sod1´s law: - diva portal

SOD1´s Law: An Investigation of ALS Provoking Properties in SOD1

Roberth Byström

Department of Chemistry 901 87 Umeå Umeå 2009 Sweden

SOD'S LAW If anything can possibly go wrong with a test or experiment, it will. Originally applied to the

natural sciences, the use of this law has been extended to cover day to day living and reads

simply, 'If anything can possibly go wrong, it will,' to which has been added, 'and it will

happen at the worst possible moment.

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Table of Contents

Table of Contents 1 Abstract 3 Abbreviations 4 Preface 5

The following papers are discussed in this thesis 5 Paper, by the author, not included in this thesis 6

Introduction 7 Proteins 7 Protein Structure 8 Primary structure 8 Secondary structure 9 Tertiary structure 10 Quaternary structure 10 Metals 11 Disulphide bond 11 Protein Folding 11 Protein stability 12 How to measure protein folding 13 Equilibrium titration 13 Protein folding kinetics 15 Stopped flow 15 Lipids bilayers 18 Lipids 18 Cell membrane 19 In vivo 20 In vitro 20 How to monitor protein-membrane interaction 21 Circular Dichroism (CD) 21 Neurodegenerative diseases: A failure of the system. 22 Amyotrophic Lateral Sclerosis (ALS) 23 Cu/Zn-Superoxide Dismutase (SOD1) and ALS 24 Cu/Zn-SOD (Cu/Zn-Superoxide Dismutase) 27 The Structure of Cu2Zn2 Superoxide Dismutase 28 Loops 29 Cu, Zn and the active site 30 Cysteins and the disulfide bond 30 Dimer Interface 30 Monomeric SOD1 31

Aim of my work 32 ALS associated SOD1 mutants analysed in this thesis 33

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Material 34 Proteins 34

Results and Discussion 34 Protein aggregation hypothesis 35 Classes of destabilisation 36

Class 1 36 Class 2 36 Class 1+2 37 Class pWT-like 37

Destabilisation of the SOD1 framework 38 Correlating disease progression to SOD1-destabilisation 38 Net charge altering ALS-associated SOD1 mutants 39 Truncation of conserved hydrogen bonds 42

D76V/Y 42 N86D/K/S 42 D90A/V 43 D101G/N 43 N139D/K 44 Concluding remarks about truncated hydrogen bonds 44

Biological membranes as aggregation matrices 44 SOD1 interacts with negatively charged membranes 45 Is the SOD1-membrane interaction irrelevant? 47

Conclusions 47 Sammanfattning 50 Acknowledgement 52 References 55

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Abstract

Proteins are the most important molecules in the cell since they take care of most of the biological functions which resemble life. To ensure that everything is working properly the cell has a rigorous control system to monitor the proper function of its proteins and sends old or dysfunctional proteins for degradation. Unfortunately, this system sometimes fails and the once so vital proteins start to misbehave or to accumulate and in the worst case scenario these undesired processes cause the death of their host. One example is Amyotrophic Lateral Sclerosis (ALS); a progressive and always fatal neurodegenerative disorder that is proposed to derive from accumulation of aberrant proteins. Over 140 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn-Superoxide Dismutase (SOD1) are linked to ALS. The key event in SOD1 associated ALS seems to be the pathological formation of toxic protein aggregates as a result of initially unfolded or partly structured SOD1-mutants. Here, we have compared the folding behaviour of a set of ALS associated SOD1 mutants. Based on our findings we propose that SOD1 mediated ALS can be triggered by a decrease in protein stability but also by mutations which reduce the net charge of the protein. Both findings are in good agreement with the hypothesis for protein aggregation. SOD1 has also been found to be able to interact with mitochondrial membranes and SOD1 inclusions have been detected in the inter-membrane space of mitochondria originating from the spinal cord. The obvious question then arose; does the misfolding and aggregation of SOD1 involve erroneous interactions with membranes? Here, we could show that there is an electrostatically driven interaction between the reduced apo SOD1 protein including ALS associated SOD1-mutants and charged lipid membrane surfaces. This association process changes the secondary structures of these mutants in a way quite different from the situation found in membrane free aqueous environment. However, the result show that mutants interact with charged lipid vesicles to lesser extent than wildtype SOD1. This opposes the correlation between decreased SOD1 stability and disease progression. We therefore suggest that the observed interaction is not a primary cause in the ALS mechanism.

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Abbreviations

A, T, C and G Adenine, Thymine, Cytosine and Guanine

ALS Amyotrophic lateral sclerosis

ApoSOD1 Metal depleted SOD1

CD Circular dichroism

DNA Deoxyribonucleic acid

EDTA Ethylene diamine tetraacetic acid

fALS Familial ALS

GdmCl Guanidinium chloride

HoloSOD1 SOD1 with the metals

pWT Pseudo wild type. SOD1 with Cys to Ala substitutions in pos 6 and 111

sALS Sporadic ALS

SOD1 Cu/Zn Superoxide dismutase

TCEP Tris(2-carboxyethyl)phosphine

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Preface

The topic of this thesis is Superoxide dismutase (SOD1) and Amyotrophic lateral sclerosis (ALS) Over 140 different mutations in the gene coding for SOD1 have been found in ALS patients. I have tried to shed some light on the possible disease provoking properties in SOD1 mutants that might be involved in the ALS mechanism. I have reviewed others work in the area and tried to explain my own result in that context. I also like to stress that the correlations that I made might change over time as new clinical data and knowledge about SOD1 properties gets unravelled.

The following papers are discussed in this thesis I. Mikael J. Lindberg, Roberth Byström, Niklas Boknäs, Peter M.

Andersen and Mikael Oliveberg. ”Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants” Proc. Nat. Ac. Sci 101(28), 9754-9759, 2005

II. Roberth Byström, Christopher Aisenbrey, Tomasz Borowik, Marcus

Bokvist, Fredrik Lindström, Marc-Antoine Sani, Anders Olofsson and Gerhard Gröbner “Disordered proteins: Biological membranes as two-dimensional aggregation matrices” Cell Biochemistry and Biophysics 52(3), 175-189, 2008

III. Roberth Byström, Peter M. Andersen, Gerhard Gröbner and Mikael

Oliveberg “Identification of property outliers among the ALS-associated mutations of SOD1” Manuscript

IV. Christopher Aisenbrey, Roberth Byström, Mikael Oliveberg and

Gerhard Gröbner “SOD1 associates to membranes in its folded apo-state”. Manuscript

V. Roberth Byström, Christopher Aisenbrey, Mikael Oliveberg and

Gerhard Gröbner “Electrostatic interactions between negatively charged phospolipid membranes and SOD1 protein: Effect of charge changing fALS mutations” Manuscript

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Paper, by the author, not included in this thesis VI. Christopher Aisenbrey, Tomasz Borowik, Roberth Byström, Marcus

Bokvist, Fredrick Lindström, Hanna Misiak, Marc-Antoine Sani and Gerhard Gröbner “How is protein aggregation in amyloidogenic diseases modulated by biological membranes?” European Biophysics Journal With Biophysics Letters 37(3), 247-255, 2008

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Introduction

Proteins I used to believe that proteins were just something that I needed to eat to stay healthy and that after a workout I needed more protein. Milk, meat and bananas were the main sources for protein. I never thought about proteins as something that we actually had already in all our cells, and even much less, that they were doing something important there. Maybe I slept during the biology classes in elementary school, but this was more or less my knowledge about proteins before I went to the university. During my years as an undergraduate student my knowledge about protein grew and so the curiosity to learn more about them. I finished my master of science in engineering biology with a degree project on Populus EST dataset. Although working with DNA was interesting I felt that it would be even more exciting to work with the product and not just the blueprint. DNA is the blueprint for life and proteins are the workers that carry out the instructions from the genetic blueprint. Every each one of the ~25000 genes that we have in our DNA produces a unique protein. DNA is built up of a combination of four nucleotides A, T, C and G. Protein on the other hand is built up from a combination of 20 available amino acids which every each one of them corresponding to a three letter code in the DNA (fig. 1). Each time a cell divides, also its DNA has to be replicated. Because the DNA is so complex, mistakes happen and the wrong nucleotide becomes inserted. Usually, the correction system in the cell is alerted and fixes the mistake. But every now and then the mistake passes unnoticed and this cell will from now on have a new blueprint. Usually this change is harmless. But if the change occurs in a coding gene, the mistake can result in a substitution of an amino acid. This change however can be serious as it can result in a defective protein that does not work properly or gains a new, even toxic function. If this happens in a gamete (sperm or egg cell) the new DNA can be inherited and passed on to the next generation. This is why many diseases are genetically inherited.

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Fig 1. The Codon table illustrates how 64 different kinds of codons can code for 20 amino acids. As an example we can look at the ALS associated missense mutation (D90A) in the gene coding for Superoxide Dismutase 1 (SOD1). The original code for the amino acid Asp (D) is GAC. A missense mutation in the second position causes a change from A to C. By looking in the table we can see that this change will cause an amino acid substitution from Asp to Ala. Hydrophobic, charged and polar refers to its chemical property. *Glycine has only a hydrogen side group and can thus fit in both, the hydrophilic and hydrophobic environment. Illustration made by [1]

Protein Structure

Primary structure All amino acids have a central carbon, the Cα that have one hydrogen(H), one amino group (NH2), one carboxyl group (COOH) and an R-group attached to it. What distinguishes one amino acid from another is the R-group. R can be any of the 20 different side chains. The carboxyl group of one amino acid condenses with the amino group of another amino acid, forming a peptide bond. The peptide bond is planar because of its double bond character which limits its possibilities to move freely around the bond (fig 2). The other two groups attached to the Cα have a single bond character which allows free rotation. The backbone torsion angle between N and Cα (called phi φ) and the angle between Cα and C1 (called psi ψ) are the degrees of freedom for the protein and they are important for the secondary structure. A protein sequence starts from a free amino group called, the N-terminal and ends with a free carboxyl group, called the C-terminal. Every each of the possible side chains has a unique chemical property which is important for the structure of the protein. The chain of covalently connected amino acids is called polypeptide and can stretch from just a few amino acids to several

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hundred. Each amino acid is then usually referred to as a residue. This is the primary structure of the protein.

