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Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Molecular Cancer Research Review Advances in Understanding the Expression and Function of Dipeptidyl Peptidase 8 and 9 Hui Zhang, Yiqian Chen, Fiona M. Keane, and Mark D. Gorrell Abstract DPP8 and DPP9 are recently identified members of the dipeptidyl peptidase IV (DPPIV) enzyme family, which is characterized by the rare ability to cleave a post-proline bond two residues from the N-terminus of a substrate. DPP8 and DPP9 have unique cellular localization patterns, are ubiquitously expressed in tissues and cell lines, and evidence suggests important contributions to various biological processes including: cell behavior, cancer biology, disease pathogenesis, and immune responses. Importantly, functional differences between these two proteins have emerged, such as DPP8 may be more associated with gut inflammation whereas DPP9 is involved in antigen presentation and intracellular signaling. Similarly, the DPP9 connections with H-Ras and SUMO1, and its role in AKT1 pathway downregulation provide essential insights into the molecular mechanisms of DPP9 action. The recent discovery of novel natural substrates of DPP8 and DPP9 highlights the potential role of these proteases in energy metabolism and homeostasis. This review focuses on the recent progress made with these post-proline dipeptidyl peptidases and underscores their emerging importance. Mol Cancer Res; 11(12); 1487–96. 2013 AACR. Introduction Proteases are efficient processing molecules involved in the physiological maintenance of all cells. They can no longer simply be thought of as essential for protein degradation and recycling; they are also vital regulators and signaling molecules (1). They control all aspects of cellular life by modifying their targets to alter location and function. In doing so, they have crucial roles in both normal and pathological processes and are thus important targets for drug treatment. One such target is the ubiquitous serine protease, dipeptidyl peptidase IV (DPPIV), with inhibitors of this enzyme now in clinical use to treat type 2 diabetes. DPPIV and related enzymes are members of the S9b protease subfamily, with a conserved catalytic triad of serine, aspartate, and histidine (2,3) and a rare ability to cleave a post-proline bond 3 residues from the N-terminus of a substrate (2, 4–7). DPPIV rapidly cleaves and inactivates insulinotropic hormones (incretins) and thus inhibitors of this enzyme are the basis for the treatment of type 2 diabetes (8,9). Other members of this family include DPP8, DPP9, and fibroblast activation protein (FAP), which are of interest in cancer research. FAP is generally expressed at low levels in normal adult tissue but is greatly upregulated in diseased and remodeling tissue as well as in tumors (10,11). Indeed, both FAP and DPPIV are multifunctional and have roles in Authors' Affiliation: Molecular Hepatology, Centenary Institute and Sydney Medical School, University of Sydney, NSW, Australia Corresponding Author: Mark D. Gorrell, Molecular Hepatology, Centenary Institute, Locked Bag No. 6, Newtown, NSW 2042, Australia. Phone: 61-295656156; Fax: 61-2-95656101; E-mail: [email protected] doi: 10.1158/1541-7786.MCR-13-0272 2013 American Association for Cancer Research. metabolic processes of obesity and diabetes, the immune system, and cancer biology (7,12–15). Structurally related to DPPIV and with overlapping proteolytic activity, DPP8 and DPP9 remain less understood but are gaining increasing research attention to decipher their unique proteolytic roles. To identify and characterize DPP8 and DPP9, both proteases were cloned, sequenced, and discovered to display considerable sequence homology with DPPIV and thus also likely structural similarity (2,3). DPP8 and DPP9, however have a different cellular localization to that of DPPIV, as well as some differences in their expression profile and are therefore likely to have distinct roles. In the past, treatment with a broad DPP family inhibitor produced functional changes in diabetes, the immune system, the inflammatory response, and cancer biology (13,16,17). Although these effects were attributed to DPPIV at the time, it is now thought that inhibition of DPP8 and DPP9 contributed to the above outcomes. With more selective DPP8/9 inhibitors being developed (18–20), there is now an opportunity to