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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
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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
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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,
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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.
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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.
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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.
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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.
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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
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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.
Received May 28, 2013; revised September 4, 2013; accepted September 5, 2013;
published OnlineFirst September 13, 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
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Molecular Cancer Research
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Published OnlineFirst September 13, 2013; DOI: 10.1158/1541-7786.MCR-13-0272
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 2013;11:1487-1496. Published OnlineFirst September 13, 2013.
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