date, it is the thinnest silver nanowires produced by using biotemplating and at least one order of magnitude thinner than nanowires manufactured by chemical synthesis. Key words: amyloid, oligomers, AFM, cytotoxicity, biotemplating, lysozyme.

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Abbreviations AFM – atomic force microscopy; CD – circular dichroism; EL – equine lysozyme; HEWL – hen egg white lysozyme; α-Lac – α-lactalbumin; Aβ – amyloid β peptide; TFE – 2,2,2-trifluoethanol; ThT – thioflavin T; DMF – N,N-dimethylformamide; HHP – high hydrostatic pressure; E. coli – Escherichia coli; NMR – nuclear magnetic resonance; TEM – transmission electron microscopy UV – ultraviolet light

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List of publications Articles included in the thesis

1. Malisauskas, M., Zamotin, V., Jass, J., Noppe, W., Dobson, C. M. and 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.

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. Malisauskas, M., Meskys, R., Morozova-Roche, L. A. Ultrathin silver nanowires produced by amyloid biotemplating. (Manuscript).

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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. and 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. 218, 165-171. 2. Morozova-Roche, L. A., Zamotin, V., Malisauskas, M., Öhman, A., Chertkova, R., Lavrikova, M. A., Kostanyan, I. A., Dolgikh, D. A. and Kirpichnikov, M. P. (2004) Fibrillation of carrier protein albebetin and its biologically active constructs. Multiple oligomeric intermediates and pathways. Biochemistry 43, 9610-9619. 3. Lavrikova, M. A., Zamotin, V. V., Malisauskas, M., Chertkova, R. V., Kostanyan, I. A., Dolgikh, D. A., Kirpichnikov, M. P. and 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., Casaite, V., Darinskas, A., Forsgren, L. and Morozova-Roche L. A. (2007) Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients. Eur. J. Neurol. 14, 327-334. 6. Darinskas, A., Gasparaviciute, R., Malisauskas, M., Wilhelm, K., Kozhevnikov, J. A., Liutkevicius, E., Pilinkiene, A. and Morozova-Roche, L. A. (2007) Engrafting fetal liver cells into multiple tissues of healthy adult mice without the use of immunosuppressants. Cell Mol. Biol. Lett. Mar 14; [Epub ahead of print]. 7. Gruden, M. A., Davidova, T. B., Malisauskas, M., Sewell, R. D., Voskresenskaya, N. I., Wilhelm, K., Elistratova, E. I., Sherstnev, V. V. and Morozova-Roche, L. A. (2007) Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: Autoantibodies to Aβ(25-35)

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oligomers, S100b and neurotransmitters. J. Neuroimmunol. May 1; [Epub ahead of print]. 8. Morozova-Roche, L.A. and Malisauskas M. (2007) A False Paradise - Mixed Blessings in the Protein Universe: The Amyloid as a New Challenge in Drug Development. Curr. Med. Chem. 14, 1221-1230.

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Introduction

Proteins and amyloidoses Living organisms are constituted of four classes of molecules: nucleic acids,

carbohydrates, lipids and proteins. The latter are molecules with the widest diversity in their structural and functional properties. Proteins are involved in all biochemical processes in vivo. The range of their functions is enormous: proteins catalyse chemical reactions, act as structural and storage agents, signalling molecules and fulfil other functions. Polypeptide chain is composed only of 20 different amino acids. However, the combination of amino acids in a primary sequence determines the spatial topology of polypeptide chain, protein function and other properties. The spontaneous transition from a disordered polypeptide chain to a unique three-dimensional structure is defined as protein folding. Under appropriate conditions nearly any protein can fold into a native conformation as all information required for the folding process is encoded in the amino acid sequence (Dobson, 2004). The process of protein folding may proceed via partially folded states, which limit the conformational search for the native state (Kim & Baldwin 1990; Dobson, 2001). In vivo the folding process occurs under a very tight quality control in order to prevent misfolding and to eliminate aggregated and misfolded species. Chaperons play an important role, supervising proteins to attain and maintain native conformation (Bukau et al., 2006) A broad range of human diseases is related to the failure of proteins or peptides to fold correctly or maintain its native conformation. These pathological conditions are referred as protein misfolding, protein conformational or amyloid diseases (Chiti & Dobson, 2006). In amyloidoses naturally occurring, normally soluble and functional polypeptides are converted into insoluble, highly ordered fibrilar material known as amyloid fibrils. Over the last decades they became the focus of extensive research, particularly in association with such widespread human diseases as Alzheimer’s, Creutzfeld-Jacob’s, Parkinson’s and Huntington’s diseases, systemic amyloidoses, amyotrophic lateral sclerosis and others. In most cases amyloid deposits are accumulated extracellularly, while in the case of Parkinson’s disease the fibrilar aggregates are found intracellularly. A list of amyloid diseases known today is presented in the Table 1 (Chiti & Dobson, 2006) together with the specific proteins, which form the deposits.

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Table 1. Human disorders associated with the formation and deposition of amyloids (adopted from Chiti & Dobson, 2006). DISEASE AGGREGATING POLYPEPTIDE Alzheimer’s disease Amyloid β peptide Spongiform encephalopathies Prion protein or fragments Parkinson’s disease α-Synuclein Dementia with Lewy bodies α-Synuclein Frontotemporal dementia with Parkinsonism Tau

Amyotrophic lateral sclerosis Superoxide dismutase Huntington’s disease Huntingtin with polyQ expansion Spinocerebellar ataxias Ataxins with polyQ expansion

Spinocerebellar ataxia 17 TATA box-binding protein with polyQ expansion

Spinal and bulbar muscular atrophy Androgen receptor with polyQ expansion Hereditary dentatorubral-pallidoluysian atrophy Atrophin-1 with polyQ expansion

Familial British dementia ABri Familial Danish dementia ADan AL amyloidosis Immunoglobulin light chains or fragments AA amyloidosis Fragments of serum amyloid A protein Familial Mediterranean fever Fragments of serum amyloid A protein Senile systemic amyloidosis Wild-type transthyretin Familial amyloidotic polyneuropathy Mutants of transthyretin Hemodialysis related amyloidosis β2-microglobulin ApoAI amyloidosis N-terminal fragments of apolipoprotein AI ApoAII amyloidosis N-terminal fragment of apolipoprotein AII ApoAIV amyloidosis N-terminal fragment of apolipoprotein AIV Finnish hereditary amyloidosis Fragments of gelsolin mutants Lysozyme amyloidosis Mutants of lysozyme Fibrinogen amyloidosis Variants of fibrinogen α-chain Icelandic hereditary cerebral amyloid angiopathy Mutant of cystatin C

