Cysteine cathepsins are proteases found in lysosomes and play a crucial role in intercellular protein degradation. During pathological states such as breast cancer they are increasingly secreted into the extracellular space, where they can contribute to disease development. One of the main mechanisms during increased proteolytic activity in the extracellular space, which was also linked to cancer development, is ectodomain shedding. Among others, some proteases classified as cysteine cathepsins are known to be sheddases. For modelling the behaviour of proteases and thus determining their role in ectodomain shedding, it is crucial to identify all substrates of each sheddase at multiple different cell lines. Until now the protein substrates of cathepsins during ectodomain shedding at the cell surface of HER2-positive cell line SK-BR-3 have not yet been identified. Using proteomic screening of the shed peptides we identified 45 protein substrates of cathepsins K, L and S on the surface of the aforementioned cell line. Western blot analysis was performed against plexin B2, EGFR and HER2 both on SK-BR-3 and MDA-MB-231 cell lines to confirm the results of the proteomic analysis. The impact of ectodomain shedding using cathepsins on cell migration was tested with Boyden Transwell test and Wound healing test on cell lines MDA-MB-231 and SK-BR-3. However, the results of the migration assays did not allow us to draw a conclusion about the influence of cathepsin shedding on cell migration of the cell line SK-BR-3.
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