Fig 2. The peptide bond is planar because of its double bond character. Illustration made by [1]

Secondary structure Secondary structure is the local spatial arrangement of the polypeptide backbone that gives rise to structural patterns. Hydrogen bonding between backbone amid and carboxyl groups stabilizes the conformation and give rise to three elements of secondary structures, α-helix, β-sheet and β-turn. The α-helix and the β-sheet structures are constrained to specific values of φ and ψ and can be found in specific regions of the Ramachandran plot (fig. 3). They were suggested by Linus Pauling and coworkers 1951 [2]. The α-helix is a right handed coiled conformation which resembles a spring. The backbone N-H group hydrogen binds to the backbone C=O group of an amino acid four residues earlier (n+4). They typically adopt backbone dihedral angles of φ = -60° and ψ = -45°. There are also left handed coils (L) but they are rare and usually occur as short segments in proteins. The β-sheet is formed by hydrogen bonds between adjacent, but not necessary close in sequence, β-strands. If the β-strands alternate directions in a successive manner so that the N-terminus of one strand is adjacent to the C-terminus of the next strand, it is called anti-parallel β-sheet. In a parallel β-sheet the N-terminal of successive strands are oriented in the same direction. The most common is the anti parallel arrangement which also is the most preferable due to the stronger planar hydrogen bond that are formed with this arrangement. Proteins can have β-sheets with a mixture of parallel and anti parallel strands. The β-strands and/or the α-helices are joined together by more flexible parts of the polypeptide called loops. A β-turn is a short tight turn of four consecutive residues that almost folds back on itself by nearly 180° and

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hydrogen binds between n+3 residues. Longer loops that connect different parts of the protein are usually rather flexible. They can be involved in binding or guidance of substrate and are often important for catalytic functions.

Fig 3. The Ramachandran plot of SOD1 showing the “allowed” torsion angles of φ and ψ for the secondary structural elements, β–sheet, α -helix and L (left handed α –helix). Illustration made by [1]

Tertiary structure In the tertiary structure the side chain conformation is very important when the elements of secondary structure folds into a compact three dimensional structure. Hydrophobic residues are usually buried inside the protein and forming the hydrophobic core. Charged and polar residues are more usually pointing outwards where it interacts with the solvent and each other to form salt and disulfide bridges. That’s why the secondary structure arrangement can be similar between two proteins whereas the tertiary structure looks completely different between the two. In the tertiary arrangement the complex surface topology that enables the protein to interact with other proteins or molecules are created. The structure is stabilized by a large number of relatively weak, polar interactions between hydrophilic groups and van der Waals interactions between non polar groups. Close packing and solvent composition decides the strength of these interactions. A folded tertiary molecule is usually fully functional by itself but it can also be a part of a bigger protein complex of several folded structures.

Quaternary structure Two or more tertiary structured units can assembly together to form a multimeric structure referred to as a quaternary structure. The stabilizing interactions are of the same type as within the individual tertiary subunits. The number of subunits in a complex can range from 2 (dimers) to 8

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(octamers). Complexes with higher numbers of monomeric units are rare, with few exceptions. One such exception is the human ribosome that is translating the genetic code from nucleic acid into a protein. The ribosome is made up of approximately 80 individual subunits. Multimers made up from identical subunits are called homo-mers and those made up of different subunits are referred to as hetero-mers.

Metals Even though the side chains in a protein can have different chemical functions that can be utilized for many biological functions their versatility is not unlimited. That’s why many proteins have one or more intrinsic metal atoms. The metals can be more suitable and more efficient for some functions. One such function is the iron in hemoglobin which is the reason for the red color of blood. Frequently used metal atoms are iron, zinc, copper, magnesium and calcium. Besides being efficient in catalytic functions the metal atoms can have an important role in stabilizing the structure of the protein. Zinc stabilizes the DNA binding region of a group of transcription factors called zinc fingers and seems to have an important role in the stability and structure of Cu/Zn Superoxide dismutase (SOD1) [3, 4] The metals are mainly bound to the side groups of histidine, cysteine, aspartic acid and glutamic acid residues. They are usually separated in the polypeptide chain in the primary sequence but come into close proximity to each other in a folded protein to form a metal binding site.

Disulphide bond Disulfide bonds are important for stability in some proteins. Two cysteine residues adjacent in the structure can oxidize and form a disulfide bridge. The disulfide bond is usually formed inside the lumen of the rough endoplasmic reticulum and the protein is then secreted to the extracellular medium outside the cell. The reducing environment in the cytosol makes the disulfide unstable which is why disulfide bonds are mostly found in secreted proteins. However there are exceptions, one such is the cytosolic protein SOD1 which has an intrasubunit disulfide bridge despite the reducing environment.

Protein Folding Some proteins need help to fold from other proteins called chaperones but most proteins fold spontaneously either during the synthesis or right after. The main driving force for this is to minimize the number of hydrophobic residues that is exposed to the surrounding water by packing them together into a hydrophobic core. This is called the hydrophobic effect. If the folding was just a sampling of all possible conformation until the right structure was found the folding would take more time than the age of the universe. Cyrus

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Levinthal made this mind experiment in 1968, called Levinthal’s paradox (fig. 4) [5]. This is of course impossible and most proteins fold within seconds to minutes. In 1973 Anfinsen stated that the final three dimensional structure of a protein is determined by the amino acid sequence [6]. This statement is supported by the fact that many proteins can be unfolded in a solvent by adding a chemical denaturant such as urea or guanidinium chloride (GdmCl). And upon removal of the denaturant from the solvent the protein spontaneously folds back into its native structure.

Fig 4. If each residue in a polypeptide chain is allowed to adopt torsion angels corresponding to three possible regions α, β and L, in the Ramachandran plot (fig. 3) and if each conversion between possible conformations took one picoseconds (10-12 seconds). Then a protein with the same number of amino acids as the SOD1 monomer would have 3153 ≈ 1073 possible conformations and would require approximately 1054 years. For comparison the age of universe is approximately 1.4 • 1010 years (~14 billion years) Illustration made by [1]

Protein stability A protein wants to be in the most energetically favoured state to be stable. The difference between its native (N) and unfolded (D) state can be described by the difference in Gibb’s free energy (∆G):

∆ ln (1)

Where,

KD

N (2)

R is the gas constant, T is the temperature in degrees Kelvin and Keq is the equilibrium constant. The state D or N with the lowest energy will be the most populated and hence decide the concentrations of D and N.

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The unfolded state has a large conformational freedom with individual residues moving relative to each other and groups easily rotating around their single bond. That means that the unfolded state has a high entropy but the enthalpy is low due to the reduced number of intramolecular bonds. The folded, native state on the other hand has low entropy due to a much more restricted conformational freedom which is balanced by a gain of enthalpy caused by close packing of side chains. The energy difference between the folded and the unfolded state is small and its value ∆G is obtained by two usually large negative values of ∆S and ∆H, equation 3:

∆ ∆ ∆ (3) However, water - the solvent which surrounds proteins - complicates things as the enthalpy and entropy of water has to be considered as well. The nature of water is still not fully understood but it will interact with both the native and the unfolded protein in a different manner. When the hydrophobic side chains of the unfolded protein are exposed to solvent the water molecules stack around them as “icebergs” by hydrogen bonds to each other [7]. The increased amount of hydrogen bonds between water molecules lowers the enthalpy and the reduced number of freely moving individual water molecules lowers the entropy [8]. Further, the hydrogen bonds by donors and acceptors in the backbone of the protein tie down even more water molecules [9]. Upon folding the water molecules are released and the gain of entropy is thought to compensate for the loss of conformational entropy in the protein [7]. Worth noting is that a typical protein is only marginally stable in solution with free energies of unfolding between 5 and 15 kcal/mol.

How to measure protein folding

Equilibrium titration If the protein folds according to a simple two-state model where the protein exists in either an unfolded (D, denatured) or folded (N, native) state the difference in concentration of D and N can be used to calculate ∆G:

D

N (4)

∆ ln 2.3 log (5) where 2.3 converts ln to log. Under physiological conditions the native state (N) is strongly favoured. To be able to measure the stability the conditions have to be changed. This can

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be done by temperature, pH changes or by adding of a chemical denaturant such as urea or GdmCl. The exact nature action of urea and GdmCl is not fully understood but the denaturant might bind to the hydrophobic groups of the unfolded polypeptides or to the water molecules surrounding it. Both ways the hydrophobic groups are shielded and thus the polypeptide chains prevented from aggregating to each other. The unfolded protein has a bigger surface area than the folded for the denaturant to bind to and as the concentration of denaturant increase the unfolded state will be favoured and stabilized. Usually chemical denaturation is reversible upon removal of the denaturant due to the shielded hydrophobic groups. This is not always possible with heat denaturation.

Fig 5. Illustration of how the intrinsic fluorescent property in proteins can be used to monitor folding events. Based on an idea from M. O. Lindberg. Illustration made by [1]

The most common way to monitor protein folding is by fluorescence. Fortunately, most proteins have an intrinsic fluorophore that can be used. Three amino acids (Trp, Tyr and Phe) have aromatic rings with delocalized pi-electrons that can be excited by light. Either one of them can be exploited as a fluorescence probe, but tryptophan is the most powerful of them. Sometimes the protein lacks a suitable fluorophore, either it has no Trp, or the Trp is badly positioned or it has several Trps. In those cases it can be good to mutate a suitable buried side chain to a Trp and create a pseudo wild type. Tryptophan is excited at 275-295 nm and re-emitting at 325-35o nm

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depending on the environment. When the protein is folded tryptophan is buried in the hydrophobic core and emits light at a high intensity while being shined on. The opposite happens when the protein is unfolded; the emitted light will have lower intensity. If we monitor the intensity of emitted light at 340 nm we will see a mixture of both populated states at any given concentration of denaturant. At low concentrations of denaturant mostly the N state will be populated and thus we have a high fluorescence intensity. When the concentration of denaturant is increased the equilibrium between N and D will change and the light intensity will decrease until almost all protein molecules are denatured and the signal does not decrease any further anymore. When the concentration of the N and D state are equal ([N] = [D]) we have the midpoint. The relation between the free energy of unfolding in denaturant and the free energy in water is empirically observed to be linear [10]. The free energy of denaturation at any given concentration is then given by:

∆ ∆ 2 (6) or,

log log 2 (7) where the slope, mD-N describes the change in accessible surface area upon denaturation.

