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Dipeptidyl Peptidases 8 and 9

Advances in Understanding the Expression and Function of Dipeptidyl Peptidase 8 and 9

Authors: Hui Zhang, Yiqian Chen, Fiona M. Keane, Mark D. Gorrell

Authors’ Affiliation:

Molecular Hepatology, Centenary Institute and Sydney Medical School, University of Sydney, NSW 2006, Australia

Running Title: Dipeptidyl Peptidases 8 and 9

Keywords: Dipeptidyl Peptidase; Expression; Function; Inhibitor; Substrate

Corresponding Author:

Associate Prof M. D. Gorrell

Molecular Hepatology, Centenary Institute, Locked Bag No. 6, Newtown, NSW 2042, Australia

Tel: +61 2 95656156

Fax: +61 2 95656101

E-mail: [email protected]

Abbreviations:

AIF: apoptosis-inducing factor; AK2: adenylate kinase 2; APN: aminopeptidase N; CLL: chronic lymphocytic leukemia;

DPP: dipeptidyl peptidase; DSS: dextran sulfate sodium; ECM: extracellular matrix; EGF: epidermal growth factor;

FAP: fibroblast activation protein; PARP-1: poly (ADP-ribose) polymerase-1; PTMs: post-translational modifications;

SUMO1: small ubiquitin-related modifier 1; TAILS: terminal amine isotopic labeling of substrates

Disclosure of Potential Conflicts of Interest

Mark Gorrell is an inventor on granted patents on mammalian DPP8 and DPP9 enzymes and cDNAs.

Grant Support: Australian National Health and Medical Research Council grant 512282; Australian Postgraduate

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Dipeptidyl Peptidases 8 and 9

Award for Hui Zhang; University of Sydney International Scholarship for Yiqian Chen.

Dipeptidyl Peptidases 8 and 9

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 while DPP9 is involved in antigen presentation and

intracellular signaling. Likewise, the DPP9 connections with H-Ras and SUMO1, and its role in AKT1 pathway

down-regulation 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.

Dipeptidyl Peptidases 8 and 9

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 two

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 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

Dipeptidyl Peptidases 8 and 9

changes in diabetes, the immune system, the inflammatory response and cancer biology (13,16,17). While 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. While 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 et al. in 2000 (2). DPP9 was then identified by

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.

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 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

Dipeptidyl Peptidases 8 and 9

DPPIV gene spans 81.8 kb and contains 26 exons ranging in size from 45 to 1386 bp (31). In contrast, the human DPP9

gene spans only 48.6 kb of genomic sequence and comprises 22 exons that are 53 bp 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 DPPIV and FAP is encoded by two exons (3,31), which

suggests that DPP8 and DPP9 are the more ancient genes of the four.

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) (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 α/β-hydrolase domain

and an eight blade β-propeller domain and the active site of the peptidase locates at the interface of these two domains

(Fig. 2). The DPPIV family of proteins share a conserved catalytic triad, with that of DPP8 consisting of Ser739, Asp817,

His849 and the equivalent residues in DPP9 being Ser730, Asp808 and His840 (3,23). A distinguishing feature of the DPPIV

protein family is that two important glutamates lie near the start of a loop that protrudes from the β-propeller domain

Dipeptidyl Peptidases 8 and 9

and borders the substrate entry site (32) and these two 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. Firstly, 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 β-propeller domain. Secondly,

single point mutations in the C-terminal loop, such as F822A, V833A, Y844A, 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. Thirdly, 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.

Post-translational Modification

Post-translational modifications (PTMs) 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

two 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 two 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.

Dipeptidyl Peptidases 8 and 9

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 preceeded 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 (2,22,23,39) (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).

