**1.2.3 Amyloid formation**

Protein aggregation is a general term that describes a number of diverse processes that culminate in the formation of non-native, multimeric complexes of varied conformations. These aggregates can range from small, soluble oligomers, to larger amorphous structures, and insoluble, well-structured fibrils (Uversky and Dunker 2010). Amyloid is a common, well characterized, type of aggregate formed by proteins associated with many diseases, including the neurodegenerative prion, Parkinson's, Alzheimer's, and Huntington's diseases (Chiti and Dobson 2006; Chiti and Dobson 2009). Extensive studies of amyloid have resulted in significant advances in understanding the underlying molecular basis of protein aggregation (Sipe and Cohen 2000; Chiti and Dobson 2006; Eisenberg et al. 2006; Chiti and Dobson 2009). Classically defined amyloid is characterized by an unbranched, fibrillar aggregate morphology, which exhibits green-gold birefringence upon binding Congo red (Sipe and Cohen 2000), a dye used in disease diagnosis, and a cross-β x-ray diffraction pattern due to the presence of β-strands oriented perpendicular to the long axis of the fibre (Serpell 2000). These large aggregates can be extremely stable and unaffected by cellular clearance machinery (Dobson 1999; Knowles et al. 2007). There is extensive evidence that most and perhaps all proteins can form amyloid under suitable, typically destabilizing, conditions (Dobson 1999; Munishkina et al. 2004; Stathopulos et al. 2004). Amyloid formation can arise from association of unstructured, partially folded, or native-like species, and can be prevented by factors that favour native folding (Chiti and Dobson 2009). These include such factors as: interactions with molecular chaperones that can stabilize partially folded conformations and increase the folding rate; and post-translational modifications or ligand binding that can stabilize the native state and prevent unfolding (Dobson 2004; Chiti and Dobson 2009). Protein size is also a factor that modulates the propensity of a protein to

Folding and Aggregation of Cu, Zn-Superoxide Dismutase 271

rate of prion replication and the strength of the disease phenotype (Verges et al. 2011). In recent years, evidence for conformational diversity, or different strains, of protein aggregates has also been described for non-infectious protein conformational disorders such as Alzheimer's, Parkinson's, Frontal Temporal Dementia, and ALS (Frost and Diamond 2009; Furukawa et al. 2010). The proteins linked to many of these diseases are natively disordered, and so can easily sample different conformations, which may facilitate aggregation via multiple pathways. Mutations and/or post-translational modifications can greatly influence the population of different conformations of a protein, and thus can largely influence the aggregation process. This point may be particularly relevant to ALS toxicity, since SOD1, although natively folded, undergoes extensive post-translational modification *in vivo* (Valentine et al. 2005), and is increasingly malleable in its less stable, immature forms (refer to section 3). How different fALS-associated mutations modulate the stability of different forms of SOD1 may largely determine the ALS phenotype (Vassall et al. 2011).

**2. Structure and function of Cu, Zn-superoxide dismutase (SOD1)** 

binds one Cu ion and one Zn ion (Figure 2A, C).

(Valentine et al. 2005).

Before discussing SOD1 folding, misfolding and aggregation, we will give a brief description of the tertiary and quaternary structure of SOD1. Human SOD1 is a 32 kDa homodimeric metalloenzyme, with each subunit consisting of a 153 amino acid chain that is often N-terminally acetylated, contains a highly conserved, intrasubunit disulfide bond, and

Each monomer folds into a Greek key β-barrel, comprised of two, four-stranded antiparallel β-sheets arranged at an angle with respect to one another. The β-barrel has a noncontinuous topology such that strands 1 through 3 together with strand 6 form the first βsheet, while strands 4, 5, 7 and 8 form the second β-sheet. The β-strands are connected by seven loops that differ greatly in length. Loop IV and loop VII are known as the metal binding and electrostatic loops, respectively, and play important roles in both stability and catalytic function by binding the metals and forming the catalytic site pocket. In addition to forming the Zn-binding site, Loop IV contains residues that are important for dimer interface and intrasubunit disulfide bond formation, which tethers Loop IV to β-strand 8 (Tainer et al. 1982). Thus, Zn binding, disulfide bond formation and dimerization together stabilize the native conformation of this long loop, greatly affecting the overall stability of the protein. Loop VII mainly plays a functional role, containing charged residues that shield the active site. These charged residues are important for guiding the superoxide anion from the surface of the protein into the active site where the redox active Cu ion is bound