Protein folding kinetics Equilibrium studies tell us about differences in free energy between the folded and the denatured state and the surface accessible area. It does not tell us anything about the pathway between the two states. For this problem we need to look at the kinetics of protein folding. A convenient and commonly used way to study protein kinetics is stopped-flow fluorescence.

Stopped flow The stopped-flow technique covers a useful time range and is based on rapid mixing of two reagents. Two syringes are used for sample and denaturant, one small and on big, usually with a ratio 10:1. The stop-syringe decides the volume of the sample that is about to be measured. To be sure the cuvette is filled up with only the new sample; the volume of the stop-syringe must be at least twice the volume of the cuvette (fig. 6).

Unfolding: Protein in the small syringe and denaturant in the

large syringe are simultaneously driven into the mixing chamber.

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When the mixed sample enters the cuvette the stop syringe fills up and hits the stop-trigger that starts the measurement. The newly entered sample is now only milliseconds old.

This procedure is done for a range of different concentrations of denaturant. Usually starting with high concentration and going down until no change is detected any longer.

Refolding: To measure refolding we fill the small syringe with a denatured unfolded protein and the large syringe with a diluting solution.

The same procedure as with unfolding only that we dilute the unfolded protein with increasing concentrations of denaturant until no further changes are detected.

Fig 6. Schematic drawing of the stopped-flow instrument. Illustration made by [1]

A reversible system where both the rate unfolding and refolding can be measured can be described as:

(8)

where ku is the rate constant of unfolding and kf is the rate constant of folding. The equilibrium constant can then be written:

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(9)

The linear relationship that was described for the equilibrium reaction is also true for the unfolding and refolding reaction:

log log 2 (10)

log log 2 (11)

where 2 and 2 are the unfolding and refolding rate in the absence of

denaturant. The values obtained from logku and logkf can be plotted against the denaturant concentration to obtain a plot referred to as the chevron plot [7] (fig 7).

Fig 7. A plot of a two-state transition showing the kinetic parameters that can be derived from a typical chevron plot. Illustration made by [1]

The V-shaped curve in the chevron plot is obtained from combining the two rate constants:

log log log 102

102 (12)

By fitting equation 12 to the chevron data (fig 7) we can obtain 2 , 2 ,

and m . The data obtained can be used for comparison between different mutant types of the same protein as I have done in this study with different ALS associated SOD1 mutants. From equation 9 we have that KD-N = ku/kf and from equation 5 that ∆G = -RTlogK which enables us to obtain stability:

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∆GD N RT log KD N RT log kH2O kH2O⁄ (13) The stability changes due to point mutations can be obtained from:

∆∆GD M ∆GD MWT ∆GD M 2.3RT ∆logkH2O ∆logkH2O (14)

Dimeric proteins basically form according to either of two models; i the unfolded monomer only folds upon dimerisation, this is a two‐state dimer, ii fully folded monomers precede the formation of dimers, which is referred to as a three‐state dimer. The SOD1 dimer is primarily formed according to the tree‐state model:

2 2 (15)

where D is the unfolded monomer, M is the folded monomer and M2 is the dimer. Loss of stability upon mutation of the SOD1 dimeric protein can be calculated from the following:

∆∆G D M 2.3RT ∆logkH2O ∆logkH2O ∆log ∆log (16)

where ka is the rate constant for monomer-monomer association and kd are the dimer dissociation rate constant.

Lipids bilayers

Lipids In biological membranes the main constituent are glycerophospholipids. They are amphipathic, meaning that they have one water loving part (hydrophilic) and one water hating part (hydrophobic). The glycerol moiety is the core with a hydrophilic phosphate head and two hydrophobic acyl chains. In this thesis the following phospholipids have been used (table 1)

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Table 1. Name and schematic representation of the lipids used in this thesis (lipid structures from www.avantilipids.com).

Cell membrane The cell is the basic unit for life in all living organisms. Some organisms such as bacteria are unicellular (consists of one cell) whereas an adult human consists of between 10 and 100 trillion cells. Each cell is surrounded by a thin double layer of amphiphatic phospholipids. They spontaneously arrange so that the hydrophilic part is facing the water and the hydrophobic tails are packed together to hide from water. This will form a lipid bilayer (fig 8) referred to as the cell membrane or plasma membrane. In a eukaryotic cell there are also internal organelles like the nucleus, mitochondria and Golgi apparatus that are also surrounded by specific lipid bilayers. The cell membrane serves as an envelope that separates the interior of a cell or an organelle from the exterior environment. The cell membrane can exist in either liquid or solid (gel) phase at any given temperature. All lipids have a characteristic temperature (melting point) at which they have their main phase transition. However, as the membrane needs to maintain its dynamic structure and to maintain its fluidity at physiological temperature the cell can alter its lipid composition.

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In vivo Cell membranes in a living organism are mainly built up from phospholipids. The membrane also contains a lot of proteins. Peripheral membrane proteins are bound to the inner or outer membrane surface; either indirectly by interactions with integral proteins or directly by interactions with lipid polar head groups. Integral membrane proteins are integrated to the inner or other membrane usually having a hydrophobic part interacting with the fatty acyl group of the phospholipids. Transmembrane proteins span from one side to the other of the bilayer. They usually work as channels for active or passive transport of molecules across the membrane. The protein content differs between different cell types and organelles, in the plasma membrane of human red blood cell the content can be 45% whereas in the inner membrane of mitochondria it can be as much as 76% of total content. Each specific membrane has a set of proteins attached to it that enables it to carry out its activities. Those proteins are usually specific membrane proteins. However soluble proteins can interact with the membrane as well to carry out different functions such as signal transduction.

In vitro Cells from a living organism can be extracted and used in the lab for experiments. However, because of the complexity of these cells the use of an artificially made model membrane is often necessary. The advantages with these lipid bilayer vesicles are several. It makes it possible to study different compositions of lipids and the membrane is free from proteins which make it possible to study protein interactions with a membrane without any interference by other proteins.

Fig 8. Schematic drawing showing the main constitutes of a plasma membrane. A) Showing a typical cell membrane in an organism. B) Showing a model membrane also referred to as a lipid vesicle. Illustration made by [1].

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How to monitor protein-membrane interaction The interaction between proteins and membranes can be monitored in several ways. With differential scanning calorimetry (DSC) it is possible to look at the phase transition of the lipid vesicle. As each lipid composition has its characteristic phase transition temperature two samples can be compared, one with just the lipid vesicles and the other with a composition of lipid vesicles and the specific protein. If the protein interacts with the membrane the phase transition will be shifted. The effect upon protein lipid interaction can also be studied with solid state magic angle spinning (MAS) NMR [11]. By looking at 31P a change in the isotropic chemical shift can be detected when an interaction between protein and lipid occurs. A third biophysical method for detecting interactions between proteins and lipid-bilayers is circular dichroism (CD).

Circular Dichroism (CD) Almost all molecules synthesized by a living organism are optically active. When scientists search for extraterrestrial life they usually start looking for optically active compounds. Plane polarised light is made up of 2 circularly polarised components. One component is rotating clockwise (right handed, R) and the other is rotated counter clockwise (left handed, L). When plane polarised light passes through an optical active sample such as a protein the plane polarised light is absorbed by different amounts:

∆ (17) where ∆A is the difference in absorbance of AL (left handed) and AR (right handed) plane polarised light. The result of the recombined components is elliptically polarised light. The occurrence of ellipticity is called Circular Dichroism (CD) and defined as θ in millidegrees (mdeg). There’s a numerical relationship between ∆A and ellipticity θ which I will not go into further here, which is 32,98:

32,98 (18) which can be converted to molar ellipticity which is reported in degrees cm2 dmol–1:

(19)

where c is the protein concentration and l is the path length of the cuvette. For comparisons with other proteins and further analyses of the spectra we need to know the mean residue ellipticity:

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(20)

where θobs is the observed value from the CD experiment and n is the number of residues in the protein. Backbone conformations in proteins are constrained to specific values of φ and ψ (Ramachandran plot fig 3) and CD is wavelength dependent. This enables us to detect structural changes of four secondary structures, α-helix, β-sheet, β-turn and random coil. Each secondary structure has its own characteristic CD spectra in far UV (~190-250 nm) (fig 9).

Fig 9. Far UV CD-plot showing the typical spectra’s of secondary structures in proteins.

This specific CD profile is especially powerful for monitoring structural changes in a protein upon interaction with membrane or for comparison of the structure between a mutated protein and its wild type.

Neurodegenerative diseases: A failure of the system. Even though the cell has its own surveillance system for monitoring the protein folding and either sends the incorrectly folded protein for refolding or degradation, this control system sometimes fails. The consequences will be an abnormal accumulation of protein aggregates which today are the pathological hallmark for neurodegenerative diseases, including Alzheimer´s disease (AD) [12], Parkinson´s disease (PD) [13], Huntington´s disease (HD) [14, 15], Creutzfeldt-Jakob disease (CJD) [16] and Amyotrophic Lateral Sclerosis (ALS) [17, 18]. The findings of those intracellular and extracellular inclusion bodies indicate that incorrectly folded or unfolded proteins are

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somehow causative in neurodegenerative diseases. Recently it has been shown that the presence of charged lipid membranes might have a role in the mechanism. Formation of fibrillar aggregates is triggered in a range of amyloidogenic proteins upon interaction with charged lipids [19]. The AD associated Aβ42 forms amyloid plaques upon interaction with specific domains in the plasma membrane [20]. A non amyloidogenic protein with misfolding properties is the ALS associated protein SOD1. Recently, it was shown to interact with membranes at the cytoplasmic face of mitochondria [21]. However, if the interactions with a membrane surface and the forming of protoaggregates are of good or evil is not clear at present. Membrane surfaces can act as chaperons and refold misfolded and unfolded proteins [22-24]. ALS-mice developed ALS like symptoms long before any detection of inclusions [25] and clinical signs precedes the findings of aggregates in AD, PD and HD (see review [26, 27]). A lot of studies have been done to understand if the protoaggregates, or some precursors are the cause. But, the exact disease causing mechanism remains elusive.

