Neuropeptidases and the metabolic inactivation of insect neuropeptides

General and Comparative Endocrinology 162 (2009) 8–17

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Neuropeptidases and the metabolic inactivation of insect neuropeptides R. Elwyn Isaac a,*, Nicholas D. Bland a,1, Alan D. Shirras b a b

Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK Department of Biological Sciences, Lancaster Environment Centre, Lancaster University, Lancaster LA1 4YQ, UK

a r t i c l e

i n f o

Article history: Received 29 May 2008 Revised 24 November 2008 Accepted 10 December 2008 Available online 24 December 2008 Keywords: Neuropeptide metabolism Neprilysin Dipeptidyl-peptidase IV Angiotensin-converting enzyme Aminopeptidase Dipeptidyl aminopeptidase III

a b s t r a c t Neuropeptidases play a key role in regulating neuropeptide signalling activity in the central nervous system of animals. They are oligopeptidases that are generally found on the surface of neuronal cells facing the synaptic and peri-synaptic space and therefore are ideally placed for the metabolic inactivation of neuropeptide transmitters/modulators. This review discusses the structure of insect neuropeptides in relation to their susceptibility to hydrolysis by peptidases and the need for specialist enzymes to degrade many neuropeptides. It focuses on five neuropeptidase families (neprilysin, dipeptidyl-peptidase IV, angiotensin-converting enzyme, aminopeptidase and dipeptidyl aminopeptidase III) that have been implicated in the metabolic inactivation of neuropeptides in the central nervous system of insects. Experimental evidence for the involvement of these peptidases in neuropeptide metabolism is reviewed and their properties are compared to similar neuropeptide inactivating peptidases of the mammalian brain. We also discuss how the sequencing of insect genomes has led to the molecular identification of candidate neuropeptidase genes. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Peptidases are key components of peptidergic signalling systems. They are required for the intracellular processing of proproteins to smaller biologically active neuropeptides by precise cleavage at well defined recognition sites within the proprotein sequence (Hook et al., 2008; Rouille et al., 1995). Not only are peptidases critical for the biosynthesis of neuropeptides, they are also important for switching-off neuropeptide signals by cleavage to inactive fragments either before or after receptor-peptide internalisation (Defea et al., 2000; Roosterman et al., 2007). Thus, peptidases hold several pivotal positions in neuropeptide signalling systems, regulating neuropeptide biosynthesis and determining the temporal and spatial range of the signal. The peptidases involved in the biosynthesis and metabolic inactivation of neuropeptides are highly conserved at both the level of protein sequence and catalytic properties, and have clearly arisen early in the evolution of neuropeptides as signalling molecules in animal nervous systems (Husson and Schoofs, 2007; Turner et al., 2001; Van de Ven et al., 1991). Many insect neuropeptides can serve as both central and peripheral signalling molecules and therefore one peptide might operate in multiple and different extracellular environments: the central neurotransmitter/modulator operating in the restricted * Corresponding author. Fax: +44 1133432835. E-mail address: [email protected] (R.E. Isaac). 1 Present address: Department of Biology (Area 11), University of York, P.O. Box 373, York YO10 5YW, UK. 0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.12.011

space of the synapse and peri-synaptic regions, and the systemic hormone released into a relatively large volume of haemolymph (Nässel, 2002; Nässel and Homberg, 2006). In the latter situation the peptide hormone, unless protected in some way, will be vulnerable to hydrolysis by peptidases present either in the haemolymph or attached to the external surface of tissues, such as fat body, that have a large surface area (Garside et al., 1997a,b; Nachman et al., 1999; Quistad et al., 1984; Starratt and Steele, 1985; Strey et al., 1993). Thus, most neuropeptide transmitters/modulators that also double as hormones possess one or more structural features that help to protect the peptide from non-specific degradation by peptidases, especially by exopeptidases that remove either N- or C-terminal amino acids. The most common protective measure is the a-amidation of the C-terminal residue, a modification that is normally critical for receptor activation (Han et al., 2004) and which also is likely to render peptides resistant to hydrolysis by carboxypeptidases that cleave the C-terminal peptide bond. These exopeptidases, the majority of which are metalloenzymes, possess a dead-end substrate binding pocket allowing the correct registering of the scissile peptide bond in the active site. This requires the recognition and stabilisation of the carboxylate group of the peptide substrate by hydrogen bonding and electrostatic interactions with an arginine side-chain in the anionic binding site (e.g., carboxypeptidase A, Auld, 2004). C-terminally amidated peptides lacking a free carboxylate group will not be recognised and consequently will not efficiently bind to the active site of most carboxypeptidases. Some peptides are protected from aminopeptidase attack either by a pyroglutamyl residue resulting

