Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions.

BACKGROUND: Cysteine cathepsins are known to primarily cleave their substrates at reducing and acidic conditions within endo-lysosomes. Nevertheless, they have also been linked to extracellular proteolysis, that is, in oxidizing and neutral environments. Although the impact of reducing or oxidizing conditions on proteolytic activity is a key to understand physiological protease functions, redox conditions have only rarely been considered in routine enzyme activity assays. Therefore we developed an assay to test for proteolytic processing of a natural substrate by cysteine cathepsins which accounts for redox potentials and pH values corresponding to the conditions in the extracellular space in comparison to those within endo-lysosomes of mammalian cells. RESULTS: The proteolytic potencies of cysteine cathepsins B, K, L and S towards thyroglobulin were analyzed under conditions simulating oxidizing versus reducing environments with neutral to acidic pH values. Thyroglobulin, the precursor molecule of thyroid hormones, was chosen as substrate, because it represents a natural target of cysteine cathepsins. Thyroglobulin processing involves thyroid hormone liberation which, under physiological circumstances, starts in the extracellular follicle lumen before being continued within endo-lysosomes. Our study shows that all cathepsins tested were capable of processing thyroglobulin at neutral and oxidizing conditions, although these are reportedly non-favorable for cysteine proteases. All analyzed cathepsins generated distinct fragments of thyroglobulin at extracellular versus endo-lysosomal conditions as demonstrated by SDS-PAGE followed by immunoblotting or N-terminal sequencing. Moreover, the thyroid hormone thyroxine was liberated by the action of cathepsin S at extracellular conditions, while cathepsins B, K and L worked most efficiently in this respect at endo-lysosomal conditions. CONCLUSION: The results revealed distinct cleavage patterns at all conditions analyzed, indicating compartment-specific processing of thyroglobulin by cysteine cathepsins. In particular, proteolytic activity of cathepsin S towards the substrate thyroglobulin can now be understood as instrumental for extracellular thyroid hormone liberation. Our study emphasizes that the proteolytic functions of cysteine cathepsins in the thyroid are not restricted to endo-lysosomes but include pivotal roles in extracellular substrate utilization. We conclude that understanding of the interplay and fine adjustment of protease networks in vivo is better approachable by simulating physiological conditions in protease activity assays.

Essential role of Pro 74 in stefin B amyloid-fibril formation: dual action of cyclophilin A on the process.

We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 degrees C, and it exhibited a prolonged lag phase at 30 degrees C and 37 degrees C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation.

Amyloid fibril formation by human stefin B: influence of pH and TFE on fibril growth and morphology.

As shown before, human stefin B (cystatin B) populates two partly unfolded species, a native-like state at pH 4.8 and a structured molten globule state at pH 3.3 (high ionic strength), from each of which amyloid fibrils grow. Here, we show that the fibrils obtained at pH 3.3 differ from those at pH 4.8 and that those obtained at pH 3.3 (protofibrils) do not transform readily to mature fibrils. In addition we show that amorphous aggregates are also a source of fibrils. The kinetics of amyloid fibril formation at different trifluoroethanol (TFE) concentrations were measured. TFE accelerates fibril growth at predenaturational concentrations of the alcohol. At concentrations higher than 10%, the fibrillar yield decreases proportionately as the population of an all alpha-helical, denatured form of the protein increases. At an optimum TFE concentration, the lag and the growth phases are observed, similarly to some other amyloidogenic proteins. Morphology of the protein species at the beginning and the end of the reactions was observed using atomic force microscopy and transmission electron microscopy. Final fibril morphologies differ depending on solvent conditions.

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.

Folding and amyloid-fibril formation for a series of human stefins' chimeras: any correlation?

To study the influence of whole secondary structure elements to the process of folding and amyloid-fibril formation, chimeras of stefins have been prepared. GdnHCl denaturation curves and folding rates (chevron plots) have been analyzed based on a two-state mechanism. The order of stability is: stefin A > aAbbbb > bAbbbb > stefin B = aBaaaa > bBaaaa, where the make up of chimeric proteins is designated by small letters representing the source of individual strands (a for stefin A, b for stefin B) and a capital letter representing the source of the helix (A for stefin A and B for stefin B). Only the fast folding reactions were included in the analysis and it has been found that stefin B folds the fastest (657 s(-1)). Similarly, fast folders are the chimeric proteins aBaaaa and bBaaaa, both of which contain the alpha-helix of stefin B. Unfolding rates correlate very well with protein stability, with the slowest rate for the most stable protein, stefin A. Amyloid-fibril growth was measured for each protein by monitoring thioflavin T fluorescence and was visualized using electron microscopy. The propensity to form amyloid-fibrils is in the order: stefin B > bAbbbb > aAbbbb > bBaaaa > aBaaaa > stefin A. This order does not correlate with stability, or with the folding or unfolding rates. Instead, the propensity to fibrillize is related to selected parts of structure, such as the beta-sheet of stefin B, and can be predicted reasonably well by calculating the beta-strand propensity of the denatured states.