Amyotrophic Lateral Sclerosis (ALS) As early as 1830 the Scottish anatomist Sir Charles Bell described it in his book “The nervous system of the human body” and in 1848 the French neurologist Francois Aran [28] described it. But it was not until 1869 the disease got its current name when the French neurologist Jean-Martin Charcot described the disease in detail. He observed a degeneration and death of both upper (UMN) and lower motor neurons (LMN) and proposed to term the disorder “sclérose latérale amyotrophique” (SLA) [29]. This is what we today know as Amyotrophic Lateral Sclerosis, (ALS, also known as Charcot’s disease, or MND) A-myo-trophic comes from the ancient Greek language and A means no, myo refers to muscle and trophic means nourishment. -No muscle nourishment-. When the muscle has no nourishment it withers away (atrophy). Lateral refers to area of the spinal cord where portions of the dying nerve cells are located. When this area degenerates, it leads to hardening (sclerosis) in the area. Motor neurons are nerve cells located in the brain, brainstem and spinal cord. They control all voluntary muscle movement in the body. Degeneration of those cells will eventually affect all voluntary muscle movement. When muscles in the diaphragm and chest wall fail to work the ability to breathe without ventilator support is no longer possible. Most people with ALS die from respiratory failure. The incidence of ALS is around 2 per 100,000 people a year over the whole population and is one of the most common neurodegenerative disorders. The risk of getting ALS increase with age and seems to peak between the ages 60 and 80 [30-32]. To be diagnosed with ALS the patient must have symptoms of damage in both UMN and LMN. The death of UMN and LMN leads to

24

muscle weakness and atrophy. At an early stage the symptoms may be unexpected tripping, dragging of a foot; difficulty with turning keys in doors or buttoning clothes; slurred speech, problems with chewing or swallowing. Unfortunately these symptoms can be so subtle in the beginning that they are overlooked. As the disease progress the complaints will develop into more obvious weakness that may cause a physician to suspect ALS but at this stage up to 50% of the motor neurons might be lost. [33]. So far only one drug is approved for treatment, riluzole (Rilutek©) which is believed to reduce damage to motor neurons and slow down the disease. As Rilutek© only modestly slows down the disease progression and does not reverse the already damaged motor neurons it is important that the patient gets a proper diagnose as early as possible. ALS is relentlessly progressive and 3 years after onset of symptoms 50% of the patients are dead. What causes ALS is still not understood. Genetic factors are implicated in the pathogenesis of ALS. Even though Charcot never accepted ALS as a plausible inherited disease the concept can be dated back to 1850 when Aran in his report commented that one of his patient’s three sisters and two maternal uncles had died from a similar disease. Today it is believed that approximately 10% of the total ALS cases are inherited in a dominant manner, meaning that there is a family history of at least two blood relatives [34]. This form is referred to as familial ALS (FALS) [35, 36]. In the remaning 90% no apparent genetic linkage has been found. This form of the disease is referred to as sporadic ALS (SALS). However similar symptoms of SALS and FALS suggest a common disease mechanism [37]. It has been suggested that SALS correspond to a combination of genetic and environmental factors.

Cu/Zn-Superoxide Dismutase (SOD1) and ALS In 1993 a major breakthrough in the understanding of the molecular mechanisms underlying ALS was taken when Daniel Rosen and colleagues [38]; [39] described 11 missense mutations in the gene encoding for Cu/Zn-Superoxide Dismutase (SOD1) in 13 families with ALS. Overall are SOD1-mutations found in about 7% of all ALS cases, even in patients with apparent sporadic ALS [40]. They might still be sporadic but some of them can be familial cases that due to insufficient data concerning family history are considered as sporadic. In 2002 Alexander et al: presented a case with a 24 year old man with an aggressive form of ALS [41]. Eighteen month after onset he dies from pneumonia. DNA from the patient showed that he had a histidine to arginine substitution in position 80 in the SOD1 protein (H80R). The unusually young age of the patient made it possible to obtain blood samples from his two siblings, both parents and the maternal grandfather. None of the tested had the SOD1 mutation. Paternity and maternity was

25

confirmed as well. This is considered a clear sporadic case were a SOD1 mutation cause ALS. Today 142 ALS associated missense mutations in the hSOD1 gene have been associated with ALS (P.M. Andersen personal communication). They are either substituting the amino acid or truncating the protein in a total of 83 positions spread all over the global structure of the SOD1 protein. Out of these 142, 134 are missense mutations causing an amino acid substitution. Eight are missense mutations causing a premature stop, and a C-terminal truncation. For one of these, the G127-truncation (G127X), a transgenic mice model has been made. Although only small quantities of mutated G127X protein were found in the central nervous system of the mice they developed a rapidly progressing disease [25]. The mutations are found in all five exons of the SOD1 gene with an almost complete scattering of the mutations in the folded protein. Only β-strand three in the β-barrel between Q22 and G37 is so far free from mutations. In 20 of those SOD1 mutations the pathogenicity have been confirmed by statistical means or by development of transgenic mice- and rat- models [42]. The low number of confirmed mutations can be assign to a number of factors. Reduced disease penetrance in some pedigrees [43-46], different PCR techniques during the 1990s and the fact that many SALS cases never have been screened for SOD1 mutations [42]. Unfortunately there is no clear pattern why these 142 mutations that are distributed over the entire protein give rise to the disorder. However, these 142 ALS associated SOD1 mutations are presumed to be causative. The close similarities to human ALS as seen in a numerous transgenic mice and rat models expressing ALS associated SOD1 mutants support the assumption. SOD1 knockout mice (null mice) where the SOD1 gene has been deleted show no symptoms of degenerated motor neurons [47]. This support the hypothesis that it is not a loss of enzymatic function that causes ALS but rather a gain of some toxic property of mutant SOD1 that is independent of the level of SOD1 activity. But what is this gain-of-function? Several lines of evidence suggest that ALS is a protein folding disease where unfolded or partially structured SOD1 proteins form toxic aggregates. The mechanism behind this is still unclear but SOD1 aggregates/inclusions are found in both ALS patients and transgenic mice [48, 49]. However, those detergent resistant aggregates found in the spinal cord of terminally ill transgenic mice and in patients with SOD1 mutations might not be causative but rather markers of something else gone terribly wrong. Transgenic mice over expressing the severely destabilized SOD1G127X mutant contained very low amounts of the mutant enzyme and still developed ALS like symptoms before any SOD1 inclusions could be detected in their spinal cords [25]. Insoluble aggregates did not appear in the CNS until the disease had progressed so far that it would be too late for them to be causative. Interestingly though is the fact that binding of soluble misfolded SOD1

26

protein to a Hydrophobic Interaction Cromatography (HIC) column was detected throughout the whole life of transgenic mice [50]. It could be that these species are more directly involved in the disease process. As the hydrophobic residues of a protein usually are buried in the interior of the protein one might expect a HIC binding SOD1 to be misfolded. Dimeric SOD1 with both copper and zinc coordinated and the intra-subunit disulfide bridge intact is an extraordinary stable protein that still shows activity at 10 M urea [3]. However if any or all of those three, i) dimer, ii) disulfide bridge and iii) metals (especially zinc) for some reason are lost it will affect the stability to some extent. Reduction of the disulfide bond with both metals properly coordinated doesn’t seem to break the dimer, neither will removal of the copper and the structurally important zinc in a disulfide intact protein. Whereas reduction of the disulfide bond in the apo protein will promote dimer dissociation [4, 51]. These monomeric disulfide reduced apo species are also trapped in the HIC column [50]. Their result shows that SOD1 species that binds to the HIC column are disulfide reduced monomers without metals. The idea of the disulfide reduced apo monomer to be the culprit in the degeneration of motor neurons is further supported by other studies: Reduction of the C57-C146 disulfide bond shifts the equilibrium toward marginally stable monomers [4, 52]. All eight C-terminal truncating SOD1-mutations that have been associated to ALS lack the possibility to form the disulfide connection due to loss of Cys-146. Thus the disulfide bond itself is not responsible for toxic properties of SOD1, but the loss of it is clearly involved to some extent. Even so, just disulfide reduction of SOD1 alone doesn’t seem to be cytotoxic as the steady state levels of disulfide reduced species are highest in transgenic mice expressing human wild-type SOD1 without any signs of motor neuronal death [53]. The role of the copper and the zinc in ALS has been extensively studied. Cu is necessary for catalytic activity and the loading of Cu to SOD1 is mediated by the Copper chaperone (CCS). Ablating the gene for CCS in mice and crossbreeding them with an ALS mice strain showed that the lack of CCS did not prevent motor neuron disease [54]. The study also shows that even in the absence of CCS, SOD1 was copper loaded to a limited degree. Loss of copper alone have no structural changes of the protein [55] whereas loss of zinc show increased mobility of the zinc binding part of loop IV. Zinc deficient SOD1 have toxic effects in cultured motor neurons [56] and zinc deficiency in transgenic SOD1G93A mice accelerated the onset of ALS symptoms [57].

27

Fig 10. The illustration shows a schematic view of the SOD1 dimer. The red spheres in the left subunit illustrate the positions of the ALS associated mutation that I have studied in this thesis. In the right subunit the orange and the grey spheres represent copper and zinc respectively. Copper is coordinated by H46, H48, H63 and H120. Zinc is coordinated by H71, H80, D83 and H63 which is shared between copper and zinc. Bright yellow spheres represent the structurally important disulfide bridge and the pale yellow spheres are the positions were C6 and C111 have been mutated to alanines to create the pseudo wild type (pWT). Illustration made by [1]

Cu/Zn-SOD (Cu/Zn-Superoxide Dismutase) Aerobic organisms, which derive their energy from the reduction of oxygen, are susceptible to the damaging actions of the small amounts of O2- , OH and H2O2 that is formed during the metabolism of oxygen and especially in the reduction of oxygen by the electron transfer system of mitochondria [58, 59]. These three species are referred to as Reactive Oxygen Species (ROS). One of these species, the superoxide radical (O2-), is very aggressive as it steal electrons from neighbouring molecules, starting a cascade of electron stealing, which will damage the cell. The number one defence mechanism to this is Cu2Zn2Superoxide Dismutase (SOD1) [60] which converts O2- to a less toxic ROS, hydrogen peroxidase (H2O2) and molecular oxygen (O2) (Reaction 1).

