Protein Complexes:

Assembly, Structure and Function

Kristina Rebecca Wilhelm

Copyright© Kristina W ilhelmNew series nr:

Department of Medical Biochemistry and Biophysics Umeå University

Sweden 2009

Copyright© Kristina R. Wilhelm New series no.: 1318 ISBN: 978 – 91 – 7264 – 913 – 2 ISSN: 0346 – 6612 Printed by: VMC – KBC, Umeå University Umeå, Sweden 2009

3

Table of contents

Table of contents........................................................................................................... 4

Abstract ......................................................................................................................... 5

List of Articles .............................................................................................................. 6

Abbreviations................................................................................................................ 7

Introduction................................................................................................................... 8

Protein misfolding......................................................................................................... 8 Amyloid formation.............................................................................................................. 10

Protein folding and misfolding in vivo......................................................................................... 11 Functional amyloids..................................................................................................................... 13

Structure of amyloid fibrils ................................................................................................. 15 Parkinson’s disease as an example of amyloid formation in vivo ................................................ 17

Parkinson’s disease and insulin............................................................................................... 17 Oligomer formation............................................................................................................. 17

Partly folded states ....................................................................................................................... 18 Mechanisms of toxicity in aggregation diseases ................................................................. 19 HAMLET as an example of oligomer formation in vivo and in vitro ................................ 21 Lysozymes .......................................................................................................................... 22

Equine Lysozyme......................................................................................................................... 23 Aims of the thesis........................................................................................................ 24

Summary of the research ............................................................................................ 25 Biomarkers for PD .............................................................................................................. 25 Papers on equine lysozyme and oleic acid complexes / ELOA. ......................................... 27

Production and characterization of ELOA................................................................................... 28 Toxicity of ELOA........................................................................................................................ 29 Revealing ELOA’s toxic agent .................................................................................................... 30 Bactericidal activity of ELOA ..................................................................................................... 31 General summary on ELOA ........................................................................................................ 31

Acknowledgements..................................................................................................... 33

References................................................................................................................... 36 Appendix I-IV

4

Abstract

Most proteins must fold into their native conformations to fulfil their biological functions. Failure of proteins to fold leads to cell pathology and a broad range of human diseases referred to as protein misfolding disease, e.g., Alzheimer’s disease, Parkinson’s disease, and type II diabetes. More than 40 proteins are known to be connected with misfolding diseases. These proteins share no sequence homology but all assemble into cross-β sheet containing insoluble fibrillar aggregates. Despite the pathological conditions that these proteins can induce, living organisms can take advantage of the inherent ability of these proteins to form such structures and to generate novel and diverse biological function, the functional amyloid. This thesis examines different aspects of cross-β sheet containing aggregates. The first paper describes the humoral response to aggregated structures of insulin and the astrocytical biomarker S100B in patients suffering from Parkinson’s disease. We show that the patients have an increased immunreactivity towards insulin and S100B in Parkinson’s disease patients compared to a control group. The second part of this work focuses on a functional amyloid. HAMLET (human α-lactalbumin made lethal for tumour cells) is a complex of α-lactalbumin and oleic acid, which kills tumour cells but not healthy differentiated cells. We wish to expand the concept of HAMLET to a structurally related protein and therefore create and characterize a complex of equine lysozyme and oleic acid (Paper II). We chose equine lysozyme because both proteins (equine lysozyme and α-lactalbumin) share common ancestors and are spatially related. The newly designed complex was named ELOA, for equine lysozyme with oleic acid. ELOA represents a functional oligomer due to its multimeric state and its ability to bind amyloid specific dyes. In the third paper, we investigate the interaction of the cytotoxic ELOA with live cells in real time to find a mechanistic model (Paper III). It is known that HAMLET is not only tumouricidal but is also toxic towards many bacteria. Therefore in the last part of the thesis, we investigated the effects of ELOA on different bacterial strains and focused on its interplay Streptococcus pneumoniae (Paper IV).

These studies have added significantly to many aspects of protein folding and misfolding from its involvement in Parkinson’s disease to the newly gained functions and structural aspects of de novo produced ELOA.

5

List of Articles

Articles included in the thesis

I Wilhelm KR, Yanamandra K, Gruden MA, Zamotin V, Malisauskas M, Casaite V, Darinskas A, Forsgren L, Morozova-Roche L. Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients. Eur. J. Neurol. 2007 Mar;14(3):327-34. II Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojevic V, Schleucher J, Morozova-Roche L. Protein oligomerization induced by oleic acid at the solid-liquid interface - equine lysozyme cytotoxic complexes. FEBS J. 2009 Aug;276(15):3975-89. III Vukojević V, Wilhelm K, Ming Y, Bowen A, Schleucher J, Hore P, Terenius L, Morozova-Roche L. ELOA interactions with the plasma membrane of live cells. (manuscript). IV Wilhelm K*, Clementi E*, Schleucher J, Morozova-Roche L**, Håkansson A **. Complexes of equine lysozyme with oleic acid with bactericidal activity against Streptococcus pneumoniae; (* shared authorship, ** joint corresponding authors).(manuscript).

(Reprints are made with the kind permission from the journal).

Articles not included in the thesis

V Darinskas A, Gasparaviciute R, Malisauskas M, Wilhelm K, Kozhevnikov JA, Liutkevicius E, et al. Engrafting fetal liver cells into multiple tissues of healthy adult mice without the use of immunosuppressants. Cell Mol. Biol. Lett. 2007. VI Gruden MA, Davidova TB, Malisauskas M, Sewell RD, Voskresenskaya NI, Wilhelm K, et al. Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Aβ (25-35) oligomers, S100b and neurotransmitters. J. Neuroimmunol. 2007. VII Nielsen S, Wilhelm K, Vad B, Morozova-Roche L, Otzen D. The molecular mechanism of the interaction equine lysozyme complexes with oleic acid with lipid membranes; (manuscript).

6

Abbreviations

Å – Angstrom

Aβ – amyloid β peptide

ANS – 8-anilino-l-naphthalenesulfonate

Ca – calcium

CNS – central nervous system

DNA – deoxyribonucleic acid

EL – equine lysozyme

ELISA – enzyme-linked immunosorbent assay

ELOA – equine lysozyme oleic acid complex

g – acceleration of gravity

HAMLET – human α-lactalbumin made lethal for tumor cells

kDa – kilo Dalton

LA – α-lactalbumin

LYS – lysozyme

M – Molar

MAL – multimers of α-lactalbumin

MG – molten globule

Na – sodium

NaCl – sodium chloride

NMR – nuclear magnetic resonance

PD – Parkinson’s disease

ThT – Thioflavin T

7

Introduction

Protein misfolding

Most proteins must fold into precise three-dimensional conformations to fulfil their

biological functions (1). Proteins fold spontaneously in vitro, confirming Anfinsen’s

pioneering insight that the linear sequence of a polypeptide chain contains all

necessary information to specify a protein’s three-dimensional structure.

In vivo the folding process requires strict quality control by the chaperone and

protease machinery. Failure of protein folding leads to cell pathology and to a broad

range of human diseases. These diseases are referred to as protein misfolding (or

protein conformational) diseases (2) (Table 1). They can be broadly divided into three

categories: (i) neurodegenerative diseases, if aggregation occurs in the brain; (ii) non-

neuropathic localized amyloidoses, if aggregation occurs in a single tissue outside the

brain; (iii) non-neuropathic systematic amyloidoses, if aggregation occurs in multiple

tissues.

In the light of our ageing society, most of these sporadic diseases are gaining more

significance. The familial cases of amyloid diseases (approximately 10%) are mostly

related to point mutations that destabilize the native state of the protein and these

diseases have an earlier onset.

