Dipeptidyl peptidase (DPP) 8 is a member of the DPP4/DPP-IV gene and enzyme family, which belongs to clan SC of serine proteases, family S9, subfamily S9B. S9B proteases have a unique ability to remove Xaa-Pro dipeptides from the N-terminus of substrates. Fibroblast activation protein (FAP), DPP4, DPP8, and DPP9 are the S9B peptidases. Several extensive reviews provide detail (Zhang et al. 2013; Waumans et al. 2015; Klemann et al. 2016; Wilson et al. 2016).
DPP8 has been localized to human chromosome 15q22.32 (Abbott et al. 2000) and 9:65032458 in the mouse. The human DPP8 gene spans 71 kb and comprises 20 exons (Abbott et al. 2000), which is shorter and with fewer exons than DPP4 but encodes more amino acids (882 versus 766 residues). In DPP8 and DPP9, the gene sequence encoding the catalytic serine and its nearby highly conserved amino acids is in a single exon, whereas in DPP4 and FAP this region is split into two exons. Therefore, perhaps the DPP4 and FAP genes were derived from a DPP8- or DPP9-like gene. DPP8 (AF221634; encoding 882 amino acids) is ubiquitous across mammalian tissues and is 27% identical to DPP4, but 35% identical to DPP4 in the enzymatic region. DPP8 has no N-linked or O-linked glycosylation (Abbott et al. 2000). The dominant form of DPP8 mRNA in adult testis is a longer form encoded by 22 exons.
The crystal structure of DPP8 has not been solved, but protein homology modeling suggests that DPP8 is very similar to DPP9 and, apart from the N-terminal extension, shares a similar tertiary structure with DPP4 and FAP (Park et al. 2008). DPP8 is dimeric, with each monomer very likely consisting of an α/β-hydrolase domain and an eight blade β-propeller domain. The active site of the peptidase lies in the interface of these two domains, consisting of Ser739, Asp817, and His849 (Abbott et al. 2000; Klemann et al. 2016). Near this catalytic pocket lie a pair of glutamates in the β-propeller domain that are essential for catalytic activity in all members of the DPP4 enzyme family. Endogenous natural DPP8 protein has not been isolated. Human recombinant DPP8 has been produced in various expression systems and is consistently enzymatically active (Abbott et al. 2000; Ajami et al. 2008; Park et al. 2008; Geiss-Friedlander et al. 2009; Yao et al. 2011; Wilson et al. 2013; Justa-Schuch et al. 2014; Zhang et al. 2015a).
Expression and Localization In Vivo
Proteins must be localized to their appropriate subcellular compartments to fully function. Unlike DPP4 and FAP, which are cell surface expressed type II membrane glycoproteins with a very small cytoplasmic tail, DPP8 lacks a transmembrane domain and is an intracellular protein (Abbott et al. 2000). DPP8 has not been seen on the plasma membrane. The intracellular distribution of DPP8 as a chimeric fluorescent protein in human 293 T cells and hepatoma Huh7 cells is diffuse, associated with endoplasmic reticulum (Abbott et al. 2000), where all proteins are manufactured. DPP9 has a long form, DPP9-L, that can enter the nucleus and contains a nuclear localization sequence near its N-terminus (Justa-Schuch et al. 2014). Like DPP9, DPP8 has been detected in the nucleus, but lacks a nuclear localization sequence (Justa-Schuch et al. 2014).
DPP8 is ubiquitously expressed in normal tissues from mouse, human, baboon, cynomolgus monkey, and Sprague Dawley rat (Yu et al. 2009; Zhang et al. 2013). Lymphocytes and epithelial cells from many organs, including lymph node, thymus, spleen, liver, lung, intestine, pancreas, muscle, and brain, express DPP8 (Yu et al. 2009). The expression of DPP8 is altered in many disease conditions such as liver disease, inflammatory bowel disease, and cancers (Zhang et al. 2013; Waumans et al. 2015).
Regulation of DPP8 Activity
DPP8 activity reversibly increases in reducing conditions (Park et al. 2008). DPP8 associates with Hras, but not as tightly as does DPP9 (Yao et al. 2011). DPP8 and DPP9 can be acetylated but whether that alters activity is unknown (Zhang et al. 2013).
Proteases regulate many physiological processes by cleaving peptide and protein substrates. Synthetic substrates with a proline at position P1 have been widely used in the discovery of DPP8 inhibitors and in the investigations of DPP8 biochemical and biological functions. DPP8 is most active on Ala-Pro-, Arg-Pro-, and Gly-Pro- containing chromogenic (p-nitroaniline; pNA) or fluorogeneic substrates, such as 7-Amino-4-methylcoumarin (AMC) or 7-Amino-4-trifluoromethylcoumarin (AFC) (Abbott et al. 2000).