(Reaction 1) 2 2

28

Cu2Zn2SOD, the product of the SOD1 gene is a homodimeric antioxidant metalloenzyme that is present in the cytosol of almost all eukaryotic cells. The active 32 kDa homodimer contains one copper and one zinc atom, sitting in the active site of each monomer. Dismutation of the super oxide radical to hydrogen peroxide and oxygen is a two step reaction, (Reaction 2 and 3)

(Reaction 2) (Reaction 3) 2

The hydrogen peroxide molecule, which is still a danger to the cell, is then further processed to nontoxic by products. In the peroxisomes, the enzyme catalase degrades hydrogen peroxide to water and oxygen (Reaction 4) and the selenium-containing glutathione peroxidase (GPX) use glutathione (GSH) to metabolize hydrogen peroxide and lipid peroxides to water and the glutathione disulfide (GSSG) (Reaction 5)[61, 62].

(Reaction 4) 2

(Reaction 5) 2

The Structure of Cu2Zn2 Superoxide Dismutase The Cu2Zn2SOD gene is localized at chromosome 21 and consists of five exons, each of 150-200 nucleotides separated by four intrones. The first crystallographic structure of human SOD1 was first reported in 1992 by Parge and colleagues [63]. The entire protein chain of 153 amino acids folds into an eight stranded β-barrel hold together by external loops in a greek key fold [64]. Two identical units form a dimer that is orientated such that their active sites face away from each other. To maintain full enzymatic activity SOD1 needs to be in its dimeric form. Even though it seems to be no cooperativity between the two monomers [65], the enzymatic activity drops to 10% in the monomeric form [66].

29

Fig 11. Topology diagram of SOD1. Illustration made by [1]

Loops Each monomer has seven loops, numbered I to VII. Loop III and VI forms the two Greek-key connections in the β-barrel. Loop I, II and V are short β-hairpin connections between adjacent strands. The two remaining loops, the metal binding loop (IV) and the electrostatic loop (VII) are functional important. Together with the metal ions they form the active channel where the electrostatic loop (residues 120–143) is of major importance for the guidance of the negatively charged superoxide (O ) substrate [67, 68] Loop IV (residues 48-83) spans over one side of the β-scaffold and harbours several functional and structural important amino acids. Residues 50 to 54 are involved in the interface of the two monomers, five out of seven metal coordinating amino acids and Cys57, that forms the intra-subunit disulfide bond with Cys146, are all in loop IV. The C-terminal end of loop IV is the also known as the Zinc binding loop.

30

Cu, Zn and the active site Each monomer binds one Cu2+ and one Zn2+. The enzymatic important copper is coordinated by hydrogen bonding to four histidine ligands (His46, His48, His63 and His120) and also binds one water molecule. Zinc does not participate in the catalytic activity but is considered to be structural important for the protein [3]. Recently it has also been found that zinc is important for maintaining the structural integrity of the Cu site [69, 70] and that removal of zinc cause dimer dissociation in disulfide reduced SOD1 [4, 71]. The zinc is coordinated by three histidine ligands and one aspartate (His63, His71, His80 and Asp83). Copper and zinc are linked to each other through the sharing of His63. As described before, the dismutation of the oxygen radical is a two step reaction. In the first round the O2-.is held in position by Arg143 with help of electrostatic forces and one electron from the oxygen radical is donated to Cu2+, reducing it to the less stable state, Cu+. The bridge between Cu-His63 is broken and the eta-nitrogen of His63 becomes protonated. The newly formed O2 is released from Arg143. During the second round Cu+ donates one electron to O2-. The superoxide anion can now form two covalent bounds by gaining two hydrogen atoms, one from the protonated His63 and the other one from the H3O+ that are present in the active site. The product of this is H2O2. With copper back to the Cu2+, oxidation state the bridge between His63 and copper is re-established. Another important residue in the active site is Asp124 sitting in the electrostatic loop. Asp124 is hydrogen binding to His46 and His71 and thus forming a second indirect bridge between Cu and Zn [63, 72]. It is believed that this bridge is important in increasing the catalytic efficiency.

Cysteins and the disulfide bond Human SOD1 has four cysteine residues, Cys6, Cys57, Cys111 and Cys146. An intra-molecular disulfide bond that anchors loop IV to the β-barrel is formed between the conserved Cys57 and Cys146. An intra molecular disulfide bond like this is rarely seen in intracellular proteins because of the reducing environment in the cell [73]. As residues 50-54 of loop IV forms part of the interface, the intra-molecular disulfide bond has shown to be very important for dimer stability. When the disulfide bridge is intact and both copper and zinc are correctly coordinated, SOD1 appears mainly in its dimeric form. When the disulfide is reduced and both metal ions are removed loop IV will “swing” out and promote dissociation of the dimer.

Dimer Interface The majority of the amino acids in the interface are highly conserved [74]. Amino acid residues 50-53, 114,148 and 151-153 are the most important ones in forming the interface between the two subunits. As mentioned earlier the

31

disulfide and the metal ions are important in preventing the dimer from monomerization [4].

Monomeric SOD1 Dimer splitting substitutions, F50E and G51E, which are frequently used for monomeric studies, reveals that loop IV and loop VII are significantly affected in the F50E/G51E mutant. NMR solution structure [75]and X-ray crystallography structure [76]. As a result, the catalytically important Arg143 loses the hydrogen bond to the carbonyl oxygen atom of Cys57. When Arg143 is disordered it is no longer in an optimal place for binding and orienting the substrate in the active site. This could explain why the enzymatic activity in F50E/G50E mutants decrease to only 10% compared to the WT enzyme. Banci with colleagues managed to restore some of the activity by substituting the negatively charged Glu133 with the neutral Gln [77].This mutation have shown to double the activity in the native dimeric protein [68]. The activity for the monomer was not fully restored but at least partly restored. The increased activity was not due to any significantly rearrangements of the loop IV and VII or Arg143 but due to increased guidance of the substrate. So even though it’s no apparent cooperativity between the two monomers [65], the interaction between the two monomers are necessary for correct arrangement of loop IV, VII as well as Arg143 [76].

32

Aim of my work

At the very beginning of this work I guess I had the hope that my result would lead to an explanation of the toxic mechanisms in ALS caused by some toxic gain of function in SOD1. Now I would say that I think that very few if any researchers solve the riddle and give a solution. If the task is to solve a protein structure or DNA-sequence I suppose you could say at some point that the question has gotten an answer. When it comes to solve such complex questions as “why do some people get neurodegenerative diseases?” I think that is better to be modest and say that:

-The aim of my work in this thesis has been to elucidate some of the gained properties in some ALS-associated SOD1 mutants (table 2) and based on the observed result propose a mechanism. Usually this gives rise to new questions which have to be shed further light upon. In the end I would say that my more specific aims were:

Measure the effect of protein stability upon ALS associated point mutations in SOD1 (paper I).

Particularly look at the effect of protein stability in net negative charge altering SOD1 mutants (paper III).

Study the interaction between lipid membranes and SOD1 (paper IV).

Specifically investigate the interaction between mitochondrial inter membrane mimicking lipid vesicles and SOD1, both destabilized and charge altering mutants (paper V).

33

ALS associated SOD1 mutants analysed in this thesis SODmut ST (years) (n) Mutant in: SOD1 structure

pWT† - - Paper I, III, IV and V

CallA* - - Paper V

A4V 1.2 205 Paper I, III, IV and V

L38V 2 22 Paper III

G41D 14.1 15 Paper I, III and V

G41S 1 16 Paper I, III and V

H43R 1.8 12 Paper I and III

H46R 17.6 49 Paper I and III

D76V 18.8 4 Paper III

D76Y 17 1 Paper III

L84V 3.2 10 Paper I and III

G85R 6 11 Paper I and III

N86D - - Paper III and V

N86K 1.7 7 Paper III and V

N86S 6.8 4 Paper III and V

D90A 8 15 Paper I and III

D90V 2.7 3 Paper III

G93A 3.1 16 Paper I, III and V

E100G 4.7 50 Paper I and III

D101G 1.9 3 Paper III

D101N 2.3 17 Paper III

I104F 21.3 3 Paper I, III and IV

S105L 3.5 7 Paper III

L106V 1.9 6 Paper I and III

I113T 4.3 38 Paper I and III

G114A 2.7 2 Paper III

N139D - - Paper III

N139K - - Paper III

L144F 11.8 15 Paper I and III

L144S 12.3 2 Paper III

V148G 2.1 11 Paper I and III

Table 2. In the table all mutants analysed in this thesis is listed together with which paper it has been discussed. ST means Survival Time and (n) is the number of patient reported with the specific mutation. In the most right column the position and mutant name is presented in the SOD1 monomer. †pseudo wild type where C6 and C111 have been substituted with Ala. *CallA, C6, C57, C111 and C146 are substituted with Ala. Illustration made by [1]

34

Material

Proteins To eliminate erroneous disulfide bond formation a pseudo wild type (SOD1pWT) were made. Two cystein residues in position 6 and 111 are mutated to alanines. The C6A/C111A mutations have insignificant effect on the folding of the monomer and the dimer [4]. For studies of the sole monomeric variant, two aditional dimer splitting substitutions were made in position 50 and 51 in the dimeric interface (F50E and G51E). All dimeric SOD1 mutants in this thesis were made on this C6A/C111A background (Paper I, III - V) and monomeric SOD1 mutants on C6A/C111A/F50E/G51E background (Paper I and III). They were co expressed with the yeast copper chaperone (yCCS) with Cu and Zn ions in the medium according to ref. [78]. A non ALS mutant with all cysteins substituted with alanines, CallA was used in paper V as a control of disulfide bridge reduction. The CallA mutant is unable to form the disulfide bond since all cysteins are removed. All mutants used in my thesis are presented in table 2. All studies were made on the apo protein where the intrinsic metals, Cu and Zn, were removed. Preparation of apo SOD1 were done by denaturing the protein for 4 hours in 4 M GdmCl in 10mM Mes buffer (pH 6.3) with 10mM EDTA. After 4 hours the GdmCl were removed by dialysis upon the protein refolds. In paper V the apo preparation were done with phosphate buffer 10mM and pH6.3. All experiment were done under oxidising conditions to maintain the disulfide bridge except in paper V were 0.5 mM TCEP were added to reduce the disulfide bond.