delineate the roles of these enzymes and further explore the therapeutic potential of their chemical inhibitors. The search for DPP8- and DPP9-specific substrates is ongoing, with the challenge remaining to determine those that are most physiologically relevant. Although DPPIV has been reviewed at length, few authors have focused specifically on DPP8 and DPP9. Here we outline the molecular and biochemical characteristics of DPP8 and DPP9 so as to gain a comprehensive understanding of these post-proline dipeptidyl peptidases (Fig. 1). Structural Properties of DPP8 and DPP9 Initial identification and cloning of DPP8 was established by Abbott and colleagues (2). DPP9 was then identified by www.aacrjournals.org Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. 1487 Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Zhang et al. Proliferation Apoptosis EGF Akt ECM interaction adhesion migration Cell behavior Immune organs Spleen Thymus Lung Immunology Colitis DPP9 DPP8 H-Ras H-Ras Alcoholic liver disease Liver fibrosis Figure 1. Overview of organs and processes in which DPP8 and DPP9 have potential functions, along with molecules that interact with these proteases. Inflammation Lymphocyte Tumor biology Liver disease Testicular tumors Biliary cirrhosis BLAST search and sequence alignment with DPP8. With growing interest in these novel homologues of DPPIV, several research groups have cloned DPP8 and DPP9 (3,21–23). Comparisons of the main physical attributes of DPPIV, FAP, DPP8, and DPP9 are summarized in Table 1. Tumor cell lines Chronic lymphocytic leukemia Gene Properties DPP8 and DPP9 have been localized by fluorescence in situ hybridization to human chromosome 15q22 and 19p13.3, respectively (2,3). A variety of diseases have been mapped to these loci, such as pulmonary fibrosis (27), ovarian adenocarcinomas (28), and Bardet–Biedl Table 1. Comparative overviews of the 4 enzyme members of the DPPIV family DPPIV FAP DPP8 short Synonyms GenBank accession References Gene location Human Mouse Number of exons Gene size (kb) mRNA (bp) Number of amino acids Monomer mobility Dimer Transmembrane domain Catalytic triad Enzyme activity CD26 M74777 (24) Seprase U09278 (4,25,26) AF221634 (2) 2q24.3 2:62330073 26 81.8 2924 766 2q23 2:62500945 26 72.8 2814 760 20 71 3127 882 110 kDa 95 kDa 100 kDa H H H H H H Ser630-Asp708-His740 Dipeptidyl peptidase Ser624-Asp702-His734 Dipeptidyl peptidase and endopeptidase Cell surface and soluble Ser739-Asp817-His849 Dipeptidyl peptidase Ser730-Asp808-His840 Dipeptidyl peptidase Intracellular Intracellular Cellular localization Cell surface and soluble DPP8-v3 long DPP9 longa Attribute DPRP1 AF354202 (21) 15q22.32 9:65032458 22 ? 3030 898 103 kDa DPP9 short DPRP2 AY374518 AF542510 (3,23) (23) 19q13.3 17:56186682 19 22 47.3 48.6 2592 3006 863 971 95–110 kDa ? yes; , no; ?, not known. The long form is a splice variant. H, a 1488 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Dipeptidyl Peptidases 8 and 9 syndrome type 4, which has the clinical manifestation of obesity (29). Thus, DPP8 and DPP9 are potentially associated with the pathogenesis of such diseases, but an underlying mechanism has not been elucidated. Adolescent idiopathic scoliosis maps to 19p13.3, but it is not associated with DPP9 (30). The human DPP4 gene spans 81.8 kb and contains 26 exons ranging in size from 45 to 1,386 bp (31). In contrast, the human DPP9 gene spans only 48.6 kb of genomic sequence and comprises 22 exons that are 53 to 1431 bp in length (23). The human DPP8 gene spans 71 kb and consists of 20 exons (2). The sequence around the active-site serine is encoded by a single exon in DPP8 and DPP9, but the homologous region in DPP4 and FAP is encoded by 2 exons (3,31), which suggests that DPP8 and DPP9 are the more ancient genes of the 4. Protein Model Protein structure is crucial for substrate binding and specificity, as well as for interacting with binding partners. The primary structure of human DPP8 contains 882 amino acids with 27% identity and 51% amino acid similarity to human DPPIV (2). Two forms of DPP9 have been cloned. A ubiquitously expressed transcript encoding 863 amino acids (short form) displays 26% identity and 47% similarity to human DPPIV (23). The most significant homology is observed between human DPP8 and DPP9 (61% identity and 79% similarity at the amino acid level; ref. 23). The amino acid sequences of both proteins are highly