Type II diabetes Amylin (islet amyloid polypeptide (IAPP)) Medullary carcinoma of the thyroid Calcitonin Atrial amyloidosis Atrial natriuretic factor Hereditary cerebral hemorrhage with Mutants of amyloid β peptide

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amyloidosis Pituitary prolactinoma Prolactin Injection-localized amyloidosis Insulin Aortic medial amyloidosis Medin Hereditary lattice corneal dystrophy C-terminal fragments of kerato-epithelin Corneal amyloidosis associated with trichiasis Lactoferrin

Cataract γ-Crystallins Calcifying epithelial odontogenic tumors Unknown Pulmonary alveolar proteinosis Lung surfactant protein C Inclusion-body myositis Amyloid β peptide Cutaneous lichen amyloidosis Keratins

In amyloidoses the appearance of clinical symptoms is commonly manifested much later than the initiation of the pathological process. Therefore, when the disease is diagnosed, it has proceeded (Alzheimer’s, Parkinson’s diseases) already to an advance stage (Blennow, 2004). Protein misfolding diseases are often associated with aging, making elderly people the most affected group in society. With an increasing life span protein amyloidoses became a serious social and financial challenge for the society and the health care system worldwide. However, some particular amyloidoses such as hereditary diseases (Pepys et al., 1993) can also develop at an young age. Under all circumstances the amyloid diseases dramatically affect the quality of life and eventually lead to fatal conditions.

Lysozyme amyloidosis In early 1990s it has been found that members of the families, carrying single-point mutations in the lysozyme gene, develop systemic hereditary amyloidosis at an early age. This condition is manifested in substantial deposits of lysozyme amyloid fibrils throughout the body, particularly in the liver; where up to kilogram of fibrils can be formed causing premature death. It has been shown that amyloid deposits consist of the mutational variants of human lysozyme, Asp67His and Ile56Thr (Pepys et al., 1993). The following studies on the mechanisms of human lysozyme amyloid formation revealed that wild-type human lysozyme is able to self-assembly into amyloid under the protein destabilising conditions (Morozova-Roche et al., 2000). Hen egg white lysozyme (HEWL), close homologues to human lysozyme, undergoes structural conversion and

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assembly into amyloids under similarly conditions. Both proteins form long (up to 10 μm) and unbrunched amyloid fibrils, typically 2-10 nm in a diameter (Krebs et al., 2000; Chamberlain et al., 2000). The studies of the amyloid formation of these two proteins significantly contributed to the understanding of the general mechanisms of amyloid formation and in particular to the identification of the sequential and structural determinants involved in the lysozyme systemic hereditary amyloidosis. It has been demonstrated that folding of human lysozyme amyloidogenic mutants is less cooperative, compared to wild type human lysozyme, and leads to a population of its partially folded states (Morozova-Roche et al., 2000). The effect of mutation can be diminished by binding to specific camel antibodies, which restore folding cooperativity of amyloidogenic variants.

Lysozyme from horse milk has a unique place within the extended family of structurally related proteins, c-type lysozymes and α-Lac, as it combines the functional, structural and folding properties of both subfamilies and can be considered as an evolutionary bridge between them. Equine lysozyme is significantly less stable and less cooperative than human lysozyme, forming numerous partially folded states (Morozova et al., 1991; Van Deal et al., 1993; Griko et al., 1995). Therefore, we expected that it should be able to assemble readily into long and mature fibrils similar to human lysozymes and subjected it to our studies described below.

General structure of amyloids

Initially soluble and functional proteins become inactive when they aggregate into insoluble amyloid fibrils (Pepys et al., 1993; Morozova-Roche et al., 2000; Bergstrom et al., 2005). The amyloid fibrils represent an insoluble material with a high protease resistance that prevents them from cellular and tissue clearance. A growing number of proteins and peptides have been shown to be able to form amyloids both in vivo and in vitro. They have no similarity in amino acid sequences and secondary or tertiary structures, however, when they self-assemble into the amyloid they share a common cross-β-sheet core as shown schematically in Figure 1. The morphology of amyloid was analysed by transmission electron and atomic force microscopy. Linear, unbrunched threads with a variable number of constituting strands are the most common amyloid species (Figure 1) (Pedersen et al., 2006; Iannuzzi et al., 2007; Morozova-Roche et al., 2000).

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Figure 1. Common amyloid structures proceed from a variety of native protein conformations. Proteins (A) with α-helical, α-helical/β-sheet and β-sheet structures form amyloid fibrils (B) shown by atomic force microscopy. (C) Structural arrangement of amyloid fibrils derived from cryo-electron microscopy.

Over the last decade a significant amount of information on the structure of fibril core has been derived from X-ray fibril diffraction analysis, solid state nuclear magnetic resonance spectroscopy (NMR), electron paramagnetic resonance spectroscopy of spin labelled derivatives and cryo-electron microscopy (Jimenez et al., 2002; Iwata et al., 2006; Makins & Serpell, 2005, Petkova et al., 2006). The detailed studies of in vitro formed amyloid fibrils as well as the examination of ex vivo material have led to a number of cross-β-sheet core models (Figure 2). (Kajava et al., 2004; 2006). It is important to note, that cross-β-sheet core packing of fibrils assembled from full-length polypeptides are different compared to the core of the fibrils assembled from short fragments of the same proteins. These findings indicate that unconstrained short peptides may undergo different fibrillation pathways than full-length ones (Nelson et al., 2004; Kajava et al., 2004; 2006)

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Figure 2. The models of cross-β-sheet core architecture in amyloid fibrils (adapted from Kajava et al., 2006).

