Contribution of the intrinsic disulfide to the assembly mechanism of islet amyloid

The oligomerization of soluble protein into large, highly ordered fibrillar structures, termed amyloids, is a common feature of a number of diseases including, for example, Alzheimer’s disease and dialysis-related amyloidosis (Rochet and Lansbury 2000). Interestingly, proteins that undergo amyloid fiber formation vary greatly in their native structures and functions, but in the fiber state, exhibit common features. Amyloid fibers form a core that is predominantly composed of β-sheets. The strands within these sheets are organized perpendicular to the long axis of the fiber with backbone hydrogen bonding oriented in parallel to the fiber axis (Eanes and Glenner 1968; Sunde and Blake 1997). The common core structure of amyloids suggests that assembly mechanisms across different systems are comparable. Indeed, the overall kinetic behaviors of amyloids also share common features. A protein placed under amyloidogenic conditions will initially remain soluble. This quiescent phase, termed the lag phase, is followed by collective assembly into the aggregated state. This implies the existence of nucleation processes akin to crystallization (Harper and Lansbury 1997). As with crystallization, nucleation reactions within the lag phase may be bypassed by providing exogenous fiber from a previously conducted reaction. A number of intermediate states likely participate in the assembly process. As amyloidogenesis occurs in a variety of biomedical systems with unrelated primary sequence, it is vital to elucidate these states in order to determine a generalized basis for assembly. In addition, recent studies suggest that it is the intermediates of fiber formation which are the cytotoxic species (Bucciantini et al. 2002). Thus, insights into amyloid assembly mechanisms are essential to developing our understanding of the intrinsic physical properties of polypeptides, and to developing effective targets for therapeutics. Islet amyloid polypeptide (IAPP), or amylin, is a peptide hormone that is normally co-secreted with insulin by the β cells of the pancreas. In patients with type II diabetes, unmodified IAPP deposits as cytotoxic amyloid in the extra-cellular spaces of the pancreatic islets (Kahn et al. 1999). In its soluble form, IAPP is an unstructured (Kayed et al. 1999; Higham et al. 2000), C-terminally amidated 37-residue peptide containing an intramolecular disulfide bond between residues 2 and 7. Generally, the energies governing amyloid formation include those associated with unfolding of the precursor and those of stabilizing the fiber. For many amyloid systems, including IAPP, the precursor is intrinsically unstructured. For such proteins, the forces that define the assembly pathway are derived from the fiber structure itself. In IAPP, for example, fluorescence resonant energy transfer (FRET) has been used to identify long-range aromatic interactions in the mature fiber. Interestingly, these interactions are also transiently sampled by prefibrillar conformations of IAPP (Padrick and Miranker 2001). Previous work on IAPP has also identified a number of events in the process of fiber assembly. In this work, we address two of these. First, we previously reported that the process of IAPP fiber formation follows a double-nucleation mechanism (DNM) of fibrillogenesis (Padrick and Miranker 2002); i.e., there are mechanisms for both fiber-independent (primary nucleation) and fiber-dependent (secondary nucleation) means for increasing the molar concentration of fiber ends. Second, we demonstrated the presence of an activation step evident during the lag phase of fiber formation (Padrick and Miranker 2002). This is visible in the kinetic profile of seeded fiber formation. Briefly, addition of seed at the beginning of a fibrillogenesis reaction gives rise to a sigmoidal reaction profile. If instead seed is added several minutes after initiation of the reaction, single-phase exponential profiles are observed. Similar two-phase kinetics for seeded polymerization have been observed in vitro for yeast prion protein Sup35p (Santoso et al. 2000). It is challenging to reconcile the presence of a minute timescale kinetic event with the dynamics of an unstructured polypeptide. Human IAPP does not possess any pro-line residues. The longest timescale available for conformational change should therefore be of the order of μsec (Plaxco and Dobson 1996; Kubelka et al. 2004) and not minutes. As the overall unfolded structure of prefibrillar IAPP has been determined by far-UV CD (Kayed et al. 1999; Higham et al. 2000), it is plausible that local structures play an important role, for example, aromatic interactions (Padrick and Miranker 2001; Gazit 2002). In this work, we examined the role of the N-terminal disulfide. In human IAPP, this disulfide bond spans residues 2–7. A five-residue span renders this region of the polypeptide incapable of forming canonical secondary structures. Nevertheless, we can report that perturbations occur to the assembly kinetics of IAPP fibers upon removal of this disulfide. These alterations shed light on the mechanism of IAPP fibrillogenesis.