homologous between human and mouse, with human DPP9 having 92% identity and 95% similarity to mouse DPP9 and human DPP8 having 95% identity and 98% similarity to mouse DPP8 (3,23). Mouse DPP8 and DPP9 enzymes have not been isolated for comparisons with their human counterparts in enzyme specificity and kinetics studies. Given that the crystal structures of DPP8 and DPP9 have not yet been solved, protein homology modeling has shown that DPP8 and DPP9 likely share a similar tertiary structure with DPPIV and FAP (32,33). DPP8 (34) and DPP9 (35) are dimeric, with each monomer likely consisting of an a/b-hydrolase domain and an 8 blade b-propeller domain and the active site of the peptidase locates at the interface of these 2 domains (Fig. 2). The DPPIV family of proteins share a conserved catalytic triad, with that of DPP8 consisting of Ser739, Asp817, and His849 and the equivalent residues in DPP9 being Ser730, Asp808, and His840 (3,23). A distinguishing feature of the DPPIV protein family is that 2 important glutamates lie near the start of a loop that protrudes from the b-propeller domain and borders the substrate entry site (32) and these 2 glutamates are essential for catalysis in DPP8 (36), DPPIV (37), and FAP (38). Despite the close sequence and structure homology with DPPIV, DPP8, and DPP9 possess several distinct structural properties. First, DPP8 has a larger substrate pocket (S2) than DPPIV (39). This suggests that DPP8 and DPP9 might have either an additional element of tertiary structure at the N-terminus and/or a larger b-propeller domain. Second, www.aacrjournals.org Figure 2. A model of DPP9 protein structure. DPP9 residues 51 to 863 are depicted in ribbon representation of the monomer. The a/b-hydrolase domain is in red and the 8-blade b-propeller domain in green. The catalytic triad (Ser-Asp-His) is shown as blue spheres and the 2 glutamates essential for catalysis as yellow spheres. An extended arm 285 304 ( VEVIHVPSPALEERKTDSYR ) in the propeller surface of DPP9 that is critical in SUMO1–DPP9 interaction is colored pink. single-point mutations in the C-terminal loop, such as F822A, V833A, Y844A, and H859A in DPP8 (34), or F842A in DPP9 (40) inactivate these proteases without disrupting dimerization. Similarly, a deletion mutation (residues 317-334) or a single-point mutation (Y334A) in a propeller loop in DPP9 can cause decreased activity without affecting dimerization (35). Thus, the C-terminal loop and the propeller loop are essential for DPP8 and DPP9 enzyme activity but not for dimerization. Third, DPP8 and DPP9 lack a transmembrane domain and are translated as intracellular proteins (2,23), whereas DPPIV and FAP are cell surface expressed type II membrane glycoproteins. Posttranslational Modification Posttranslational modifications (PTM) regulate protein activity, localization, and interaction with other cellular molecules. The DPP8 sequence contains no N-linked or O-linked glycosylation sites (2) and, despite identification of 2 potential N-glycosylation sites in DPP9, no evidence of glycosylation has been found (3,23,40). Lysine 51 and Lysine 314 in DPP9 have been identified as potential acetylation targets in 2 separate proteomic and mass spectrometric acetylation studies (41,42), but this has not been verified in vivo. Further investigation is thus necessary to understand PTMs of DPP8 and DPP9, which may lead to alteration of their localization, function, and enzyme activity. Mol Cancer Res; 11(12) December 2013 Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. 1489 Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Zhang et al. DPP8 and DPP9 Activity Regulation DPP8 and DPP9 specifically interact with SUMO1 (43) and a novel SUMO1-binding arm in the propeller domain of DPP9 (Fig. 2) has been shown to be important for its enzymatic regulation, whereby SUMO1 binding results in elevated DPP9 activity in vitro and gene silencing of SUMO1 leads to reduced DPP8 and DPP9 activity in cell extracts (43). SUMO modification mediates numerous intracellular processes including transcription, DNA repair, chromatin remodeling, and nuclear translocation (44). Therefore, it would be interesting to explore the consequences of SUMO1 interactions with DPP8 and DPP9. Furthermore, the activity of purified DPP9 from bovine testes (45) and recombinant DPP8 and DPP9 produced by insect cells (33) is dependent on the redox state of their cysteines, whereby