Protein folding, misfolding and aggregation

Protein folding is a process in which a newly expressed and post-translationally modified polypeptide chain adopts a structural conformation in which the hydrophobic elements are buried inside the fold while the hydrophilic ones are exposed on the surface (Crick, 1970). This transition is crucial as the right fold is a prerequisite for a protein’s long term stability and the ability to selectively interact with its partners (Dobson, 2003). But what is the mechanism of folding and how is the native structure adopted? The pioneering work on protein folding in the 1970s (Anfinsen, 1973) concluded that the three-dimensional structure is encoded in the amino acid sequence of the polypeptide chain and that this functional fold should represent a global minimum on the energy landscape. Based on this finding, enormous effort has been put in order to understand the process of folding and how the minimum energy state is achieved. It became evident that a random search for the right conformation cannot be performed on a microsecond to millisecond biological timescale of protein folding, and that the protein is rather “funneled” down the energy landscape by energy barriers, kinetic traps and narrow pathways (Figure 1-1) (Wolynes et al., 1995; Dill and Chan, 1997).

Figure 1-1. Schematic representation of folding energy landscapes.

(a) Folding is achieved by a random search through all possible conformations.

(b) Protein is funneled down the energy landscape through intermediate stages (adapted from Dill and Chan, 1997).

The presence of metastable intermediates may lead to intermolecular interactions between the partially unfolded states and self association into large assemblies known as aggregates (Dobson, 2003; Brockwell and Radford, 2007). Therefore, it is necessary for the cell that folding is controlled in order to prevent misfolding (Figure 1-2). In a normal biological state this is accomplished by molecular chaperones (Hsp) and the ubiquitin-proteasome pathway which prevent protein unfolding and aggregation by facilitating refolding and degradation, respectively (Jahn and Radford, 2005). Impairment of these factors results in malfunctioning of the living organism and hence in disease (Thomas et al., 1995;

Dobson, 2001; Horwich, 2002).

Figure 1-2. Factors influencing protein folding and aggregation in vivo.

In normal state, molecular chaperones (Hsp) and proteasomal degradation pathway prevent protein unfolding or misfolding. Failure of this mechanisms results in increased protein aggregation. By self-association of partially folded proteins small oligomers are formed which may further assemble into pores, protofibrils and mature fibril deposits (adapted from Jahn and Radford, 2005).

To current knowledge, more than a 100 diseases in both humans and animals involve aggregation of any of twenty non-homologous proteins (Kisilevsky, 1983; Pepys, 1988;

Jacobson and Buxbaum, 1991; Selkoe, 1997; Kisilevsky, 2000). One of the classes of protein aggregation disorders are amyloidoses (see Table 1-1). This group of diseases is directly associated with deposition of proteinaceous aggregates in tissues, including the brain, heart, spleen, pancreas and skeletal tissue (Kelly and Lansbury, 1994; Tan and Pepys, 1994;

Horwich, 2002). In many cases, the protein assemblies found in vivo contain a variety of other molecules, such as carbohydrates or additional proteins (Dobson, 2003).

Table 1-1. Examples of amyloidoses.

disease related protein

Alzheimer’s disease β-amyloid peptide, tau

Parkinson’s disease α-synuclein

familial amyloid polyneuropathy transthyretin transmissible spongiform encephalopathies prion protein

Huntington’s disease huntingtin

frontotemporal dementia tau

dialysis related amyloidosis β-2-microglobulin diabetes mellitus type II islet amyloid polypeptide

Even though the monomeric soluble forms of disease related proteins range from compact globular folds to intrinsically unstructured polypeptides, the aggregated forms share many common features. They are able to bind certain dyes such as Thioflavin T or Congo red (Elghetany and Saleem, 1988) and exhibit similar morphologies – namely long, unbranched and often twisted fibers a few nanometers in diameter, which show a characteristic X-ray diffraction pattern. These patterns display typical meridional reflections at ~4.7 Å and equatorial reflections at ~10 Å, indicative of a silk-like structure composed of stacked β-sheets with the strands running perpendicular to the axis of the fibril (cross-β structure). The common features of amyloid fibrils are summarized in Figure 1-3.

Alternative to the cross-β structure, β-helix and nanotube models have been proposed, in which one or more β-sheets wrap around a hollow core in a helical manner. The β-helical model was first suggested for fibrils of a short model peptide and showed a cylindrical antiparallel β-helix 20 Å in diameter (Lazo and Downing, 1997). The nanotube model, based on absence of the 10 Å equatorial reflection, suggested a watter filled tube with internal and

external diameters of 12 Å and 32 Å, respectively (Perutz et al., 2002). Beta-helical models have also been proposed for the prion protein, based on 2D crystals of the protein extracted from brain tissue (Govaerts et al., 2004), and for amyloid-beta peptide, based on hydrogen-deuterium exchange data and a proline scan experiment (Kheterpal et al., 2000; Williams et al., 2004).

Figure 1-3. Characteristics of amyloid proteins.

(a) EM images of negatively stained amyloid fibrils. (b) X-ray fibre diffraction pattern from aligned fibrils formed by islet amyloid polypeptide showing the 4.7 Å and ~10 Å reflections (adapted from(Makin and Serpell, 2005). (c) A model of the cross-β architecture showing the interstrand distance of 4.7 Å and intersheet distance of 10 Å (modified from(Jack et al., 2006).

Regarding the origin of the structure of the fibrillar state, the ability to form aggregates is regarded an intrinsic physicochemical property of the polypeptide chain, as the amyloid structures are mostly stabilized by hydrogen bonds involving the protein backbone (Dobson, 1999). This is supported by evidence that under specific conditions also non-disease related proteins can form fibrils (Guijarro et al., 1998; Chiti et al., 1999).

Evidence emerges that low molecular weight aggregates are indeed causing the toxicity, while appearance of mature fibril deposits is suggested to play a protective role as a removal mechanism for the toxic oligomeric intermediates (Snyder et al., 1994; Harper et al., 1997; Lambert et al., 1998; Klein et al., 2001; Bucciantini et al., 2002; Walsh et al., 2002;

Yong et al., 2002). In fact, by targeting regions of the protein responsible for further self-association of oligomeric and protofibrillar species, it is possible to generate mutants with increased toxicity which do not form amyloid fibrils (Karpinar DP, unpublished data).

However, irrespective of whether the amyloid fibrils or intermediates are the toxic species, it is of extreme importance to investigate the structural properties of all stages on the aggregation pathway, in order to fully understand the process of amyloid formation and enable the development of treatment strategies. It is also very likely, that the structure of mature fibrils will shed light on the structure of their precursors.