Results and Discussion

Amyotrophic lateral sclerosis (ALS) is an always fatal adult onset neurodegenerative disease. Despite intensive research, we still cannot explain which mechanisms cause this disorder. Over 140 mutations in the cytosolic enzyme Cu/Zn-Superoxide dismutase (SOD1) have been linked to ALS. The inclusions containing SOD1 which have been observed in the brain and spinal cord in both patients and transgenic mice indicate towards protein aggregation as a part of the mechanism that causes the devastating disorder ALS. [25, 50, 79]. This is in good accordance with the findings of similar aggregates in other neurodegenerative diseases as AD and PD [80]. It have recently been observed that SOD1 can form large, soluble and stable protein aggregates under physiological conditions (cytosol), i.e., 37˚C, pH 7.0 and 100µM protein concentration [81]. Of approximately 25-thousand proteins that could be synthesized in the cell, there are so far only a handful of proteins that are known to aggregate under those conditions [82]. Chiti and Dobson showed in their famous equation (Chiti-Dobson equation) [83]

35

that there are certain criteria that needed to be fulfilled for a disease causing mutation to push an otherwise non aggregating protein towards forming aggregates. Proteins with lowered stability, decreased protein net charge, increased β-sheet propensity or decreased α-helix propensity will be more prone to form aggregates [83]. We have therefore the hypothesis that ALS associated mutations in SOD1 might trigger the else stable SOD1-protein to form aggregates.

Fig 12. The positions of the 15 ALS associated SOD1 mutants that were tested in Paper I are marked with spheres in the structure. Illustration made by [1]

Protein aggregation hypothesis To test our hypothesis we started with a set of 15 ALS associated SOD1 mutants with reliable clinical data (fig.12 and table 2). The aim of the study was to determine what impact the mutations had on protein stability (paper I). The dimeric apoSOD1pWT folds according to a three state behaviour, (scheme 1) [84] where the monomer folds independently following a classical two-state process (scheme 2) [4]. After folding the structured monomers will assemble to homo-dimers [4, 85].

2 2 Scheme 1)

36

Scheme 12)

where D is the unfolded monomer, M is the folded monomer and M2 is the dimer. ku is the rate constant of unfolding and kf is the rate constant of folding. ka is the rate constant for monomer-monomer association and kd are the dimer dissociation rate constant

Classes of destabilisation Our data show that the destabilisation affect on the protein by mutation can be divided into four classes. Class 1: mutations selectively destabilize the free monomer, class 2: selectively destabilize the dimer interface. Class 1+2 are affecting both the single monomer and the dimer interface and the last class, called pWT-like have almost no detectable affect on neither the monomer or on the dimer interface.

Class 1 Selective destabilisation of the free monomer was shown in 5 of the 29 analysed mutants (paper I and III). Characteristic for this group of mutants is a pronounced decrease of their monomer stability through an increase in the unfolding rate constant (ku). However, no effect on the dimer interface is observed with a dissociation constant (kd) identical to the apoSOD1pWT. The chevron plot of SOD1D90A as presented in fig 13 shows a typical class 1 behaviour.

Class 2 This class of mutants have so far only been observed for 4 of the 29 studied mutants (paper I and III). In contrast to the class 1 mutants this group does not show any destabilisation effect at all on the monomer but a clear destabilisation of the dimer interface. In its purest form the affected protein is incapable to form dimers. The chevron plot (fig 11) of L144F shows a pure class 2 mutation. Its inability to form a stable dimer was further investigated by size exclusion chromatography. L144F was more or less eluting together with SOD1 monomer. A stable dimer relies on an intact disulfide bridge [4, 52] and fully coordinated metal sites [70, 86, 87]. The substitution of Leu with the bulky Phe in position 144 probably affects the adjacent disulfide bond between C146 and C57 in a disturbing manner and explains why this mutant is unable to form a dimer in its apo state.

37

Class 1+2 The majority, namely 15 of 29 ALS associated SOD1 mutants which have been studied by us sum up in this group (paper I and III). Class 1+2 behaviour correspond to both monomer and dimer interface destabilisation. The chevron plots (fig 11) of G41S and I104F are two example of class 1+2 destabilisation. Both show a pronounced destabilisation of the dimer interface with a logkd = -1.10 and -1.75 respectively. Therefore the dimer dissociation of apoSOD1G41S is almost 10 times faster than for the apoSOD1pWT (logkd = -2,13 ).

Fig 13. A selected set of chevron plots show the characteristics of class 1, 2 and 1+2 destabilisation.

Class pWT-like This group of ALS associated mutants are almost indistinguishable from the pWT in terms of destabilisation (paper III). So far 4 of the 29 mutants that we have studied are pWT-like. Common to this group is that they all affect surface exposed positions in the protein. The two mutants in position 76 are examples of such structural benign mutations. The chevron plots (fig 14) show that unfolding limb and refolding limb of the monomer and dimer are identical to the pWT.

Fig 14. Chevron plots of D76V and D76Y represent pWT-like effect on the folding.

38

Destabilisation of the SOD1 framework A common denominator of the mutations of hydrophobic residues in the interior and loop glycines is that they all shift the folding equilibrium towards the ensemble of unfolded and partly structured species preceding the folded apo monomer. Class 1, 2 and 1+2 are all shifting the balance in the same direction (fig 15). Class 1 with its selective destabilisation of the monomer increase the denatured population and the selective destabilisation in class 2 mutants will decrease the population of dimeric protein and hence shift the equilibria towards the monomeric and denatured states. The combined destabilization effect on both, the monomer and dimer interface (characteristic class 1+2 mutants) will equally increase monomer as well as denatured populations. Mutations in the pWT-like group (paper III) have no obvious effect on the folding equilibrium and cannot be explained in this context. They will be further discussed later in this thesis.

Fig 15. The diagram is showing the shift of the folding equilibrium towards the proposed starting material for toxicity.

Overall there is no pronounced effect on the refolding for any of the studied mutants which suggest that the impact on the early folding event is only marginally. The effect on stability seems to be on the downhill side of the folding barrier[7]. This late effect on the folding will make the folded monomer a little loose in the structure and more prone to sample the partly structured states.

Correlating disease progression to SOD1-destabilisation The amount of reliable clinical data is far from satisfying. Even so it is tempting to try to correlate the effect on destabilisation (∆∆G) with mean survival time after onset of the first ALS disease symptoms. The first set of analysed mutations (fig 12, table 2) were chosen on a basis of as good and reliable clinical data as possible. Surprisingly a correlation (paper I) between severity of the disease and the degree of destabilisation could be observed. Even though the correlation is not linear, a clear link between rapid disease progression and severely destabilizing mutations could be seen.

39

Fig 16. The correlation between protein stability change (∆∆G) and survival time after diagnosis is disrupted of disease associated mutations that is altering the net charge.

Net charge altering ALS-associated SOD1 mutants The mutations that fall out as outliers (fig 16, paper I) are all affecting the net charge of the SOD1 molecule to some extent. Because of a pI of 4.8 – 5.1, human SOD1 has net negative charge (-6) under physiological pH [88]. Interestingly 48 of 142 today known ALS associated SOD1 mutants alter the net charge. 40 reduce the net charge whereas only 8 produce an increase (fig 16). To further investigate the effect of net charge altering mutations an additional set of ALS associated SOD1 mutant were studied (paper III).

40

Fig 17. 142 ALS associated SOD1 mutations are reported to the Alsod database [89]. 40 of those reduce the net negative charge of SOD1 and only 8 increase the charge.

This new set of mutants was selected on the basis of altering the net charge in the protein. Unfortunately clinical data for some of the tested mutations are missing and for several others the data is based on just a few patients. Nevertheless they are all reported as ALS associated mutations in the SOD1 gene on the ALS online database (Alsod) [89]. The correlation observed in paper I (fig 16) between decreased ∆∆G and shorter survival times agree with the aggregation hypothesis [83]. Decrease of a proteins repulsive charge induces changes of the proteins biophysical and biochemical properties; changes which can promote aggregation [83]. As a simple test of the aggregation hypothesis we made an extended version (paper III) of the correlation plot (fig 16) and used all our stability data together with data from five other studies made by other groups [90-94]. To allow comparison between urea and thermal denaturation experiments we normalized the destabilisation data as described in [95]. The normalized destabilisation (∆∆Gnorm) was then plotted against survival time (fig 18). High numbers of ∆∆Gnorm imply higher degree of destabilisation, i.e., H46R is most stable and G93V are the most unstable. The data is heavily scattered and no obvious correlation can be observed in the plot. However, a pattern based on the mutations affect on charges can be observed. If we just look at the none-charge altering mutations (black circles) it is apparent that the most destabilised mutation correlate to short survival time. Mutations that decreases the net negative charge (red circles) are scattered in survival time but biased to lower values of ∆∆Gnorm. Mutations with an increase of the repulsive charge are underrepresented in the list of ALS associated mutations. Furthermore, an increase of net negative charge has been

41

proposed (paper I) to have a mitigating effect on aggregation of severely destabilised mutants, i.e., G41D have a much longer survival time than the equally destabilised L106V.

Fig 18. Correlation plot between survival time and ∆∆Gnorm . The horizontal dotted line denotes the division between short and medium/long survival time. Open circles n<5 and solid circles n>5. Red means a decrease of net negative charge i.e., change to more positive charge and blue denotes an increase of net negative charge. Black circles are not affecting any charges. The three arrows are set to show the medium destabilisation in each group. To the calculation of medium destabilisation of each group, ∆∆Gnorm from additional mutants where no survival times were reported were used.

We also checked how the charge altering mutations were divided between short (<5 years) and long (≥5 years) survival time after first diagnosis (paper III). For this study we used 75 confirmed ALS associated SOD1 mutation with clinical data reported (number of patients >0) [95] and (P. M. Andersen personal communication). 30% of the charge altering mutation is in the group with a fast disease progression and 50% in the group with slower disease progression. The absolute number of patients for the groups over and under five years of survival time is equal for the charge altering mutation (14 vs. 14) but with a bias of none charge affecting mutations in the group under five years (33 vs. 14). Therefore, it seems likely that the charge changing mutations can provoke ALS without global stability decrease. And often they correspond to a slower disease progression. However, there are also clear outliers which will be discussed next.