enzyme activity is reversibly decreased by oxidation and increased by reduction (33). Synthetic Substrates for Activity Determination Proteases regulate various physiological processes by cleaving substrates to cause inactivation or modified function. Therefore, identifying the substrates for DPP8 and DPP9 is essential for understanding their biological functions. DPP8 and DPP9 have similar substrate specificities, with both enzymes preferring substrates with an aromatic or branched aliphatic amino acid (46) or basic residue (34,40) in the P2 position. An in vivo substrate study confirmed that the majority of substrates of DPP8 and DPP9 contain a proline in the P1 position preceded by an alanine in the P2 position (47). DPP8 and DPP9 hydrolyze the synthetic chromogenic substrates Gly-Pro-pNA, Ala-Pro-pNA, and Arg-Pro-pNA equally well (refs. 2,22,23, and 39; Table 2). Using substrates Ala-Pro-pNA and Gly-Pro-pNA, the pH optimum for DPP9 is between pH 7.5 and 8.0 (40). DPP8 has a neutral pH 7.4 optimum with little activity below pH 6.3 (2), similar to the pH 7.8 optimum of DPPIV (48). Natural Substrate Proteins Certain naturally occurring peptides and chemokines can be cleaved by DPP8 and/or DPP9 in vitro but significantly slower than DPPIV (refs. 22 and 49; Table 2). However, the physiological relevance of these substrates is uncertain because they are predominantly extracellular whereas DPP8 and DPP9 are intracellular. However, in rat brain extract, Neuropeptide Y (NPY) can be cleaved in the presence of a DPPIV inhibitor, indicating that DPP8 and DPP9 can cleave NPY in vivo (50). NPY-driven tumor cell death has been achieved by inhibiting DPP8 and DPP9, indirectly indicating that DPP8 and DPP9 can cleave releasable NPY (51). Other natural substrates have also been identified. Silencing or inhibition of DPP9 in intact cells results in increased presentation of the RU134-42 peptide Table 2. Kinetic parameters of DPP8 and DPP9 substrates Km (mmol/L) Substrate a H-Ala-Pro-pNA H-Gly-Pro-pNA H-Ala-Pro-AFCb H-Gly-Pro-pNA H-Ala-Pro-pNA H-Val-Ala-pNA H-Ala-Pro-pNA H-Gly-Pro-pNA H-Arg-Pro-pNA DPP8 DPP9 References 0.991 0.171 0.467 0.064 0.18 0.02 0.33 0.01 0.34 0.10 0.76 0.08 0.30 0.02 0.4 0.04 0.11 0.005 N/A N/A 0.18 0.07 0.37 0.003 0.31 0.13 1.22 0.13 N/A N/A N/A (2) (23) (22) (39) t1/2 (h) Substrate DPP8 DPP9 References GLP-1 GLP-2 NPY PYY CXCL 12/SDF-1a (1-67) CXCL 12/SDF-1b (1-72) CXCL 10/IP10 (1-77) CXCL 11/ITAC (1-73) 3.8 4.0 0.2 50 4 0.5 2 0.6 13 2.4 >24 6.1 6.7 0.2 8.0 N/A N/A N/A N/A (22) (49) Abbreviation: N/A, not available. a pNA ¼ para-nitroaniline. b AFC ¼ 7-Amino-4-trifluoromethyl coumarin. 1490 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Dipeptidyl Peptidases 8 and 9 (VPYGSFKHV), which was the first natural substrate of DPP9 to be identified (46). It is possible that many more proline-containing antigens present in the cytoplasm are substrates for DPP9. Recently, a number of potential natural substrates for DPP8 and DPP9 have been discovered using a sophisticated terminal amine isotopic labeling of substrates proteomic method (47). In that study, calreticulin and adenylate kinase 2 (AK2) were identified as potential substrates of both DPP8 and DPP9 and acetyl-CoA acetyltransferase, which is important in fatty acid metabolism, was identified as a potential substrate of DPP8 (47). As AK2 plays a key role in maintaining cellular energy homeostasis, DPP8 and DPP9 may regulate this process by cleaving AK2. Although the impact of cleavage by DPP8 and DPP9 on the functions of these substrates remains to be studied, their identification highlights the potential roles of DPP8 and DPP9 in energy metabolism and homeostasis. Inhibitor Development for DPP8 and DPP9 The properties of potent and selective inhibitors for DPP8 and DPP9 are listed in Table 3. The compounds alloIle-isoindoline and 1G244 are the most commonly used DPP8/9 selective inhibitors. Allo-Ile-isoindoline had IC50 values of 50 nmol/L against DPP8 and DPP9 when first reported (52) but later IC50 values have been found to be 200 nmol/L (18, 53–55). The most potent inhibitory effect is observed with 1G244 (IC50 values of 14 and 53 nmol/L against DPP8 and DPP9, respectively), which does not inhibit DPPIV (18). 