Dipeptidyl Peptidases 8 and 9

Natural Substrate Proteins

Certain naturally occurring peptides and chemokines can be cleaved by DPP8 and/or DPP9 in vitro but significantly

slower than DPPIV (22,49) (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 (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 TAILS (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. While 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

allo-Ile-isoindoline and 1G244 are the most commonly used DPP8/9 selective inhibitors. Allo-Ile-isoindoline had IC50

Dipeptidyl Peptidases 8 and 9

values of ~50 nM against DPP8 and DPP9 when first reported (52) but later IC50 values have been found to be ~200 nM

(18,53-55). The most potent inhibitory effect is observed with 1G244 (IC50 values of 14 nM and 53 nM 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 nM (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 towards either DPP8 or DPP9 (19). Limited selectivity for

DPP8 over DPP9 (roughly 10-fold) has been achieved in two 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, while (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 towards 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

Dipeptidyl Peptidases 8 and 9

and their ubiquitous expression pattern has been confirmed at mRNA and protein levels (Table 4).

DPP9 cDNA has two forms, with sizes of 2589 bp and 3006 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 abundant 5 kb transcript (AF542510; long form encoding

971 amino acids) is abundant in skeletal muscle (23). Similarly, three 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 two

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 two 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 (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 non-renal 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 while DPPIV is the predominant

Dipeptidyl Peptidases 8 and 9

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).

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 to 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 (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

Dipeptidyl Peptidases 8 and 9

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 (PARP-1) and

apoptosis-inducing factor (AIF) (51) and enzyme inhibition of DPP8 and DPP9 by 1G244 can enhance macrophage

apoptosis (69). Therefore, experiments in which DPP9 is manipulated by overexpression or gene silencing or enzyme

inhibition have provided non-concordant data and thus the pro- or anti-apoptotic activity of DPP9 may depend on the

cell type and cell culture environment.

DPP9 mRNA is generally abundant in human tumor cell lines, such as melanoma (G-361), colorectal adenocarcinoma

(SW480), chronic myelogenous leukemia (K-562) 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 two estrogen receptor negative breast cancer cell lines that are

negative for DPPIV mRNA (70). In many tumor tissues, including skin melanotic melanoma, neuroblastoma and

leukaemia, 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) (72). DPP8 mRNA and protein expression are significantly upregulated in CLL

compared to 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 anti-leukemic activity in leukemia

Dipeptidyl Peptidases 8 and 9

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-boro-Pro (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

anti-proliferative 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 above, extensive in vivo expression of 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 appear 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 IL-2 (52). Moreover, DNA synthesis of mitogen-stimulated splenocytes

Dipeptidyl Peptidases 8 and 9

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 down-regulated while DPP8 mRNA

is slightly decreased at day 3 post-cerebral ischemia. DPPIV, DPP8 and APN are in the activated microglia and

macrophages at day 3 post-ischemia and in astroglial cells at day 7 post-ischemia (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

Dipeptidyl Peptidases 8 and 9

pro-inflammatory cytokines IL-6 and TNFα secretion activated by lipopolysaccharide and IFNγ, suggesting an

anti-inflammatory effect of the DPP8/9 inhibitor (69).

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. Additionally, 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, while only DPP9 is predicted to be acetylated. Recent advances in this field have begun to show

functional differences between the two proteases. DPP9 has a stronger association with H-Ras, attenuates the

EGF-mediated 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

Dipeptidyl Peptidases 8 and 9

pathogenesis.

Acknowledgments

We thank Ms Margaret G. Gall for kind suggestions and proofreading. We are grateful for support from the Australian

National Health and Medical Research Council, grant 512282, Australian Postgraduate Award for Hui Zhang and

University of Sydney International Scholarship to Yiqian Chen.