The detailed packing of amino acid residues within the core of amyloid

fibrils has been observed by high-resolution solid state NMR (Petkova et al., 2005; 2006), by X-ray diffraction studies (Iwata et al., 2006; Nelson et al., 2004) and by using amide hydrogen exchange studies coupled with NMR and mass-spectrometry (Olofsson et al., 2007). Amyloid β (Aβ) peptide is one of the most studied objects in a last decade, which is related to the Alzheimer’s disease. Aβ peptide is one of a very few amyloidogenic polypeptides for whom the architecture of fibrilar core has been clarified. In the model of Aβ(1-40) profilament, each Aβ(1-

40) molecule contributes to a pair of β-strands connected by the loop, the β-strands participate in the formation of two β-sheets within the same polymer (Olofsson et al., 2007). The model demonstrates that the Aβ peptide fibrils are constituted of two protofilaments, consisting of four β-sheets separated by a distance of 10 Å

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(Petkova et al., 2006; Olofsson et al., 2007). Different groups working with these polypeptides have suggested similar models of the Aβ fibril architecture. The data indicates that only a fraction of the polypeptide sequence is involved in the fibrilar core formation. Moreover, the small difference in the peptide size (Aβ(1-40) or Aβ(1-

42)) and/or the aggregation conditions may lead to different packing within amyloid core and different amyloid morphology (Figure 3).

Generally, the amyloid core is supported by a dense network of inter- and intramolecular hydrogen bonds formed between the amino and carbonyl groups. The amyloid is also stabilised by interactions of side-chains, including π-bonding between adjacent hydrophobic rings and salt-bridges between charged pairs (Petkova et al., 2005; Nelson et al., 2004; Bemporad et al., 2006).

Figure 3. Amino acid hydrogen protection pattern in Aβ fibrilar model. The solvent amide proton protection ratios determined for the residues within Aβ (1-40) and Aβ(1-42) fibrillar core are mapped in the corresponding fibrillar models. The colour code is varied between the following extremes: navy blue for complete

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and red for no solvent protection. Residues with no protection ratios available are depicted in grey. Main-chain hydrogen bonds are directed along the fibril axis, perpendicular to the plane of the paper. A and C. Ball and stick models show a dimmer consisting of four cross-β sheets shown in a cross-section of the Aβ(1-40) and Aβ(1-42) fibrils, respectively. Assignments are indicated in some positions by one letter codes. B and D. Models of the fibrillar assembly for Aβ(1-40) and Aβ(1-42), respectively. (Adapted from Olofsson et al., 2007). The mutation analysis of amyloid fibrils by alanine, proline and cysteine mutagenesis of Aβ(1-40) peptide shows forgiving nature of amyloid structure. Amyloid fibrils are able readily to attain different arrangements within amyloid core to accommodate the mutations (Williams et al., 2004; 2006; Peim et al., 2006). These experiments have revealed a remarkable malleability of the Aβ(1-40) fibrilar structure in which radical changes of the individual contacts can occur without changing the stability of amyloid fibrils (Wetzel et al., 2007). When the fibrils are exposed to mutations of the core forming residues, they structurally adjust and accommodate the mutation rather than undergo breaking. These results have indicated that there are multiple modes of the amyloid core formation and this may be the origin of the amyloid fibril polymorphism (Wetzel et al., 2007). Indeed, all amyloids are characterised by a significant polymorphism of their species, which is observed even within the same sample (Morozova-Roche, Zamotin et al., 2004). Hierarchy of amyloid structures

The formation of amyloid occurs in a hierarchical manner. The first step is

formation of nuclei (Figure 4, 5B), which develop further into small protofibrils (Morozova-Roche, Zamotin et al., 2004). An elongation of the protofibrils lead to appearance of single protofilaments which able to wind around each other to form mature fibrils (Morozova-Roche et al., 2000; Morozova-Roche, Zamotin et al., 2004) (Figure 4, 5A). Even larger amyloid structures have been discovered recently defined as spherulites. Due to their dimensions, the scientists analysing amyloid structures by AFM and transmission electron microscopy (TEM) at the tens of micrometer scale has overlooked them previously (Figure 5C) (Krebs et al., 2004).

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Figure 4. Schematic presentation of amyloid pathways. This includes the formation of non-amyloidogenic oligomers without cross-β-sheet core, which give rise to the larger cross-β-sheet containing oligomers and proceed further to fibrils.

Long unbrunched fibrils represent the most common type of amyloid. The fibrils can grow up to ten micron length as shown in the cases of albebetin (Morozova-Roche, Zamotin et al., 2004), HypF (Chiti et al., 2001; Relini et al., 2004), insulin (Jimenez et al., 2002) Aβ peptide (Olofsson et al., 2007), human lysozyme (Morozova-Roche et al., 2000) and other polypeptides. The diameter of amyloid fibrils can vary from 1.5 nm to more than 10 nm, depending on the number of protofilaments winded around each other. It has been demonstrated that same proteins, such as calcitonin, Aβ, glucagon, lysozyme and others, can assemble into amyloid with different morphology depending on the solution conditions (Pedersen et al., 2006; Petkova et al., 2005; Malisauskas et al., 2003). This may occur due to the altered cross-β-sheet core or fibrillar interface packing (Pedersen et al., 2006; Petkova et al., 2005; Naito et al., 2004)

Recently, a growing attention has been drawn to transient amyloid oligomers (Malisauskas et al., 2005; Relini et al., 2004, Kayed et al., 2003; 2004, Plakoutsi et al., 2006; Zamotin et al., 2006).These species are also denoted as

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prefibrils and protofibrils. The amyloid oligomers can assemble into linear and ring-shaped chains with a “bead-on-string” morphology (Morozova-Roche, Zamotin et al., 2004) and co-exist with the fibrilar species. It has been suggested that circular assemblies are the major cause of pathogenicity in Alzheimer’s and Parkinson’s diseases (Lashuel et al., 2002; 2003; Arispe et al., 2007). The on-going inter-conversion of smaller oligomers into larger ones and their subsequent assembly into filamentous structures contribute to the intrinsic heterogeneity of amyloids (Morozova-Roche, Zamotin et al., 2004).

At the largest range of amyloid spherulites lays, spherical assemblies of amyloid fibrils, which can reach a diameter of up to a few hundred microns. The distinct property of these structures is a “Maltese Cross” appearance under the polarised light. The formations of spherulites have been described for insulin and other proteins as well as for synthetic polymer polypropylene (Krebs et al., 2004). Given the chemical difference between the protein and synthetic polymers, the formations of similar structures suggest close structural physical similarities among them (Wetzel et al., 2007).

