CHAPTER 1
`
`Dipeptidyl Peptidases: Substrates
`and Therapeutic Targeting in
`Human Health and Disease
`
`CLAIRE H. WILSON AND CATHERINE A. ABBOTT
`
`School of Biological Sciences, Flinders University, GPO BOX 2100,
`Adelaide 5001, South Australia, Australia
`
`1.1 Introduction
`
`Dipeptidyl peptidase 4 (DP4), fibroblast activation protein (FAP), DP8, and
`DP9 are the enzymatic members of the serine protease S9b DP4-like gene
`family. One of the most important features of the DPs is their ability to pre-
`ferentially cleave the N-terminal post-prolyl bond of regulatory peptides and
`small protein substrates. DP4 proteolysis results in the inactivation, activation,
`or alteration of its substrates function via changes in receptor selectivity; thus
`DP4 plays an important role in regulating biological function. Together, DP4
`and FAP have been implicated in a number of diseases including liver disease,
`obesity, type II diabetes, arthritis, inflammatory bowel disease and cancer.
`Recently, evidence has emerged to implicate both DP8 and DP9 in innate
`immunity, and DP8/9 in vitro cleavage of well-known DP4 substrates, including
`neuropeptide Y (NPY), glucagon-like peptide (GLP)-1, and a number of che-
`mokines, has been demonstrated. Despite this, the true pathophysiological
`roles of DP8/9 and their involvement within human biology and disease are still
`to be elucidated. Identification of the in vivo substrate repertoire of each DP will
`be an important step toward elucidating the biochemical pathways in which
`each protease is involved. This will allow us to unravel further the roles that the
`
`RSC Drug Discovery Series No. 18
`Proteinases as Drug Targets
`Edited by Ben Dunn
`r Royal Society of Chemistry 2012
`Published by the Royal Society of Chemistry, www.rsc.org
`
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`Chapter 1
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`DPs play in human biology and disease, and evaluate further their suitability as
`therapeutic targets.
`In this chapter, we will provide an introduction to the significance of post-
`proline cleavage, the DP4-like gene family enzymatic members, and the related
`enzymes prolyl endopeptidase (PEP) and DP2. This will be followed by an in-
`depth examination of the biochemical characteristics of DP4, FAP, DP8, and
`DP9, their natural substrates, and biological relevance following DP cleavage.
`Lastly, the suitability of targeting the DP family for the development of ther-
`apeutics for the treatment of human health and disease will be discussed.
`
`1.2 Post-Proline-Cleaving Enzymes
`
`A number of biologically active proteins and regulatory peptides such as
`cytokines, chemokines, growth hormones, and neuropeptides are protected
`from general proteolysis due to an evolutionary conserved N-terminal proline
`residue.1–3 Such protection results from the unique cyclic and imino structure of
`proline imposing conformational restrictions on the polypeptide backbone.3
`Even proteases exhibiting a very broad substrate specificity are unable to attack
`peptide bonds where the prolyl residue is situated, and hence degradation of
`such peptides requires the use of proline specific peptidases.1–3
`Although a number of proline-cleaving enzymes have been identified, only a
`limited number of these proteases are capable of cleaving the N-terminal post-
`prolyl, X-Pro-, bond2,3 (Figure 1.1). The most notable of these enzymes are the
`N-terminal-specific, dipeptidyl peptidases of the serine protease SC clan, S9b
`sub-family such as DP4 (EC 3.4.15.5), the endopeptidase of the parent S9
`family prolyl-endopeptidase (PEP; EC 3.4.21.26), and the S28 family member
`DP2 (EC 3.4.14.2). Lysosomal prolylcarboxypeptidase (PCP) (EC 3.4.16.2)
`also belongs to the S28 family, but in contrast to DP2, it functions as a C-
`terminal-specific protease cleaving the Pro-Xaa bond at the C-termini of pro-
`teins to release a single C-terminal amino acid. Carboxypeptidase P (EC
`3.4.17.16) is a membrane-localized protease with similar cleavage specificity to
`lysosomal PCP; however, it is a metallo-, as opposed to serine, protease.
