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. 2013 May 17;288(20):13936-13949.
doi: 10.1074/jbc.M112.445841. Epub 2013 Mar 21.

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 (V体育官网入口)

Claire H Wilson  1 , Dono Indarto  2 , Alain Doucet  3 , Lisa D Pogson  2 , Melissa R Pitman  4 , Kym McNicholas  4 , R Ian Menz  4 , Christopher M Overall  5 , Catherine A Abbott  6
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V体育平台登录 - 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

Claire H Wilson et al. J Biol Chem. .

Abstract (VSports app下载)

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 VSports手机版. .

Keywords: Adenylate Kinase; Aminopeptidase; Calreticulin; Cell Metabolism; DP8/DPP8; DP9/DPP9; Dipeptidyl Peptidase; Energy Metabolism; Enzymes; Proteomics. V体育安卓版.

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Figures

FIGURE 1.
FIGURE 1.
Characterization of stable wild-type and mutant DP8 and DP9 SKOV3 cell lines. A, cells transfected with wild-type (DP8-EGFP and DP9-EGFP) or mutant (DP8(S739)-EGFP and DP9(S729A)-EGFP) constructs (filled histograms) or nontransfected cells (open histograms) were analyzed by fluorescent flow cytometry on a FACScan. B and C, cell lysates (50 μg) from DP8-EGFP, DP8(S739A)-EGFP, DP9-EGFP, DP9(S729A)-EGFP, vector-transfected, and nontransfected SKOV3 cells were analyzed by 8% (w/v) SDS-PAGE and immunoblotting for detection of DP8 (B) or DP9 (C). Recombinant purified DP8 and DP9 were included as controls and are indicated by arrows. For a loading control, β-actin was detected. D and E, specific activity against the synthetic DP substrate H-Ala-Pro-p-nitroanilide (0.5 mm) was determined in whole cells (D) and in membrane and soluble fractions (E). Values in D are expressed as means ± S.E. (n = 10). Values in E are from a single experiment.
FIGURE 2.
FIGURE 2.
Summary of total peptides and proteins identified by TAILS analysis. A and B, three-way Venn diagrams of the number of unique peptides, unique stripped peptides, and unique proteins (identified by more then one spectra) identified by MASCOT searches in experiments (Exp.) 1, Exp. 2, and Exp. 3 for DP8 (A) and DP9 (B). C, two-way Venn diagram of unique proteins (identified by more then one hit) identified from all datasets for DP8 and DP9. The intersection of C displays the number of overlapping proteins identified in both the DP8 and DP9 datasets. The number of unique peptides for each experiment takes into account all possible modifications of a given peptide, including variable oxidation of methionine residues, although the number of unique stripped peptides for each experiment refers to unique peptides after the removal (stripping) of all possible modifications. The total number of unique proteins excludes any “single hit” proteins, i.e. a protein that is not identified by any other peptide/spectra in the dataset. All peptides were identified with ≥95% confidence according to PeptideProphet. All MASCOT searches were performed against the human IPI database (version 3.16, 62,322 entries, release date 4/2006).
FIGURE 3.
FIGURE 3.
Peptides of several candidate substrates identified by TAILS. Peptides identified by MS/MS are indicated by bold underlined letters. Light colored letters indicate precursor peptides of mature proteins or their initiator Met residues that are known to be removed as annotated in UniProtKB. The dipeptide residues identified as being cleaved, or as having the potential for cleavage, are outlined by a box. Where two peptides were identified by MS/MS as being the noncleaved DP8/DP9 precursor peptide (N-terminal dipeptide intact) and the DP8/DP9 cleavage product peptide (N-terminal dipeptide removed), an arrow is used to indicate the site of DP8/DP9 proteolysis. Peptides for C-1-tetrahydrofolate synthase and cytoplasmic and lysosomal protective proteins were identified from manual parsing of the DP8 dataset. All other proteins were identified in both DP8 and DP9 datasets. The peptides corresponding to three of the potential cleavage sites indicated for fructose-bisphosphate aldolase A were only identified from manual parsing of the DP9 dataset (see Table 1). Full-length protein sequences of the above and other potential substrates can be found in supplemental Figs. S2 and S3.
