Abstract
Cystatin C (CysC) is a versatile and ubiquitously-expressed member of the cysteine protease inhibitor family that is present at notably high concentrations in cerebrospinal fluid. Under mildly denaturing conditions, CysC forms inactive domain-swapped dimers. A destabilizing mutation, L68Q, increases the rate of domain-swapping and causes a fatal amyloid disease, hereditary cystatin C amyloid angiopathy. Wild-type (wt) CysC will also aggregate into amyloid fibrils under some conditions. Propagated domain-swapping has been proposed as the mechanism by which CysC fibrils grow. We present evidence that a CysC mutant, V57N, stabilized against domain-swapping, readily forms fibrils, contradicting the propagated domain-swapping hypothesis. Furthermore, in physiological buffer, wt CysC can form oligomers without undergoing domain-swapping. These non-swapped oligomers are identical in secondary structure to CysC monomers and completely retain protease inhibitory activity. However, unlike monomers or dimers, the oligomers bind fluorescent dyes that indicate they have characteristics of pre-amyloid aggregates. Although these oligomers appear to be a pre-amyloid assembly, they are slower than CysC monomers to form fibrils. Fibrillation of CysC therefore likely initiates from the monomer and does not require domain-swapping. The non-swapped oligomers likely represent a dead-end offshoot of the amyloid pathway and must dissociate to monomers prior to rearranging to amyloid fibrils. These prefibrillar CysC oligomers were potent inhibitors of aggregation of the Alzheimer's-related peptide, β-amyloid. This result illustrates an example where heterotypic interactions between pre-amyloid oligomers prevent the homotypic interactions that would lead to mature amyloid fibrils.
Highlights
Cystatin C (CysC)2 is a soluble basic protein belonging to the cysteine protease inhibitor family
To uncover reasons why oCys was less prone to amyloid fibril formation, we searched for differences in its structural characteristics compared with mCys and the domain-swapped dimeric CysC
Observed mechanisms for amyloid aggregation include the nucleationelongation model, where a rare event provides a nucleus for further growth by monomer addition, and the aggregation-conformational conversion model, where association of natively folded or intrinsically disordered proteins into soluble oligomers is followed by subsequent rearrangement within the hydrophobic core of the oligomer to the cross--sheet structure typical of amyloid fibrils (59 – 62)
Summary
For all known cases of inherited cerebral amyloid angiopathy (Icelandic type), a lethal disease that causes brain hemorrhaging and strikes young adults [21, 22]. This mutation destabilizes CysC, making it prone to aggregate into amyloid fibrils. It has been proposed that CysC amyloid fibrils form via propagated domain-swapping (Fig. 1c) [15, 21, 23, 24], this view has been challenged recently [25, 26]. We demonstrate that these oligomers inhibit A aggregation better than monomeric or domain-swapped dimeric CysC
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