`Aminopeptidase P (EC 3.4.17.16), prolidase (EC 3.4.13.19) and prolinase are
`additional proline-cleaving metalloproteases. Aminopeptidase P is an N-
`terminal-specific protease, cleaving the pre-prolyl bond to release single amino
`acids at the N-termini of proteins. Importantly, aminopeptidase P is involved in
`cooperative activities with DP4 and related enzymes.1 Prolidase and prolinase
`cleave dipeptides with pre- and post-proline respectively to release two amino
`acids.1 The metalloprotease angiotensin-converting enzyme (ACE; EC 3.4.15.1)
`is also capable of cleaving prolyl bonds, although it is not renowned for its
`ability to do this. DP4, first discovered in 1966 by Hopsu-Havu and Glenner as
`the dipeptidyl cleaving glyclproline napthylamidase,4 was the first N-terminal
`post-proline-cleaving enzyme identified and hence the most readily investi-
`gated. Since its discovery, related enzymes have been identified including
`fibroblast activation FAP,5 then DP8 and DP9.6
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`Figure 1.1 Proline-bond cleaving proteases. ACE, angiotensin-converting enzyme;
`DP, dipeptidyl peptidase; FAP, fibroblast activation protein; PCP, prolyl-
`carboxypeptidase; PEP, prolyl-endopeptidase.
`
`1.3 DP4-Like Gene Family and Related Enzymes
`
`Members of the DP4-like gene family, S9b, make up a sub-family of the
`prolyl-oligopeptidase (POP) S9 family within the serine protease clan SC.7,8
`In total the S9b family consists of six homologous members; the four pro-
`teases, DP4, FAP, DP8 and DP9 and two inactive protease homologs, DP6
`and DP10 (Figure 1.2). Sharing similar features, PEP from the parent S9
`family is also capable of cleaving the N-terminal post-prolyl bond but with
`endopeptidase specificity.9,10 In contrast to DP4, PEP is limited in its ability
`to cleave unblocked, free N-terminal dipeptides from substrates;10 however,
`inhibitors designed specifically for DPs can still bind to, and block, the
`enzymatic function of PEP.11,12 FAP, in addition to its DP activity, also
`functions as an endopeptidase.13,14 Structurally, the three crystallized S9b
`family members DP4, FAP and DP6 contain an a/b hydrolase domain and
`eight-bladed b-propeller domain (Figure 1.2)13,15–18 distinguishing it from
`that of parent PEP which contains a seven-bladed b-propeller domain.19
`Protease members of this family and PEP all contain the non-classical
`arrangement of the serine protease catalytic triad (Ser, Asp, and His), located
`within the C-terminal portion of the a/b hydrolase domain (Figure 1.2). The
`inactive protease homologs DP6 and DP10 contain a mutation of the cata-
`lytic serine residue to an aspartic acid20 and glutamine residue21 respectively.
`All enzyme members contain the catalytic serine protease motif around the
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`Figure 1.2
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`Schematic of the DP4-like gene family and related prolyl endopeptidase.
`Domains and residues of interest are not arranged to scale. Arrows indi-
`cate the catalytic residues required for enzyme activity. Only the main
`isoforms are represented. The majority of splice variants differ in regard to
`the length of their N-terminal cytoplasmic domain. Being intracellular
`proteases, DP8, DP9, and PEP do not contain a transmembrane domain.
`The integrin-binding domain of DP9 is also displayed within the N-
`terminal cytoplasmic domain. Figure adapted from Gorrell.61 Reproduced
`with permission, from Gorrell (2005), Clinical Science, 108, 277–292.
`r The Biochemical Society. DP, dipeptidyl peptidase; FAP, fibroblast
`activation protein; PEP, prolyl-endopeptidase.