0

0

5

(∆∆Gnorm)0.80.60.40.2

15

10

Sur

viva

l Tim

e (y

ears

)

20

H46R

D76V

D76Y

I104F

G37R

D101ND125H

S134N

D90A

N86S

L144S

E100K

L144F

G85R

D101GN86K

H48Q

V7E

E100G

G93S

D90VS105L

I113T

G93R

L84V

G41D

G93D

G93V

V148G

G41SH43R

A4VL106V

G114AG93A

L38V

42

Fig 19. Distribution of charge mutations among patients with short and medium/long survival time.

Truncation of conserved hydrogen bonds In paper III we characterized 9 ALS associated SOD1 mutants that affected four positions with little or no effect on the global protein stability (fig 20).

D76V/Y This mutation pair has pWT-like stability (fig 14). The position is conserved across the SOD1 family. A clue to its structural role could be that it is hydrogen binding to G73 which is close to the Zn ligand H71. There are also indications of a conserved salt-bridge between D76 and K128 in the electrostatic loop VII. This bond can be observed in SOD1 x-ray structure from other organisms, i.e., Bovine SOD1 (2Z7U) but not in human structures (1HL5) [96]and (1PU0) [97]. The structural effect of the mutation could cause increased dynamics in loop IV and/or VII. This benign effect of the protein correlate with the slow disease progression for D76V (19 years, n=4) and D76Y (9 years, n=2) patients.

N86D/K/S The most puzzling effect of those mutations is that they all seem to weaken the dimer interface even though they are positioned far from the interface. The side chain of N86 has two hydrogen bonds to D124 that anchors loop VII to the β-barrel. D124 is also acting as a bridge between the Cu ligand H46 and the Zn ligand H71. It is also known that the disulfide link between C57 in loop IV and C146 in β8 is important for the interface stability [4, 52]. Furthermore, loop VII extends to the interface via β8. It is thus possible that increased dynamics in loop VII due to the N86D/K and S mutations of the apo protein will affect the interface and/or the disulfide bond. Disease progression for the three mutations in pos 86 follows the trend of mutational perturbation. N86K has the fastest disease progression (1.7 years) and also the largest effect on stability with a pronounced effect on both dimer and

43

monomer stability. N86D/S have class 2 behaviour with a pronounced effect on the interface but almost no effect on the monomer stability. The survival time for N86S is 6.8 years and the one patient with the increased repulsive charge mutation N86D is still alive and displays “slow” disease progression.

Fig 20. ALS associated SOD1 mutants in four different positions that all are having some effect on the hydrogen bonds in loop IV and VII. A) Structure showing the hydrogen bonding network in this area. B) Showing hydrogen bonds from the affected positions. Illustration made by [1]

D90A/V The aspartic acid side chain in position 90 supports the tight loop between β5 and β6 with hydrogen bonds to the backbones of D92 and G93. Substitutions to Ala or Val truncate the supporting hydrogen bonds in both cases. The destabilising effect for the substitution is mainly on the monomer, the chevron plot for the dimer differs insignificant from the wildtype. The larger destabilisation effect of the D90V substitution could be caused by the fact that the bulky side chain in addition to truncation of H-bonds can cause steric conflicts on the protein surface. The bulkiness of Val is also known to restrict the degree of freedom for the protein backbone which could affect the tight junction between β5 and β6.

D101G/N The destabilizing effects from those two mutations are quite different. D101G shows increased destabilisation for both the dimer and the monomer whereas D101N has a wild-type like behaviour with a slight stabilisation of the monomer. The most clear structural impact from both those mutation is the disruption of the conserved salt-bridge between D101 in β5 and R79 in loop IV. The D101N substitution is only affecting this electrostatic interaction whilst the D101G substitution also truncates several van der Vaals contacts. This could be a possible explanation for the observed difference in the destabilisation behaviour for these two mutations. Despite

44

the difference between the two species in terms of stability both yield very short survival times after onset, 2.3 years (n=17) and 1.9 years (n= 3) respectively.

N139D/K Those two mutations were chosen because they involve both an increase and a decrease of the net negative charge. Unfortunately no clinical data about disease progression or survival time have been reported for those two. Mutations of N139 in loop VII will mainly affect two hydrogen bonds to N131. Nine of 22 amino acids in the electrostatic loop VII are charged (4 negatively and 5 positively). A change from the neutral Asn to either negative Asp or positive Lys will experience repulsive and/or attractive forces. However, it is hard to tell if this has an effect on local stability of loop VII.

Concluding remarks about truncated hydrogen bonds The common effect of those nine mutations is that they all truncate hydrogen bonds in conserved hydrogen bonding networks (fig 21). The effect on global protein stability is overall small and where the effect is somewhat larger (N86K and D101G) it still don’t agree with the rapid disease progression. The truncation of hydrogen bonds and reduction of the net repulsive charge seems to be enough to trigger the disease.

Fig 21. A consensus sequence from multiple alignments of 23 eukaryotic SOD1 protein sequences (Homologene: 392)[98]. The degree of conservation is visualized with the size of the letter, i.e., bigger letter higher degree of conservation. Especially the negatively charged amino acids D and E (blue) show a high degree of conservation. Illustration of consensus sequence made with WebLogo[99] Alignment done with T-Coffee using the default parameters [100]

Biological membranes as aggregation matrices Protein interactions with membrane have been showed to play a role in the conversion of structurally protein into amyloidogenic assemblies in both Alzheimer’s disease (Aβ) and Parkinson’s disease (α-synuclein)[13, 101] (paper II). The interaction between proteins and the membrane surface is mainly initiated by electrostatic interactions. Positively charged amino acid

45

side chains interact with negatively charged moieties in the membrane. A membrane surface is close to two-dimensional if compared to the three-dimensional space in solution. The shift from 3D space to 2D space when the proteins interact with membranes will drastically shorten the relative distance between the individual protein molecules (fig 22). As aggregation of proteins is expected to arise from partly folded or misfolded molecules the reduced distance upon membrane interaction could accelerate the aggregation process.

Fig 22. Illustration of the change in relative distance between individual proteins when going from 3D space (cytosol) to 2D space (membrane interface).

SOD1 interacts with negatively charged membranes SOD1 has been found to interact with cytoplasmic interface of mitochondria [21] and to charged lipid vesicles [102]. SOD1 have also been observed to be imported to the intermembrane space of mitochondria [103]. In our studies we observed an interaction of disulfide reduced apoSOD1 to negatively charged lipid vesicles but not to neutral vesicles (paper IV). We can thus conclude that the interaction is electrostatically driven as also have been observed for Aβ [11, 19]. The interaction induces conformational changes in SOD1pWT with pronounced helical features as, CD observations clearly reveal (fig 24). Conformational modification upon membrane contact were seen for the two ALS associated SOD1 mutants A4V and I104F as well. The induced change is less for SOD1I104F and almost unnoticeable for the severely destabilized SOD1A4V at elevated temperatures. However, it seems like a correct fold is necessary for SOD1 membrane interaction. CD (circular dichroism) measurements that we made at 25˚C show higher degree of conformation changes of A4V than at 37˚C. The disulfide reduced apoSOD1A4V is expected to be more or less unable to populate the folded state in 37˚C which is a possible explanation to the reduced membrane interaction of A4V at this temperature.

46

Fig 23. Schematic presentation of the SOD1 membrane association process. Native structured apoSOD1 interacts with the membrane and conformational changes are induced. None native folded proteins do not interact with the lipid surface. The amount of interacting protein corresponds to the equilibrium between unstable and stable. Illustration made by G. Gröbner

The observed trend of decreased interaction for destabilized ALS associated SOD1 mutants opposes the correlation between decreased stability and shorter survival time. We therefore propose that the observed interaction to membranes is not a part of the ALS mechanism.

Fig 24. A,B and C show CD spectra of 8µM protein concentration with increasing concentration of lipid vesicles (DMPG) Solid line is the protein without lipid vesicles, whereas the dashed lines correspond to 100µM (dash) and to 500µM (dash-dot lines) of lipid vesicles. The dotted line was measured in the presence of 1000µM lipid vesicles. Measurements show the largest conformational change in the pWT. The induced conformational change is decreasing with decreasing ∆G. Panel D, E and F show the change in secondary structure upon interaction. -helical structures (closed triangles, apex up), -sheet structure (open triangles apex down), random structures (circles) and turn structures (squares).

47

Is the SOD1-membrane interaction irrelevant? We know that SOD1 can be imported to mitochondria via an unknown mechanism, with the TOM mechanism discussed earlier in this context [103]. Clearly, this translocation process requires unfolding and refolding of the protein, a process easily generating large populations of unfolded proteins prone to potential misfolding. Nevertheless, charged lipid membranes like the mitochondrial inner membrane can act like chaperones and refold imported proteins. Clearly proteins that do not interact with these chaperone-like membranes due to pronounced instability might therefore possibly accumulate in the inter membrane space. And accumulations of SOD1 have been observed in outer membrane, intermembrane space and also in matrix of brain mitochondria from transgenic mice [104]. The environment in the intermembrane space of mitochondria has a pH that is ~0.7 pH units lower than in the cytosol [105]. This will decrease the net repulsive charge of SOD1. In paper V we looked further into the previously observed interaction between SOD1 and negatively charged membranes. We used a composition of lipids (POPC/POPE/Cardiolipin) that resembles the lipid composition found in parts of the inter membrane environment. The mutant “pair” N86D and N86K was tested as well as SOD1pWT. They are of special interest as N86D increases the net negative charge and N86K decrease the repulsive charge. Both pWT and N86D shows a pronounced increase in helical content upon interaction. However, no significant change in structure was seen for N86K. It seems like the decrease in net repulsive charge is overcompensated by the decrease in stability for the aggressive N86K mutant. If the observed interaction is relevant for ALS or not is difficult to say but the absence of interaction for one of the most aggressive ALS mutants (A4V) indicates a non-causative role for membranes in the ALS mechanism. However, the lower pH (~0.7 units) present at the inter membrane space can still be of interest. Bringing the protein closer to neutrality will increase the aggregation propensity [83] and the enhanced effect from the net charge decreasing mutants will be even more pronounced in such environment.