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from the cyclicisation of an N-terminal glutamine or by the presence of a Pro close to the N-terminus. For example, all members of the AKH family of insect peptides have both an amidated C-terminal amino acid and an N-terminal pyroglutamate residue, both of which are critical for biological activity and will provide metabolic protection and stability in the circulation appropriate for the homeostatic role of AKHs in regulating energy reserves (Gade, 1997; Oudejans et al., 1996). The lack of a positive charge at the Nterminus of peptides, such as insect AKH, is likely to confer protection by not allowing electrostatic interaction at the cationic binding site of aminopeptidases. The frequency with which Pro is found towards the N-terminus of some insect peptide families is probably due to the fact that this amino acid offers good protection from indiscriminate aminopeptidases attempting to cleave the first peptide bond (Vanhoof et al., 1995). Proline is unique amongst the naturally occurring amino acids in that the side-chain is attached to both the a-carbon and the nitrogen atom that forms the peptide bond. The resulting imino ring structure prevents free rotation around the aC–N bond and the conformational restraint placed on peptide bonds in the vicinity of the proline residue can confer resistance to non-specialised peptidases (Yaron and Naider, 1993). For example, the majority of characterised insect tachykinins have Pro at position 2, irrespective of the length of the peptide and insect order, and this Pro is not critical for receptor activation (Nässel, 1999). This strong conservation of the penultimate N-terminal residue of insect tachykinins suggests that a role in limiting unwanted metabolism by aminopeptidases is important for the proper functioning of insect tachykinins as signalling molecules. Larger peptides often have additional strategies for protecting bioactivity, such as secondary structure and the presence of one or more disulfide bonds leading to cyclic peptides. The size and shape of the peptide will often restrict entry to the enzyme active site making the peptide less susceptible to proteolysis. Many insect peptides have a core C-terminal sequence required for eliciting a biological response and which often comprises fewer than six residues plus a C-terminal a-amide group (e.g., peptides of the tachykinin, FMRFamide and allatostatin families), which means that the N-terminal region can vary in composition and in length without compromising fundamental signalling properties (Bendena et al., 1999; Gade, 1997; Nässel, 1999; Thorpe et al., 1995). It is not uncommon for a single pro-hormone to contain the sequences of several peptides that on processing will give rise to a set of bioactive peptides with identical or very similar C-terminal structure, but with a variable N-terminal region (Gade et al., 2008; Muren and Nässel, 1996; Siviter et al., 2000). The variation in the primary sequence of these peptides is likely to result in differences in susceptibility to peptidases, while conserving signalling properties. For example, the pro-hormone of cockroach (Diploptera punctata) allatostatins gives rise to a large family of peptides that have a conserved Y/FXFGLamide C-terminal sequence required for inhibition of juvenile hormone synthesis, but which can vary greatly in length and in amino acid residues beyond this core region (Bendena et al., 1999; Tobe et al., 2000). The release of cocktails of functionally homologous peptides with different N-terminal sequences and possibly different rates of metabolism might therefore provide some security in the uncertain environment of the haemolymph where proteolytic activities will vary both quantitatively and qualitatively, depending upon the physiological state and developmental stage of the insect. It follows that if neuropeptides have built-in defensive features against ‘‘run-of-the-mill” peptidases, then specialist enzymes capable of overcoming the aforementioned structural defences will be required for the proteolytic cleavage of neuropeptides into inactive fragments. This is of particular relevance to peptides operating as central transmitters/modulators since metabolism will be an important mechanism for timely clearance of neuropeptides in