`
`serine residue of GWSYG.7 Another distinguishing characteristic of the S9b
`sub-family that differentiates them from PEP is the pair of conserved gluta-
`mate residues situated within the b-propeller domain found to be essential for
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`catalytic activity of DP422 and DP823 (Figure 1.2). DP2, belonging to the
`serine protease S28 family, is a non-structural homolog sharing a number
`of similarities with the DP4-like gene family including the conserved non-
`classical catalytic triad and similar substrate specificity with its ability to
`cleave N-terminal dipeptides from peptide substrates with proline in the
`penultimate position.24 As for PEP, a number of inhibitors designed for
`targeting DP4 are also known to interact and inhibit DP2.11,24,25 Hence, when
`investigating the expression and roles of the S9b family, due consideration
`must also be given to the potential involvement (or targeting in the case of
`inhibition) of this related enzyme. The subcellular localization of each
`enzyme is diverse and sometimes overlapping; they are found membrane-
`bound (DP4, FAP, PEP), in lysosomes (DP2), in the cytoplasm (DP8/9, PEP,
`DP2), or in secretory vesicles (DP2), so it is likely that their substrate
`degradomes will mostly differ.
`Containing a trans-membrane domain within their N-termini and a number
`of glycosylation sites, DP4, FAP, DP6, and DP10 are all type II-membrane
`glycoproteins localized to the cell surface.26 Plasma-soluble forms of both
`DP427 and FAP28,29 have been identified with their presence in circulation
`believed to result from shedding events at the plasma membrane. DP8, DP9,
`and PEP lack a transmembrane domain and are cytosolic proteins.6,30,31 A
`number of membrane associated forms of PEP have been identified.31,32
`Unique to DP9 is the presence of one of the best-known integrin-binding
`motifs the RGD (arginine-glutamine-aspartic acid; Arg-Gly-Asp) motif,
`within its N-termini33 (Figure 1.2). DP9 also contains two potential N-linked
`glycosylation sites;23,34 however, there is no evidence at present of DP9
`glycosylation,35 so the significance of these features is yet to be revealed.
`DP4, FAP, DP8/9, and PEP all have neutral pH optimums of pH 7–8.6,21 In
`contrast, DP2 is a smaller, 492-amino-acid, lysosomal enzyme, active across a
`broad pH range with an acidic pH optimum of 5.5.25 DP2 also localizes within
`intracellular vesicles distinct from lysosomes in resting quiescent cells.36 It is
`likely that these vesicles contain a secretory component due to the release of
`fully functional DP2 in response to calcium stimulation.36 All S9b family
`members and the S28 member DP2 form homodimers with dimerization being
`essential for the catalytic activity of DP4,37,38 FAP13,39 and DP2.40 In con-
`trast, PEP is a monomeric enzyme with SDS-PAGE mobility of 70–80
`kDa.9,41 S9b proteins have a monomer mobility on SDS-PAGE gel of 90–110
`kDa and dimer mobility ranging between 150 and 200 kDa.7 Being much
`smaller, DP2 migrates in its glycosylated form as a B60 kDa monomer on
`SDS-PAGE and forms a B120 kDa homodimer.40, 42–44
`A brief discussion of the homology and gene structure of the DP4-like gene
`family is provided below. Although both DP2 and PEP are of importance when
`discussing therapeutic targeting of DP4, FAP, and DP8/9, they will not be
`discussed here in detail; thus, the reader should refer to critical reviews on DP2
`by Maes et al.24 and on PEP by Garcia-Horsman et al.45. The inactive protease
`homologs DP6 and DP10 will be discussed in brief, as they are not a focus of
`this review (recently reviewed in McNicholas et al.46).