Table 1: Human diseases associated with the formation of extra-cellular amyloid deposits or intracellular inclusions with amyloid-like characteristics (from reference (2)).

Disease Aggregating protein or peptide Neurodegenerative diseases Alzheimer's disease Amyloid β peptide Spongiform encephalopathies Prion protein or fragments thereof Parkinson's disease α-Synuclein Dementia with Lewy bodies α-Synuclein Frontotemporal dementia with Parkinsonism Tau Amyotrophic lateral sclerosis Superoxide dismutase 1 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

8

Disease Aggregating protein or peptide Familial British dementia Abri Familial Danish dementia ADan Nonneuropathic systemic amyloidoses 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 Nonneuropathic localized diseases Type II diabetes Amylin, also called islet amyloid polypeptide (IAPP)Medullary carcinoma of the thyroid Calcitonin Atrial amyloidosis Atrial natriuretic factor Hereditary cerebral haemorrhage with amyloidosis

Mutants of amyloid β peptide

Pituitary prolactinoma Prolactin Injection-localized amyloidosis Insulin Aortic medial amyloidosis Medin Hereditary lattice corneal dystrophy Mainly C-terminal fragments of kerato-epithelin Corneal amylodosis 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

The above proteins do not share any obvious sequence identity or structural or

functional homology, but they all form similar higher ordered protein assemblies with

common features. These proteinaceous structures are called amyloids and are highly

insoluble and difficult to isolate. The main amyloid components are elongated

unbranched protein fibrils (Figure 1), composed of one of the proteins listed in Figure

1. For pathologists to classify a disease as amyloid, the fibrils should be deposited

extracellularly and should bind the dye Congo red, giving an 'apple-green'

birefringence (3) and also bind Thioflavin T (ThT). Amyloid structures that do not

fulfil these criteria, extracellular localisation and Congo red binding of the amyloidal

structures, are referred to as “amyloid – like”.

9

Figure 1: Electron microscopy image of amyloid fibrils. Lower panels show enlarged sections of a twisted rope arrangement showing individual protofilaments. (Figure adapted from reference (4)).

Other components of amyloid material are non-fibillar compounds including the

serum amyloid component P, apolipoprotein E, as well as other proteins such as

glycosaminoglycans and proteoglycans (5). These non-fibrillar materials are

independent of the fibril precursor protein and are supposed to protect fibrils from

degradation via still unknown mechanisms.

The term amyloid was coined by the German medical doctor Rudolf Virchow in 1854.

The term amyloid is derived from amylo- (starch) and -oid (like), a somewhat

misleading term as it reflects the mistaken identification of the substance as a starch

when using crude staining techniques.

Amyloid formation

It is widely established that fibril formation has many characteristics of a nucleation-

dependent growth mechanism, typically including a lag phase, a growth phase, and a

steady state phase. Thus it is proposed that in the first phase, the lag-phase, nuclei are

formed. The formation of nuclei may take from seconds to days, depending on the

protein, its concentration and the environmental conditions. During the second phase,

the growth phase, fibrils grow by association either monomers or oligomers to the

nuclei. In the last phase, the steady state phase, ordered polymers and monomers

10

remain in equilibrium. The time course of the conversion from native protein into

fibrillar species can be monitored by specific fluorescence dyes whose fluorescence

increases in the fibrillar-bound form, e.g., by ThT fluorescence. A characteristic

amyloid formation kinetic curve, monitored by ThT binding, is shown in Figure 2.

Figure 2: Idealized kinetic curve for amyloid formation via nuclear-growth mechanism.

In recent years, it has been found that proteins not associated with human diseases

also can form amyloid fibrils in vitro. The fibrils are uniformly stabilized by the

hydrogen bondings of their polypeptide chains. On this basis, the ability to form

amyloid fibrils has been suggested to be a general property of the polypeptide chain

(6).

Protein folding and misfolding in vivo After release from the ribosome, proteins fold in three-dimensional structures,

geometries that make them functional. Small single domain proteins (< 100 amino

acids) fold in seconds. The folding landscape of these proteins is usually relatively

smooth, resulting in only two species being stably populated during the folding

reaction, namely unfolded states and the folded state, without populating any

significant intermediate states (1, 7).

11

Folding intermediates are the rule for larger proteins of (>100 amino acids) (ca. 90%

of all proteins in a cell) before the formation of completely folded states. Larger

proteins have a greater tendency to rapidly collapse in aqueous solution into compact

non-native structures, because of a variety of factors, including a higher proportion of

hydrophobic residues, which provide a greater driving force for chain collapse

(intramolecular contacts). It is still under debate whether these intermediates are

helping the protein find its correct fold or whether they are unavoidable traps that

slow down folding (7).

The association of two or more non-native protein molecules (intermolecular

contacts), also mainly driven by hydrophobic forces, can result in the formation of

amorphous structures. Alternatively, the formation of highly ordered fibrils can occur;

their formation corresponds to the deepest folding minimum on the energy landscape

(Figure 3). Every protein displays an individual energy landscape that depends on the

amino acid sequence of the protein as well as on the external factors such as

temperature and pH.

Generally, the propensity to misfold increases with (i) a topologically complex fold, a

fold that is stabilized by long-range interactions, or (ii) when proteins contain multiple

domains, domains that are separate in the native state but may interact during folding.

12

Figure 3: Energy landscape scheme of protein folding and aggregation. The purple surface shows the multitude of conformations 'funnelling' to the native state via intramolecular contacts and the pink area shows the conformations moving toward amorphous aggregates or amyloid fibrils via intermolecular contacts. Both parts of the energy surface overlap (Figure from reference (1)).

The parameters that influence aggregation can be divided into intrinsic

(hydrophobicity, propensity to form β-sheet structure and net charge) and extrinsic

factors (pH, ionic strength, and protein concentration) (8). Interestingly, these

parameters not only determine aggregation, but also are equally important for folding.

The differentiation between protein folding and aggregation occurs in different parts

of the protein (9).

Functional amyloids The classical view that amyloid is considered as exclusively disease associated has

been challenged in recent years: growing evidence indicates that amyloid may also be

a productive part of cell biology and contribute to normal physiology. In fact, amyloid

formation seems to be an intrinsic propensity of polypeptides in general and the

amyloid β-fold is an evolutionarily highly conserved structure. Functional amyloids

13

have been found in a wide range of organisms, from bacteria to mammals, with

functions as diverse as biofilm formation, development of aerial structures,

scaffolding, regulation of melanin synthesis, epigenetic control of polyamines, and

information transfer (2, 10-11) (Table 2). Due to the unique stability and insolubility

of fibrils, it has even been suggested that they can be used for nanotubular scaffolding

in Bio Nanotechnology (12).

Table 2: Proteins forming naturally nonpathological amyloid-like fibrils with specific functional roles (from reference (2)).