Conclusions

Reduced stability shifts the folding equilibrium towards denatured monomers. The severity of the mutant induced destabilizing effect of the SOD1 molecule seems to correlate fairly well with disease progression. Decrease of the net repulsive charge result in less aggressive disease progression unless they involve considerable stability losses or truncation of hydrogen bonds. Truncations of conserved salt links and H-bonds upon ALS associated point mutations however seems to be a yet unresolved ALS

48

triggering question. A possible explanation to their ALS provoking effect is the increased breathing motions in loop IV and VII caused by the loss of H-bonds. SOD1 interacts with negatively charged lipid vesicles but not with neutral ones. The electrostatic association to membrane surfaces however opposes the correlation between decreased stability and disease progression. We suggest that the observed interaction is not a primary part of the ALS mechanism. Conformational changes in the ALS associated mutant N86D are seen upon interactions with mitochondria mimicking lipid vesicles, while for the aggressive N86K changes were less severe. Decrease of net repulsive charge does not enhance the electrostatic interaction for the destabilized N86K.

49

Appendix

Appendix Table 1 Stability data for all mutants studied in this thesis. * The value of the unfolding rate constant at 5.8 M urea.† The disease survival time is the time from onset of first symptom to death or tracheotomy

50

Sammanfattning

Amyotrofisk Lateral Skleros (ALS) är en neurodegenerativ sjukdom. Det innebär att de nervceller som styr all frivillig muskelrörelse förtvinar och man blir förlamad. Eftersom andningen är en muskel som vi kan styra med viljan så innebär det tyvärr att andning till sist kommer att sluta fungera. Medelåldern för insjuknande i ALS är 55 år och 50% av de drabbade dör inom 26 månader. Det finns några bromsmediciner men tyvärr så är dom inte särskilt effektiva. ALS beskrevs redan under 1800-talet och 1869 så fick den sitt nuvarande namn då den franske neurologe Jean-Martin Charcot beskrev sjukdomen. Om man nu känt till sjukdomen så länge skulle man ju kunna tycka att man borde ha nått längre i forskningen. Problemet med sjukdomar som ALS är att dom är väldigt komplexa och det är inte alltid så lätt att ställa rätt diagnos i början av sjukdomsförloppet. Ett betydade genombrott gjordes dock 1993 då man fann att mutationer i genen som kodar för proteinet Superoxide dismutase gick att koppla till ALS. Sedan dess har man hittat över 140 olika mutationer i SOD hos ALS patienter. Värt dock att påpeka är att det endast är 5 – 10% av alla ALS fall där man hittar en mutation i SOD genen. Icke desto mindre så är upptäckten viktig för att förstå vilka mekanismer det är i kroppens celler som orsakar ALS. Man brukar skilja på ärftlig och icke ärftlig ALS. Enkelt förklarat så är det ärftlig ALS om det finns någon annan i släkten som har en mutation av samma slag i SOD-genen. Hittar man ingen genetisk koppling så är det icke ärftlig ALS. Man vet att ALS inte beror på att SOD slutar fungera eftersom försöksmöss inte utvecklar ALS när man helt tar bort genen som kodar för SOD. Däremot så har man klistrat in den mänskliga genen för SOD med en ALS mutation i möss och då har dom fått ALS liknande symptom. ALS har gemensamt med flera andra neurodegenerativa sjukdomar såsom Alzheimers och Parkinsons sjukdom att man hittar klumpar av proteiner i celler från hjärnan och ryggmärgen. Man tror att dessa proteinklumpar kan vara giftiga för cellerna. SOD är ett protein som består av två likadana delar (monomerer). När dom sitter ihop med varandra så kallas det för en dimer. I vardera monomer så sitter något som vi kallar disulfidbrygga. Det är två stycken speciella aminosyror som sitter ihop med varandra i en stark binding. Varje monomer har en kopparatom och en zinkatom. Dessa metaller och disulfidbryggan är viktiga för proteinets stabilitet. Min egen forskning har handlat om att försöka förstå hur ALS mutationer påverkar SOD. Vi har med olika biokemiska och biofysikaliska metoder studerat hur proteinet får sin rätta form och om dessa ALS mutationer påverkar denna process. Om en ALS mutation påverkar proteinet så att den förlorar en del av sin stabilitet så tror vi att de lättare klumpar ihop sig med varandra så att det blir

51

proteinklumpar. Vi har visat att flera av dessa mutationer påverkar stabiliteten till väldigt hög grad. Vi har också kunna se en försiktig korrelation mellan förlorad stabilitet och överlevnadstid räknat från då diagnos har ställts. Vidare har vi sett att det finns en grupp av ALS mutationer som ändrar proteinets laddning. I sin vanliga form i cellen så har proteinet en laddning på -6 och ungefär 30% av ALS mutationerna ändrar denna laddning så att den blir lägre, dvs mera positiv. Beräkningar gjorda av andra forskare inom detta område har visat att om proteinets laddningen blir mera positiv så kan de lättare kladda ihop sig med varandra. Vi kan säga att de mutationer som ändrar laddningen oftare har väldigt liten påverkan på proteinets stabilitet. Vi tycker oss dock se en trend mot att dessa mutationer i högre grad finns hos patienter där sjukdomförloppet är långsammare. Fortfarande är det väldigt mycket vi inte vet och en sådan komplex sjukdom som ALS går knappast att förklara enbart med enkla jämförelser mellan överlevnadstid efter diagnos och proteinstabilitet. Däremot är det en liten tråd att dra i för att på sikt kunna nysta ut de komplexa mekanismer som orsakar denna fruktansvärda sjukdom.

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Acknowledgement

Ett speciellt tack till:

Gerhard Gröbner, som tog hand om mig då jag drygt ett år in på min doktorandtid plötsligt stod utan både handledare och forskningsgrupp. Jag var nog inte den lättaste att motivera till en början, så jag vill rikta ett stort tack till dig Gerhard för att du inte gav upp på mig och att du envisats med att fråga mig ”har du skrivit något Nature-pek ännu?” och när jag blivit tvungen att vara hemma med sjuka barn så har du alltid varit förstående även om du hävdat att deras förkylningar och magsjukor beror på dåliga gener.

Mikael Oliveberg, vill jag tacka för att du faktiskt antog mig som doktorand från första början. Tyvärr så hann vi inte prata så mycket forskning medans du var kvar i Umeå men desto mera de senaste månaderna. Tack för effektivt samarbete i elfte timmen.

Stefan Marklund, tack för dina ekonomiska insatser. Jag vill också tacka för den trevliga “alla ALS-doktorander ska ha sabrerat champange innan dom blir godkända” ceremonin. Mycket trevlig tillställning.

Peter Andersen, tack för värdefull information om olika ALS mutationer, även om du ibland är lite hemlig med ny information så är din insats en värdefull tillgång för oss i ALS-Umeå.

Resten av ALS-gänget i Umeå: Tomas Brännström, Pelle Z vi kanske ses i Vasaloppsspåret, Andreas för en komplett sammanfattning av alla ALS-referenser skrivna fram till 2005. Finns dom inte i din avhandling så är dom skrivna efteråt, Daniel, Sabine, Caroline, Anna B, Anna W, Karin och alla som jag glömt, tack för trevliga stunder på konferenser och fester.

Biofysikalkemi, Christopher, thanks for all the help that I got and for all the work that you have done, I owe you. Marco thanks for the help I got with CD and preparation of lipid vesicles. Marcus, du får ta vid med det som jag inte hann med. Tobias tack för ditt envisa frågade om jag skulle spela innebandy, jag började ju faktiskt till sist och kul var det, P-O min egen korridorsopponent, det är bra att jag får öva att argumentera,tack! Tack till mina rumskamrater Matteus och Therese som gör kontoret till en trevlig arbetsplats, även om jag jobbar effektivare hemma så är det inte lika trevligt Greger, Britta, MWW, Tofeeq, Erik, Lennart, Göran, Leif, Nils,

53

Ida, Radek, Oleg, Jörgen, Ulrika. Tack till er alla (även icke omnämda gamla och nya) för ert bidrag till en trevlig korridor

Anna-Märta och Anita för all hjälp med blanketter och annat administrativt som man inte förstår som ni alltid har stenkoll på. Nog blev det tråkigare på korridoren utan er sekreterare där. Tack till Kenneth för att du anförtrodde mig med nyckel till den lilla verkstaden, tack vare det så kunde jag fixa det sista inför MC-säsongen -08.

Biokemi (Nu och då), Lars Backman ständig belysare av att allt har en baksida ;) Tack för allt stöd och givande diskussioner och för gemensamt intresse att stopped-flow utrustningen ska funka. Anders Öhman Tack för din hjälp med Molmol i tidernas begynnelse. Barbara, for loveable hugs, The Spanish inquisition: Anna, Jose, Maribel and Miguel. Hélder, Wolfgang, Christiane, Liudmila Irene, Harald, Thomas, Patrik, Erik och Michael. Tack Viola för värdefull kunskap från år av labvana, extra tack till Katarina för all hjälp med mutation, odling och rening av ALS-SOD1 mutanter + värdefull mångårig erfarenhet. Tack till alla som under åren gjort sitt för en trevlig korridor.

Gamla “Folding gruppen”, Mikael “Micke” Lindberg (jag har läst sönder din avhandling, kan jag få en ny?), Magnus och Linda kommer ni och hälsar på snart?, Anna vi tänker på dig här uppe, kämpa på! Maria den träningsvärk jag fick efter workoutpasset på doktoranddagen på IKSU-Spa glömmer jag aldrig, Tack för den! Tommy min rumskamrat i “confined space”, Ellinor det verkar som om vi blir färdiga till sist...

Tack till Dig som jag glömt men som vet att jag borde vara tacksam för din hjälp. Jag är tacksam men tyvärr så tror jag att det ramlat ut en del ur huvudet då jag fyllt på med ALS-forskning i andra änden

Morsfarsan och mina syskon Jenny och Rickard. Även om ni inte har någon vidare aning om vad jag gör och varför så kännar jag att ert stöd finns där, Tack!....och om ni eller någon annan undrar så hade jag inte tänkt plugga mer nu

Mina älskade barn, William, Olivia, Seth och Miralie. Jag är så stolt över er alla fyra och jag älskar er mest av allt.

Linda, du är den som känner mig bäst och förstår mig mest. Min kärlek till dig är exponentiell. Tack för ditt stöd, jag älskar dig.

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I also want to acknowledge SOD1, the most persistent of all enzymes: Enzymatic SOD1 activity was found in brain tissue from the Egyptian mummy of a 16 year old male from 1800 BC [106]. After almost 4000 years, the active part of SOD1 was still intact enough to perform enzymatic activity.

55

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