1G244 is a slow-tight binding competitive inhibitor of DPP8 and a reversible competitive inhibitor of DPP9 (18). It would be of great value to distinguish DPP8 from DPP9 activity in many biological processes using selective inhibitors as well as molecular tools. Isoindoline derivatives have been reported as potent DPP8 inhibitors with IC50 values below 20 nmol/L (56,57), but were later found to inhibit both DPP8 and DPP9 (18). Recently, 1G244 has been modified into several substructures to obtain some selectivity toward either DPP8 or DPP9 (19). Limited selectivity for DPP8 over DPP9 (roughly 10-fold) has been achieved in 2 compounds (12m and 12n), using methylpiperazine analogues of 1G244 (19). The boroProline-based dipeptidyl boronic acids are potent DPPIV inhibitors (20). Various substitutions at the 4-position of the boroProline ring alter the inhibitory activity. Arg-(4S)-boroHyp (4q) shows the most potent inhibition against DPPIV, DPP8, and DPP9, whereas (4S)-Hyp-(4R)boroHyp (4o) exhibits the greatest selectivity for DPPIV over DPP8 and DPP9 (20). Such studies point to the structure– activity relationship that will facilitate the future design of inhibitors toward either DPP8 or DPP9 alone and thus help to differentiate DPP8 and DPP9 activity from each other and also from that of DPPIV. These enzyme studies used human DPP8 and DPP9. It is likely that the homologues from other species would have very similar properties, but this has not been directly examined. Expression Profiles of DPP8 and DPP9 Expression profiles of DPP8 and DPP9 have been an important focus of interest in characterizing these new proteases and their ubiquitous expression pattern has been confirmed at mRNA and protein levels (Table 4). DPP9 cDNA has 2 forms, with sizes of 2,589 and 3,006 bp (23). Using Northern blot analysis, a predominant DPP9 mRNA transcript of 4.4 kb (AY374518; short form encoding 863 amino acids) was detected ubiquitously with the highest levels in liver, heart, and skeletal muscle (3,23). A less Table 3. Properties of potent and selective inhibitors for DPP8 and DPP9 IC50 (nmol/L) Ki (nmol/L) Compound DPP8 DPP9 DPPIV DPP8 DPP9 References Allo-Ile-isoindoline (DPP8/9-selective) 38 120 145 14 12 21 55 910 (irreversible) 55 290 242 53 84 260 540 N/A 30,000 90,000 >100,000 >100,000 >50,000 >100,000 >50,000 8,000 N/A N/A 13.7 0.9 N/A N/A N/A N/A N/A N/A 33.7 4.2 N/A N/A N/A N/A (52) (54,55) (18) (18) (19) (19) (19) (57) 530 (irreversible) N/A >500, 000 N/A N/A (57) 530 1.2 240 0.31 16 0.3 N/A N/A N/A N/A (20) (20) 1G244 1G244 analogue (compound 12 m) 1G244 analogue (compound 12 n) Bis(4-acetamidophenyl) Pyrrolidin-2-yl Phosphonate with Lys in P2 position (compound 7e) Bis(4-acetamidophenyl) Isoindolin-2-yl Phosphonate with Lys in P2 position (compound 2e) (4S)-Hyp-(4R)-boroHyp (compound 4o) Arg-(4S)-boroHyp (compound 4q) Abbreviation: N/A, not available. www.aacrjournals.org Mol Cancer Res; 11(12) December 2013 Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. 1491 Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Zhang et al. Table 4. Expression features of DPP8 and DPP9 Characteristic DPP8 DPP9 References mRNA expression in normal adult tissues Protein expression in normal adult tissues Expression in tumor tissues Expression by hepatocytes Expression by activated hepatic stellate cells Expression by lymphocytes Ubiquitous Ubiquitous Ubiquitous Ubiquitous H H H H (3,23,58) (59) (23,59) (59–61) (59) (59,61,62) H, H H yes; , no. abundant 5 kb transcript (AF542510; long form encoding 971 amino acids) is abundant in skeletal muscle (23). Similarly, 3 mRNA transcripts of DPP8 have been identified with intense signals in testis, prostate, and muscle (2,58). In particular, a transcript variant of human DPP8, with 16 more amino acids at the N-terminus (898 amino acids in total), is abundant in the adult testis (2,21). DPP8 and DPP9 enzymatic activities are predominant in bovine and rat testis and immunohistochemical studies have localized these 2 proteases in spermatozoids (63). A natural form of DPP9 has been purified from bovine testes and identified as the short form (45). Overall, the abundance of DPP8 and DPP9 in testis suggests that these 2 proteases might participate in the regulation of the male reproductive system. The ubiquitous expression of DPP8 and DPP9 in mammals has been shown by in situ hybridization and enzyme assays of mouse, human, and baboon organs (59). Notably, lymphocytes and epithelial cells from many organs, including lymph node, thymus, spleen, liver, lung, intestine, pancreas, muscle, and brain, express DPP8 and DPP9 (ref. 59; Fig. 3). Generally, concordant data on DPP8 and DPP9 expression has been obtained from the cynomolgus monkey and Sprague-Dawley rat (64), in which mRNA and protein levels correlated with specific enzymatic activity (64). In addition, DPP8 and DPP9 activity is approximately 10-fold lower than DPPIV activity in nonrenal rat and monkey tissues (64). DPPIV and DPP8 are more abundant than DPP9 protein in rat primary endothelial cells, however, about 60% of the total DPP enzyme activity is derived from DPP8/9 rather than DPPIV in these cells (65). DPP9 is the only DPPIV-like enzyme detectable by immunohistochemistry in human carotid artery endothelial cells, whereas DPPIV is the predominant DPPIV-like enzyme in ventricular microvasculature by in situ hybridization (65). Thus, the regulated expression of individual DPPs in these cells is indicative of a role in endothelia. Altered DPP8 and DPP9 expressions, at both mRNA and protein levels in diseased liver, point to important regulatory roles in the pathogenesis of liver diseases (59–61). DPP8/DPP9 enzyme activity in mouse organs 140 Highest 120 High mU/g 100 Medium Low 80 60 40 20 Negligible Ly mp Liv er hn o d Pa nc e rea Ut s eru s Lu Th ng ym us Br ain Co lon Te sti Sp s lee Ad n ren PB al MC Kid s ne Sk y ele tal Hea mu rt s Ad cle ipo Pr se os tat e Ov Su ary bm ax illa Ski n ry gla nd P Bo las Sm ne m ma all arr int ow es tin e 0 Figure 3. Ranking mouse organs by approximate intensity of DPP8 and DPP9 enzyme activity. DPP activity in DPPIV gene knockout mice with/without N-ethylmaleimide (NEM) was measured by Yu and colleagues (59). NEM is an inhibitor of DPP8/9 enzyme activity. Subtracting NEM-inhibited activity from total DPP activity was used here to estimate the activity derived from DPP8 and DPP9. 1492 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Dipeptidyl Peptidases 8 and 9 Biological Functions of DPP8 and DPP9 Enzymes of the DPPIV family are likely to participate in normal homeostasis as well as in the pathology of disease conditions that include tumor growth, type 2 diabetes, liver cirrhosis, and inflammatory and autoimmune diseases (13,16,66,67). As knowledge of the precise expression and localization of DPP8 and DPP9 advances, distinct physiological functions of these proteases are emerging. DPP8 and DPP9 in Cell Behavior and Tumor Biology Intracellularly distributed DPP8 and DPP9 have previously been shown to influence cell behavior, including cell–extracellular matrix (ECM) interactions, proliferation, and apoptosis. HEK293T epithelial cells overexpressing DPP8 and DPP9 exhibit impaired in vitro cell adhesion, migration, and monolayer wound healing, independent of enzyme activity (68). In HEK293T cells, DPP8 and DPP9 can enhance induced apoptosis independent of enzyme activity and DPP9 overexpression causes spontaneous apoptosis (68). In HepG2 cells, the levels of active forms of caspase 9 and caspase 3 can be significantly elevated following overexpression of enzyme active DPP9 compared with an enzyme inactive mutant DPP9, indicating that the intrinsic apoptosis induced by DPP9 is mediated by the caspase 9/ caspase 3 pathway (60). DPP9 overexpression attenuates the epidermal growth factor (EGF)–mediated PI3K/Akt pathway, resulting in augmented apoptosis and suppressed cell proliferation (ref. 60; Fig. 4). This inhibitory effect on Akt phosphorylation is largely dependent upon DPP9 enzyme activity (60), which is the first indication of DPP9 enzyme activity regulating cell behavior through the EGF signaling pathway. Perhaps surprisingly, knockdown of DPP8 or DPP9 can induce NPY-driven tumor cell death, mediated by poly (ADP-ribose) polymerase-1 and apoptosis-inducing factor (51) and enzyme inhibition of DPP8 and DPP9 by 1G244 can enhance macrophage apoptosis (69). Therefore, EGF e ran mb e am sm Pla PI3K EGFR H-Ras DPP9 Raf MEK Akt ERK Figure 4. Diagram of potential involvement of DPP9 in the EGF signaling pathway. Cytoplasmic DPP9 is associated with H-Ras. DPP9 attenuates the EGF mediated PI3K/Akt pathway but does not influence the Raf/MEK/ ERK pathway. www.aacrjournals.org experiments in which DPP9 is manipulated by overexpression or gene silencing or enzyme inhibition have provided nonconcordant data and thus the pro- or antiapoptotic activity of DPP9 may depend on the cell-type and cellculture environment. DPP9 mRNA is generally abundant in human tumor cell lines, such as