Reference 1. Turk B, Turk D, Turk V. Protease signalling: The cutting edge. EMBO J 2012;31:1630-43. 2. Abbott CA, Yu DMT, Woollatt E, Sutherland GR, McCaughan GW, Gorrell MD. Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8. Eur J Biochem 2000;267:6140-50. 3. Olsen C, Wagtmann N. Identification and characterization of human Dpp9, a novel homologue of dipeptidyl peptidase IV. Gene 2002;299:185-93. 4. Aertgeerts K, Levin I, Shi L, Snell GP, Jennings A, Prasad GS, et al. Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J Biol Chem 2005;280:19441-4. 5. De Meester I Dipeptidyl peptidase 9. In: Rawlings NL, Salvesen G, editors. Handbook of Proteolytic Enzymes 3rd Edition. San Diego: Elsevier; 2013.p.3384-9. 6. Abbott C, Gorrell M Dipeptidyl peptidase 8. In: Rawlings NL, Salvesen G, editors. Handbook of Proteolytic Enzymes 3rd Edition. San Diego: Elsevier; 2013.p.3379-84. 7. Gorrell MD, Park JE Fibroblast activation protein alpha. In: Rawlings NL, Salvesen G, editors. Handbook of Proteolytic Enzymes 3rd Edition. 3 edn. San Diego: Elsevier; 2013.p.3395-401. 8. Deacon CF, Danielson P, Klarskov L, Olesen M, Holst JJ. Dipeptidyl peptidase IV inhibition reduces the degradation and clearance of GIP and potentiates its insulinotropic and antihyperglycemic effects in anesthetized pigs. Diabetes 2001;50:1588-97. 9. Mentlein R, Gallwitz B, Schmidt WE. Dipeptidyl-peptidase IV hydrolyses gastric inhibitory polypeptide, glucagon-like peptide-1(7-36)amide, peptide histidine methionine and is responsible for their degradation in human serum. Eur J Biochem 1993;214:829-35. 10. Mersmann M, Schmidt A, Rippmann JF, Wuest T, Brocks B, Rettig WJ, et al. Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas. Int J Cancer 2001;92:240-8. 11. Wuest T, Moosmayer D, Pfizenmaier K. Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein. J Biotechnol 2001;92:159-68. 12. Gorrell MD, Song S, Wang XM. Novel metabolic disease therapy. International patent application 2010/083570. 2010.