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the synaptic and peri-synaptic spaces, and to switch off the agonistic activity of neuropeptides in an analogous fashion to the role of acetylcholinesterase at cholinergic synapses (Defea et al., 2000; Kenny and Hooper, 1991; Turner et al., 1985). These enzymes are often integral plasma membrane proteins with their active sites accessible from the extracellular milieu. Metabolic inactivation might be achieved either by overcoming the block to hydrolysis by non-specific peptidases or by directly attacking internal peptide bonds in the core sequence critical for biological activity. An important catalytic property of many of these peptidases and one which differentiates them from peptidases involved in general protein turnover is that their activity is largely restricted to oligopeptides (Antczak et al., 2001; Kenny and Hooper, 1991). This feature, together with their anchored position on cell membranes, means that they are unlikely to cause unwanted proteolysis of extracellular proteins (Mentlein, 2004). Studies aimed at understanding how neuropeptides are inactivated in invertebrate nervous systems have identified peptidase activities that are similar to those responsible for metabolic inactivation of peptides in mammalian brain. Prominent amongst the various peptidases are members of the neprilysin (NEP), dipeptidyl-peptidase IV (DPP4), angiotensin-converting enzyme (ACE) and aminopeptidase families. In this review, we will focus attention mainly on these peptidase families regarded as specialists capable of overcoming the protective defences of insect neuropeptides. 2. Neprilysin (NEP)-like endopeptidases Brain NEP and its role in neuropeptide metabolism was discovered in studies aimed at identifying new analgesics that block the degradation of enkephalin ‘opioid’ neuropeptides in mammals (Malfroy et al., 1978; Roques et al., 1993). This work led initially to the name ‘enkephalinase’ being adopted for the enzyme, but it was soon realised that this peptidase was not selective for enkephalins and indeed was identical to a kidney enzyme called neutral endopeptidase 24.11 (Matsas et al., 1983; Turner et al., 1985). The enzyme is the archetypical member of the M13 family of zinc-metalloendopeptidases (Rawlings et al., 2006). It is a type II integral membrane protein with a short N-terminal cytoplasmic domain, a transmembrane hydrophobic region and a large lumenal domain containing the active site (Turner, 2004). As an ectoenzyme, NEP catalyses peptide hydrolysis at the extracellular face of the plasma membrane. It is a promiscuous peptidase, but with a strong preference for hydrolysing relatively short oligopeptides (<30 amino acids) by cleaving on the N-terminal side of hydrophobic amino acid residues, P10 in Fig. 1, and it is potently inhibited by the Streptomyces product, phosphoramidon, and several synthetic inhibitors (e.g., thiorphan) (Roques et al., 1993). The ectodomain of the human protein is composed of two separate a-helical lobes joined by intertwining peptide segments (Oefner et al., 2000). The two domain structure of NEP reveals why the enzyme functions exclusively as an oligopeptidase. The smaller second domain appears to restrict access to the active site and thereby limits the size of the peptide substrate that can enter (Oefner et al., 2000). Other related mammalian NEP-like enzymes that are also expressed in the mammalian brain include the endothelin-converting enzymes (ECE-1 and ECE-2), and DINE (damage-induced neuronal endopep-

Fig. 1. A schematic model of a peptidase active site showing the relative positions of the enzyme sub-sites (S3, S2, S1, S10 , S20 , S30 ), the residues of the peptide substrate (P3, P2, P1, P10 , P20 , P30 ). ;, Site of cleavage; , catalytic site.

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tidase) and a soluble secreted endopeptidase (MMEL2, SEP/NEP2) (Turner et al., 2000, 2001). Alternative promoters drive expression of different ECE-1 isoforms, one of which is targeted to endosomal membranes in neuroendocrine cells (Muller et al., 2000). Recently, it has been shown that endosomal ECE-1 can degrade internalised neuropeptides (tachykinin, calcitonin gene-related peptide and somatostatin-14) and that the enzyme regulates receptor recycling (Padilla et al., 2007; Roosterman et al., 2007, 2008). These results have led to the proposal that cleavage of the peptide agonist is necessary to disrupt the peptide-receptor-b-arrestin complex and to recycle and desensitise peptide receptors (Padilla et al., 2007; Roosterman et al., 2007). For the other neuronal NEP-like peptidases, there is a paucity of information about peptide substrates and functioning of these proteins in the nervous system. The first demonstration of a neuronal NEP-like peptidase in insects was obtained using synaptic membranes isolated from the nervous tissue of the desert locust Schistocerca gregaria, to cleave insect peptides (Isaac, 1988). This activity cleaved the Asn3-Phe4 peptide bond of locust AKH at neutral pH and was inhibited by chelators of divalent metal ions and by low micromolar concentrations of phosphoramidon and thiorphan. The same peptide bond of AKH was cleaved by pig kidney NEP. Thus, the locust neuronal peptidase has the hallmark properties of mammalian NEP. A similar peptide-degrading enzyme activity that cleaves [D-Ala2, Leu5]enkephalin at the Gly-Phe peptide bond is present in membranes prepared from the heads of Musca domestica and Drosophila melanogaster, and from the brains of Leuocphaea maderae (Isaac, 1990; Isaac and Nässel, 2003; Lamango and Isaac, 1993b). NEP-like enzymes have also been found in neural tissues from two lepidopteran species (Lymantria dispar and Lacanobia oleracea) and the cockroach, D. punctata, emphasising the widespread occurrence of this class of membrane peptidase in insect nervous systems (Garside et al., 1997b; Isaac et al., 2002; Masler et al., 1996). There are also several reports that NEP-like peptidases of peripheral tissues (fat body, muscle and Malpighian tubules) are responsible for the degradation of circulating AKH and HrTH (hypertrehalosemic hormone) in S. gregaria and the cockroach, Blaberus discoidalis, respectively (Rayne and O’Shea, 1992; Strey et al., 1993). Although these studies identified NEP-like peptidases as potential neuropeptide-inactivating enzymes in insects, information on the cellular distribution of the enzyme activity in the insect brain and the matching this distribution to that of neuropeptide receptors is important for providing clues as to the identity of in vivo substrates. Progress in this direction was made using a histochemical method developed for detection of NEP activity in mammalian brain to map phosphoramidon-sensitive NEP-like activity in the brain of several insect species (Isaac and Nässel, 2003). Peptidase activity occurs in many, but not all, neuropil regions of the brain of the cockroach L. maderae and the migratory locust Locusta migratoria. In the cockroach, NEP-like activity is present in the upper division of the central body, the protocerebral lip anterior to the central body and certain areas of neuropil in the lateral protocerebrum. Enzyme activity is also present in the lobula of the optic lobe and in several regions of the tritocerebrum. Several prominent regions of the L. maderae brain do not contain any NEP-like activity: the mushroom bodies, the antennal lobes, the medulla and lamina of the optic lobes and tritocerebral chemosensory neuropil. In the locust brain, the NEP expression pattern is slightly different from that of the cockroach. NEP activity was found in parts of the mushroom bodies (posterior portion of the calyx, in the b-lobes, in the dorsal a-lobe) and in the lower division of the central body. Also the optic lobe pattern was different in the locust with a set of layers in the basal medulla and the lobula containing strong activity. The brain of L. migratoria also displayed neuropil with no NEP activity, including many regions of the dorsal and lateral protocerebrum, the antennal lobes, the lamina of the