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`1.3.1 Homology and Gene Structure
`
`A high degree of homology exists within the DP4-like gene family with all
`members sharing 25–60% amino acid sequence identity and 47–77% amino
`acid sequence similarity.7 Notably, FAP and DP4, located adjacent to each
`other on the long arm of chromosome 2 (2q24.2 and 2q24.3 respectively), share
`52% amino acid identity. The two non-enzyme members, DP6 and DP10,
`located on chromosomes 7q36.2 and 2q14.1 respectively, share 53% amino acid
`sequence identity, and the most recently identified enzyme members, DP8 and
`DP9, located respectively on chromosomes 15q22.32 and 19q13.3, share 61%
`amino acid sequence identity. The close proximity and high level of similarity
`between the DP4 and FAP genes suggest that they have arisen from a recent
`gene duplication event.7 Likewise, it is possible that DP10, also located on the
`long arm of chromosome 2, was derived from either DP4 or FAP, followed by
`the divergence of DP6 on chromosome 7. Similarly, the high sequence identity
`and shared cytosolic localization suggest that DP8 and DP9 have arisen from a
`gene duplication event.7 Although of varying gene length, the DP4 (82 kb),
`FAP (73 kb), DP6 (935 kb), and DP10 genes (1402 kb) all contain 26 exons,7
`further supporting the likelihood of a common gene ancestor. In contrast, the
`DP8 (72 kb) gene contains 20 exons,6 and the DP9 gene contains 19 or 22 exons
`dependent on whether the gene is expressed in its short (863-amino-acid) or
`long (971 amino acid) forms.34 Like DP9, short and long transcript variants
`with variable length N-termini arising from alternate first exon use exist for
`DP647,48, DP10,49,50 and DP8.6,51 Differing patterns of expression and tissue
`specificity are often associated with these alternative transcripts, but a com-
`prehensive study investigating their significance to human biology is yet to be
`carried out.
`
`1.4 Protease Members of the DP4-Like Gene Family
`
`1.4.1 DP4 and FAP: Extracellular Proteins
`
`DP4 is the best-characterized member of the DP enzyme family, followed to a
`lesser extent by FAP, which was identified almost 20 years after DP4. Both
`enzymes are implicated in a number of roles, including tissue remodeling/
`wound healing, inflammatory bowel disease, arthritis, type II diabetes, obesity,
`and cancer (reviewed in several previous studies52–55). In cancer, both DP4 and
`FAP play conflicting roles acting as either a tumour suppressor or tumour
`promoter depending on the cancer type.56 DP4 plays a key role in glucose
`homeostasis, regulating the activities of GLP-1 and glucose-dependent insuli-
`notropic peptide (GIP) via its proteolytic activity, and has thus become a
`clinically validated target in the management of type II diabetes (reviewed in
`several previous studies57–59). FAP is also of clinical relevance, particularly
`within a cancer and liver disease setting as discussed below. Here, the expres-
`sion patterns and biological importance of DP4 and FAP will be introduced.
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`Human DP4 is ubiquitously expressed in a wide variety of tissues and cell
`types, exerting its functions on a broad range of physiological processes
`within the human body. DP4 is expressed at high levels on the surface of
`several endothelial, epithelial, and fibroblast cells of the lung, kidney, liver,
`intestine, bile duct, and other organs, thus affecting the gastrointestinal/
`digestive, cardiovascular, endocrine, neurological, and immunological sys-
`tems.60,61 In kidney, DP4 is one of the major microvillus membrane/brush-
`border membrane proteins.62 Relatively high levels of soluble DP4 activity are
`observed in human bodily fluids,63–65 and thus the activity of soluble, circu-
`lating DP4 is likely to also contribute to effects observed in many of these
`systems. Variations in the level of serum DP4 activity have been associated
`with numerous diseases, and the effectiveness of measuring soluble DP4
`activity levels as a biomarker for diagnosis, monitoring, and prognosis of
`diseases such as cancer, arthritis, and psychiatric disorders has been investi-
`gated.66–72 In the immune system, expression of DP4, known as the surface
`antigen CD26, is detectable at low levels on the surface of some resting T cells,
`B cells, and natural killer (NK) cells.73–75 DP4 functions as a co-stimulatory
`molecule of T-cell activation, displaying increased expression on the cell
`surface of activated T-cells.73 On the surface of B cells, DP4 expression is
