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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
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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
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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.
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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).
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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
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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
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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”.
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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
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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).
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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.
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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
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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?
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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
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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)).
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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
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(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
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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
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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).
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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
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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.
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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
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