Dipeptidyl Peptidases 8 and 9

13. Yu DMT, Yao T-W, Chowdhury S, Nadvi NA, Osborne B, Church WB, et al. The Dipeptidyl Peptidase IV family in cancer and cell biology. FEBS J 2010;277:1126-44. 14. Keane FM, Chowdhury S, Yao T-W, Nadvi NA, Gall MG, Chen Y, et al. Targeting Dipeptidyl Peptidase-4 (DPP-4) and Fibroblast Activation Protein (FAP) for diabetes and cancer therapy. In: Dunn B, editor Proteinases as Drug Targets. RSC Drug Discovery Series, vol 18. Cambridge, UK: Royal Society of Chemistry; 2012.p.119-45. 15. Liu R, Li H, Liu L, Yu J, Ren X. Fibroblast activation protein: A potential therapeutic target in cancer. Cancer Biol Ther 2012;13:123-9. 16. Yazbeck R, Howarth GS, Abbott CA. Dipeptidyl peptidase inhibitors, an emerging drug class for inflammatory disease? Trends Pharmacol Sci 2009;30:600-7. 17. Reinhold D, Goihl A, Wrenger S, Reinhold A, Kühlmann UC, Faust J, et al. Role of dipeptidyl peptidase IV (DPIV)-like enzymes in T lymphocyte activation: investigations in DPIV/CD26 knockout mice. Clin Chem Lab Med 2009;47:268-74. 18. Wu JJ, Tang HK, Yeh TK, Chen CM, Shy HS, Chu YR, et al. Biochemistry, pharmacokinetics, and toxicology of a potent and selective DPP8/9 inhibitor. Biochem Pharmacol 2009;78:203-10. 19. Van Goethem S, Matheeussen V, Joossens J, Lambeir AM, Chen X, De Meester I, et al. Structure-activity relationship studies on isoindoline inhibitors of dipeptidyl peptidases 8 and 9 (DPP8, DPP9): is DPP8-selectivity an attainable goal? J Med Chem 2011;54:5737-46. 20. 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;22:5536-40. 21. Zhu H, Zhou ZM, Lu L, Xu M, Wang H, Li JM, et al. Expression of a novel dipeptidyl peptidase 8 (DPP8) transcript variant, DPP8-v3, in human testis. Asian J Androl 2005;7:245-55. 22. Bjelke JR, Christensen J, Nielsen PF, Branner S, Kanstrup AB, Wagtmann N, et al. Dipeptidyl peptidase 8 and 9 specificity and molecular characterization compared to dipeptidyl peptidase IV. Biochem J 2006;396:391-9. 23. Ajami K, Abbott CA, McCaughan GW, Gorrell MD. Dipeptidyl peptidase 9 has two forms, a broad tissue distribution, cytoplasmic localization and DPIV-like peptidase activity. BBA - Gene Structure Expression 2004;1679:18-28. 24. Tanaka T, Camerini D, Seed B, Torimoto Y, Dang NH, Kameoka J, et al. Cloning and functional expression of the T cell activation antigen CD26. J Immunol 1992;149:481-6. 25. Scanlan MJ, Raj BK, Calvo B, Garin-Chesa P, Sanz-Moncasi MP, Healey JH, et al. Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc Natl Acad Sci U S A 1994;91:5657-61. 26. Edosada CY, Quan C, Tran T, Pham V, Wiesmann C, Fairbrother W, et al. Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly(2)-Pro(1)-cleaving specificity. FEBS Lett 2006;580:1581-6. 27. Fingerlin TE, Murphy E, Zhang W, Peljto AL, Brown KK, Steele MP, et al. Genome-wide association study identifies multiple susceptibility loci for pulmonary fibrosis. Nat Genet 2013;45:613-20. 28. Wang ZJ, Churchman M, Campbell IG, Xu WH, Yan ZY, McCluggage WG, et al. Allele loss and mutation screen at the Peutz-Jeghers (LKB1) locus (19p13.3) in sporadic ovarian tumours. Br J Cancer 1999;80:70-2. 29. Bruford EA, Riise R, Teague PW, Porter K, Thomson KL, Moore AT, et al. Linkage mapping in 29 Bardet-Biedl syndrome families confirms loci in chromosomal regions 11q13, 15q22.3-q23, and 16q21. Genomics 1997;41:93-9. 30. Qiu XS, Tang NLS, Yeung HY, Qiu Y, Cheng JCY. Association study between adolescent idiopathic scoliosis and the DPP9 gene which is located in the candidate region identified by linkage analysis. Postgrad Med J 2008;84:498-501. 31. Abbott CA, Baker E, Sutherland GR, McCaughan GW. Genomic organisation, exact localization, and tissue