`upregulated following the immunogenic stimulation of B cells with pokeweed
`mitogen76 or the Staphlococcus aureus-derived immunogen streptokinase.77
`Despite the widespread involvement of DP4 in animal biology, the absence of
`DP4 expression does not result in any known adverse defects, as demon-
`strated by the apparent healthy phenotype of knockout DP4 mice (DP4–/–)78
`and their favorable protection against diet-induced obesity and associated
`insulin resistance.79 Recently, studies investigating the long-term chronic loss
`of DP4 expression in genetically induced DP4 deficient rats have identified
`some age-dependent alterations in immune system composition and stress-
`regulatory responses.80,81 Of interest to human pathological conditions,
`particularly cancer, is the apparent loss of DP4 expression during disease
`progression. Loss or alteration of DP4 surface expression has been observed
`in several malignant cells and carcinomas such as lung, breast and ovarian,
`prostate,82,83 and colon adenocarcinoma84 and during the progression of
`melanocytes to melanoma.85,86 It should be made clear, however, that loss of
`DP4 expression is not a hallmark of all cancers with many studies examining
`the expression and roles of DP4 in cancer producing conflicting results. In
`fact, DP4 upregulation has been observed in differing cancer studies to con-
`tribute to the metastatic cancer phenotype, thus acting as a tumor promoter
`contributing to disease progression.
`In contrast to DP4, FAP displays a more restricted and specific pattern of
`expression in adult humans. Detection of translated human FAP in normal
`adult tissue with the F19 monoclonal antibody is limited to the occasional
`fibroblasts and a subset of pancreatic islet cells.87 Results of a master RNA
`blot provide evidence for there being a more ubiquitous pattern of FAP
`expression at the transcript level
`in tissues where protein has not been
`detected.52 FAP protein is most widely expressed in reactive stromal
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`fibroblasts of epithelial cancers including lung, colorectal, breast and ovarian
`carcinomas, subsets of bone and soft-tissue sarcomas, granulating tissue at
`sites of wound-healing, and foetal mesenchymal tissues.87,88 Additionally,
`FAP localizes to the advancing portion (‘invadopodia’) of cultured malignant
`LOX melanoma cells where it contributes to the invasive phenotype.89
`Like the DP4–/– mice, FAP knockouts display a normal phenotype; thus
`a critical role for FAP in foetal development is unlikely.90 FAP is expressed
`by hepatic stellate cells at the tissue remodeling interface in human cirrhosis91
`where its expression is strongly and significantly correlated with the severity
`of liver fibrosis.92 Further studies in human liver have co-localized FAP
`with collagen fibers, fibronectin, and type I collagen.93 Heterologous over-
`expression of FAP results in reduced adhesion and migration of human
`embryonic kidney (HEK)-293T cells.93 In contrast, the same study found that
`over-expression of FAP in the human hepatic stellate line, LX-2, resulted in
`increased adhesion and migration of cells to extracellular matrix proteins and
`invasion across transwells,
`independent of transforming/tumour growth
`factor beta (TGF-b).93 In both HEK-293T and LX-2 cell lines, FAP over
`expression resulted in enhanced staurosporine streptomyces-stimulated
`apoptosis.93 It may be possible that, because of its roles in tissue remodeling
`and wound healing, FAP mRNA is kept at low levels in most cell/tissue types
`to initiate a quick response in the event of external damage. FAP expression
`can be upregulated by TGF-b, 12-O-tetradecanoyl phorbol-13-acetate (TPA)
`and retinoids (chemical compounds related to vitamin A).94 TGF-b is a
`ubiquitously expressed cytokine shown to play central roles in numerous
`processes including FAP relevant wound healing, tissue repair, fibrosis, and
`cancer (reviewed in several previous studies95–98). FAP is also expressed at the
`remodeling interface of idiopathic pulmonary fibrosis (IPF),99 a progressive
`and debilitating disease of the lung. TGF-b is likely to play a key role in the
`mediation of IPF.100 Soluble FAP has been purified from bovine serum29 and
`has been identified as the antiplasmin-cleaving enzyme (APCE) in humans.101
`FAP is responsible for cleavage and increased activity of the human plasmin
`inhibitor, a2-antiplasmin (a2AP),28 thus further supporting the pro-fibrotic
`role of FAP and its early involvement in wound healing.