Figure 5. A hierarchy of amyloid structures. A – Amyloid fibrils of hen egg white lysozyme; B – Amyloid oligomers of equine lysozyme; C – Spherulites of human insulin.

Amyloid precursor state

In order to assemble into amyloid, native proteins often undergo a dramatic structural conversion. Generally, the precursor protein requires destabilisation or unfolding of its native conformation to enter amyloid formation pathways (Figure 4) (Armen & Daggett, 2005; Qin et al., 2007; Malisauskas et al., 2003; Morozova-Roche et al., 2000; Pepys et al., 1993). There is a substantial evidence that

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partially folded intermediates serve the role of amyloid precursor, as this has been shown for immunoglobulin light chain (Qin et al., 2007), human and equine lysozymes (Morozova-Roche et al., 2000; Malisauskas et al., 2003), cytochrome b (Pertinhez et al., 2001) and others. Among the amyloid precursor states, molten globule intermediates are of particular interest. These states are characterised by destabilized tertiary contacts, but retained specific secondary structural elements. In particularly, the object of our studies - equine lysozyme enters amyloid formation pathways under conditions of acidic pH and elevated temperatures, where the molten globular conformation is populated. A large number of natively unfolded proteins have been found to be amyloidogenic, including α-synuclein (Lashuel et al., 2002), mammalian prothymosin α (Pavlov et al., 2002), a 71 amino acid fragment of gelsolin mutant (Ratnaswamy et al., 1999) and others. In the case of proteins with a complex quaternary structure such as insulin and transthyretin, the native complexes dissociate and form smaller, usually non-native building blocks, which subsequently assemble into amyloid fibrils (Liu et al., 2000; Nielsen et al., 2001).

Amyloid properties

There is no structural or sequence homology among amyloid forming proteins. That includes disease-related proteins and proteins whose amyloidogenic properties have been discovered in vitro. Despite this, amyloids share a number of common features such as cross-β-sheet core of fibrils, tinctorial properties – binding to ThT and Congo red, nucleation-dependent mechanisms of amyloid formation, high stability of amyloid fibrils and amyloid cytotoxicity.

The ThT and Congo red dyes have been primarily used in diagnostics to detect amyloid deposition in histological samples (Westermark et al., 1999). In vitro experiments they are often employed to identify amyloid and to monitor the kinetics of its formation. It has been suggested that these dyes bind to the core of amyloid fibrils perpendicular to β-strands (described below).

Protein crystallization and polymerization are highly ordered processes, occurring via nucleation-dependent pathways (Kelly, 2000; Blow et al., 1994). These processes are characterized by (a) a slow nucleation phase, in which proteins undergo a series of unfavourable association steps to form oligomeric nuclei, (b) a growth phase, in which nuclei rapidly grow to form larger polymers, and (c) a steady state phase, in which ordered polymers and monomers remain at equilibrium (Figure 6) (Jarrett & Lansbury, 1993; Harper & Lansbury, 1997). The

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characteristic features of a simple nucleation-dependent polymerization are as follows: (a) No aggregation occurs at a protein concentration below the critical concentration. (b) At protein concentrations that exceed the critical concentration, there is a lag-time before the polymerization occurs. (c) During the lag time, an addition of seeds results in shortening lag-phase or immediate polymerization.

Figure 6. A nucleation dependant polymerization model of amyloid

formation. A – Nucleation phase; B –Growth phase; C- Steady state phase (adapted from Harper & Lansbury, 1997).

The kinetics of amyloid polymerization for different proteins may vary

significantly. For example, the length of lag phase may vary form seconds to days. Even for the same protein, such as insulin, the lag phase can differ form minutes to hours (Jansen et al., 2005; Ahmad et al., 2004), depending on environmental conditions and presence of preformed seeds. The addition of preformed amyloid fibrils – seeds, may significantly speed up the aggregation process eliminating the lag phase and eventually leading to a larger quantity of amyloids (Conway et al., 2000; Ahmad et al., 2004; Harper & Lansbury, 1997). In in vitro studies of lysozyme, the close sequential homology between the fibrilar seeds and the seeded sample was an essential factor for effective cross-seeding, suggesting that sequence similarity is required for establishing long-range interactions within the fibrils (Krebs et al., 2004 (a)). Nevertheless, non-homologues sequences still exhibited a seeding potency (Krebs et al., 2004 (a)). It was demonstrated that α-synuclein amyloid formation can be initiated by adding GroES amyloid from E. coli, preformed fibrils of insulin and lysozyme (Yagi et al., 2005). Thus, the cross-seeding can initiate fibrillation, though its effectiveness depends on the nature of

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the precursor and the seed, as well as on the fibrillation mechanisms involved and in general is less efficient than the self-seeding.

The amyloid fibrils under some conditions are more stable than the same precursor protein in its native conformation and therefore correspond to the global energy minimum of the system (Figure 7). A range of chemical and physical denaturants such as pH, organic co-solvents, high hydrostatic pressure (HHP) and high temperature have been used to probe stability of various amyloid structures. The early amyloid oligomers and fibrils, assembled from the transthyretin 105-115 peptide, can be dissociated by hydrostatic pressure, in the contrary to the mature amyloid fibrils (Dirix et al., 2005). The thermal stability of amyloids is usually larger than that of the precursor proteins. This has been shown by assessing the thermal stability of the amyloid fibrils of β2-microglobulin and Aβ peptide, which dissociate at the temperatures above 100 °C (Sasahara et al., 2005). It is important to note that amyloid fibrils not always disassemble into monomers, but often to the aggregates of a smaller size and with different properties compared to the monomeric protein or amyloid fibrils (Hirota-Nakaoka et al., 2003).

Figure 7. A schematic energy landscape for protein folding and

aggregation. The surface shows the multitude of conformations 'funnelling' towards the native state via intramolecular contact formation, or towards the formation of amyloid fibrils via intermolecular contacts. Recent experiments have allowed the placement of different 'intermediate' structures on both pathways

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although detailed structural models for many of these species are not yet available. Furthermore, the species involved in converting kinetically stabilized globular structures into the thermodynamic global free energy minimum in the form of amyloid fibrils for different proteins is currently not defined (adapted from Jahn & Radford, 2005).