optic lobe and parts of the mushroom body calyx. The NEP expressing regions of the cockroach and locust also stain strongly with antibodies recognising insect tachykinin-related peptides (Muren et al., 1995; Nässel, 1993), suggesting that the insect NEP-like enzyme might form part of a tachykinin signalling system with responsibility for inactivating neuromodulatory tachykinin peptides at synapses. The molecular identity of a locust NEP-like protein was obtained from the cloning of an ECE cDNA homologue from the brain of L. migratoria (Macours et al., 2003). Comparison of the deduced amino acid sequence with mammalian M13 family members suggests that the insect protein is a homologue of ECE-1. The predicted protein has all the residues known to be important for the specific conversion of big endothelin precursor to the bioactive endothelin peptide (Ahn and Johnson, 2004). The conservation of most of the cysteines involved in the formation of disulfide bonds suggests that the general tertiary structure of the locust protein is similar to that of mammalian NEP and ECE, but the absence of the equivalent of Cys412 of human ECE-1, which is utilised for the dimerisation of ECE proteins (Ahn and Johnson, 2004), suggests that the locust enzyme does not form covalently linked protein dimers. The gene is highly expressed in the locust brain and in the midgut where neuropeptides are synthesised in gut endocrine cells (Macours et al., 2003). Although no endothelin homologue has been isolated from insects, endothelin immunoreactivity has been detected in the midgut endocrine cells of L. migratoria (Montuenga et al., 1994). It is not known whether locust ECE is co-localised with the endothelin immunoreactivity and has a role in the maturation of this putative peptide hormone. A great deal more information on the number and evolutionary relationships of insect M13 peptidases can be gleaned from the deduced amino acid sequences obtained from the various insect genome sequencing projects now being completed apace. Phylogenetic analysis places the insect NEP-like proteins into five main clades (Fig. 2, clusters I–V) that delineate with D. melanogaster NEP1, NEP4, NEP3, NEP5 and NEP2, respectively (Bland et al., 2008). Transcripts of NEP1, NEP2, NEP3 and NEP4 are enriched in both the adult brain and the thoracicoabdominal ganglion (Chintapalli et al., 2007), suggesting an important role for these predicted peptidases in neuronal signalling. D. melanogaster NEP2 differs from the main group of M13 peptidases as it is a soluble secreted enzyme. D. melanogaster NEP1, NEP3 and NEP2 are also strongly expressed in peripheral tissues (NEP1, midgut endocrine cells; NEP2 stellate cells of the Malphighian tubules and testes; NEP3, principal cells of the Malphighian tubules) that either make peptide hormones or whose physiology is regulated by peptide hormones (Bland et al., 2007) (Thomas, Bland, Isaac and Shirras, unpublished results). Representatives of the five clades are present in Apis mellifera, indicating that these gene groups appeared prior to the divergence of Hymenoptera and Diptera. Indeed, four of the insect groups can be identified in the genome of the crustacean Daphnia pulex and are therefore likely to have been present in early arthropods (A.D. Shirras, unpublished observations). D. pulex has a gene homologous to insect genes in Group VI. The duplication of this single gene to giving rise to the two clades within this group is therefore presumed to have occurred during early insect evolution. Group III includes the L. migratoria ECE-like protein and all group members possess the ECE-like VNAYY motif which is important for determining the specificity of mammalian ECE-1 for big endothelin. This group clusters with the extended clade containing mammalian ECE proteins. The crystal structure of human NEP has greatly improved our understanding of the molecular structure of the active site of this and related enzymes by identifying the amino acids whose sidechains form the S10 and S20 sub-sites (Oefner et al., 2000). The S10 sub-site is a deep hydrophobic pocket, which explains the prefer-