Protein Organism Function of the resulting amyloid-like fibrils Curlin Escherichia coli

(bacterium) To colonize inert surfaces and mediate binding to host proteins

Chaplins Streptomyces coelicolor (bacterium)

To lower the water surface tension and allow the development of aerial hyphae

Hydrophobin EAS Neurospora crassa (fungus)

To lower the water surface tension and allow the development of aerial hyphae

Proteins of the chorion of the eggshell

Bombyx mori (silkworm)

To protect the oocyte and the developing embryo from a wide range of environmental hazards

Spidroin Nephila edulis (spider) To form the silk fibers of the web Intralumenal domain of Pmel17

Homo sapiens To form, inside melanosomes, fibrous striations upon which melanin granules form

Ure2p (prion) Saccharomyces cerevisiae (yeast)

To promote the uptake of poor nitrogen sources ([URE3])

Sup35p (prion) Saccharomyces cerevisiae (yeast)

To confer new phenotypes ([PSI+]) by facilitating the readthrough of stop codons on mRNA

Rnq1p (prion) Saccharomyces cerevisiae (yeast)

Not well understood ([RNQ+], also known as [PIN+], phenotype)

HET-s (prion) Podospora anserina (fungus)

To trigger a complex programmed cell death phenomenon (heterokaryon incompatibility)

Neuron-specific isoform of CPEB (prion)

Aplyisia californica (marine snail)

To promote long-term maintenance of synaptic changes associated with memory storage

The identification of amyloid forms that are not associated with disease raises two

questions (13): Should the term “misfolded protein” still be applied to some types of

alternatively folded species? Does the term alternative-folding more accurately

reflect these folded species?

14

Structure of amyloid fibrils

The morphology of amyloid fibrils is independent of their origin (in vivo, in vitro or

ex situ) and in general of their protein sequence. Amyloid fibrils show linear un-

branched threads with a variable number of constituting strands as seen by atomic

force or electron microscopy. The height varies between 60-100 Å, while the length

can reach up to micrometers (4).

On the molecular level assembly of such thread-like fibrillar structure typically

involves the formation of a large degree of β-sheet structure and with it an extensive

hydrogen-bonded network (Figure 4).

Figure 4: The basic structure of all amyloid fibrils is substantially the same: polypeptide regions comprising 5-12 amino acids constitute β-strands, which are hydrogen-bonded to form a β-sheet.

The cross-β sheet structures give rise to amyloid specific characteristic – cross β-sheet

reflections in X-ray diffraction analysis (4) (Figure 5). The X-ray diffraction pattern

contains two predominant reflections. The first reflection corresponding to the fibre

axis at 4.7 Å (meridian) is thought to arise from the hydrogen-bonding distance

between β-strands in a β-sheet. The second reflection represents a more diffuse

reflection (equator) on the equator around 10-12 Å that likely corresponds to the

varying distance between β-sheets, which depends on the side-chain content of the

peptide. These X-ray diffraction patterns are regarded as one of the main

15

characteristic feature of amyloid fibrils (4), together with the above mentioned Congo

red binding and fibrillar morphology (Figure 1).

A B

Figure 5: (A) X-ray diffraction pattern from partially aligned amyloid fibrils formed in vitro from Aβ(1-42), showing the characteristic cross-β diffraction signals on the meridian and equator at 4.7 Å and 10 Å , respectively; (B) schematic presentation of the hydrogen-bonded β-sheet structure (Figures from references (4) and (13)).

Determination of the full molecular structure of amyloid fibrils is lacking because of

their noncrystalline, insoluble structure although some models have been published

(Figure 6), e.g., for Aβ1-40 (14-15). In these models, an Aβ1-40 peptide forms two β-

strands that are part of one β-sheet: residues 1-9 are structurally disordered, residues

10-22 and 30-40 form each a β-strand. According to the model these β-sheets possess

a hydrophobic core and form intra- and intermolecular hydrophobic interactions. Thus

four of these β-sheets form protofilaments that assemble into fibrils.

Figure 6: Representation of a full Aβ1-40 fibril viewed parallel and perpendicular (adapted from reference (14)).

16

Parkinson’s disease as an example of amyloid formation in vivo

Parkinson’s disease (PD) represents the second most common age-related

neurodegenerative disease after Alzheimer’s disease (Table 1). The pathological PD

symptoms include slow movements (bradykinesia), tremor, rigidity, and characteristic

gait (parkinsonian gait). Symptoms of PD usually begin to be apparent on one side of

the body. PD may progress quickly or gradually over the years. Many patients become

profoundly disabled and others continue to function relatively well.

Histochemically, PD is characterized by the loss of dopaminergeric neurons from the

substantia nigra (a part of the midbrain), and the formation of fibrillar intraneuronal

inclusions (Lewy bodies) (16), which consist of fibrillar α-synuclein. Alpha-synuclein

is a small (14 kDa) heat cytoplasmic protein that is found distributed in the

presynaptic terminals of neurons throughout the central nervous system (17). The

function of α-synuclein is unknown. Alpha-synuclein is natively unfolded, but folds

in the presence of membranes (18-19).

Parkinson’s disease and insulin PD as well as more than 20 other degenerative syndromes are associated with diabetes

mellitus, increased insulin resistance and obesity, disturbed insulin sensitivity, and

excessive or impaired insulin secretion (20-22). Diabetes mellitus, the fourth biggest

cause of death worldwide, exhibits a higher prevalence in patients with

neurodegenerative disorders (21) compared to non- neurodegenerative controls.

Oligomer formation Oligomers, intermediates in fibril formation, occur on the pathway to amyloid fibrils.

Oligomers can represent transient and intermediate species on the pathway to amyloid

formation, or a dead-end in protein folding. The oligomers are of special interest since

they, rather than mature fibrils, are primarily responsible for amyloid pathogenesis

17

(23). However, they are often transient and present in low concentrations under

physiological conditions (24).

In vitro prepared oligomers are soluble spherical aggregates of approximately 3 to 10

nm diameter, ranging from dimers up to particles of a million Dalton or more. The

general feature of oligomers seems to be a common epitope that does not exist in the

monomeric protein (25). The abundance of amyloid oligomers never exceeds a few

percent and they are in dynamic exchange with their monomeric background (26).

The definition of soluble amyloid oligomers is based on their consistent presence in

the supernatant after very high-speed centrifugation (> 370,000g). By contrast, fibrils

are found in the pellet after centrifugation at < 100,000g (27).

A growing body of evidence suggests that membrane permabilization by amyloid

oligomers may represent the common primary mechanism of pathogenesis (28-29),

whereby exposed hydrophobic groups that are normally buried in the folded state may

play a key role (24, 30).

Because of their proposed relevance to disease progression, many attempts have been

undertaken to populate and stabilize amyloidic oligomers in vitro on their way to fibril

formation, e.g., stabilizing these structures with the help of additives (31), or on

specially prepared polymer surfaces, or with increased pressure (24).

The first step in oligomer formation of a globular protein is destabilization of protein

molecule and population of its partially unfolded conformation. This can be easily

achieved during incubation above a critical concentration at an increased temperature

most often combined with a decreased pH.

Partly folded states One of the partly folded states are the molten globule (MG) states, a folding

intermediate possessing a secondary structure but lacking the tertiary contacts of the

native state. It is believed that molten globules are universal intermediates in protein

18

folding (32), ranging from states very similar to native-like states to those fully

approaching unfolded states, emphasizing that a variety of MGs do exist. MGs can be

generated by exposing the protein to mild acid solutions, in the presence of moderate

concentrations of protein denaturants, by removing protein-bound ligands (metal ions

or prosthetic groups) as well as by chain truncations or amino acid replacements (33).

However, analysis of molecular features is not an easy task since these states are

heterogeneous and usually flexible. 

The model protein to study MG is α-lactalbumin, a small acidic Ca2+ binding protein

with a fold similar as lysozyme. Alpha-lactalbumin forms the classical MG state

under different conditions (18). The MGs of some lysozymes and α-lactalbumins

share common features; their α-domains are more stable compared to the β-domain,

which is more flexible or disordered. The most significant tertiary structure

perturbation in acid solution was observed in bovine α-lactalbumin and equine and

pigeon egg-white lysozyme, as shown by CD measurements (33).