SOD1 is abundant and ubiquitously expressed in the cytosol of aerobic organisms (Brown et al. 2004). Maturation of the protein involves a series of post-translational modifications, which are understood to varying extents. When it is initially synthesized in the reducing environment of the cytosol the protein is thought to adopt a marginally stable, folded, monomer structure with a reduced disulfide bond and no bound metals. How the protein acquires Zn is not known; however, Cu can be acquired by interaction with the copper chaperone for SOD1 (CCS) or by a CCS-independent mechanism that may involve glutathione, but that is not well understood (Leitch et al. 2009). CCS also catalyzes intrasubunit disulfide bond formation in the reducing cellular environment (Leitch et al. 2009). Although the most abundant form of SOD1 is usually the native, fully mature,

form amyloid fibrils, as it is less energetically favourable for large proteins to form an amyloid core, compared to smaller proteins (Baldwin et al. 2011; Ramshini et al. 2011). It should be noted that ALS is not classified by pathologists as an amyloid disease (Kerman et al. 2010). Recent studies have reported the formation of SOD1 aggregates *in vitro* that exhibit some features of amyloid (Banci et al. 2008; Furukawa et al. 2008; Oztug Durer et al. 2009); however, the relevance of such studies to human disease is not known. Typically, there is considerable structural heterogeneity in amyloid (Platt and Radford 2009) and in other amorphous or ordered aggregate structures formed by many peptides and proteins (Fink 1998; Seshadri et al. 2009) (see 1.2.4). Careful analyses using multiple probes are required to distinguish between these different aggregate structures. Appropriately characterizing mixtures of aggregate structures is a major, ongoing challenge in the study of protein aggregation.

### **1.2.4 Protein aggregation heterogeneity and disease complexity**

Neurodegenerative disorders characterized by protein misfolding and aggregation, including ALS, commonly display phenotypic diversity, such as variation in the age of onset, the rate of neuronal dysregulation, and the area of the nervous system affected (Armstrong et al. 2000; Goedert et al. 2001; Frost and Diamond 2009; Williamson et al. 2009). Although the molecular origins of such phenotypic diversity are complex and may differ between diseases, in recent years it has been shown that protein aggregates, including amyloid fibrils, exhibit extensive structural heterogeneity both *in vivo* and *in vitro* (Berryman et al. 2009; Frost and Diamond 2009). Not only do fibrils formed by different amino acid sequences adopt conformations that differ in length and twist, but the structure of fibrils formed by the *same* sequence can vary depending on solution conditions (Berryman et al. 2009). Fibres can vary in the number of amino acids that participate in forming the amyloid core, the arrangement of β-strands in a parallel or antiparallel conformation within each protofilament, and the alignment of β-sheets along the protofilament axis (Tycko 2006). The structure that a particular protein adopts prior to aggregation influences the structure of the aggregate formed and the conformational plasticity of a native protein may play a large role in determining the number of structurally different aggregates produced (Jones and Surewicz 2005; Natalello et al. 2008). Although aggregate structures formed from the same protein can be quite diverse (ie. amorphous versus amyloid structures), in many cases the formation of such structures is energetically favourable and therefore switching between aggregate conformations can require a large amount of energy. As a result, a particular fibril can become trapped in a single conformation (Berryman et al. 2009).

Structural heterogeneity of protein aggregates has been known for many years for amyloid fibrils derived from prion proteins, infectious protein agents that give rise to a number of neurodegenerative disorders known as spongiform encephalopathies or prionopathies. In these diseases, the infectious agent is a misfolded prion protein (PrPSc, S referring to Scrapie, the disease caused by this infectious agent), which once introduced into a host cell can bind to the native prion protein (PrPc, c referring to cellular) and induce conversion to the PrPSc form, inevitably resulting in the spread of the disease phenotype (Tuite and Serio 2010). Because a prion protein can adopt a number of conformations, there is considerable heterogeneity in the structure of the amyloid fibrils that are formed from these proteins. Prion amyloid fibrils can differ in stability, surface charge and degree of polypeptide incorporation into the amyloid core, differences that may play a large role in determining

