title: 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.
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.
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia; Departments of Biochemistry and Molecular Biology and Oral Biological and Medical Sciences, Centre for Blood Research and Faculty Dentistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Claire H
authors.initials: CH
authors.lastname: Wilson
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia; Flinders Centre for Innovation in Cancer, Flinders University, Adelaide, South Australia 5001, Australia.
authors.forename: Dono
authors.initials: D
authors.lastname: Indarto
authors.affiliation: Departments of Biochemistry and Molecular Biology and Oral Biological and Medical Sciences, Centre for Blood Research and Faculty Dentistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Alain
authors.initials: A
authors.lastname: Doucet
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia; Flinders Centre for Innovation in Cancer, Flinders University, Adelaide, South Australia 5001, Australia.
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Pogson
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia.
authors.forename: Melissa R
authors.initials: MR
authors.lastname: Pitman
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia.
authors.forename: Kym
authors.initials: K
authors.lastname: McNicholas
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia.
authors.forename: R Ian
authors.initials: RI
authors.lastname: Menz
authors.affiliation: Departments of Biochemistry and Molecular Biology and Oral Biological and Medical Sciences, Centre for Blood Research and Faculty Dentistry, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Electronic address: chris.overall@ubc.ca.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
authors.affiliation: School of Biological Sciences, Flinders University, Adelaide, South Australia 5001, Australia; Flinders Centre for Innovation in Cancer, Flinders University, Adelaide, South Australia 5001, Australia. Electronic address: cathy.abbott@flinders.edu.au.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Abbott
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism*; D000595:Amino Acid Sequence; D037282:Calreticulin / Q000378:metabolism*; D002412:Cations; D045744:Cell Line, Tumor; D002469:Cell Separation; D003593:Cytoplasm / Q000378:metabolism; D004150:Dipeptidases / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism*; D004734:Energy Metabolism; D005434:Flow Cytometry; D006706:Homeostasis; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D037282:Calreticulin; D002412:Cations; D000263:Adenylate Kinase; C110938:adenylate kinase 2; D004150:Dipeptidases; C471241:DPP9 protein, human; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C487997:DPP8 protein, human
keywords: Adenylate Kinase; Aminopeptidase; Calreticulin; Cell Metabolism; DP8/DPP8; DP9/DPP9; Dipeptidyl Peptidase; Energy Metabolism; Enzymes; Proteomics