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51

Tb11972

100 99

Ame1 Aga1 DmeNEP1

95

I

Dps1 Ame4 Tb04762

89

Aga4

54

82

DmeNEP4

79 100

Dps4 Tb15207

100

Dps3 DmeNEP3

100

Ame3

II

III

LmiECE 68 100

Aga3 Ame6 Tb10845 Ame5

64

Tb08205 96

Aga5

IV

DmeNEP5

99 100

Dp5 DmeCG8358

99

71

DmeCG5527 Dps7 DmeCG15485 Tb15284

51

Tb13215 Tb15285 DmeCG14529

100

DmeCG14526 DmeCG14527

100

DmeCG4721 Dps9 DmeCG14528

54 99

Dps10 Tb06174

53

54 84

Ame7 Ame8 Tb09012 Tb00565 Tb13113 Tb13112 Tb13114

55

Tb14700 100

Tb14701 DmeNEP2 Dps2

53

Aga2 100 72

BmNEP MseNEP

V

Ame2. Tb13030 Fig. 2. Phylogenetic analysis of insect NEP-like peptidases. A neighbour-joining phylogenetic tree was generated from a multiple sequence alignment of insect peptidase sequences predicted from genome sequence data using the method described in (Bland et al., 2008). All nodes are labelled with percentage bootstrap (1000 replicates).

ence for amino acids with bulky neutral aliphatic and aromatic side-chains in the P01 position of substrates and inhibitors. The S20 sub-site is relatively large and accommodates the indole sidechain of phosphoramidon. By constructing homology models of insect NEP-like proteins using the crystal structure of human NEP as a template it is possible to compare the substrate/inhibitor binding

sites between the human and insect enzymes. A molecular model of the active site of D. melanogaster NEP2 active site suggests that the S10 pocket of the insect enzyme has a smaller hydrophobic pocket compared to human NEP and a polar rather than the chargedS20 siteinthehumanprotein;thiswouldexplainobserveddifferences in the cleavage of LomTK-1 (GPSGFYGVRamide) – human

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NEP2 cleaves at the Gly7-Val8 peptide bond whereas human NEP cleaves the Gly4-Phe5 bond (Thomas et al., 2005). These structural differences at the S10 and S20 sub-sites may also explain the reduced potency of phosphoramidon and thiorphan as inhibitors of NEP2 when compared with mammalian NEP. Molecular modelling of the interaction of NEP2 active site with D. melanogaster tachykinin-like peptide (DTK2, APLAFVGLRamide) shows a similar conformation to that of LomTK-1 (GPSGFYGVRamide) with the penultimate residue of the DTK2, occupying the S10 sub-site (Fig. 3) and therefore we would predict that DTK2 will be cleaved at the Gly7-Leu8 bond by NEP2, which has been confirmed biochemically (Bland et al., 2007). Similar modelling and prediction of substrate specificities has not as yet been performed on other insect NEPs, but might be very informative, especially since there is significant diversity in the amino acids that are predicted to form the S20 substrate binding site. Many insect neuropeptides have hydrophobic residues either close to or within core sequences for biological activity and these amino acids are highly conserved within neuropeptide families

(Gade, 1997). It is therefore not surprising that insect NEP-like peptidases that also exploit bulky hydrophobic side-chains for substrate binding are well suited as a general mechanism for switching-off neuropeptide signalling and that these enzymes have been highly conserved during the course of insect evolution. 3. Dipeptidyl-peptidase IV-like (DPP IV)-like peptidases Dipeptidyl-peptidase IV-like (DPP IV) is a cell-surface serine peptidase (S9 family) that specialises in removing dipeptides from the N-terminus of oligopeptides (<8000 Da) with either Pro or Ala in position 2 (P1) (Mentlein, 2004; Misumi and Ikehara, 2004; Rawlings et al., 2006). The mammalian enzyme is being studied intensively because it is a target for the development of inhibitors as drugs to treat type 2 diabetes. The therapeutic potential of DPP IV inhibitors results from their blocking of the degradation of glucagon-like peptide (GLP-1), a potent stimulator of insulin biosynthesis and secretion (Arulmozhi and Portha, 2006). Dipeptidyl-peptidase IV, like NEP, is primarily a type II integral membrane