`Co-expression of FAP and DP4 results in heteromeric complex formation on
`the cell surface of COS-1 cells.102 DP4–FAP expression is found in reactive
`fibroblasts of healing wounds102 and has been localized to invadopodia of
`migratory cells.103 In vivo DP4 and FAP colocalize on capillary endothelial
`cells, but not large blood vessels in invasive breast ductal carcinoma.104 DP4–
`FAP complex formation is important in the development of the tissue-invasive
`phenotype in migratory cells during wound healing103 and plays an important
`role in the migration and invasion of migratory fibroblasts and human endo-
`thelial cells in collagenous matrices.103,104 This is likely to involve localized
`DP4–FAP gelatin degradation. Blocking of the gelatin-binding domain of DP4
`in the DP4–FAP complex blocks local gelatin degradation by endothelial and
`migratory fibroblasts and alters migration and invasion of cells on collagenous
`matrices.104
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`1.4.1.1 Non-Enzymatic Roles of DP4 and FAP
`
`In addition to their enzymatic roles, both DP4 and FAP exhibit a number of
`important functions independent of their enzyme activity, many of which
`involve binding with other proteins and indirect regulation of other proteases
`such as matrix metalloproteinase (MMPs) and plasmin. Of particular ther-
`apeutic interest are the non-enzymatic functions of DP4 and FAP that con-
`tribute to their involvement in cell adhesion and migration. Both DP4 and
`FAP interact with components of the extracellular matrix (ECM). The
`observed roles of DP4 in adhesion and migration of cancer cells may be
`attributable to its binding with cell surface fibronectin.105 Notably, DP4 also
`functions as a binding partner and adhesion molecule for other proteins
`including adenosine-deaminase (DP4 is also known as ADA-binding
`protein),106,107 the kidney Na1/H1 exchanger isoform NHE3,108 and the
`T-cell antigen CD45,109 a protein tyrosine phosphatase. DP4 has also been
`identified as a cell surface receptor for plasminogen.110 DP4 binding with
`ADA and CD45 initiates a signal transduction pathway via tyrosine phos-
`phorylation contributing to the co-stimulatory effect of DP4 on T-cells.73,111
`Binding of the ADA–DP4 complex with plasminogen has been demonstrated
`in 1-LN human prostate-cancer cell lines112 and is thought to contribute to
`FAP and DP4-mediated fibrinolysis.61 Despite homology between the two
`enzymes, no evidence exists for the binding of FAP with ADA.91 In renal
`brush-border membranes where the multimedia complex between DP4 and
`Na1/H1 exchanger isoform NHE3 is formed, inhibition of DP4 activity
`results in the downregulation of NHE3 activity via inhibition of a tyrosine
`kinase signaling pathway, thus suggesting that DP4 plays an important role in
`modulating Na1H1 exchange mediated by NHE3.113,114 The exact mechan-
`ism by which DP4 inhibitors affect downstream tyrosine kinase signaling
`pathways is unknown, but it may be possible that inhibitor-binding results in
`a conformational change to DP4, altering downstream signaling pathways or
`that inhibition of DP4 activity prevents DP4 substrate-mediated signaling.113
`In the human 1-LN prostate-tumor cell line, it has been shown that the
`association of plasminogen with DP4 and NHE3 is involved in regulating the
`invasive phenotype.115 Direct binding of plasminogen type II (Pg 2) with DP4
`on the surface of prostate cancer cells results in the induction of an intra-
`cellular Ca21 signaling cascade that in turn results in the increased expression
`of MMP9.116 DP4-plasminogen binding is thought to ‘lock’ plasminogen into
`a favorable configuration for its cleavage and activation by the urokinase