Dipeptidyl Peptidases 8 and 9

expression of the human CD26 (dipeptidyl peptidase IV) gene. Immunogenetics 1994;40:331-8. 32. Rummey C, Metz G. Homology models of dipeptidyl peptidases 8 and 9 with a focus on loop predictions near the active site. Proteins 2007;66:160-71. 33. 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. 34. Lee HJ, Chen YS, Chou CY, Chien CH, Lin CH, Chang GG, et al. Investigation of the dimer interface and substrate specificity of prolyl dipeptidase DPP8. J Biol Chem 2006;281:38653-62. 35. Tang HK, Chen KC, Liou GG, Cheng SC, Chien CH, Tang HY, et al. Role of a propeller loop in the quaternary structure and enzymatic activity of prolyl dipeptidases DPP-IV and DPP9. FEBS Lett 2011;585:3409-14. 36. Ajami K, Abbott CA, Obradovic M, Gysbers V, Kähne T, McCaughan GW, et al. Structural requirements for catalysis, expression and dimerisation in the CD26/DPIV gene family. Biochemistry 2003;42:694-701. 37. Abbott CA, McCaughan GW, Gorrell MD. Two highly conserved glutamic acid residues in the predicted beta propeller domain of dipeptidyl peptidase IV are required for its enzyme activity. FEBS Lett 1999;458:278-84. 38. Wang XM, Yu DMT, McCaughan GW, Gorrell MD. Fibroblast activation protein increases apoptosis, cell adhesion and migration by the LX-2 human stellate cell line. Hepatology 2005;42:935-45. 39. Pitman MR, Menz RI, Abbott CA. Hydrophilic residues surrounding the S1 and S2 pockets contribute to dimerisation and catalysis in human dipeptidyl peptidase 8 (DPP8). Biol Chem 2010;391:959-72. 40. Tang HK, Tang HY, Hsu SC, Chu YR, Chien CH, Shu CH, et al. Biochemical properties and expression profile of human prolyl dipeptidase DPP9. Arch Biochem Biophys 2009;485:120-7. 41. Choudhary C, Kumar C, Gnad F, Nielsen ML, Rehman M, Walther TC, et al. Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions. Science 2009;325:834-40. 42. Zhao S, Xu W, Jiang W, Yu W, Lin Y, Zhang T, et al. Regulation of Cellular Metabolism by Protein Lysine Acetylation. Science 2010;327:1000-4. 43. Pilla E, Möller U, Sauer G, Mattiroli F, Melchior F, Geiss-Friedlander R. A novel SUMO1-specific interacting motif in Dipeptidyl peptidase 9 (DPP9) that is important for enzymatic regulation. J Biol Chem 2012;287:44320-9. 44. Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol 2007;8:947-56. 45. Dubois V, Lambeir A-M, Vandamme S, Matheeussen V, Guisez Y, Scharpé S, et al. Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry. BBA - Proteins & Proteomics 2010;1804:781-8. 46. 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. 47. 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. 48. Fukasawa KM, Fukasawa K, Hiraoka BY, Harada M. Characterization of a soluble form of dipeptidyl peptidase IV from pig liver. Experientia 1983;39:1005-7. 49. 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. 50. Frerker N, Wagner L, Wolf R, Heiser U, Hoffmann T, Rahfeld J-U, et al. Neuropeptide Y (NPY) cleaving enzymes: Structural and functional homologues of dipeptidyl peptidase 4. Peptides 2007;28:257-68. 51. Lu C, Tilan JU, Everhart L, Czarnecka M, Soldin SJ, Mendu DR, et al. Dipeptidyl peptidases as survival factors in Ewing sarcoma family of tumors: implications for tumor biology and therapy. J Biol Chem 2011;286:27494-505.