melanoma (G-361), colorectal adenocarcinoma (SW480), chronic myelogenous leukemia (K562), and HeLa cells (3). DPP8 mRNA expression is significantly greater than all the other DPPs in breast cancer and ovarian cancer cell lines, but DPP8 and DPP9 protein expression is ubiquitous across those cell lines (70). DPP9 mRNA is also greatly upregulated in human testicular tumors (59). Interestingly, the highest level of DPP9 protein is in 2 estrogen receptor negative breast cancer cell lines that are negative for DPPIV mRNA (70). In many tumor tissues, including skin melanotic melanoma, neuroblastoma, and leukemia, the long form of human DPP9 mRNA is expressed (23). DPP8 and DPP9 are more abundantly expressed than DPPIV and FAP in human meningiomas (71) and constitutive expression of DPP8 and DPP9 is found in B-cell chronic lymphocytic leukemia (CLL; ref. 72). DPP8 mRNA and protein expression are significantly upregulated in CLL compared with normal tonsil B lymphocytes (72). The ubiquitous but differential expression of DPP8 and DPP9 in tumor cell lines and tissues indicates that they may have roles in tumor pathogenesis, perhaps in particular stages or circumstances. Recently published data more clearly point to the roles of DPP8 and DPP9 in tumor growth. A high-throughput screen has found that the DPP inhibitor vildagliptin synergistically enhances parthenolide's antileukemic activity in leukemia and lymphoma cell lines as well as in primary human acute myeloid leukemia (73). Inhibition of DPP8 and DPP9, but not DPPIV, is responsible for vildagliptin's enhancement of parthenolide's cytotoxicity (73). In addition, inhibition of DPP8 and DPP9 probably contributes to the tumor regression induced by the compound Val-boroPro (74). Moreover, a separate study suggests that the DPPs act as survival factors in the Ewing Sarcoma Family of Tumors (51). In such sarcoma cell lines, cell death can be enhanced by blocking DPPIV, DPP8, and DPP9 with either selective enzyme inhibition or siRNA knockdown. These effects were blocked by NPY receptor antagonists, indicating that the cell survival might be dependent on the NPY pathway (51). Perhaps paradoxically, overexpression of DPP9 is antiproliferative and enhances intrinsic apoptosis in epithelial tumor cell lines (60,68), which might indicate that high levels of DPP9 can be detrimental. More probably, different responses of DPP8 and DPP9 under different pathological conditions imply that the roles of DPP8 and DPP9 may vary with tumor stage or tumor type. DPP8 and DPP9 in the Immune System Good evidence, from both in vivo and in vitro studies, indicates that DPP8 and DPP9 participate in immunoregulation. As described earlier, extensive in vivo expression of Mol Cancer Res; 11(12) December 2013 Downloaded from mcr.aacrjournals.org on August 10, 2017. © 2013 American Association for Cancer Research. 1493 Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Zhang et al. DPP8 and DPP9 occurs in normal immunological tissues (59). DPP8 and DPP9 are expressed by all major lymphocyte subpopulations and are upregulated upon B or T lymphocyte activation (2,61). Intriguingly, there is one report suggesting that a minor fraction of typically intracellularly localized DPP8 and DPP9 might also be loosely bound on the surface of immune cells under certain circumstances (75). In addition, enzyme activity of DPP8 and DPP9 is present in cultured human and mouse primary leukocytes and B and T cell lines (2,52,75,62). Effects of DPP8 and DPP9 on the activation and proliferation of immune cells seem to be mediated by enzyme activity. A selective DPP8 and DPP9 inhibitor can attenuate both proliferation of peripheral blood mononuclear cells and the release of cytokine interleukin (IL)-2 (52). Moreover, DNA synthesis of mitogenstimulated splenocytes of both wild-type and DPPIV knockout mice is suppressed by the selective inhibition of DPP8 and DPP9 (17). Collectively, these data point to DPP8/9 enzyme activity having important roles in immunoregulation, but the mechanisms involved require additional investigations. DPP8 and DPP9 in the Inflammatory Response DPPIV activity is clearly related to the induction of the inflammatory response (76,77). Therefore, it would be interesting to know the extent of involvement of DPP8 and DPP9, which have DPPIV-like activity, in inflammation. Both mRNA and the enzyme activities of DPP8 and DPP9 are upregulated after