It should be mentioned that the calcium free form (apo-form) of either α-lactalbumins

or equine lysozyme does not necessarily resemble the one of MGs (32). Only upon

heating, at low ionic strength, or in the presence of a denaturing agent do these

proteins adopt a MG state. Furthermore, the conformational MG differences between

the calcium form (holo-form) and apo-form are confined at the level of the calcium

binding site (33).

A second intermediate state that may represent the most likely fibrillogenic

intermediates is the pre-molten globule state (18). The pre-molten globule state is less

compact than the MG state and posses a low content of secondary structure although

it is still more compact than the random coil structure (summarized in (18)).

Mechanisms of toxicity in aggregation diseases

In general, a few different types of cell death have been described: apoptosis,

necrosis, autophagy (34), and some additional sub-types (35). Apoptosis is defined by

19

stereotypic morphological changes where the chromatin condenses. Other typical

features are phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis (plasma

membrane blebbing), and the formation of apoptotic bodies. In the most classic form,

apoptosis is associated with caspase activation (35).

Necrosis does not involve chromatin condensation and usually does not involve

caspase activation. Because rapid cytoplasmic swelling is characteristic for necrosis, it

is often referred to as oncosis (condition characterized by swelling) (34). Necrosis is

the direct effect of stress on cells and therefore called “uncontrolled” or “accidental”

death. Cells undergoing death associated with autophagy are characterized by the

presence of double membrane autophagic vacuoles. Autophagy is mostly associated

with nutrient or growth factor deprivation (34-35).

It is widely accepted, that the pathway by which amyloidic structures lead to cell

death is apoptosis or less frequently necrosis (36). This is even true for amyloid

formed from proteins not associated with disease (36-37). The exact mechanism by

which amyloid structures act to cause death is still not understood, but there is

increasing evidence that the main toxic agents are soluble precursors of the proteins

rather than the mature insoluble fibrils (38). It has been postulated that the toxic

oligomers share a common structure and that as a consequence there is a generic

pathological mechanism at work (23).

Commonly it is accepted that the toxic effects of oligomers are due to their interaction

with the cell membranes. There the structural organization and selective ion

permeability are affected, leading eventually to cell death (37). The first step towards

cell death seems to be membrane destabilization, which is one of the key events in

amyloid toxicity (37). However, the mechanism of amyloid toxicity is still unknown,

even though a number of factors have been identified that influence the susceptibility

of different cell lines to toxic oligomers. Amyloid mediated cell death depends on the

membrane content of cholesterol (inverse correlation) as well the ability of the cells to

counteract early modifications in the intracellular free Ca2+, the redox states of the

cells, and the degree of cell differentiation (positive correlation) (39-40).

20

HAMLET as an example of oligomer formation in vivo and in vitro

In 1995, a most astonishing complex was discovered, named HAMLET as an

acronym for human α-lactalbumin made lethal for tumour cells (41). HAMLET is a

complex of α-lactalbumin and oleic acid (C18:1:9cis). What is remarkable about

HAMLET is its ability to discriminate between healthy differentiated and cancer cells.

HAMLET is characterized by a partially unfolded structure as shown by circular

dichroism, intrinsic and ANS-fluorescence, resembling the MG state of α-

lactalbumin.

Two approaches have been used to produce HAMLET. It can be purified from human

milk (42) or it can be obtained by eluting monomeric Ca2+-deleted α-lactalbumin

from human milk whey through an oleic acid-preconditioned column (43). The

purified fraction of human milk contains multimers of α-lactalbumin (MAL); α-

lactalbumin is one of the components of human milk whey, as shown by SDS-PAGE

(42, 44). MAL crosses the plasma membrane and the cytosol enters the cell nucleus,

where it induces DNA fragmentation. MAL also interacts with the mitochondria

where it induces cytochrom c releases and caspase activation (41, 45).

When producing HAMLET with the help of a column, EDTA was added to remove

bound Ca2+ from the protein. This now less stable protein structure was then stabilized

by oleic acid or other unsaturated fatty acids in cis-conformation (46).

It has been proposed that HAMLET presents a naturally occurring substance that is

present in the stomach of babies and protects the nursed child from cancer (47), since

both components of HAMLET, namely α-lactalbumin and oleic acid, are components

of human milk. It was shown that breast fed children have a lower risk of getting

cancer and that breast-feeding also lowers the risk of breast cancer for the mother. It is

tempting to speculate that “Mother Nature” provided the best for its offspring and that

HAMLET represents one form of protection.

In addition to its tumouricidal activity, HAMLET also kills various bacteria (48), with

Streptoccocus pneumoniae being the most sensitive species. The unique ability of

21

HAMLET to discriminate between tumour and healthy differentiated cells (49), its

astonishing success as a potential therapeutic agent in vivo (50-51), and its

antimicrobial activity makes HAMLET one of a kind.

Lysozymes

Lysozyme is an ubiquitous enzyme, catalyzing the hydrolysis of the β 1-4 glycosidic

bond between N-acetylmuramic and N-acetylglucosamine acid residues that make up

the bacterial peptidoglycan, a major component of the bacterial cell wall. Human

lysozyme is found in a variety of organs, including spleen, lung, and kidney, and in

extra-cellular fluids such as plasma, milk, salvia, and tears.

Lysozyme is an excellent system to study fibril formation as its structure and folding

mechanism are well known (52). Lysozyme is an ancient protein whose origin goes

back an estimated by 400 to 600 million years (53). It was originally a bactericidal

defence agent and has been adapted to serve as a digestive agent. The gene encoding

lysozyme also gave rise by gene duplication (300 to 400 million years ago) to a gene

that currently codes α-lactalbumin. Alpha-lactalbumin is a protein expressed in the

lactating mammary gland of most mammals. Despite their functional diversity,

lysozymes and α-lactalbumins are 40% homologous in sequence and share a close

spatial structure, which is the reason why lysozymes and α-lactalbumins belong to the

same structural family. The major structural difference between the two families lies

in its ability to bind calcium for α-lactalbumins but not for most members of the

lysozymes family.

In general, c-type (c for chicken) lysozymes are separated into two groups with

respect to their calcium-binding ability, namely calcium binding lysozyme and

conventional non-calcium binding lysozymes, e.g., human and hen-egg white

lysozyme. Equine, pigeon, canine, donkey, and goose belong to the calcium binding

group (54-55), being the structural link between the α-lactalbumin and the lysozyme

families.

22

Equine Lysozyme Similar to other c-type lysozymes equine (horse), lysozyme (EL) contains two

domains separated by a cleft. One domain is rich in α-helixes and the other one is in

β-conformation. In the case of EL, the β-sheet containing domain is in contact with

inter-domain interface containing a calcium binding site (Figure 7).

Figure 7: Schematic diagram of equine lysozyme molecule demonstrating the folding areas with local cooperativity. They are colour-coded as follows: violet, corresponding to the rapidly folded A, B, and D α-helical core; blue, the 59–61 residue β-strand stabilized with 50–100 ms time constants; green, the C-helix, 120–150 ms time constants; yellow, the elements of secondary structures attaining persistent structure with 200–250 ms time constants; orange, the middle 51–55 residue β-strand, ca. 400 ms time constants; and red, denoting the most slowly stabilized loops. The disulphide bridges are shown in ball-and-stick representation. (Figure adapted from (56)) The presence of the calcium binding site most likely contributes to EL’s significantly

lower stability and cooperativity compared to c-type lysozymes (57-59). The two

domains of EL unfold separately in its apo-form; the first unfolds the β-domain,

changing the fold of the protein from native to MG-like. The second transition drives

from the MG-like state to the unfolded state (57, 60). The first transition strongly

depends on calcium concentration (57-58, 60-61), suggesting that calcium binding

stabilizes the protein, leading to more cooperative unfolding. A special feature of EL

is the extremely stable α-helical MG core, formed by the A-, B-, and D-helices. These

helices are stabilized via hydrophobic interactions based on their native interactions.

