TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

Abstract

Deregulated cathepsin proteolysis occurs across numerous cancers, but invivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS), a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Zinvivo substrates and cleavage sites with the use ofsix different cathepsin knockout genotypes in theRip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114N termini identified by TAILS, stable proteolyticproducts were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%-44% of 139 cleavages). This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%-83%) for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.