FIGURE 4.
FIGURE 4.
Frequency distribution of residues in P1 and P2 position of DP8 and DP9 candidate substrates. The frequency distribution of amino acids in the P1 (A and B) and P2 (C and D) position of candidate substrates identified in Table 3 are given for DP8 (A and C) and DP9 (B and D). The exact number of peptides in which each residue is found in the P1 or P2 position is given along with the percentage relating to frequency distribution.
FIGURE 5.
FIGURE 5.
DP8 and DP9 cleave the N-terminal peptide of adenylate kinase 2 in vitro. The N-terminal peptide of mature adenylate kinase 2 is displayed. The arrow indicates the site of DP8/DP9 proteolysis with the gray text indicating the dipeptide that is removed following DP8/DP9 proteolysis. A, 10 μm of the adenylate kinase 2 peptide was incubated alone or with 1.7 milliunits of active purified recombinant DP8 or DP9. Samples were collected and stopped with 1% TFA (v/v) final at 0, 1, and 4 h. MS spectra were acquired for noncleaved (m/z) and DP8/DP9-cleaved (m/z) peptides with a mass accuracy of 0.01 to 0.05% error. Theoretical masses of noncleaved and cleaved peptides are 1681.89 and 1512.79 Da, respectively. B, specificity of cleavage by DP8/DP9 of these peptides was confirmed by performing catalysis reactions in the presence of 10 μm of the nonselective DP inhibitor, Val-Boro-Pro (PT-100/Talabostat). Displayed spectra are representatives from three independent cleavage experiments. AK2, adenylate kinase 2.
FIGURE 6.
FIGURE 6.
DP8 and DP9 cleave the N-terminal peptide of calreticulin in vitro. The N-terminal peptide of mature calreticulin is displayed. The arrow indicates the site of DP8/DP9 proteolysis, and the gray text indicates the dipeptide that is removed following DP8/DP9 proteolysis. A, 10 μm of the calreticulin was incubated alone or with 1.7 milliunits of active and purified recombinant DP8 or DP9. Samples were collected and stopped with 1% TFA (v/v) final at 0, 1, and 4 h. MS spectra were acquired for noncleaved (m/z) and DP8/DP9-cleaved (m/z) peptides with a mass accuracy of 0.01 to 0.05% error. Theoretical masses of noncleaved and cleaved peptides were 2245.45 and 2017.95 Da, respectively. B, specificity of cleavage by DP8/DP9 of these peptides was confirmed by performing catalysis reactions in the presence of 10 μm of the nonselective DP inhibitor, Val-Boro-Pro (PT-100/Talabostat). Displayed spectra are representatives from three independent cleavage experiments. CRT, calreticulin.
FIGURE 7.
FIGURE 7.
Expression and co-localization of adenylate kinase 2 and calreticulin with DP8 and DP9 in SKOV3 cells. A, DP8-EGFP- and DP9-EGFP SKOV3-expressing cells lines were analyzed by confocal microscopy and immunofluorescence using anti-calreticulin (1:50) and anti-adenylate kinase 2 (1:50) polyclonal antibodies. As labeled, red panels display adenylate kinase 2 or calreticulin, and green panels display DP8-EGFP or DP9-EGFP expression. Merged images are shown in the far right-hand panels. B, cell lysates (25 μg) from DP8-EGFP, DP8(S739A)-EGFP, DP9-EGFP, DP9(S729A)-EGFP, vector-transfected, and nontransfected SKOV3 cells were analyzed by 10% (w/v) SDS-PAGE and immunoblotting using DP8 (1:5000), anti DP9 (1:5000), calreticulin (1:10,000), adenylate kinase 2(1:5000), and β-actin (1:10,000) as a loading control. AK2, adenylate kinase 2; CRT, calreticulin.
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