`plasminogen activator receptor (uPAR).110 It is this activation/interaction
`with uPAR that results in release of intracellular calcium stores and induction
`of MMP9 expression.110 Interestingly, FAP is thought to interact directly
`with uPAR via complex formation.117 UPAR is an important cell surface
`protease, converting plasminogen to plasmin, contributing to regulation of
`ECM proteolysis.118 Numerous studies have investigated physiological roles
`and therapeutic potential of uPAR in human diseases such as cancer
`(reviewed in several previous studies119–121). Co localization of FAP and
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`uPAR in the plasma membrane of LOX malignant melanoma cells and FAP–
`uPAR complex formation has been demonstrated,117 likely increasing
`proteolytic progression of cells through the tumor microenvironment. Such
`non-enzymatic roles of FAP are thought to contribute to its roles in pro-
`fibrinolysis.52 In 2005, Wang et al. found that an enzyme negative FAP
`mutant over-expressed in the LX-2 human hepatic stellate cell (HSC) resulted
`in enhanced cell adhesion, migration and staurosporine streptomyces-stimu-
`lated apoptosis, providing further evidence for the importance of FAPs non-
`enzymatic roles in the facilitation of tissue remodeling in chronic liver
`injury.93 Similar to DP4, FAP expression has also been linked with changes in
`MMP expression. Over expression of either FAP or DP4 in 293T cells resulted
`in an increase in MMP-2 and CD44 expression accompanied by a reduction in
`integrin-b1.93 MMPs are well known for their roles in cell adhesion and
`migration via proteolytic degradation of the ECM, and the association of
`DP4 and FAP with altered MMP activities most likely contributes to their
`observed roles in cancer.
`
`1.4.2 DP8 and DP9: Intracellular Proteins
`
`DP8 was first identified and cloned by Abbott et al. in 2000,6 almost 40 years
`after the initial discovery of DP4. Within the Abbott et al. study, DP9 was first
`identified in silico by BLAST alignment with DP8.6 In 2002, Olsen and
`Wagtmann reported the first cloning of the short 863-amino-acid form of
`DP930 followed by repeated cloning of short DP9 by Qi et al. in 200321 and the
`subsequent cloning of both short and long 892-amino-acid forms by Ajami
`et al. in 2004.34 Due to the high structural homology of both DP8/9 with DP4
`and FAP, investigations into their biochemical functions have rapidly increased
`since their discovery. At present, studies have led to the implication of both
`DP8/9 in immune functions, but clear physiological roles are yet to be defined
`for either of the enzymes. DP8 and DP9 are closely related molecules sharing
`61% amino acid identity,
`intracellular localization, and ubiquitous tissue
`expression. Intuitively, it seems likely that DP8/9 play essential roles in human
`biology, and an overlap or redundancy in function may exist between the two.
`That said, it is also likely that each may have unique and independent functions
`that are yet to be revealed. An inherent problem that arises when investigating
`DP8 and DP9 is differentiating between the observed effects of these two
`enzymes using current research tools. Until knockout animals are generated
`and/or crystal structures resolved to enable the design of DP8 and DP9 selec-
`tive inhibitors, this will continue to be an issue. If DP8 and DP9 are essential
`intracellular enzymes with redundant functions, it will be highly beneficial for a
`double DP8/9 knockout to be generated for an assessment of its viability.
`Likewise, if redundant/compensatory mechanisms exist between DP8 and DP9,
`then individual knockout animals should presumably result in viable offspring.
`At present, only speculations can be made.