Dipeptidyl Peptidases 8 and 9

52. Lankas G, Leiting B, Roy R, Eiermann G, Beconi M, Biftu T, et al. Dipeptidyl peptidase IV inhibition for the treatment of type 2 diabetes - Potential importance of selectivity over dipeptidyl peptidases 8 and 9. Diabetes 2005;54:2988-94. 53. Chen SJ, Jiaang WT. Current advances and therapeutic potential of agents targeting dipeptidyl peptidases-IV, -II, 8/9 and fibroblast activation protein. Curr Top Med Chem 2011;11:1447-63. 54. Van der Veken P, De Meester I, Dubois V, Soroka A, Van Goethem S, Maes MB, et al. Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads. Bioorg Med Chem Lett 2008;18:4154-8. 55. Van Goethem S, Van der Veken P, Dubois V, Soroka A, Lambeir AM, Chen X, et al. Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors. Bioorg Med Chem Lett 2008;18:4159-62. 56. Jiaang WT, Chen YS, Hsu T, Wu SH, Chien CH, Chang CN, et al. Novel isoindoline compounds for potent and selective inhibition of prolyl dipeptidase DPP8. Bioorg Med Chem Lett 2005;15:687-91. 57. Van der Veken P, Soroka A, Brandt I, Chen YS, Maes MB, Lambeir AM, et al. Irreversible inhibition of dipeptidyl peptidase 8 by dipeptide-derived diaryl phosphonates. J Med Chem 2007;50:5568-70. 58. Qi SY, Riviere PJ, Trojnar J, Junien JL, Akinsanya KO. Cloning and characterization of dipeptidyl peptidase 10, a new member of an emerging subgroup of serine proteases. Biochem J 2003;373:179-89. 59. Yu DMT, Ajami K, Gall MG, Park J, Lee CS, Evans KA, et al. The in vivo expression of dipeptidyl peptidases 8 and 9. J Histochem Cytochem 2009;57:1025-40. 60. Yao T-W, Kim W-S, Yu DM, Sharbeen G, McCaughan GW, Choi K-Y, et al. A Novel Role of Dipeptidyl Peptidase 9 in Epidermal Growth Factor Signaling. Mol Cancer Res 2011;9:948-59. 61. Chowdhury S, Chen Y, Yao T-W, Ajami K, Wang XM, Popov Y, et al. Regulation of dipeptidyl peptidase 8 and 9 expression in activated lymphocytes and injured liver. World J Gastroenterol 2013;19:2883-93. 62. Maes M-B, Dubois V, Brandt I, Lambeir A-M, Van der Veken P, Augustyns K, et al. Dipeptidyl peptidase 8/9-like activity in human leukocytes. J Leukoc Biol 2007;81:1252-7. 63. Dubois V, Ginneken CV, De Cock H, Lambeir A-M, Van der Veken P, Augustyns K, et al. Enzyme activity and immunohistochemical localization of dipeptidyl peptidase 8 and 9 in male reproductive tissues. J Histochem Cytochem 2009;57:531-41. 64. Harstad EB, Rosenblum JS, Gorrell MD, Achanzar WE, Minimo L, Wu J, et al. DPP8 and DPP9 expression in cynomolgus monkey and Sprague Dawley rat tissues. Regul Pept 2013;186:26-35. 65. Matheeussen V, Baerts L, De Meyer G, De Keulenaer G, Van Der Veken P, Augustyns K, et al. Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries. Biol Chem 2011;392:189-98. 66. Nagy LE. Recent insights into the role of the innate immune system in the development of alcoholic liver disease. Exp Biol Med (Maywood) 2003;228:882-90. 67. Deacon CF. Dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes: a comparative review. Diabetes Obes Metab 2011;13:7-18. 68. Yu DMT, Wang XM, McCaughan GW, Gorrell MD. Extra-enzymatic functions of the dipeptidyl peptidase (DP) IV related proteins DP8 and DP9 in cell adhesion, migration and apoptosis. FEBS J 2006;273:2447-61. 69. Matheeussen V, Waumans Y, Martinet W, Van Goethem S, Van der Veken P, Scharpe S, et al. Dipeptidyl peptidases in atherosclerosis: expression and role in macrophage differentiation, activation and apoptosis. Basic Res Cardiol 2013;108:350. 70. Wilson C, Abbott C. Expression profiling of dipeptidyl peptidase 8 and 9 in breast and ovarian carcinoma cell lines.

Dipeptidyl Peptidases 8 and 9

Int J Oncol 2012;41:919-32. 71. Stremenova J, Mares V, Lisa V, Hilser M, Krepela E, Vanickova Z, et al. Expression of dipeptidyl peptidase-IV activity and/or structure homologs in human meningiomas. Int J Oncol 2010;36:351-8. 72. Sulda ML, Abbott CA, Macardle PJ, Hall RK, Kuss BJ. Expression and prognostic assessment of dipeptidyl peptidase IV and related enzymes in B-cell chronic lymphocytic leukemia. Cancer Biol Ther 2010;10:180-9. 73. Spagnuolo PA, Hurren R, Gronda M, Maclean N, Datti A, Basheer A, et al. Inhibition of intracellular dipeptidyl peptidases 8 and 9 enhances parthenolide's anti-leukemic activity. Leukemia 2013;27:1236-44. 74. 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. 75. Bank U, Heimburg A, Wohlfarth A, Koch G, Nordhoff K, Julius H, et al. Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects. Biol Chem 2011;392:169-87. 76. Kruschinski C, Skripuletz T, Bedoui S, Tschernig T, Pabst R, Nassenstein C, et al. CD26 (dipeptidyl-peptidase IV)-dependent recruitment of T cells in a rat asthma model. Clin Exp Immunol 2005;139:17-24. 77. Ohnuma K, Dang NH, Morimoto C. Revisiting an old acquaintance: CD26 and its molecular mechanisms in T cell function. Trends Immunol 2008;29:295-301. 78. Schade J, Stephan M, Schmiedl A, Wagner L, Niestroj AJ, Demuth HU, et al. Regulation of expression and function of dipeptidyl peptidase 4 (DP4), DP8/9, and DP10 in allergic responses of the lung in rats. J Histochem Cytochem 2008;56:147-55. 79. Yazbeck R, Sulda ML, Howarth GS, Bleich A, Raber K, von Horsten S, et al. Dipeptidyl peptidase expression during experimental colitis in mice. Inflamm Bowel Dis 2010;16:1340-51. 80. Röhnert P, Schmidt W, Emmerlich P, Goihl A, Wrenger S, Bank U, et al. Dipeptidyl peptidase IV, aminopeptidase N and DPIV/APN-like proteases in cerebral ischemia. J Neuroinflam 2012;9:44.