Cytotoxicity is one of the newly gained amyloid properties absent in the

monomeric precursor. A number of the disease-related and non-related proteins become cytotoxic when they self-assemble into amyloid. In the dawn of amyloid studies, it was believed that amyloid fibrils were the main cause of the disease and the main cytotoxic agents. Nowadays, it is accepted that the low molecular weight, soluble amyloid oligomers and pre-fibrils are culprit of diseases and cause cytotoxicity rather than mature amyloid fibrils. In 1999, Selkoe’s group separated amyloid into the different fractions including low molecular weight oligomers (mostly monomers and dimmers), pre-fibrils and fibrils and tested their toxicity on rat primary cortical neuron cell cultures (Hartley et al., 1999). The results showed that all fractions were cytotoxic in concentration and time-dependant manners. The authors have suggested that earlier Aβ assemblies formed during the process of fibrillogenesis may also play a role in AD pathogenesis. For transthyretin amyloid, the cytotoxicity is associated only with early stages of fibril formation and mature amyloid fibrils represent an inert end stage, which might serve as a rescue mechanism (Andersson et al., 2002). The discovery of toxicity of early amyloid oligomers from disease non-related proteins such as SH3 domain from bovine phosphatidyl-inositol-3’-kinase, the N-terminal domain of the E.coli HypF protein (Bucciantini et al., 2002), equine lysozyme (Malisauskas et al., 2005) and albebetin (Zamotin et al., 2006) culminated in the concept that amyloid toxicity is associated with the common conformational properties of amyloid assemblies, rather than with the sequences of the precursor. A lot of attention has focused on the cellular mechanism of amyloid cytotoxicity. The main mechanisms explore amyloid-lipid interactions leading to the formation of channels/pores (Lashuel et al., 2002; 2003; Quist et al., 2007) or disrupting the integrity of membrane (Relini et al., 2004). The toxicity of mature amyloid fibrils have been demonstrated with a number of amyloid forming proteins including pharmaceuticals products such as glucagon (Pedersen, et al., 2006), calcitonin (Naito et al., 2004), insulin (Nielsen et al., 2001), Aβ peptide (Petkova et al., 2005) and others. Thus, there is strong evidence that accumulation of amyloid, primarily fibrilar species, in the various tissues and organs cause them to malfunction (Harrison et al., 1996; Pepys et al., 1993).

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Applications of amyloid in nanotechnology

The growing field of nanotechnology requires new approaches for manufacturing nano-scale units with controlled dimensions and parameters. Nature provides us with an inspiration how to solve this problem. Naturally occurring biological complexes and biopolymers can be successfully utilised as the templates for controlled nanoparticle assemblies as well as for the production of novel nanobiomaterials. The biomolecular assemblies are attractive nanostructures due to their biocompatibility, straight forward chemical modifiability, inherent molecular recognition properties and their availability for bottom-up fabrication (Scheibel et al., 2003; Berti et al., 2005; Reches & Gazit, 2003).

Indeed, the amyloid fibrils are non-covalent polypeptide polymers with highly structured core, the self-assembly of which is controlled by environmental conditions. They also can be assembled from virtually any polypeptide. These well ordered structures have attracted attention of many researchers working in the field of protein engineering. A number of polypeptides have been used to design amyloid-like fibrils and nanotubes in order to manufacture conductive metal nanowires. Genetically modified prion amyloid fibrils after the metallization with silver and gold yielded into conductive nanowires of ca. 80-100 nm in a diameter (Scheibel et al., 2003). The casting silver nanowires inside the hollow part of dipeptide-based nanotubes with amyloid-like properties have resulted in the silver wire of 20 nm in a diameter (Rechet & Gazit, 2003). The horizontal alignment of the di-phenylalanine peptide nanotubes on the solid surface was achieved through non-covalent coating of the tubes with a ferrofluid and the application of an external magnetic field. The formation of a vertically aligned nanoforest was produced by axial unidirectional growth of a dense array of the peptide tubes. These developments open new venues for the controlled formation of 2D and 3D protein matrixes and the creation of variety of sensors with a very high density of functional units (Reches & Gazit, 2006).

The deposition of preformed amyloid-like peptide nano-tubes on the surface of carbon electrode has significantly improved the electrode performance. Based on this finding, the improved glucose biosensor was created by cross-linking the nanotubes with glucose oxidase and deposition of the complex on the gold electrode. The new indirect enzyme immobilization by using amyloid nanotubes highly increased the biosensor stability, sensitivity and lifetime (Yemini et al., 2005).

The co-assembly of semiconducting oligoelectrolytes with bovine insulin under amyloid forming conditions resulted in the formation of electroactive

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luminescent bioorganic nanowires, exhibiting amyloid properties. These structures showed a tendency for lateral assembly into the larger tape-like structures, which can coil into tubes with 10-30 μm in a diameter, still retaining the luminescence and amyloid properties (Herland et al., 2005).

Despite the fact that amyloid fibrils essentially are non-functional, they provide manometer size proteinaceous scaffolds for the immobilization of other biological molecules and functional groups. Significant efforts have been undertaken in order to functionalise the amyloid scaffolds. The molecular wires were created by displaying the multiheme cytochromes on the surface of amyloid fibrils. This was achieved by means of protein engineering. The chimeric protein (SH3-SH3-Cyt) was created from a tandem repeat of the SH3 domain and the functional domain – cytochrome b562 from E. coli. Under the amyloid forming conditions, the chimeric protein has assembled into typical amyloid fibrils with cytochromes on the surface. The functional properties of cytochromes were restored by adding heme to the fibrilar solution. The spectroscopic characterisation of these polymers have shown that cytochromes on the fibrilar surface were positioned close enough to perform as the electron transferring elements (Baldwin et al., 2006).