asthma induction. Thus, the increased expression of DPP8 and DPP9 and their specific localization suggests that these enzymes have important roles during allergic reactions (78). An experimental colitis model in mice has provided further evidence for the participation of DPP8 in the inflammatory responses. After dextran sulfate sodium (DSS) treatment for 6 days to induce colitis, DPP8, but not DPP9, mRNA, and enzyme activity is elevated in the colons of both wild-type and DPPIV knockout mice (79), with inhibition of DPP activity impairing neutrophil infiltration at the site of inflammation (79). These data implicate DPP8 in the inflammatory response in colitis. DPPIV, aminopeptidase N (APN), and DPPIV/APN-like proteases are involved in ischemia-trigged inflammation (80). DPP9 mRNA is transiently and significantly downregulated whereas DPP8 mRNA is slightly decreased at day 3 postcerebral ischemia. DPPIV, DPP8, and APN are in the activated microglia and macrophages at day 3 postischemia and in astroglial cells at day 7 postischemia (80). Therefore, DPPIV, DPP8, and DPP9 have potential roles in cerebral inflammation. Most recently, abundant DPP8 and DPP9 have been found in macrophage-rich regions of human atherosclerotic plaques (69). Upregulated DPP9 and unchanged DPP8 expression have been observed during macrophage differentiation and activation (69). Inhibition of DPP8 and DPP9 decreases proinflammatory cytokines IL-6 and TNF-a secretion activated by lipopolysaccharide and IFN-g, suggesting an antiinflammatory effect of the DPP8/9 inhibitor (69). 1494 Mol Cancer Res; 11(12) December 2013 Many studies have the limitation that DPP8 and DPP9 activities have been examined in the presence of DPPIV. DPPIV-deficient mice and DPP8/9 selective inhibitors can overcome this issue. In addition, most studies are confined to in vitro models. More in vivo evidence and clinical data are needed. The development of DPP8 or DPP9-deficient animals would be a breakthrough for such functional studies. Concluding Remarks The recent insights into the emerging members of the DPPIV family reveal very interesting biochemical and biological traits of DPP8 and DPP9. Both DPP8 and DPP9 interact with SUMO1. DPP9 has a novel SUMO1-specific interacting motif that is important for its allosteric enzymatic regulation. Both proteases are redox regulated and probably not glycosylated, whereas only DPP9 is predicted to be acetylated. Recent advances in this field have begun to show functional differences between the 2 proteases. DPP9 has a stronger association with H-Ras, attenuates the EGFmediated PI3K/Akt pathway, and regulates cell behavior. The enzyme activity of DPP9, but not DPP8, is rate limiting for degradation of antigenic proline-containing peptides and thereby may have an important role in antigen presentation, whereas DPP8 is more important in gut inflammation. Some potential natural substrates of DPP8 and DPP9 have been identified recently using proteomics, one of which was specific for DPP8 only. These exciting new functional data reinforce the importance of expanding our knowledge of DPP8 and DPP9 expression patterns in both rodent and primate species. The development of selective inhibitors and substrates for DPP8 versus DPP9 is needed and would be instrumental in the future exploration and exploitation of the roles that DPP8 and DPP9 exhibit in pathogenesis. Disclosure of Potential Conflicts of Interest Mark D. Gorrell has an ownership interest in patents on DPP8 and DPP9. No potential conflicts of interest were disclosed by the other authors. Authors' Contributions Conception and design: H. Zhang, Y. Chen, M.D. Gorrell Writing, review, and/or revision of the manuscript: H. Zhang, Y. Chen, F.M. Keane, M.D. Gorrell Study supervision: F.M. Keane, M.D. Gorrell Acknowledgments The authors thank Ms. M.G. Gall for kind suggestions and proofreading. The authors are grateful for support from the Australian National Health and Medical Research Council, grant 512282, Australian Postgraduate Award for H. Zhang and University of Sydney International Scholarship to Y. Chen. Grant Support This work is supported by Australian National Health and Medical Research Council Grant 512282; Australian Postgraduate Award for H. Zhang; and University of Sydney International Scholarship for Y. Chen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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