1. Identifying Natural Substrates for Dipeptidyl Peptidases 8 and 9 Using Terminal Amine Isotopic Labeling of Substrates (TAILS) Reveals in Vivo Roles in Cellular Homeostasis and Energy Metabolism*♦
- Author
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Kym McNicholas, Claire H. Wilson, Catherine A. Abbott, Lisa D. Pogson, R. Ian Menz, Alain Doucet, Christopher M. Overall, Dono Indarto, Melissa R. Pitman, Wilson, Claire H, Indarto, Dono, Doucet, Alain, Pogson, Lisa D, Pitman, Melissa R, McNicholas, Kym, Menz, R Ian, Overall, Christopher M, and Abbott, Catherine A
- Subjects
Proteomics ,Cytoplasm ,Dipeptidases ,Cellular homeostasis ,Cell Separation ,Biochemistry ,DP9/DPP9 ,Mass Spectrometry ,dipeptidyl peptidase ,Substrate Specificity ,calreticulin ,Dipeptidyl Peptidase 9 ,cell metabolism ,energy metabolism ,Homeostasis ,chemistry.chemical_classification ,0303 health sciences ,aminopeptidase ,030302 biochemistry & molecular biology ,Terminal amine isotopic labeling of substrates ,Flow Cytometry ,Cell biology ,Enzymes ,Isotope Labeling ,DP8/DPP8 ,Aminopeptidase ,enzymes ,Dipeptidyl Peptidase ,Molecular Sequence Data ,Adenylate kinase ,Biology ,Dipeptidyl peptidase ,adenylate kinase ,03 medical and health sciences ,proteomics ,In vivo ,Cations ,Cell Line, Tumor ,Humans ,Amino Acid Sequence ,Dipeptidyl-Peptidases and Tripeptidyl-Peptidases ,Molecular Biology ,Dipeptidyl peptidase-4 ,030304 developmental biology ,Adenylate Kinase ,Cell Biology ,Cell Metabolism ,Protein Structure, Tertiary ,Enzyme ,chemistry ,Enzymology ,Energy Metabolism ,Calreticulin - Abstract
Background: 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. Conclusion: These substrates indicate roles for DP8 and DP9 in metabolism and energy homeostasis. Significance: Being the first proteomics screen for DP8/DP9 substrates, unexpected new cellular roles were revealed., 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.
- Published
- 2013