Equine lysozyme properties

Glycoside hydrolase family 22 comprises enzymes with two known activities; lysozyme type C (EC:3.2.1.17) and α-Lac. Lysozyme is a muramidase that hydrolyses beta-1,4-links between N-acetyl-muramic acid and N-acetyl-D-glucosamine in the peptidoglycan of bacterial cell walls (Cauerhff et al., 2004), thus destroying invading bacteria. In this capacity, the enzyme is found in tears, milk and saliva (Hankiewicz & Swierczek, 1974). Lysozymes are structural homologous to α-lactalbumins, showing a conserved tertiary fold and about 50% of similarity in their primary sequences. There is, however, no similarity in function, by contrast to lysozymes α-Lac is involved in the biosynthesis of lactose and is essential for milk production. Another significant difference between the two proteins is that all α-Lac have the ability to bind calcium, however, this property is restricted to only a few lysozymes such as pigeon, canine, donkey and equine lysozymes (Nitta et al., 1988). Therefore, it has been suggested that α-Lac have evolved from mammalian calcium-binding lysozymes (Nitta et al., 1989).

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The proteins of this extended family are comprised of two domains; the α-domain is rich in α-helices and the β-domain includes a triple-stranded β-sheet and several loops. Calcium-binding site is located in the domain interface (Figure 8).

Figure 8. Equine lysozyme is close structural homologues to non-calcium binding lysozymes and α-Lac. A - Calcium-binding equine lysozyme, B - Non-calcium binding hen lysozyme and C - Bovine α-lactalbumin. They are structurally related proteins comprised of 123-129 amino acid residues and displaying a similar fold (adapted from Blanch et al., 2000) The key differences between non-calcium binding lysozymes, calcium binding lysozymes and α-Lac were revealed by studying protein folding and unfolding mechanisms. It has been demonstrated that unfolding of α-Lac and EL is a typical two stage process, which proceeds via molten globular intermediates, while HEWL unfolds via a single two state transition (Griko et al., 1995; Polverino de Laureto et al., 2002).

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The molten globular intermediate was described for the first time for α-Lac by Kuwajima (Kuwajima, 1977) and Ptitsyn and co-authors (Dolgikh et al., 1981). They noticed that the changes in near and far UV circular dichroism (CD) ellipticity do not change simultaneously upon protein acid denaturation indicating that the protein initially looses its tertiary structure, but retains almost all of its secondary structural elements. A similar behaviour was observed upon temperature induced denaturation. These data suggest that α-Lac undergoes unfolding via folding intermediate – a molten globule state, which lacks 3D structure (absence of the ellipticity in the near UV CD), but retains its secondary structure (remaining ellipticity in the far UV CD). From the viscosimetry studies it become evident that the volume of the molten globular state exceeds the native

state not more than 20% (Dolgikh et al., 1981), indicating that the molten globule is a compact protein state.

In EL the calcium-binding site is formed by three Asp residues located in the interface of the α and β domains, as it has been demonstrated by Stuart et al., 1986. EL unfolds in two distinct steps as shown in Figure 9 (Morozova et al., 1991; Griko et al., 1995). The first stage reflects the unfolding from the native-state to the molten globular and the second transition corresponds to unfolding of the molten globule to denatured state. The first transition strongly depends on calcium concentration (Morozova et al., 1991; Van Dael et al., 1993; Griko et al., 1995; Permyakov et al., 2006), suggesting that calcium binding stabilises the protein, thus leading to more cooperative protein unfolding and lowering the population of molten globular intermediates (Figure 9).

Figure 9. Thermal unfolding of equine lysozyme at different calcium concentration monitored by differential scanning microcalorimetry. Calcium- binding increases the folding cooperativity of equine lysozyme and shifts the first unfolding transition towards higher temperatures (adapted from Permyakov et al., 2006).

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The molten globule state of equine lysozyme possesses an extremely stable α-helical core, which is formed by the A, B and D helixes (Morozova et al., 1995; Morozova-Roche et al., 1997). By using amide hydrogen exchange studies coupled with NMR it has been shown that in the molten globule state these helices retain native-like interactions via the hydrophobic side chain contacts in their

interfaces, providing high core stability. The core structure of equine lysozyme is apparently more stable than the corresponding structures of human and hen egg white lysozymes (Filimonov & Morozova-Roche, unpublished data).

Methods and techniques used in the study

Amyloid specific dye binding

The investigation of amyloid formation requires adequate methods to distinguish amyloids from amorphous protein aggregates and observe the nano-scale structures. A lot of information regarding the morphology of amyloid aggregates was obtained by using AFM and TEM. In order to discriminate the cross-β-sheet containing structured protein aggregates from the amorphous ones we have also applied spectroscopic methods. These methods are based on the changes of the dyes optical properties upon binding to amyloid. In particularly, we have extensively used benzothiazole dye – thioflavine T.

.

Figure 10. The excitation and emission fluorescence spectra of free and amyloid-bound ThT (adapted from LeVine, 1999).

ThT undergoes the characteristic spectral changes upon binding to variety

of amyloid fibrils that do not occur upon binding to the precursor polypeptides or amorphous aggregates. This dye was extensively used in the histological determinations of amyloid in various tissues and in the last decade was adapted for in vitro studies of amyloids. ThT is a unique dye compare to others (Congo red,

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Thioflavine S) as in the amyloid-ThT complex it shows altered spectroscopic properties compare to the free dye in solution. The free dye has an excitation (absorption) maximum at 342 nm and emission at 430 nm, as opposed to the bound form in which the excitation is shifted to a maximum at 450 nm and the emission at 482 nm (Figure 10).

The precise biophysical mechanisms by which amyloid fibrils induce the notable 115 nm hypochromic ThT excitation spectral red shift is still not entirely understood. The pronounced excitation spectral changes, coupled with the relatively smaller effect on the emission spectrum, suggest that the ground state of the chromophore is altered by binding to amyloid fibrils rather than the excited state, which is usually associated with the emission spectrum. The possible models of the ThT binding to the amyloid fibrils is summarised in the Figure 11.

Figure 11. The model of ThT binding to the amyloid (adapted from Krebs et al., 2005 and LeVine, 2005).

Atomic force microscopy

The atomic force microscope is one of about two dozen types of scanned-proximity probe microscopes. All of these microscopes operate by measuring local properties, such as height, local absorption or magnetism, with a probe or "tip" placed very close to the sample. To acquire an image the microscope raster scans the probe over the sample, while measuring the local property in question. The resulting image consists of many rows or lines of information placed one after each other.

