A mechanistic model to predict effects of cathepsin B and cystatin C on ß-amyloid aggregation and degradation.

ß-Amyloid (Aß) aggregation is thought to initiate a cascade of neurodegenerative events in Alzheimer's disease (AD). Much effort is underway to develop strategies to reduce Aß concentration or inhibit aggregation. Cathepsin B (CatB) proteolytically degrades Aß into non-aggregating fragments but is potently inhibited by cystatin C (CysC). It has been suggested that decreasing CysC would facilitate Aß clearance by relieving CatB inhibition. However, CysC binds Aß and inhibits Aß aggregation, suggesting that an intervention that increases CysC would prevent Aß aggregation. Both approaches have been tested in animal models, yielding contradictory results, possibly because of the opposing influences of CysC on Aß degradation versus aggregation. Here, we sought to develop a model that quantitatively predicts the effects of CysC and CatB on Aß aggregation. Aß aggregation kinetics in the absence of CatB or CysC was measured. The rate constant for Aß degradation by CatB and the equilibrium constant for binding of CysC to Aß were determined. We derived a mathematical model that combines material balances and kinetic rate equations. The model accurately predicted Aß aggregation kinetics at various CatB and CysC concentrations. We derived approximate expressions for the half-times of degradation and aggregation and show that their ratio can be used to estimate, at any given Aß, CatB, or CysC concentration, whether Aß aggregation or degradation will result. Our results may be useful for designing experiments and interpreting results from investigations of manipulation of CysC concentration as an AD therapy.

Insights into the mechanism of cystatin C oligomer and amyloid formation and its interaction with ß-amyloid.

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, ß-amyloid. This result illustrates an example where heterotypic interactions between pre-amyloid oligomers prevent the homotypic interactions that would lead to mature amyloid fibrils.

Expression, purification, and characterization of human cystatin C monomers and oligomers.

Human cystatin C (cysC) is a soluble basic protein belonging to the cysteine protease inhibitor family. CysC is a potent inhibitor of cathepsins--proteolytic enzymes that degrade intracellular and endocytosed proteins, remodel extracellular matrix, and trigger apoptosis. Inhibition is via tight reversible binding involving the N-terminus as well as two ß-hairpin loops of cysC. As a significant component of cerebrospinal fluid, cysC has numerous other functions, including support of neural stem cell growth and differentiation. Several studies suggest that cysC may bind to the Alzheimer-related protein beta-amyloid (Aß), and inhibit its aggregation and toxicity. Because of an increasing recognition of its important biological roles, there is considerable interest in methods to produce full-length recombinant human cysC. Several researchers have reported success, but with processes that require multiple purification steps. Here we report successful production of human cysC using an intein-based expression system and a simple one-column purification scheme. The recombinant protein so obtained was natively folded and active as an enzyme inhibitor. Unexpectedly, even mild concentration by ultrafiltration caused significant oligomerization. The oligomers are noncovalent and retain the native secondary structure and inhibitory activity of the monomer. The oligomers, but not the monomers, were highly effective at inhibiting aggregation of Aß. These results demonstrate the critical importance of careful physicochemical characterization of recombinant cysC protein prior to evaluation of its biological functions.