Furthermore, equine lysozyme can form fibrils at acidic pH and at elevated

temperatures and these structures are cytotoxic to a number of cell lines (62).

23

Aims of the thesis

This thesis examines different aspects of protein folding. One part of the thesis is

dedicated to immunoreactivity, while the main part is dedicated to the studies of

equine lysozyme and oleic acid complexes and their structural and functional and

possible therapeutic properties.

The specific aims are as follows:

• To elucidate a correlation between immunoreactivity to amyloid insulin

structures, autoimmune reaction to biomarkers of PD and PD development.

• Preparation and structural characterization of a complex of equine lysozyme

and oleic acid.

• Elucidation of a mechanism for ELOA cytotoxicity.

• Investigation of the effect of ELOA complex on the bacteria Streptococcus

pneumoniae.

24

Summary of the research Paper I Immune reactivity towards insulin, its amyloid and protein S100B in blood sera of Parkinson's disease patients. Wilhelm KR, Yanamandra K, Gruden MA, Zamotin V, Malisauskas M, Casaite V, Darinskas A, Forsgren L, Morozova-Roche L., Eur. J. Neurol., 2007

Biomarkers for PD Biomarkers are generally considered to be plasma measurements of molecules that

provide independent diagnostic or prognostic value by reflecting an underlying

pathological conditions (63).

Potentially, S100B can be used as a neurologic screening marker for injuries in the

central nervous system. The S100B level is elevated in blood sera as S100B is

released into the circulation in a variety of central nervous system (CNS) disorders,

among them Alzheimer’s disease, Down Syndrome, and traumatic brain injuries (63).

S100 proteins are found in abundance in astroglial and Schwann cells (64). S100B is a

low molecular weight (10.4 kDa), acidic, calcium-binding protein that is highly

conserved throughout the vertebrate species. S100B was thought to be the first and

most abundant brain specific protein (65). At least 80-90% of the total S100B is found

in the brain; the rest is located in other non-neuronal tissues. Historically, the name

“S100” referred to the protein’s 100% Solubility in saturated ammonium sulphate

solution at neutral pH (65).

In the present study, we explored the link between PD progression and S100B

level in blood sera.

PD, a degenerative disease of the CNS, is diagnosed according to patient’s movement

disorders. Unfortunately, there are no diagnostic tests for PD; the disease is diagnosed

according to cardinal features (rest tremor, muscle rigidity, bradykinesia, and

asymmetric onset).

The clinical diagnosis can be difficult, especially at the beginning of the disease;

therefore, it would be very desirable to have a biomarker or at least a strong indicator

25

for PD. We evaluated the presence of S100B biomarkers in the blood sera of 26 PD

patients compared with controls by using ELISA. We found a statistically significant

increase of the autoimmune responses to S100B in PD patients compared with

controls with a mean increase of 50% in the autoimmune reactions towards S100B.

In the present study, we tried to link PD and the autoimmune responses to

generic epitope of fibrils.

It was discussed earlier that oligomers and fibrils posses a common generic epitope

(25) that does not exist in the monomeric protein. In the brain of PD patients,

proteinaceous amyloidic plaques are found. Our hypothesis was that patients that

suffer from PD develop antibodies against these structures, as shown for (66)

Alzheimer’s patients. For this purpose, we formed amyloid structures from human

insulin. The immune reactivity of insulin monomers, insulin oligomers, and fibrils

was tested. The results showed a decreased immune reactivity toward insulin fibril

compared to oligomers and monomers. The immune reactivity towards monomers

was highest, followed by the reactivity towards oligomers, and the least reaction was

against fibrils. It might be speculated that the decreased immune reactivity towards

higher amyloidic structures coincides with decreased monomer presence.

The overall conclusion from this study is that immune reactions towards S100B and

insulin may reflect the neurodegenerative brain damaging processes and impaired

insulin homeostasis occurring in PD, while an immune reaction towards generic fibril

epitope was not detected.

26

Papers on equine lysozyme and oleic acid complexes / ELOA. Paper II Protein oligomerization induced by oleic acid at the solid-liquid interface--equine lysozyme cytotoxic complexes. Wilhelm K, Darinskas A, Noppe W, Duchardt E, Mok KH, Vukojevic V, Schleucher J, Morozova-Roche L. (FEBS J. 2009) Paper III ELOA interactions with the plasma membrane of live cells. Vukojević V, Wilhelm KR, Ming Y, Bowen A, Schleucher J, Hore P, Terenius L, Morozova-Roche L. (manuscript) Paper IV Complexes of equine lysozyme with oleic acid with bactericidal activity against Streptococcus pneumoniae. Wilhelm K*, Clementi E*, Schleucher J, Morozova-Roche L**, Håkansson A**. (* shared authorship, ** joint corresponding authors); (manuscript)

Inspired by the HAMLET findings, a protein complex that kills tumour cells but not

healthy differentiated cells, we focused on the complexes of equine lysozyme with

oleic acid. Indeed, HAMLET consists of partially unfolded α-lactalbumin and oleic

acid. We used a protein structurally related to α-lactalbumin, namely equine

lysozyme, with a sequence homology of 61.8% and an identity of 41.2% (Table 3).

Table 3: Sequence variation in protein sequence.

Sequence homology %

Human LA

Human LYS

Equine LYS

Human LA 100 Human LYS 59.2 100 Equine LYS 61.8 73.1 100

Hen egg white LYS 54.5 76.9 67.7 Sequence

identity % Human LA 100

Human LYS 37.4 100 Equine LYS 41.2 50.8 100

Hen egg white LYS 35.1 60.0 49.2 Despite the low sequence homology, there is a conserved tertiary fold. Both proteins

consist of two domains: a larger α-helical domain and a smaller β-sheet domain.

27

These domains are connected by a calcium-binding loop. The binding constant is

2x106 M-1 for equine lysozyme and 3x108 M-1 for α-lactalbumin. Both structures are

stabilized by four disulfide bridges (56, 67).

A B

Figure 8: (A) α-lactalbumin (PBD name: 1A4V) and (B) equine lysozyme (PBD name: 2QEL) images produced by Pymol (http://delsci.com/rel/099/)

Production and characterization of ELOA ELOA was produced by passing holo-lysozyme over an ion exchange column

conditioned with oleic acid. The protein-lipid complex was eluted with a linear salt

gradient consisting of 0-1.5 M NaCl. We assume that with the help of the column

oleic acid molecules are kept soluble, and due to hydrophilic and electrostatic

interactions between the protein and the fatty acid, the protein unfolds and binds to

oleic acid and as a result the ELOA was formed.

Just like HAMLET, ELOA shows a less folded protein structure as shown by CD,

ANS-binding, and NMR spectroscopy. (Paper II). Using NMR, we characterized the

stoichiometry of ELOA and revealed that the oleic acid protein ratio varied from 9-48

bound oleic acid per molecule of protein. Diffusion NMR measurements revealed that

ELOA forms a range of oligomers from tetra up to 30 mers. These results are

schematically presented in Figure 9. It might be noted that all the experiments

presented here are performed at pH 9.

28

Figure 9: Schematic representation of the ELOA formation at the solid–liquid interface within column chromatography. The exposed hydrophilic residues are denoted in purple and the buried hydrophobic residues in grey. (During interaction with the solid–liquid interface in the column, the hydrophobic residues (grey) are exposed and its molecules assemble with each other and with oleic acids to form ELOA (encircled schematically) (adapted form Paper II).

