Abstract
Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease.
Highlights
Biological roles for intracellular dipeptidyl peptidases 8 and 9 are unknown
In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression
All cell lines were found to express basal levels of endogenous DP8 and DP9 protein (ϳ98 kDa in mobility) (Fig. 1B) that may contribute to background proteolysis and affect the discovery of substrates by terminal amine isotopic labeling of substrates (TAILS)
Summary
Biological roles for intracellular dipeptidyl peptidases 8 and 9 are unknown. Results: By degradomics, 29 new in vivo substrates were identified (nine validated) for DP8/DP9, including adenylate kinase 2 and calreticulin. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis These results demonstrate remarkable in vivo substrate overlap between DP8/ DP9, suggesting compensatory roles for these enzymes. Ubiquitous but differential expression of DP8/DP9 has been observed in breast and ovarian carcinoma cell lines [14], and a study has identified DP8/DP9 as survival factors for the Ewing sarcoma family of tumors [15]. As DP8 and DP9 are intracellular enzymes, it is unlikely that these secreted substrates will be of physiological relevance in vivo Clues for their in vivo roles come from studies blocking DP8/DP9 or DPIV expression, which leads to neuropeptide Y-driven cell death within the Ewing sarcoma family of tumors cells [15]. This work reveals the involvement of DP8 and DP9 in cellular energy homeostasis pathways in this ovarian cancer cell line
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