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Abstract

Cysteine cathepsins are endosomal proteases that are involved in lysosomal protein degradation. Additionally, they engage in specific biological processes, like bone degradation (CatK), antigen presentation (CatS) and pro-hormone processing (CatV and CatL). Their enhanced activity was described in numerous pathological states, like cancer and viral activation, which made them promising drug targets. Yet their specificity remains elusive because methodology and tools applied could not match the complexity of their processing patterns. Recent statistical analysis of cleavage patterns from large-scale proteomics data enabled us to address this issue. Thirty peptides, representing a variety of all seven major clusters of CatV substrates, were selected and synthesized. Their interactions with the active site mutant of CatV were analyzed in crystal structures, and their cleavage patterns by the native CatL, K, and V were studied in solution.
Over 20 crystal structures of CatV-peptide complexes were determined. They were grouped into four binding patterns, based on their binding location or cleavage event. They interacted with the cathepsin surface at the sites S4 – S6’. Superimposition of structures showed that the residues at positions with non-normal distribution of residues, called heterogeneous positions, reflect the cathepsin specificity. Peptidyl residues at such positions bound to the rigid cathepsin regions. In contrast, the residues at positions with normal distribution of residues, called homogeneous positions, do not reflect cathepsin specificity. Peptidyl residues at such positions exploited the protease structural variability, sometimes also inducing it. By analyzing the structures of CatK, L, and S, from PDB database, we showed that the same conclusions can be applied to them, too. Moreover, we were able to pinpoint to the structural areas and the contributing residues responsible for specificity of CatL substrates at P3 and CatL, V, and F substrates at P1’. These areas can be targeted to enhance selective inhibition of cathepsin endopeptidases.
We compared cleavages of peptides and proteins, carrying the same primary sequence, and found that several were cleaved at different places when they were in the peptidyl form or as a part of the protein structure. The comparison of peptide complexes with the sole crystal complex of CatL and a protein substrate, published in the PDB database, suggested that substrate specificity of cathepsins cannot be explained by contributions of individual amino acids, but by the combined effect of the substrate as a whole. Hence, the knowledge gathered from the peptide processing does not necessarily apply to the processing of protein substrates and vice versa.
In addition, we determined the crystal complex of CatK and an alkyne-based inhibitor. The electron density indisputably confirmed the covalent bond between the catalytic Cys of CatK and the alkyne of inhibitor, showing that the alkyne warhead can be used as latent electrophile for targeting proteases. In addition, we showed that calpeptin and compounds alike, which inhibit Mpro protease of the SARS-CoV-2 virus and suppress viral activation in cell-based assays, strongly inhibit the human CatL, K, V, and to a lesser extent also CatB. This suggests that the viral suppression mediated by calpeptin may primarily be due to the cathepsin inhibition rather than inhibition of Mpro. To provide further support, we determined the crystal structure of CatV-calpeptin complex.