Toxicity of ELOA We tested the cytotoxicity of ELOA on a number of different cell lines:

o rat pheochromocytoma of the rat adrenal medulla cells (PC12); o human neuroblastoma cells (SHSY - 5Y); o mouse embryonic liver cells; and o mouse embryonic fibroblasts.

ELOA showed toxicity to all tested cell lines in a concentration and time dependent

manner in an apoptosis-like mechanism as shown by acridine-orange staining (Paper

II).

29

Revealing ELOA’s toxic agent Proteins interact with their reaction – partner like substrate, cofactors, and ligands.

After the interaction, a protein can fulfil an additional function, a phenomena known

as “moonlighting” (68). In our cytotoxicity experiments, we wanted to reveal whether

the components (namely the oleic acid and the protein equine lysozyme) are the toxic

agents or whether the toxicity is a newly gained function – a “moonlighting” function.

In all performed experiments, the native protein never showed any toxicity toward

cells (Paper II and Paper III). The oleic acid-treated cells showed a higher viability

compared to ELOA treated cells.

To further analyze the mechanism of cytotoxicity, we labelled ELOA with fluorescent

dye Alexa@488 and recorded the interaction in real time. We observed an

accumulation of ELOA at the vicinity of the cell membrane. Surprisingly, we never

saw an uptake of ELOA into the cell. Further analysis of the fluorescently-labelled

cell membrane revealed that the membrane was ruptured (Figure 10, B) after about

one hour incubation with ELOA. After the complete rupture of the cell, ELOA

“streamed” inside the cells. Rupture of the cell membrane was never detected after

incubation of cells with equine lysozyme (Figure 10,A).

A B

Figure 10: Plasma membrane staining with DiIC5(18) of cells treated with (A) equine lysozyme and (B) ELOA.

In summary, ELOA presents a model of synergy between oleic acid and proteins; the

oleic acid is kept “soluble” by the protein. It is tempting to speculate that micelle

forming oleic acid has a higher energy barrier to release single oleic acid molecules

than ELOA, which might explain the lower degree of toxicity caused by oleic acid

alone.

30

Bactericidal activity of ELOA It has been known for a long time that HAMLET shows bactericidal activity (48),

being the most active against the gram positive bacterium Streptococcus pneumoniae

D39 (69) (pneumococcus). Pneumococcal contagion is the leading cause of morbidity

and mortality from respiratory tract and invasive infections in children and the elderly

worldwide (70). Like HAMLET, ELOA killed the bacteria in a concentration-

dependent manner and via an apoptotic-like mechanism as shown by DNA

fragmentation (Paper IV). Adding equine lysozyme had no effect on the viability

although sonicated oleic acid decreased viability compared to controls but to a

statistically significant lower degree than equine lysozyme. Non-sonicated oleic acid

had no affect on bacterial viability.

Furthermore, we could detect rupture and depolarization of the bacterial wall during

the interaction of the bacteria with ELOA. Interestingly, we could reveal the

involvement of different specific channels (calcium, sodium/calcium, and calcium

activated calcium channels) in the bactericidal response to ELOA; by inhibiting these

channels, we could restore the depolarization, membrane rupture viability of S.

pneumoniae. These are the same channels that are involved in HAMLET

depolarization, rupture of the pneumococcal membrane, and death (Clementi and

Håkansson, personal communication). Overall, these results demonstrate that ELOA

possesses anti-bacterial activity.

General summary on ELOA

Cancer is the leading cause of death according to the World Health Organization: it

accounts for 7.4 million deaths (2004), among them 160.000 children (World Child

Cancer Foundation). HAMLET seems to be a potential therapeutic natural occurring

agent against cancer. Using HAMLET as a model, we produced our own complex of

equine lysozyme and oleic acid, ELOA.

The multimeric complexes ELOA were produced by ion-exchange chromatography

(Figure 9) on a column preconditioned with oleic acid. The properties of ELOA were

characterized using NMR, spectroscopic methods, and atomic force microscopy.

31

ELOA showed similarity with both amyloid oligomers such as ThT-binding and

spherical appearance using atomic force microscopy revealed HAMLET-like

properties such as bound oleic acid and a partially unfolded protein. Similar to

HAMLET, ELOA showed bactericidal activity against Streptococcus pneumoniae.

Studies of well-populated ELOA shed light on the nature of the amyloid oligomers

and HAMLET complexes, suggesting that they constitute one large family of

cytotoxic proteinaceous species.

32

Acknowledgements

I want to take this opportunity to thank you all - I have always felt fortunate and

privileged to be surrounded by researchers. I find it very enjoyable to work with

people from all around the world, people who see more in science than just a job,

people like you. The further I am really happy to have experienced the Swedish way of

living with “fika”& “lagom”.

So dear reader – Thank you.

From reading a lot of acknowledgements, I have found that all supervisors/

collaborators/ colleges are enthusiastic, supportive and generally of the friendly sort.

What I feel though is that those words, although mostly true, are not very personal –

which is why I will give a more personal view, describing what makes all these

persons special to me.

In Umeå:

My supervisor Ludmilla: Religion is a way of life within a larger framework of

meaning – I admire that you live up to that.

My present and past colleagues:

Vladimir, thank you so much for your patience in teaching me AFM; Kiran, the

positive spirit in the lab, my great project students Judith & Jan; Anna for being more

than a colleagues; and all the “newer” members of the group for their many friendly

smiles.

My second supervisor Gerhard G.: Rough surface – otherwise only positive and

lovable attributes.

33

My NMR-guy Jürgen S.: THE BACKPACK - what kind of character does it need to

pull that off – you manage…..

The crystal-guy Tobias H.: Crystals or not – it was great to discuss life with you.

Marinna G.: Compassion in combination with a warm personality.

The heart of the department: Ingrid R.; the warrior against bureaucracy/ my chaos:

Clas W.; Urban B.: your practical teaching that set standards.

Outside Umeå:

the start:

Ken Mok: Diplomacy is an art that you master perfectly, thank you so much for introducing me to the HAMLET project, and your advice.

friendly bacteria:

Anders Håkansson: Caring for people made you care about science – the noblest of all intentions. I really enjoyed staying with your family, working in your lab. Emily Clementi: Whenever I give a positive statement about the kindness of people in the US, I refer to you and your sister & thanks for your many nice, kind, friendly and calming e-mails they were always a little ray of sunlight during the writing period. five cell imaging:

Vladana Vukojević: Just some of the things I learned form you: “science can be as exiting Christmas presents, you determine your own destiny, and there is a nearly endless amount of energy fitting into a tiny body”.

“biophysics – pure”:

Daniel Otzen, Søren B. Nielsen: Thanks for the opportunity to stay with you in the lab I decided the next European country I want to life in is Denmark, thanks for the huge amount of smiley faces in your mails☺.

