On the nucleation of amyloid β-protein monomer folding

Abstract

The amyloid β-protein (Aβ) is a normal, soluble component of human plasma and cerebrospinal fluid (Haass et al. 1992; Seubert et al. 1992; Shoji et al. 1992). Aβ exists predominantly as a 40- or 42-residue peptide (for review, see Teplow 1998). Historically, the assembly of Aβ into amyloid fibrils was thought to initiate a pathogenic cascade resulting in AD (the amyloid cascade hypothesis) (Hardy and Allsop 1991). However, progressively smaller assemblies have been discovered (Oda et al. 1995; Harper et al. 1997; Walsh et al. 1997; Lambert et al. 1998; Bitan et al. 2003a), and each has been found to be neurotoxic (Lambert et al. 1998; Hartley et al. 1999; Walsh et al. 1999; Sun et al. 2003; Taylor et al. 2003). This accumulating evidence supports a revision of the amyloid cascade hypothesis such that Aβ assembly into neurotoxic oligomers, and not into fibrils, is the seminal event in AD pathogenesis (Haass and Steiner 2001; Klein et al. 2001, 2004; Kirkitadze et al. 2002; Walsh et al. 2002). If the hypothesis is true, then preventing the folding of nascent Aβ monomer into toxic conformers or oligomers would have therapeutic benefit.

Certain regions of Aβ exert strong control over assembly kinetics and biological activity. The dipeptide Ile41–Ala42 at the C terminus of Aβ, which distinguishes Aβ(1–42) from Aβ(1–40), is responsible for distinct biophysical (Jarrett et al. 1993; Bitan et al. 2003a), physiologic (Klein et al. 2004), and clinical (Iwatsubo et al. 1995) behaviors of the longer peptide. These behaviors include oligomerization into pentamer/hexamer units (paranuclei) (Bitan et al. 2003a), nucleation of amyloid formation from shorter variants such as Aβ(1–39) and Aβ(1–40) (Jarrett et al. 1993), formation of Aβ-derived diffusible ligands (ADDLs) (Oda et al. 1995; Lambert et al. 1998), early deposition in senile plaques in Down’s syndrome patients (Iwatsubo et al. 1995), and strong linkage to AD (Younkin 1995). Amino acid substitutions within and adjacent to the central hydrophobic cluster (CHC) of Aβ, Leu17–Ala21, cause cerebral amyloid angiopathy and AD-like diseases (Levy et al. 1990; Kamino et al. 1992; Hendriks and Vanbroeckhoven 1996; Tagliavini et al. 1999; Grabowski et al. 2001; Nilsberth et al. 2001). In vitro studies have probed the biophysical consequences of these mutations. For example, the Glu22→Gln substitution increases both the rates of nucleation and elongation of Aβ fibrils (Wisniewski et al. 1991; Teplow et al. 1997) and the Glu22→Gly substitution facilitates protofibril formation (Nilsberth et al. 2001; Päiviö et al. 2004). Met35 may be important both in Aβ-associated redox chemistry (Butterfield 2002) and in the peptide assembly (Snyder et al. 1994; Seilheimer et al. 1997; Watson et al. 1998; Butterfield 2002; Hou et al. 2002a; Palmblad et al. 2002). Recent studies suggest that formation of methionine sulfoxide (Met[O]) or methionine sulfone (Met[O₂]) block oligomerization of Aβ(1–42), preventing fibril assembly (Hou et al. 2002b; Palmblad et al. 2002; Bitan et al. 2003b). Taken together, these data have revealed how small changes in Aβ primary structure can alter intermolecular interactions among Aβ monomers.

To examine assembly-dependent features of intramolecular peptide organization, Aβ secondary structure characteristics have been determined spectroscopically. The conformational changes occurring during fibril assembly involve random coil (RC)→α-helix, RC→β-strand, and α-helix→β-strand transitions (Barrow et al. 1992; Soto et al. 1995; Sticht et al. 1995; Coles et al. 1998; Shao et al. 1999; Walsh et al. 1999; Zagorski et al. 2000; Zhang et al. 2000; Kirkitadze et al. 2001). The involvement of turn elements early in fibril formation is less clear. Studies of Aβ fragments, including Aβ(19–28) (Gorevic et al. 1987; Sorimachi et al. 1990), Aβ(15–28) (Gorevic et al. 1987), and Aβ(25–35) (Laczko et al. 1994), suggest turns are present. However, no turns were found in studies of Aβ(10–35) fibrils (Benzinger et al. 2000). In contrast, modeling based on established examples of β-helical conformation, and on hydrophobic and hydrogen bonding effects on protein folding, suggests a β-turn at Val24–Asn27 (Lazo and Downing 1998). In silico modeling of fibrils from Aβ(1–43) reveals a β-turn at Gly25–Lys28 (George and Howlett 1999). Solid-state NMR studies of Aβ(1–40) fibrils suggest a bend at Gly25–Gly29 (Petkova et al. 2002).

As illustrated above, a deepening understanding of Aβ peptide oligomerization and fibril formation is being obtained. The aim of the work reported here was to establish how these important assembly processes are initiated at the monomer level. To do so, we probed the conformation of Aβ monomer using limited proteolysis (Fontana et al. 1997; Hubbard 1998) in combination with mass spectrometry. This approach is useful in the study of conformational changes in proteins that have a strong propensity to aggregate (Kheterpal et al. 2001; Polverino de Laureto et al. 2003; Monti et al. 2004). Results suggest, in both Aβ(1–40) and Aβ(1–42), that a nucleus for intramolecular folding exists within the decapeptide region Ala21–Ala30. Solution-state NMR studies of the corresponding Aβ(21–30) decapeptide revealed a turn element formed by residues Val24–Lys28 and provided a resolved structure of the nucleus.

Acknowledgments

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Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041292205.

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