form amyloid fibrils, as it is less energetically favourable for large proteins to form an amyloid core, compared to smaller proteins (Baldwin et al. 2011; Ramshini et al. 2011). It should be noted that ALS is not classified by pathologists as an amyloid disease (Kerman et al. 2010). Recent studies have reported the formation of SOD1 aggregates *in vitro* that exhibit some features of amyloid (Banci et al. 2008; Furukawa et al. 2008; Oztug Durer et al. 2009); however, the relevance of such studies to human disease is not known. Typically, there is considerable structural heterogeneity in amyloid (Platt and Radford 2009) and in other amorphous or ordered aggregate structures formed by many peptides and proteins (Fink 1998; Seshadri et al. 2009) (see 1.2.4). Careful analyses using multiple probes are required to distinguish between these different aggregate structures. Appropriately characterizing mixtures of aggregate structures is a major, ongoing challenge in the study of

Neurodegenerative disorders characterized by protein misfolding and aggregation, including ALS, commonly display phenotypic diversity, such as variation in the age of onset, the rate of neuronal dysregulation, and the area of the nervous system affected (Armstrong et al. 2000; Goedert et al. 2001; Frost and Diamond 2009; Williamson et al. 2009). Although the molecular origins of such phenotypic diversity are complex and may differ between diseases, in recent years it has been shown that protein aggregates, including amyloid fibrils, exhibit extensive structural heterogeneity both *in vivo* and *in vitro* (Berryman et al. 2009; Frost and Diamond 2009). Not only do fibrils formed by different amino acid sequences adopt conformations that differ in length and twist, but the structure of fibrils formed by the *same* sequence can vary depending on solution conditions (Berryman et al. 2009). Fibres can vary in the number of amino acids that participate in forming the amyloid core, the arrangement of β-strands in a parallel or antiparallel conformation within each protofilament, and the alignment of β-sheets along the protofilament axis (Tycko 2006). The structure that a particular protein adopts prior to aggregation influences the structure of the aggregate formed and the conformational plasticity of a native protein may play a large role in determining the number of structurally different aggregates produced (Jones and Surewicz 2005; Natalello et al. 2008). Although aggregate structures formed from the same protein can be quite diverse (ie. amorphous versus amyloid structures), in many cases the formation of such structures is energetically favourable and therefore switching between aggregate conformations can require a large amount of energy. As a result, a particular fibril

Structural heterogeneity of protein aggregates has been known for many years for amyloid fibrils derived from prion proteins, infectious protein agents that give rise to a number of neurodegenerative disorders known as spongiform encephalopathies or prionopathies. In these diseases, the infectious agent is a misfolded prion protein (PrPSc, S referring to Scrapie, the disease caused by this infectious agent), which once introduced into a host cell can bind to the native prion protein (PrPc, c referring to cellular) and induce conversion to the PrPSc form, inevitably resulting in the spread of the disease phenotype (Tuite and Serio 2010). Because a prion protein can adopt a number of conformations, there is considerable heterogeneity in the structure of the amyloid fibrils that are formed from these proteins. Prion amyloid fibrils can differ in stability, surface charge and degree of polypeptide incorporation into the amyloid core, differences that may play a large role in determining

**1.2.4 Protein aggregation heterogeneity and disease complexity** 

can become trapped in a single conformation (Berryman et al. 2009).

protein aggregation.

rate of prion replication and the strength of the disease phenotype (Verges et al. 2011). In recent years, evidence for conformational diversity, or different strains, of protein aggregates has also been described for non-infectious protein conformational disorders such as Alzheimer's, Parkinson's, Frontal Temporal Dementia, and ALS (Frost and Diamond 2009; Furukawa et al. 2010). The proteins linked to many of these diseases are natively disordered, and so can easily sample different conformations, which may facilitate aggregation via multiple pathways. Mutations and/or post-translational modifications can greatly influence the population of different conformations of a protein, and thus can largely influence the aggregation process. This point may be particularly relevant to ALS toxicity, since SOD1, although natively folded, undergoes extensive post-translational modification *in vivo* (Valentine et al. 2005), and is increasingly malleable in its less stable, immature forms (refer to section 3). How different fALS-associated mutations modulate the stability of different forms of SOD1 may largely determine the ALS phenotype (Vassall et al. 2011).