34

Alexandra: Super-Schwaben Spätzle. Angela: The hard effort to get to know you was paid back triple-million-billion times (and occasions). Anna G.: I am very proud and honored to be your daughters’ aunt, now we are connected for life. Barbara: Beauty, Charm and Intelligence the only thing missing till perfection is the step-dance class with me. Birgit: Danke! Björn: Bästa office-kompis. Christopher: you could mostly restore my data & unlock my bike with liquid nitrogen & went skiing with me because I had to train and had no car at -28ºC & accepted that my part of the gardening was to eat the strawberries & you are my “Umeå Hero”. Elke: It is always great to talk to a person with an independent spirit like yours. Familie: Danke das Ihr da wart - in Trauer und immerwährenden Gedänken. Franziska: Die mir liebste Deiner vielen guten Eingenschaften ist mir dein grosses Herz. Gautam: A friend is one who believes in you when you have ceased to believe in yourself, thanks for believing in me. Harry-Sweetie: nicht unter ferner liefen (- bei dem Knie ?!!?). Marija P.: Camping in deep snow and felt like -157ºC could only have been fun with you. Miriam: Every time I drive my car I thank you for your patience dealing with Swedish bureaucracy. Thank you for being my connection to the department. Svensk Tjeijklassiker girls Isabelle and Miriam: it was great to travel through Sweden with you, and to meet your families; managing the (tjeij-) vasaloppet with you was definitely one of the high-points of my stay here. Some great people that had a special impact I want to express my gratitude: Dörthe – nice that you are here; Bine - immer gut Leute zu kennen die eine andere Einstellung zum Leben haben; Cora & Mirko - Heimspiel oder Auswärts, egal Hauptsache mit Euch; Waltraud & Erich – danke für Eure moralische Unterstützung und Euer Intresse, my dear Italian sister Katja. Yours Kristina

35

References 1. Hartl, F. U., and Hayer-Hartl, M. (2009) Converging concepts of protein

folding in vitro and in vivo, Nature Structural & Molecular Biology 16, 574-581.

2. Chiti, F., and Dobson, C. M. (2006) Protein misfolding, functional amyloid, and human disease, Annual Review of Biochemistry 75, 333-366.

3. Westermark, P., Benson, M. D., Buxbaum, J. N., Cohen, A. S., Frangione, B., Ikeda, S. I., Masters, C. L., Merlini, G., Saraiva, M. J., and Sipe, J. D. (2005) Amyloid: Toward terminology clarification - Report from the Nomenclature Committee of the International Society of Amyloidosis, Amyloid-Journal of Protein Folding Disorders 12, 1-4.

4. Stromer, T., and Serpell, L. C. (2005) Structure and morphology of the Alzheimer's amyloid fibril, Microsc Res Tech 67, 210-217.

5. MacRaild, C. A., Stewart, C. R., Mok, Y. F., Gunzburg, M. J., Perugini, M. A., Lawrence, L. J., Tirtaatmadja, V., Cooper-White, J. J., and Howlett, G. J. (2004) Non-fibrillar components of amyloid deposits mediate the self-association and tangling of amyloid fibrils, J Biol Chem 279, 21038-21045.

6. Dobson, C. M. (2003) Protein folding and misfolding, Nature 426, 884-890. 7. Dobson, C. M. (2004) Principles of protein folding, misfolding and

aggregation, Seminars in Cell & Developmental Biology 15, 3-16. 8. Bemporad, F., Calloni, G., Campioni, S., Plakoutsi, G., Taddei, N., and Chiti,

F. (2006) Sequence and structural determinants of amyloid fibril formation, Acc Chem Res 39, 620-627.

9. Chiti, F., Taddei, N., Baroni, F., Capanni, C., Stefani, M., Ramponi, G., and Dobson, C. M. (2002) Kinetic partitioning of protein folding and aggregation, Nat Struct Biol 9, 137-143.

10. Maury, C. P. (2009) The emerging concept of functional amyloid, J Intern Med 265, 329-334.

11. Otzen, D., and Nielsen, P. H. (2008) We find them here, we find them there: functional bacterial amyloid, Cell Mol Life Sci 65, 910-927.

12. Waterhouse, S. H., and Gerrard, J. A. (2004) Amyloid fibrils in bionanotechnology, Australian Journal of Chemistry 57, 519-523.

13. Herczenik, E., and Gebbink, M. F. (2008) Molecular and cellular aspects of protein misfolding and disease, Faseb J 22, 2115-2133.

14. Petkova, A. T., Yau, W. M., and Tycko, R. (2006) Experimental constraints on quaternary structure in Alzheimer's beta-amyloid fibrils, Biochemistry 45, 498-512.

15. Petkova, A. T., Ishii, Y., Balbach, J. J., Antzutkin, O. N., Leapman, R. D., Delaglio, F., and Tycko, R. (2002) A structural model for Alzheimer's beta -amyloid fibrils based on experimental constraints from solid state NMR, Proc Natl Acad Sci U S A 99, 16742-16747.

16. Lashuel, H. A., Petre, B. M., Wall, J., Simon, M., Nowak, R. J., Walz, T., and Lansbury, P. T., Jr. (2002) Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils, J Mol Biol 322, 1089-1102.

17. Norris, E. H., Giasson, B. I., Ischiropoulos, H., and Lee, V. M. (2003) Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications, J Biol Chem 278, 27230-27240.

36

18. Uversky, V. N., and Fink, A. L. (2004) Conformational constraints for amyloid fibrillation: the importance of being unfolded, Biochim Biophys Acta 1698, 131-153.

19. Uversky, V. N. (2009) Intrinsic disorder in proteins associated with neurodegenerative diseases, Front Biosci 14, 5188-5238.

20. Sandyk, R. (1993) The relationship between diabetes mellitus and Parkinson's disease, The International journal of neuroscience 69, 125-130.

21. Ristow, M. (2004) Neurodegenerative disorders associated with diabetes mellitus, J Mol Med 82, 510-529.

22. Ma, H. I., Kim, J. H., Chu, M. K., Oh, M. S., Yu, K. H., Kim, J., Hahm, W., Kim, Y. J., and Lee, B. C. (2009) Diabetes mellitus and drug-induced parkinsonism: a case-control study, Journal of the neurological sciences 284, 140-143.

23. Glabe, C. G., and Kayed, R. (2006) Common structure and toxic function of amyloid oligomers implies a common mechanism of pathogenesis, Neurology 66, S74-S78.

24. Ferreira, S. T., De Felice, F. G., and Chapeaurouge, A. (2006) Metastable, partially folded states in the productive folding and in the misfolding and amyloid aggregation of proteins, Cell Biochem Biophys 44, 539-548.

25. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science 300, 486-489.

26. Frare, E., Mossuto, M. F., de Laureto, P. P., Tolin, S., Menzer, L., Dumoulin, M., Dobson, C. M., and Fontana, A. (2009) Characterization of oligomeric species on the aggregation pathway of human lysozyme, J Mol Biol 387, 17-27.

27. Sharon, R., Bar-Joseph, I., Frosch, M. P., Walsh, D. M., Hamilton, J. A., and Selkoe, D. J. (2003) The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease, Neuron 37, 583-595.

28. Bucciantini, M., Calloni, G., Chiti, F., Formigli, L., Nosi, D., Dobson, C. M., and Stefani, M. (2004) Prefibrillar amyloid protein aggregates share common features of cytotoxicity, Journal of Biological Chemistry 279, 31374-31382.

29. Kayed, R., Sokolov, Y., Edmonds, B., McIntire, T. M., Milton, S. C., Hall, J. E., and Glabe, C. G. (2004) Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfolding diseases, Journal of Biological Chemistry 279, 46363-46366.

30. Frare, E., Mossuto, M. F., Polverino de Laureto, P., Dumoulin, M., Dobson, C. M., and Fontana, A. (2006) Identification of the core structure of lysozyme amyloid fibrils by proteolysis, J Mol Biol 361, 551-561.

31. Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed, R., Liosatos, M., Morgan, T. E., Rozovsky, I., Trommer, B., Viola, K. L., Wals, P., Zhang, C., Finch, C. E., Krafft, G. A., and Klein, W. L. (1998) Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins, Proc Natl Acad Sci U S A 95, 6448-6453.

32. Polverino de Laureto, P., Frare, E., Gottardo, R., Van Dael, H., and Fontana, A. (2002) Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis, Protein Sci 11, 2932-2946.