A

F578 I 573

T 706 S 138

V 595 Y 594

F 134 S 135 W 707 S 728 S 130 Y 711 R 719

B

F578

S 138

I 573 V 595

T 706 F 134

S 135

Y 594 Zn2+

W 707 R 719

S 728

S 130 Y 711

Fig. 3. A model of NEP2 with the peptide substrate DTK2 (APLAFVGLRamide) docked at the active site was constructed as described elsewhere (Thomas et al., 2005). The ribbon diagrams show views from two angles of the active-site space with metal ion co-ordinating residues. DTK2 is shown in yellow and the ligand-binding sub-sites S10 and S20 are shown with their side-chains in grey and blue, respectively. Associated helices and zinc co-ordinating ligands are shown in magenta. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.

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glycoprotein that exists as a homodimer (Mentlein, 2004). The crystal structure of human DPP IV has revealed that the catalytic triad (Ser, Asp and His) is in a large cavity accessed by a relatively open cleft formed between the beta propeller and the catalytic domain (Engel et al., 2003; Rasmussen et al., 2003; Thoma et al., 2003; Weihofen et al., 2004).. This allows DPPIV to accommodate larger peptides compared with NEP-like enzymes. Mammalian DPP IV is inhibited by diisopropylfluorophosphate, an irreversible general inhibitor of serine peptidases, but only weakly by phenylmethylsulfonylfluoride (Mentlein, 2004). Ile-Pro-Ile and Val-Pro-Leu are highly selective competitive inhibitors and, recently, a variety of designed inhibitors belonging to different chemical classes have been discovered that are highly potent as well as selective for DPP IV (Demuth et al., 2005). DPP IV-like activity has also been found in neuronal membranes from the blowfly Calliphora vicina, D. melanogaster, L. migratoria, L. maderae and L. oleracea, providing evidence for the widespread occurrence of neuronal DPP IV in insects (Chihara et al., 2005; Isaac et al., 2002; Martensen et al., 1998; Nässel et al., 2000). A membrane-associated DPP IV-like activity was first reported in insect brain by Martensen et al. using Gly-Pro-4-nitroanilide as a chromogenic substrate to assay for activity in the blowfly, C. vicina, but this membrane enzyme was not studied in any detail (Martensen et al., 1998). The same substrate was used by Nässel et al. to show the presence of DPP IV-like activity in homogenates of the brain and midgut from L. maderae and that this peptidase activity was enriched in a membrane fraction (Nässel et al., 2000). The DPP IV-like activity is strongly inhibited by Ile-Pro-Ile, but only weakly by the serine protease inhibitor, phenylmethylsulfonylfluoride. Inhibitors of aspartyl and cysteine peptidases are ineffective as is zinc, a metal that strongly inhibits mammalian DPP IV. Leucophaea maderae DPP IV-like enzyme, partially purified from midgut membranes, cleaves N-terminal dipeptides from L. maderae LemTRP-1 (APSGFLGVRamide), Calliphora vomitoria CavTK-I (APTAFYGVRamide) and Neb TMOF (NPTNLH), but not C. vomitoria CavTK-II (GLGNNAFVGVRamide), L. maderae LemTRP-3 (NGERAPGSKK APSGFLGTRamide), human bradykinin (RPPGFSPFR) and Anodonta cygnea ANcTK (pQYGFHAVRamide) (Nässel et al., 2000). These results establish the specificity of the enzyme for Xaa-Pro N-termini and also show that a Pro-Pro peptide bond is resistant. The cockroach enzyme also cleaves the dipeptide, Thr-Ala, from TAFYGVRamide, the primary product of CavTK-I hydrolysis by DPP IV, indicating that the enzyme, like its mammalian counterpart, also has a preference for peptide bonds where Ala is in the P1 position. Removal of the N-terminal dipeptide from LemTRP-1 and the sequential loss of two dipeptides from the N-terminus of CavTK1 result in progressive loss of biological activity when the cleavage products were tested in a cockroach hindgut contraction assay (Nässel et al., 2000). Thus, although DPP IV is unlikely to serve as an instant off-switch for many insect tachykinins, its activity can attenuate signalling and make the once protected peptide susceptible to attack by aminopeptidases. In L. maderae, DPP IV-like enzyme activity is present at high levels in the double calyces of the mushroom bodies, a region devoid of NEP activity (Nässel et al., 2000). Intermediate DPP IV activity is located in the central body, neuropils adjacent to the a- and b-lobes of the mushroom bodies and in the antennal lobes. Most of these brain regions are richly innervated by neurons expressing Pro2 containing neuropeptides (LemTRPs and leucokinin-like peptides), suggesting that DPP IV is involved in clearing and inactivating neuropeptides. BLAST analysis of the D. melanogaster genome using human DPP IV protein sequence as the query identifies four candidate fly homologues (OME, CG11319, CG3744 and CG11034) that possess the catalytic triad essential for peptidase activity, but only CG11319 and OME are strongly predicted to be type II membrane proteins. There is a 1.8-fold enrichment of OME transcripts in the