Dipeptidyl Peptidases 8 and 9

Table 1. Comparative overviews of the four enzyme members of the DPPIV family

DPP8-v3 DPP9 DPP9 Attribute DPPIV FAP DPP8 short long short long a

Synonyms CD26 Seprase DPRP1 DPRP2

GenBank M74777 U09278 AF221634 AF354202 AY374518 AF542510 Accession

References (24) (4,25,26) (2) (21) (3,23) (23)

Gene Location

- Human 2q24.3 2q23 15q22.32 19q13.3

- Mouse 2:62330073 2:62500945 9:65032458 17:56186682

Number of Exons 26 26 20 22 19 22

Gene Size (kb) 81.8 72.8 71 ? 47.3 48.6

mRNA (bp) 2924 2814 3127 3030 2592 3006

Number of Amino 766 760 882 898 863 971 Acids

Monomer Mobility 110 kDa 95 kDa 100 kDa 103kDa 95-110 kDa ?

Dimer √ √ √ √

Transmembrane √ √ × × Domain

Ser630-Asp708- Ser624-Asp702- Catalytic Triad Ser739-Asp817-His849 Ser730-Asp808-His840 His740 His734

dipeptidyl dipeptidyl Enzyme Activity peptidase and dipeptidyl peptidase dipeptidyl peptidase peptidase endopeptidase

Cellular cell surface cell surface cytoplasmic cytoplasmic Localization and soluble and soluble

√, yes; ×, no; ?, not known; a, the long form is a splice variant

Dipeptidyl Peptidases 8 and 9

Table 2. Kinetic parameters of DPP8 and DPP9 substrates

Km (mM)

Substrate DPP8 DPP9 References

H-Ala-Pro-pNAa 0.991±0.171 N/A (2) H-Gly-Pro-pNA 0.467±0.064 N/A

H-Ala-Pro-AFCb 0.18±0.02 0.18±0.07 (23)

H-Gly-Pro-pNA 0.33±0.01 0.37±0.003

H-Ala-Pro-pNA 0.34±0.10 0.31±0.13 (22)

H-Val-Ala-pNA 0.76±0.08 1.22±0.13

H-Ala-Pro-pNA 0.30±0.02 N/A

H-Gly-Pro-pNA 0.42±0.04 N/A (39)

H-Arg-Pro-pNA 0.11±0.005 N/A

t1/2 (h)

Substrate DPP8 DPP9 References

GLP-1 ~3.8 ~6.1

GLP-2 ~4.0 ~6.7 (22) NPY ~0.2 ~0.2

PYY ~50 ~8.0

CXCL 12 / SDF-1α (1-67) 4±0.5 N/A

CXCL 12 / SDF-1β (1-72) 2±0.6 N/A (49) CXCL 10 / IP10 (1-77) 13±2.4 N/A

CXCL 11 / ITAC (1-73) >24 N/A

N/A: not available

a pNA = para-nitroaniline

bAFC = 7-Amino-4-trifluoromethyl coumarin

Dipeptidyl Peptidases 8 and 9

Table 3. Properties of potent and selective inhibitors for DPP8 and DPP9

IC50 (nM) Ki (nM)