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`Originally identified as monomeric enzymes, both DP8 and DP9 are now
`readily known to form catalytically active dimers.122–124 It has recently been
`shown that DP8 monomers still retain activity.125 Recombinant human DP8
`and DP9 display optimal enzyme activity between pH 7.0 and 8.5 with low
`levels of residual activity still observed at acidic pH 5.0.6,21,124 Recently, a
`natural form of DP9 was purified from bovine testes126 and identified as being
`the short form of DP9 by matrix-assisted laser desorption ionization time of
`flight mass spectrometry (MALDI-TOF/TOF MS) analysis of the N-terminal
`sequence.35 This natural form of bovine DP9 displays a similar pH profile to
`the human DP9 expressed and purified from insect cells.126 Enzymatic activities
`of both recombinant DP8 and DP9 can be inhibited in the presence of
`Zn21.6,126 DP9 activity is also reduced in the presence of Ni1 and almost
`completely inhibited by the presence of Cu21 and Hg1. These results suggest a
`physiological role for intracellular Zn21 or other metal ions in the regulation of
`cytosolic DP8/9 activity. Recently, in vitro evidence has emerged suggesting
`that the activity of DP8/9 might also be regulated by intracellular redox
`events.126,127 Evidence of redox-state regulation of related PEP enzyme activity
`has been previously demonstrated.128,129
`Both DP8 and DP9 are ubiquitously expressed at the mRNA level in all
`human adult and foetal tissues as assayed by master RNA blot or Northern
`blot hybridization.6,34 High levels of DP8 transcript can be found in the testis,
`heart, liver, skeletal muscle, kidney, pancreas,6 and a number of endocrine-
`related tissues.52 In testis, a smaller alternative transcript of DP8 with
`high expression has been detected.6,51 Displaying a similar pattern of
`expression, the highest levels of DP9 transcript can be found in the heart,
`liver, skeletal muscle, spleen, prostate, testis, ovary, small intestine, colon,
`peripheral blood leukocytes, and also a number of other endocrine related
`tissues.34 In comparison with DP8, a much lower level of DP9 mRNA was
`detected in the testis;52 thus, it was surprising that natural DP9 was purified
`from bovine testis and not DP8.35 The presence of different mRNA tran-
`scripts for DP8 and DP9 also suggests their importance in the male repro-
`ductive system. Expression of DP8/9 in this system has thus been further
`investigated.
`Using individual DP specific inhibition profiles, Dubois et al. found that the
`majority of detectable DP activity in bovine and rat testis could be attributed to
`the activities of DP8/9, whereas in the epipididymis, DP4 was the predominant
`contributor.130 Immunohistochemistry revealed specific, and distinct staining
`of DP8 and DP9 in sperm cells from late stages of spermatogenesis; however,
`little to no DP8/9 activity was detectable in preparations of bovine sperm,130
`suggesting that DP8/9 play important roles in the late stages of development
`but play no role in mature male gametocytes. In support of these findings, a
`recent study by Yu et al. using microarray data analysis to determine the levels
`of DP8 and DP9 mRNA during the different stages of murine sperm cell
`development found a high abundance of DP9 in later stage spermatocytes.131
`In addition, in situ hybridization of baboon testis found DP8/9 distributed
`in spermatogonia and spermatides but only weak staining of DP9 in
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`Petitioner GE Healthcare – Ex. 1046, p. 11
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`12
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`Chapter 1
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`spermatocytes suggesting species-specific differences.131 If DP8 and DP9 play
`crucial roles in the development of sperm maturation, it can be speculated that
`loss of both DP8/9 expression within the male reproductive system could lead
`to sterility. Future studies analyzing human samples and using knockout ani-
`mals will be useful to answer this question and to assess whether DP8 and/or
`DP9 are potential targets for the development of therapeutics in the treatment
`of infertility.
`As above, individual DP specific inhibition profiles have also been used to
`assess the level of DP8/9 specific activity in human brain, rats and mice.
`Stremenova et al. identified DP8/9 as the major enzymes contributing to
`detectable DP activity in non-malignant human brain tissue.132 DP8/9 have
`also been identified as major enzymes contributing to DP activity in rat
`brains, with comparisons made between crude brain extracts isolated from
`wild-type and DP4 deficient rats.133 Using DP selective inhibitors, Freker et
`al. found that in brain, the level of DP8/9 activity was higher than that of DP4
`and lower than the activity attributable to the related DP2.133 In a similar
`study using DP4 wild type and DP4–/– mice, DP8/9 were identified as strong
`contenders for the observed DP activity in murine brain.134 In addition, this
`Ansorge