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adult brain compared to the whole fly, but the majority of expression takes place in gut tissues and Malpighian tubules (Chintapalli et al., 2007). In contrast, expression of CG11319 is greatly up-regulated in both the adult brain (18.2-fold enrichment) and the thoracicoabdominal ganglion (19.6-fold increase) relative to the whole fly suggesting a specific neuronal role for DPP IV (Chintapalli et al., 2007). CG11319 is therefore a strong candidate for a DPP IV peptidase involved in the metabolic regulation of neuropeptides in the fly central nervous system. A close homologue of the CG11319 gene can be found in the genomes of the mosquito Anopheles gambiae, A. mellifera and the beetle Tribolium castaneum and it would be interesting to know if these genes display a strong neuronal pattern of expression. 4. Angiotensin-converting enzymes (ACE) Angiotensin-converting enzyme (peptidyl-dipeptidase A, ACE) is a member of the M2 family of zinc metallopeptidases (Rawlings et al., 2006). It is a promiscuous enzyme, cleaving dipeptides and sometimes dipeptideamides from the C-terminus of oligopeptides and is best known for its role on the surface of endothelial cells in the synthesis of the vasoconstrictor peptide, angiotensin II (Corvol et al., 2004). Mammalian somatic ACE is a type I integral membrane protein comprising two highly similar fused peptidase units, often called, the N- and C-domains joined by a short peptide linker sequence (Turner and Hooper, 2002). Both units possess peptidase activity, but their substrate specificity and susceptibility to inhibitors differs. There are two other forms of mammalian ACE: a soluble form found in body fluids, which is generated by proteolytic cleavage of the somatic membrane enzyme, and a germinal ACE that is a single domain protein largely identical to the C-domain of somatic ACE and generated in male germ cells by use of an alternative intragenic transcription initiation site (Corvol et al., 2004). A neuronal form of ACE is found in synaptic membranes from mammalian brain which appears to differ from endothelial ACE only by the extent of glycosylation (Hooper and Turner, 1987). ACE can hydrolyze a wide range of neuropeptides and peptide hormones, including enkephalins, substance P, LH-RH and CCK-8 – the last three peptides have an amidated C-terminus and therefore for these substrates ACE should be described as an endopeptidase (Corvol et al., 2004). The localisation of neuronal ACE to regions of the mammalian brain that express substance P led to the suggestion that ACE might have a role in regulating tachykinin signalling (Yokosawa et al., 1983). The first report of an insect ACE came from a study of the metabolism of [D-Ala2, Leu5]enkephalin by head membranes from M. domestica (Lamango and Isaac, 1993a). The housefly ACE, unlike mammalian somatic ACE, was primarily a soluble protein with an Mr of 67,000, corresponding to the size of a protein with a single catalytic unit. The insect enzyme has similar enzymatic properties to mammalian ACE, e.g., activation by chloride and potent inhibition by inhibitors of mammalian ACE (Lamango et al., 1997, 1996). Purified housefly ACE and recombinant D. melanogaster ACE (also known as ANCE) cleave dipeptides or dipeptideamides from a range of mammalian neuropeptides and hormones (angiotensin I, bradykinin, [Leu5]enkephalin, [Met5]enkephalin, substance P, LH-RH, [Leu5]enkephalinamide, [Met5]enkephalinamide) and a range of insect neuropeptides (leucokinin I, leucokinin II, LomTK-1, LomTK-II, DTK 4 and 5, allatostatin I, and AKH I) (Lamango et al., 1997; Siviter et al., 2002). Although insect ACE displays activity towards a spectrum of peptide structures, the rate of hydrolysis of individual substrates can vary greatly. For example, LomTK-1 is rapidly hydrolysed, but other insect tachykinin-like peptides are hydrolysed much more slowly or even not at all (Siviter et al., 2002). Proctolin with a proline at the penultimate position is completely resistant to attack by insect ACE. The ability of insect