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AFM can operate in a number of different modes. While the contact mode provides more accurate imagining, the acoustically oscillating mode is more frequently used. In acoustically oscillating mode, the cantilever operates near its resonant frequency. The interaction between the tip and the sample is predominately vertical, thus negligible lateral forces are encountered. Consequently, acoustically oscillating mode AFM does not suffer from the tip or sample degradation effects, which are sometimes observed after many scans in contact mode AFM. Thus, this technique is very helpful for imaging soft, biological samples. In acoustically oscillating mode, the tip-sample interactions cause changes in the tip amplitude, phase and resonance frequency. The variations can be shown in height (topography) and amplitude or phase (interaction) images, which can be collected simultaneously.

Figure 12. The principal scheme of atomic force microscopy. The tip is placed at the end of the cantilever, which deflects when the tip encounters features on the sample surface. This deflection is sensed with an optical lever (red line): a laser beam reflecting from the end of the cantilever onto a segmented photodiode magnifies small cantilever deflections into larger changes in the relative intensity of the laser light on the two segments of the photodiode. In this way, the AFM makes a topographic map of the sample surface.

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There are two ways to drive the cantilever into oscillations. Firstly, this can be achieved by indirect vibration, in which high frequency acoustic vibrations from a piezoelectric transducer attached to the cantilever holder (Figure 12) excite the cantilever. This is called the acoustic mode. Another method in which the cantilever is excited directly without causing to vibrate the cantilever housing or other parts and is called magnetic mode (MAC Mod). To achieve MAC Mod imaging, the cantilever coated with a magnetic material is driven into oscillation by magnetic field generated by a solenoid positioned close to the cantilever housing. MAC Mod has even greater advantages when the cantilever is vibrating in liquid due to minimising the noise produced by the system (www.molec.com).

Spherical cap model

Schneider and co-workers showed that AFM can be used to determine the molecular volumes of single protein molecules deposited on the mica surface (Schneider et al., 1998). We employed this algorithm to identify the molecular volumes of amyloid oligomers formed by equine lysozyme. By using, AFM two parameters of the particles were determined such as height and width at a half-maximum height, which were obtained by multiple cross-section measurements. Instead of the particle diameter at the base, the half-height diameter was chosen to avoid an error induced by AFM tip curvature and broadening of lateral dimensions.

Based on the collected data and by using “spherical cap” (Figure 13) model we were able to calculate the molecular volumes of the particles and to identify a number of monomers arranged in one particle.

Figure 13. The “spherical cap” model used to calculate the volumes of amyloid oligomers deposited on a mica surface. The volume of the particles (VAFM) was calculated from the AFM measurements by using equation (1) 32

VAFM = (πh/6)(3r2+h2) (1) Where VAFM a volume; h is a height and r is a radius of the particle at a half maximum height. Molecular volume of monomeric protein was calculated by using equation (2) Vm = (Mo/No)(V1+dV2) (2) Mo is the protein molecular weight, No is Avogadro’s number, d is the extent of protein hydration (0.4 mol of H2O/mol of protein), and V1 and V2 are the partial specific volumes of the individual protein (0.74 cm3g-1) and water (1 cm3g-1) molecules, respectively (Schneider et al., 1998). The number of monomers in the oligomeric species was determined by the equation (3) n = VAFM / Vm (3) This approach proved to be very productive in the analysis of the amyloid samples of equine lysozyme allowing us to determine the volumes of monomers, tetramers, 8-mers and 20-mers as well as it has been applied to analyse other amyloid systems.

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Aims of the thesis

The aim of the thesis is to investigate the structural aspects of amyloid formation and to relate it to the functional properties of amyloid.

It can be subdivided into the following tasks:

• Investigation of the amyloidogenic properties of equine lysozyme; • Characterisation of transient oligomeric intermediates of equine

lysozyme; • Functional characterisation of the amyloidogenic species of equine

lysozyme; • Nanotechnological applications of amyloid polymers.

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Summary of the present study

This section summarises three research papers (I-III) comprising this thesis. The figures and numbers correspond to those used in the original papers.

Paper I. Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration.

In this study, we have demonstrated that equine lysozyme exposed to the condition where its molten globular state is populated can enter the amyloid formation pathway. The amyloid formation was studied at pH 4.5, 57 °C in the presence of 10 mM EDTA or 10 mM of CaCl2 and at pH 2.0, 57 °C. The morphological studies of amyloids were performed by using AFM. We found that at pH 4.5 and 57 °C in the presents of Ca2+ ions equine lysozyme assembles into short and straight linear amyloid protofilaments with a typical diameter of ca. 2 nm and length of ca. 300 nm (Figure 1). In the presence of EDTA, we found that EL forms donut-like structures, similar to those observed in the case of Aβ peptide and α-synuclein. Their diameters were of ca. 40-50 nm measured in AFM cross-sections, taking the highest points on the surface of the rings (Figure 2). When equine lysozyme was incubated at pH 2.0 and 57 °C, it formed linear amyloid protofilaments with appearance similar to those formed at pH 4.5 and in the presence of calcium ions and the rings of larger diameter of ca. 200 nm (Figure 4). All analysed structures exhibited Congo red and ThT binding properties typical for amyloids (Figure 3).

We have found that during the exposure to the low pH and high temperature equine lysozyme undergoes partial acidic hydrolysis. However, we were able to find appropriate conditions and the time frame during which EL assembled into amyloid without being hydrolysed. This indicates that a full length protein is able to assemble into amyloid structures and partial hydrolysis is not required for this process, though acidic hydrolysis can contribute to the amyloid formation.

We have suggested that ring-shaped amyloid protofilaments represent a new generic type of amyloid in addition to more common linear type. The presence of a very stable α-helical core in equine lysozyme structure resulted in the formation of amyloid with distinct morphology. The presence of the stable 35

core prevents equine lysozyme from an assembly into micron length mature fibrils by providing an unsticky fibrilar interface. This property defers equine lysozyme form hen and human lysozymes and bovine α-lactalbumin.

Paper II. Does the cytotoxic effect of transient amyloid oligomers from common equine lysozyme in vitro imply innate amyloid toxicity?

In this study, we continued to explore amyloidogenic properties of equine lysozyme. We have analysed the EL amyloid intermediates under amyloid forming conditions as previously described (Malisauskas et al., 2003). We have provided an insight into amyloid formation pathway of EL, which appeared to by different from human and hen egg white lysozyme self-assembly. We have evaluated the size of oligomeric intermediates and shown that they are cytotoxic.