Compound DPP8 DPP9 DPPIV DPP8 DPP9 References

allo-Ile-isoindoline 38 55 30,000 N/A N/A (52)

(DPP8/9-selective) 120 290 90,000 N/A N/A (54,55)

145 242 >100,000 13.7 33.7 (18)

1G244 14 53 >100,000 0.9 4.2 (18)

12 84 >50,000 N/A N/A (19)

1G244 analogue 21 260 >100,000 N/A N/A (19) (compound 12m)

1G244 analogue 55 540 >50,000 N/A N/A (19) (compound 12n)

Bis(4-acetamidophenyl) Pyrrolidin-2-yl 910 Phosphonate with Lys in P2 position N/A 8,000 N/A N/A (57) (irreversible) (compound 7e)

Bis(4-acetamidophenyl) Isoindolin-2-yl 530 Phosphonate with Lys in P2 position N/A >500, 000 N/A N/A (57) (irreversible) (compound 2e)

(4S)-Hyp-(4R)-boroHyp (compound 4o) 530 240 16 N/A N/A (20)

Arg-(4S)-boroHyp 1.2 0.31 0.3 N/A N/A (20) (compound 4q)

N/A: not available

Dipeptidyl Peptidases 8 and 9

Table 4. Expression features of DPP8 and DPP9

Characteristic DPP8 DPP9 References

mRNA expression in normal adult tissues ubiquitous ubiquitous (3,23,58)

Protein expression in normal adult tissues ubiquitous ubiquitous (59)

Expression in tumor tissues √ √ (23,59)

Expression by hepatocytes √ √ (59-61)

Expression by activated hepatic stellate cells × × (59)

Expression by lymphocytes √ √ (59,61,62)

√, yes; × , no

Dipeptidyl Peptidases 8 and 9

Legends for tables and figures

Table 1. Comparative overviews of the four enzyme members of the DPPIV family

Table 2. Kinetic parameters of DPP8 and DPP9 substrates

Table 3. Properties of potent and selective inhibitors for DPP8 and DPP9

Table 4. Expression features of DPP8 and DPP9

Figure 1. Overview of organs and processes in which DPP8 and DPP9 have potential functions, along with molecules

that interact with these proteases.

Figure 2. A model of DPP9 protein structure. DPP9 residues 51 to 863 are depicted in ribbon representation of the

monomer. The α/β-hydrolase domain is in red and the 8-blade β-propeller domain in green. The catalytic triad

(Ser-Asp-His) is shown as blue spheres and the two glutamates essential for catalysis as yellow spheres. An extended

arm (285VEVIHVPSPALEERKTDSYR304) in the propeller surface of DPP9 that is critical in SUMO1-DPP9 interaction

is colored pink.

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 et al (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.

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.

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DPP8 DPP9

H-Ras H-Ras Tumour Alcoholic liver disease Liver Tumour Biology cell lines Liver fibrosis Disease Chronic Biliary cirrhosis Testicular lymphocytic tumours leukemia

Figure 1 Author Manuscript Published OnlineFirst on September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

DPP8/DPP9 Enzyme Activity in Mouse Organs 140 Highest 120 High 100 Medium 80 Low

60 mU/g

20 negligible

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Subm

Downloaded from mcr.aacrjournals.org on September 24, 2021.Figure © 2013 3American Association for Cancer Research. Downloaded from Author ManuscriptPublishedOnlineFirstonSeptember13,2013;DOI:10.1158/1541-7786.MCR-13-0272 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. mcr.aacrjournals.org

EGF

DPP9 PI3K Raf

MEK Akt

ERK

Figure 4 Author Manuscript Published OnlineFirst on September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Advances in Understanding the Expression and Function of Dipeptidyl Peptidase 8 and 9

Hui Zhang, Yiqian Chen, Fiona M. Keane, et al.

Mol Cancer Res Published OnlineFirst September 13, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-13-0272

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