The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gln (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 Angstrom resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.
