Structural biology provides insights in the mechanisms of biological processes by delivering structures of macromolecules that link biological sequence information to their position in 3-dimensional space. This allows us to combine structural information with data obtained through biochemical, molecular, cell biology, and computational approaches and integrate them into increasingly comprehensive research schemes.
The primary role of endosomal/lysosomal system is protein degradation (Turk et al., 2012). In addition to nonspecific proteolysis in the acidic environment of endosomes/lysosomes, proteolytic enzymes cysteine cathepsins also have specific roles, such as the cleavage of collagen by cathepsin K in bone remodeling, the processing of thyroglobulin in the production of thyroid hormones. They are also involved in the progression of tumors and in the activation of viruses such as SARS-CoV-2 and Ebola. Because of their specific physiological and pathological functions, cysteine cathepsins are important targets for drug discovery.
Our recently published study and statistical analysis of approximately 30,000 substrates of cathepsins K, V, B, L, S and F revealed a large number of proteins in the cell lysate, of which 941 were cleaved only once (Tušar et al., 2023). This list of substrates included transcription factors with cleavages at the sites where they bind DNA (Li et al., 2016) or kinases at the sites where they perform phosphorylation (Lolli et al., 2012). The analysis was performed by the software platform SAPS-ESI (“Statistical Approach to Specific Peptide Substrate-Enzyme Interaction”) developed in our laboratory.
The aim of this research project is to reveal new physiological roles of cysteine cathepsins based on the determination of features of shared substrates of cysteine cathepsins and other enzymes using new approaches in statistics and bioinformatics (upgrading SAPS – ESI) and in experimental methods. To confirm the physiological role of enzymes combinations and their shared substrates, some selected protein substrates will be expressed and their degradation and possible complex formation analyzed by biochemical, structural and molecular biology, and proteomic approaches in vitro and also in cells. The research also includes the determination of the structures of complexes of peptide fragments with enzymes and their structural analysis.
Reference
- Turk et al., Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochimica et biophysica acta 1824, 68-88 (2012).
- Tušar L., Loboda J., Turk D. et al., Proteomic data and structure analysis combined reveal interplay of structural rigidity and flexibility on selectivity of cysteine cathepsins. Communications biology 6, 450 (2023).
- Li et al., Structural basis for DNA recognition by STAT6. Proceedings of the National Academy of Sciences of the United States of America 113, 13015-13020 (2016).
- Lolli, L. A. Pinna, R. Battistutta, Structural determinants of protein kinase CK2 regulation by autoinhibitory polymerization. ACS chemical biology 7, 1158-1163 (2012).