The cleavage of the peptide hormones gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) by DPP4 makes DPP4 inhibitors an effective treatment for type II diabetes (Klemann et al. 2016). Therefore the identification of DPP8 natural substrates can produce insights into biological function. The first natural substrates identified for DPP8 were hormones and chemokines that are also cleaved by DPP9 and DPP4 (Ajami et al. 2008). DPP4 has many other natural substrates and binding partners (Klemann et al. 2016) that have not been investigated as potential DPP8 substrates and binding partners.
Mass spectrometry–based proteomic strategies have been successfully applied to identify substrates of DPP8. Using N-terminal amine isotopic labeling of substrates (TAILS), proteomic analysis of cytosolic proteins has revealed many putative DPP8 substrates in cells stably expressing either enzymatically active or inactive DPP8 on a background of endogenous DPP8 (Wilson et al. 2013). In addition, using 2D DIGE in a cell line expressing enzyme inactive DPP9, proteins were confirmed as DPP9 substrates by MALDI-TOF or immunoblotting and all of these substrates are also DPP8 substrates (Zhang et al. 2015b; Wilson et al. 2016).
A compound that selectively inhibits DPP8 would be a useful biochemical tool to identify DPP8 substrates and to study the functions of DPP8. However, all the synthetic compounds that inhibit DPP8 also inhibit DPP9 to significant extents, with 1G244 being the most potent and selective (IC50 values of 14 nM and 53 nM against DPP8 and DPP9, respectively) (Wu et al. 2012). Thus, although 1G244 has low activity on DPP4 and FAP, a limitation for studies using 1G244 is that DPP8 and DPP9 cannot be discriminated. Therefore, the development of a selective DPP8 inhibitor is very important for progressing DPP8 research. The identification of dipeptide Val-Ala as a site for DPP9 cleavage not recognized by DPP8 (Zhang et al. 2015b) may facilitate the development of selective inhibitors.
DPP8 in Metabolism
The TAILS proteomic analysis of DPP8 substrates included several mitochondrial proteins. Most notably, the mitochondrial enzyme adenylate kinase 2 (AK2) is a confirmed substrate of both DPP8 and DPP9 (Wilson et al. 2013). Therefore, DPP8 probably has a role in energy metabolism. Adipogenesis in preadipocytes can be blocked by the DPP8/DPP9 inhibitor 1G244 (Han et al. 2015). In addition, the knockdown of DPP8 or DPP9 significantly impairs adipocyte differentiation from preadipocytes in vitro. Blocking the expression or activities of DPP8 and DPP9 attenuates PPARγ2 induction during preadipocyte differentiation. Addition of PPARγ agonist or ectopic expression of PPARγ2 is able to rescue the adipogenic defect caused by DPP8/DPP9 inhibition in preadipocytes (Han et al. 2015). These data indicate a possible role of DPP8 in adipogenesis.
DPP8 in Cellular Functions and Implications for Cancer
Emerging evidence points to the role of DPP8 in a number of cellular processes, including extracellular matrix interactions and epithelial growth factor–driven proliferation (Yao et al. 2011; Zhang et al. 2015a). DPP8 is ubiquitously expressed in tumor cell lines and has so far been investigated in several types of tumors, including Ewing sarcoma, testicular, liver, breast and ovarian cancer, and leukemia and lymphoma (Yu et al. 2009; Yao et al. 2011; Spagnuolo et al. 2013; Zhang et al. 2015a). HEK293T cells overexpressing DPP8 have impaired cell adhesion and monolayer wound healing, suggesting a role for DPP8 in cell adhesion and migration, but the underlying mechanisms remain elusive (Yao et al. 2011; Zhang et al. 2015a). In Ewing Sarcoma, cell death can be enhanced by blocking DPP8 and DPP9 with either enzyme inhibition or siRNA knockdown and DPP8/DPP9 inhibition enhances parthenolide’s tumor cell cytotoxicity (Spagnuolo et al. 2013). Similarly, the inhibition of DPP8 and/or DPP9 probably contributes to the tumor regression induced by the compound Val-boro-Pro (Talabostat) (Walsh et al. 2013). These findings may thus implicate DPP8 and/or DPP9 in tissue and tumor growth and metastasis. Possibly, the regulation of DPP8 in cell proliferation is mediated by inhibiting Akt activation involving the epidermal growth factor signaling pathway (Yao et al. 2011). However, selective inhibitors targeting DPP8 and conditional DPP8 knockout mice are needed to differentiate DPP8 from DPP9 for future investigations.
