Proteolysis of prion protein by cathepsin S generates a soluble beta-structured intermediate oligomeric form, with potential implications for neurotoxic mechanisms.

Formation of PrP aggregates is considered to be a characteristic event in the pathogenesis of TSE diseases, accompanied by brain inflammation and neurodegeneration. Factors identified as contributing to aggregate formation are of interest as potential therapeutic targets. We report that in vitro proteolysis of ovine PrP(94-233) (at neutral pH and in the absence of denaturants) by the protease cathepsin S, a cellular enzyme that also shows enhanced expression in pathogenic conditions, occurs selectively in the region 135-156. This results in an unusually efficient, concentration-dependent conformational conversion of a large subfragment of PrP(94-233) into a soluble beta-structured oligomeric intermediate species, that readily forms a thioflavin-T-positive aggregate. N-terminal sequencing of the proteolysis fragments shows the aggregating species have marked sequence similarities to truncated PrP variants known to confer unusually severe pathogenicity when transgenically expressed in PrP(o/o) mice. Circular dichroism analysis shows that PrP fragments 138-233, 144-233 and 156-233 are significantly less stable than PrP(94-233). This implies an important structural contribution of the beta1 sequence within the globular domain of PrP. We propose that the removal or detachment of the beta1 sequence enhances beta-oligomer formation from the globular domain, leading to aggregation. The cellular implications are that specific proteases may have an important role in the generation of membrane-bound, potentially toxic, beta-oligomeric PrP species in pre-amyloid states of prion diseases. Such species may induce cell death by lysis, and also contribute to the transport of PrP to neuronal targets with subsequent amplification of pathogenic effects.

Essential role of proline isomerization in stefin B tetramer formation.

Here we present the tetrameric structure of stefin B, which is the result of a process by which two domain-swapped dimers of stefin B are transformed into tetramers. The transformation involves a previously unidentified process of extensive intermolecular contacts, termed hand shaking, which occurs concurrently with trans to cis isomerization of proline 74. This proline residue is widely conserved throughout the cystatin superfamily, a member of which, human cystatin C, is the key protein in cerebral amyloid angiopathy. These results are consistent with the hypothesis that isomerization of proline residues can play a decisive role in amyloidogenesis.

Crystal structure of NS-134 in complex with bovine cathepsin B: a two-headed epoxysuccinyl inhibitor extends along the entire active-site cleft.

The crystal structure of the inhibitor NS-134 in complex with bovine cathepsin B reveals that functional groups attached to both sides of the epoxysuccinyl reactive group bind to the part of active-site cleft as predicted. The -Leu-Pro-OH side binds to the primed binding sites interacting with the His110 and His111 residues with its C-terminal carboxy group, whereas the -Leu-Gly-Meu (-Leu-Gly-Gly-OMe) part (Meu, methoxycarbonylmethyl) binds along the non-primed binding sites. Comparison with the propeptide structures of cathepsins revealed that the binding of the latter part is least similar to the procathepsin B structure; this result, together with the two-residue shift in positioning of the Leu-Gly-Gly part, suggests that the propeptide structures of the cognate enzymes may not be the best starting point for the design of reverse binding inhibitors.

The role of cathepsin C in Papillon-Lefèvre syndrome, prepubertal periodontitis, and aggressive periodontitis.

We have previously reported that loss-of-function mutations in the cathepsin C gene (CTSC) result in Papillon-Lefèvre syndrome, an autosomal recessive condition characterized by palmoplantar keratosis and early-onset, severe periodontitis. Others have also reported CTSC mutations in patients with severe prepubertal periodontitis, but without any skin manifestations. The possible role of CTSC variants in more common types of non-mendelian, early-onset, severe periodontitis ("aggressive periodontitis") has not been investigated. In this study, we have investigated the role of CTSC in all three conditions. We demonstrate that PLS is genetically homogeneous and the mutation spectrum that includes three novel mutations (c.386T>A/p.V129E, c.935A>G/p.Q312R, and c.1235A>G/p.Y412C) in 21 PLS families (including eight from our previous study) provides an insight into structure-function relationships of CTSC. Our data also suggest that a complete loss-of-function appears to be necessary for the manifestation of the phenotype, making it unlikely that weak CTSC mutations are a cause of aggressive periodontitis. This was confirmed by analyses of the CTSC activity in 30 subjects with aggressive periodontitis and age-sex matched controls, which demonstrated that there was no significant difference between these two groups (1,728.7 +/- SD 576.8 micro moles/mg/min vs. 1,678.7 +/- SD 527.2 micro moles/mg/min, respectively, p = 0.73). CTSC mutations were detected in only one of two families with prepubertal periodontitis; these did not form a separate functional class with respect to those observed in classical PLS. The affected individuals in the other prepubertal periodontitis family not only lacked CTSC mutations, but in addition did not share the haplotypes at the CTSC locus. These data suggest that prepubertal periodontitis is a genetically heterogeneous disease that, in some families, just represents a partially penetrant PLS.