37

33. Fontana, A., de Laureto, P. P., Spolaore, B., Frare, E., Picotti, P., and Zambonin, M. (2004) Probing protein structure by limited proteolysis, Acta Biochim Pol 51, 299-321.

34. Krysko, D. V., Vanden Berghe, T., D'Herde, K., and Vandenabeele, P. (2008) Apoptosis and necrosis: detection, discrimination and phagocytosis, Methods 44, 205-221.

35. Leist, M., and Jaattela, M. (2001) Four deaths and a funeral: from caspases to alternative mechanisms, Nature reviews 2, 589-598.

36. Stefani, M. (2008) Protein folding and misfolding on surfaces, International journal of molecular sciences 9, 2515-2542.

37. Stefani, M. (2007) Generic cell dysfunction in neurodegenerative disorders: role of surfaces in early protein misfolding, aggregation, and aggregate cytotoxicity, Neuroscientist 13, 519-531.

38. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases, Nature 416, 507-511.

39. Cecchi, C., Baglioni, S., Fiorillo, C., Pensalfini, A., Liguri, G., Nosi, D., Rigacci, S., Bucciantini, M., and Stefani, M. (2005) Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates, J Cell Sci 118, 3459-3470.

40. Cecchi, C., Pensalfini, A., Liguri, G., Baglioni, S., Fiorillo, C., Guadagna, S., Zampagni, M., Formigli, L., Nosi, D., and Stefani, M. (2008) Differentiation increases the resistance of neuronal cells to amyloid toxicity, Neurochemical research 33, 2516-2531.

41. Hakansson, A., Andreasson, J., Zhivotovsky, B., Karpman, D., Orrenius, S., and Svanborg, C. (1999) Multimeric alpha-lactalbumin from human milk induces apoptosis through a direct effect on cell nuclei, Exp Cell Res 246, 451-460.

42. Svensson, M., Sabharwal, H., Hakansson, A., Mossberg, A. K., Lipniunas, P., Leffler, H., Svanborg, C., and Linse, S. (1999) Molecular characterization of alpha-lactalbumin folding variants that induce apoptosis in tumor cells, J Biol Chem 274, 6388-6396.

43. Svensson, M., Hakansson, A., Mossberg, A. K., Linse, S., and Svanborg, C. (2000) Conversion of alpha-lactalbumin to a protein inducing apoptosis, Proc Natl Acad Sci U S A 97, 4221-4226.

44. Hakansson, A., Zhivotovsky, B., Orrenius, S., Sabharwal, H., and Svanborg, C. (1995) Apoptosis induced by a human milk protein, Proc Natl Acad Sci U S A 92, 8064-8068.

45. Kohler, C., Hakansson, A., Svanborg, C., Orrenius, S., and Zhivotovsky, B. (1999) Protease activation in apoptosis induced by MAL, Exp Cell Res 249, 260-268.

46. Svensson, M., Mossberg, A. K., Pettersson, J., Linse, S., and Svanborg, C. (2003) Lipids as cofactors in protein folding: stereo-specific lipid-protein interactions are required to form HAMLET (human alpha-lactalbumin made lethal to tumor cells), Protein Sci 12, 2805-2814.

47. Svensson, M., Fast, J., Mossberg, A. K., Duringer, C., Gustafsson, L., Hallgren, O., Brooks, C. L., Berliner, L., Linse, S., and Svanborg, C. (2003) Alpha-lactalbumin unfolding is not sufficient to cause apoptosis, but is

38

required for the conversion to HAMLET (human alpha-lactalbumin made lethal to tumor cells), Protein Sci 12, 2794-2804.

48. Hakansson, A., Svensson, M., Mossberg, A. K., Sabharwal, H., Linse, S., Lazou, I., Lonnerdal, B., and Svanborg, C. (2000) A folding variant of alpha-lactalbumin with bactericidal activity against Streptococcus pneumoniae, Mol Microbiol 35, 589-600.

49. Pettersson-Kastberg, J., Aits, S., Gustafsson, L., Mossberg, A., Storm, P., Trulsson, M., Persson, F., Mok, K. H., and Svanborg, C. (2009) Can misfolded proteins be beneficial? The HAMLET case, Ann Med 41, 162-176.

50. Hallgren, O., Aits, S., Brest, P., Gustafsson, L., Mossberg, A. K., Wullt, B., and Svanborg, C. (2008) Apoptosis and tumor cell death in response to HAMLET (human alpha-lactalbumin made lethal to tumor cells), Adv Exp Med Biol 606, 217-240.

51. Gustafsson, L., Leijonhufvud, I., Aronsson, A., Mossberg, A. K., and Svanborg, C. (2004) Treatment of skin papillomas with topical alpha-lactalbumin-oleic acid, N Engl J Med 350, 2663-2672.

52. Redfield, C., and Dobson, C. M. (1990) H-1-Nmr Studies of Human Lysozyme - Spectral Assignment and Comparison with Hen Lysozyme, Biochemistry 29, 7201-7214.

53. Qasba, P. K., and Kumar, S. (1997) Molecular divergence of lysozymes and alpha-lactalbumin, Crit Rev Biochem Mol Biol 32, 255-306.

54. Nitta, K., Tsuge, H., Shimazaki, K., and Sugai, S. (1988) Calcium-Binding Lysozymes, Biological Chemistry Hoppe-Seyler 369, 671-675.

55. Nitta, K., Tsuge, H., Sugai, S., and Shimazaki, K. (1987) The calcium-binding property of equine lysozyme, FEBS Lett 223, 405-408.

56. Morozova-Roche, L. A. (2007) Equine lysozyme: the molecular basis of folding, self-assembly and innate amyloid toxicity, FEBS Lett 581, 2587-2592.

57. Morozova, L., Haezebrouck, P., and Van Cauwelaert, F. (1991) Stability of equine lysozyme. I. Thermal unfolding behaviour, Biophysical chemistry 41, 185-191.

58. 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.

59. Morozova, L. A., Haynie, D. T., Arico-Muendel, C., Van Dael, H., and Dobson, C. M. (1995) Structural basis of the stability of a lysozyme molten globule, Nature structural biology 2, 871-875.

60. 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, Journal of molecular biology 252, 447-459.

61. 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.

62. 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.

39

40

63. Sen, J., and Belli, A. (2007) S100B in neuropathologic states: the CRP of the brain?, J Neurosci Res 85, 1373-1380.

64. Bloomfield, S. M., McKinney, J., Smith, L., and Brisman, J. (2007) Reliability of S100B in predicting severity of central nervous system injury, Neurocrit Care 6, 121-138.

65. Moore, B. W. (1965) A soluble protein characteristic of the nervous system, Biochem Biophys Res Commun 19, 739-744.

66. 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 Abeta(25-35) peptide and human lysozyme in the serum of patients with progressive Alzheimer's disease, Dement Geriatr Cogn Disord 18, 165-171.

67. Permyakov, E. A., and Berliner, L. J. (2000) alpha-Lactalbumin: structure and function, FEBS Lett 473, 269-274.

68. Jeffery, C. J. (1999) Moonlighting proteins, Trends Biochem Sci 24, 8-11. 69. Avery, O. T., Macleod, C. M., and McCarty, M. (1944) Studies on the

Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types : Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type Iii, J Exp Med 79, 137-158.

70. O'Brien, K. L., Wolfson, L. J., Watt, J. P., Henkle, E., Deloria-Knoll, M., McCall, N., Lee, E., Mulholland, K., Levine, O. S., and Cherian, T. (2009) Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates, Lancet 374, 893-902.