DPP8 in Immunology
The expression patterns of DPP8 and DPP9 in immune organs are similar (Yu et al. 2009). DPP8 may have a role in the immune system and the inflammatory response, but where it has been compared with DPP9 the latter enzyme has had a much more significant role (Geiss-Friedlander et al. 2009). DPP8 and DPP9 have been implicated in lymphocyte proliferation and activation and are expressed in ill-defined subsets of leucocytes, including lymphocytes and macrophages. DPP8/DPP9 inhibition attenuates macrophage activation, possibly due to the reduced interleukin-6 secretion (Waumans et al. 2016).
The identified substrates of DPP8 also suggest potential immune roles of DPP8. Two important immune regulators, CXCL10 and IL1RA, have been shown to be DPP8 substrates (Ajami et al. 2008; Zhang et al. 2015b). Cells and molecules of the immune system are a fundamental component of the tumor microenvironment. The chemokine CXCL10 is converted to its antagonist form by DPP4 in certain tumors (Rainczuk et al. 2014) in which the active form of CXCL10 attracts lymphocytes that can attack tumor cells. CXCL10 can be cleaved by DPP8 (Ajami et al. 2008), so DPP8 potentially also inhibits CXCL10-mediated processes. Moreover, the putative DPP8 and DPP9 substrates S100-A10, SET, and NUCB1 are mediators of immunity and/or inflammatory response (Zhang et al. 2015b). However, a functional or physiological outcome of DPP8 mediated cleavage has not been investigated.
DPP8 is a large, dimeric nonglycosylated intracellular polypeptide with a narrow specificity postproline proteolytic activity. This protease is ubiquitous and abundant, and yet the understanding of DPP8 in biology is rudimentary. DPP8 might have a role in nuclear events, in energy metabolism, in the brain, and in cancer and immunology. Further investigations are essential.
MDG is supported by grants 1105238 and 1113842 from the Australian National Health and Medical Research Council.
- Ajami K, Pitman MR, Wilson CH, Park J, Menz RI, Starr AE, et al. Stromal cell-derived factors 1 alpha and 1 beta, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8. FEBS Lett. 2008;582:819–25. doi: 10.1016/j.febslet.2008.02.005.PubMedCrossRefGoogle Scholar
- Geiss-Friedlander R, Parmentier N, Moeller U, Urlaub H, Van den Eynde BJ, Melchior F. The cytoplasmic peptidase DPP9 is rate-limiting for degradation of proline-containing peptides. J Biol Chem. 2009;284:27211–9. doi: 10.1074/jbc.M109.041871.
- Justa-Schuch D, Möller U, Geiss-Friedlander R. The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus. Cell Mol Life Sci. 2014;71:3611–26. doi: 10.1007/s00018-014-1591-6.PubMedCrossRefGoogle Scholar
- Park J, Knott HM, Nadvi NA, Collyer CA, Wang XM, Church WB, et al. Reversible inactivation of human dipeptidyl peptidases 8 and 9 by oxidation. The Open Enz Inhib J. 2008;1:52–61. http://www.bentham.org/open/toeij/openaccess2.htm
- Walsh MP, Duncan B, Larabee S, Krauss A, Davis JP, Cui Y, et al. Val-BoroPro accelerates T cell priming via modulation of dendritic cell trafficking resulting in complete regression of established murine tumors. PLoS One. 2013;8:e58860. doi: 10.1371/journal.pone.0058860.PubMedPubMedCentralCrossRefGoogle Scholar
- Waumans Y, Baerts L, Kehoe K, Lambeir A-M, De Meester I. The dipeptidyl peptidase family, prolyl oligopeptidase and prolyl carboxypeptidase in the immune system and inflammatory disease, including atherosclerosis. Front Immunol. 2015;6:387–405. doi: 10.3389/fimmu.2015.00387.PubMedPubMedCentralCrossRefGoogle Scholar
- Wilson CH, Indarto D, Doucet A, Pogson LD, Pitman MR, Menz RI, et al. Identifying natural substrates for dipeptidyl peptidase 8 (DP8) and DP9 using terminal amine isotopic labelling of substrates, TAILS, reveals in vivo roles in cellular homeostasis and energy metabolism. J Biol Chem. 2013;288:13936–49. doi: 10.1074/jbc.M112.445841.PubMedPubMedCentralCrossRefGoogle Scholar
- Wu W, Liu Y, Milo Jr LJ, Shu Y, Zhao P, Li Y, et al. 4-Substituted boro-proline dipeptides: synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activities. Bioorg Med Chem Lett 2012;22:5536–5540. 10.1016/j.bmcl.2012.07.033.