The EL amyloid formation was induced by lowering pH to 2.0 or 4.5 and increasing the incubation temperature to 57 °C. Under those conditions, significant amounts of amyloid polymers were detected after 120 and 200 hours of incubation, respectively (Figure 1). We followed the kinetic of amyloid formation by using ThT binding assay and AFM (Figure 1). The EL samples containing the oligomeric intermediates corresponding to the amyloid growth phase were selected after 24h and 72 h at pH 2.0 57 °C and pH 4.5 57 °C respectively. The samples exhibit the same ThT binding level, what indicates a similar cross-β-sheet content in both samples. The morphological characterisation and the oligomers size determination in both samples was performed be using AFM. Two approaches were used to evaluate the size of amyloid oligomers. The first one was based on the volume measurements of a large number (more then 12000) of particles at the same time by applying the grain analysis module of the Scanning Probe Image Processor (SPIP) software (Image Metrology). The second method was based on the manual cross-section analysis of the particles and using “spherical cap model” for the volume calculations. The results are presented in the Figure 2 and the summary in the Table 1. We found that even the samples, having similar amount of amyloids and both being on the pathway to the formation of amyloid fibrils, were composed of different types of oligomeric particles. In the sample which was incubated at pH 2.0 and 57 °C for 24 h we identify monomers, 4-mers and 8-mers, while in the sample that was incubated at pH 4.5 and 57°C for 72 h we found monomers, 4-mers, 8-mers and 20-mers (Table 1). The well-defined samples containing the monomeric protein, amyloid oligomers, and EL amyloid

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protofilaments were subjected to the cytotoxicity assays. The cell viability of primary neuronal and fibroblast cell cultures was monitored by using ethidimium bromide staining of damaged cells. The cytotoxic effects produced by amyloids on human neurablastoma cell line were evaluated by MTT, ethidium bromide and TUNEL assays. The results clearly showed that soluble equine lysozyme amyloid oligomers are cytotoxic in contrast to the monomeric protein and amyloid protofilaments. The primary cell cultures appeared to be more susceptible to damaging effect of the amyloids compared to neuroblastoma cell line. It is important to note, that the amyloid sample containing the larger amyloid oligomers was more toxic towards all cells, causing apoptotic cell death as it has been demonstrated by TUNEL assay (Figure 4 and 5). In the samples with amyloid oligomers, a fraction of donut-like structures was always observed. They were assembled from the oligomers present in the sample. The number of ring-like structures was varied from one sample preparation to another and there was no correlation between the amounts of these structures and the sample cytotoxicity. This implies that the cytotoxicity is a common property of amyloids. The amyloid cytotoxicity does not depend of the primary amino acid sequence of the native precursor protein but rather on the morphology and size of the toxic species. The oligomers were the primary toxic agents, but not the rings, which can be viewed as a secondary effect of the oligomeric assembly.

Paper III. Ultrathin silver nanowires produced by amyloid biotemplating. The fast developing field on nanotechnology affects many aspects of our daily life. The applications of nanotechnology span from novel self-cleaning windows and water resistant clothing to new nanoelectronics and micro/nanochips. The classical “top-down” approach, which based on the miniaturization of existing devises and technologies, reaches its limits and a new strategy in the manufacturing processes called a “bottom-up” approach is emerging. The “bottom-up” design is based on self-assembly of nanosize components into functional devises. The inspiration for this process can come from nature, as the principal of separate manufacturing and later self-assembly of the complexes is the basic principal of living organisms. In the current study we explored the property of polypeptide chains to self-assemble into non-covalent proteinaceous polymers know as amyloid, which were used as a scaffold for casting ultrathin silver nanowires.

37

The amyloidogenic properties of lysozymes have been described in numerous studies. This factor as well the availability and price of the protein makes it an attractive candidate for the application in nanobiotechnology. The amyloid formation of hen egg white lysozyme was induced by lowering pH and increasing the incubation temperature as describe previously. The key component of the process was 2,2,2-trifluoroethanol, which reduced silver ions to colloidal silver and induced the formation of amyloid scaffold. We found that 2,2,2-trifluoroethanol is acting as reducing agent as shown in the equation (1). TFE can be used for the production of uniform silver nanoparticles. This process can be tightly controlled as shown in Figure 2. TFE induced thickening of amyloid polymers and the formation of the proteinaceous scaffold, within which the reduction of silver ions occur leading to the formation of silver nanowires with a diameter ca. 1.0-2.5 nm and up to 1 μm in length (Figures 3, 4). The application of others well known silver reducing agents did not produced the same results. The dimetilformamid and citric acid were tested. Their silver reducing capabilities have been described previously. In contrast to the TFE, DMF and citric acid did not induce amyloid scaffold formation, but caused the fragmentation of initial amyloid material (Figure 5). The replacing of TFE by DMF and citric acid in the nanowire manufacturing process did not lead to production of silver nanowires, indicating that the single amyloid threads do not poses a cavity within the core and the assembly of larger amyloid scaffolds is necessary for silver nanowire formation.

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Acknowledgments

I would like to thank many people with whom I spent time in Umeå. First of all, I am very grateful to my mentor Ludmilla Morozova-Roche who was always supportive of my new ideas and initiatives. I have learned how to “swim” in the rough seas of science and you always supported my interest in science.

I would like to thank Arūnas Leipus and Jaunius Urbonavičius who are responsible for my arrival to Umeå and for their tremendous support during the first days in this “new world”. Vladimir Zamotin, who was my colleague and close friend all these years in Umeå, for nice trips, parties and time we spent together. Rima and Karolis for being so different and similar at the same time and for our long trips home. My very close friends Osvaldas and Jurga who accept me as I am, for their friendship and support at any time and in all situations thank you! My special thanks for Rolandas Meškys who was the first to introduce me to the “dirty” biochemistry and showed how funny and challenging science can be.

I am very grateful to all the people I met in Umeå during these years. We shared the good and bad times together; these memories are the biggest treasure I will take with me from Umeå. Ačiū Mamai ir Tėčiui už jūsų paramą mano ilgu studijų metu ir tikėjimą manimi. Šis darbas yra skirtas jums. AČIŪ!!!

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Articles included in the thesis

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