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ACE to cleave dipeptideamides from the C-terminus of some substrates is of special interest, since for most insect neuropeptides the C-terminus is amidated and critical for biological activity. Neuropil regions of brains from several insect species (Neobellieria bullata, L. maderae, Carausius morosus, Leptinotarsa decemlineata, Mamestra brassica and Bombyx mori) stain with an antibody raised to housefly ACE, suggesting a possible role in extracellular metabolism of neuropeptides in the nervous systems of these insects (Schoofs et al., 1998). Most of the ACE peptidase activity towards an insect tachykinin (Lom TK-1) in neuronal membranes from L. maderae and L. oleracea fractionates with integral membrane proteins when membranes are subjected to phase separation experiments using the detergent Triton-X114 (Isaac et al., 2002). This property of neuronal ACE in these two species is consistent with ACE being anchored to cell membranes and being well placed for the extracellular metabolism of neuropeptides. However, ACE activity does not appear to be important for tachykinin metabolism in neuronal membranes from L. migratoria and D. melanogaster (Isaac et al., 2002). Strong immunoreactivity has also been found within neurosecretory cells of L. migratoria, and S. gregaria, M. brassica and B. mori, where the antigen is co-localised with locustamyotropin-like peptides (Isaac et al., 1998a; Schoofs et al., 1998). This enzyme might be a soluble form involved in pro-hormone processing by trimming C-terminal pairs of basic amino acids from the C-terminus of biosynthetic intermediates generated by the activity of pro-hormone convertases (Isaac et al., 1998b). Analysis of the sequenced insect genomes shows that the number of ACE-like genes can vary amongst species with the mosquitoes Aedes aegypti and A. gambiae having the most expansive family so far, each with nine genes, of which one encodes a protein comprising two peptidase units linked in tandem by a short peptide (Burnham et al., 2005; Isaac et al., 2007). Most of the insect ACEs are predicted to be secreted proteins with the full complement of catalytic residues expected for a metallopeptidase of the M2 family, but some lack critical active site residues (e.g., D. melanogaster ANCE-2, ANCE-4, ANCE-5 and ANCE-3) and therefore are unlikely to have peptidase activity (Coates et al., 2000). Only the two-unit mosquito proteins possess a C-terminal hydrophobic region, which has the potential to serve as an integral membrane anchor. If these predictions are correct, these mosquito proteins resemble mammalian somatic ACE in both their domain structure and membrane topology. The membrane-associated ACE activity found in neuronal membranes from L. maderae and L. oleracea might well be tethered to the membrane by a similar C-terminal peptide anchor (Isaac et al., 2002).

5. Aminopeptidases Aminopeptidases are exopeptidases that have been implicated in the metabolism of a variety of neuropeptides by removing the N-terminal amino acid (dipeptidylaminopeptidases remove N-terminal dipeptides and are discussed below) from peptides with a free amino terminus (Hui, 2007; Kitabgi, 2006; Llorens-Cortes and Kordon, 2008; Molinaro et al., 2004). They can display specificity for either the first (P1) or second residue (P01 ). For example, aminopeptidase P (APP) cleaves peptides that have Pro at P01 , but has little selectivity for different P1 residues. The presence of a Pro adjacent to the N-terminus makes these peptides resistant to cleavage by other aminopeptidases (Simmons and Orawski, 1992). Aminopeptidase A has a preference for peptides with an acidic N-terminal amino acid (e.g., angiotensin II and CCK-8) (Llorens-Cortes and Kordon, 2008), whereas puromycin-sensitive aminopeptidase has a preference for peptides with either a hydrophobic or basic residue at the amino terminus (e.g., enkephalin peptides) (Thompson and Hersh, 2004). In mammals, there is

both a soluble cytoplasmic and a membrane ectopeptidase form of aminopeptidase P that share a high degree of sequence similarity (Molinaro et al., 2004). Bradykinin and substance P are good in vitro substrates for mammalian APP and other peptides with an N-terminal Xaa-Pro sequence are expected also to be readily cleaved by these enzymes. Since Pro is present at the penultimate N-terminal position of some insect neuropeptides, an aminopeptidase P at the cell surface could play an important role in initiating metabolic inactivation of several neuropeptide transmitters/modulators. It is therefore surprising that there is no evidence for a membrane-associated aminopeptidase P activity in insects, although all the insect genomes sequenced to date contain genes encoding both membrane and soluble forms of APP (Shirras and Isaac, unpublished data). However, only the soluble cytoplasmic form has the full complement of active site residues necessary for APP activity (Kulkarni and Deobagkar, 2002). Why a gene for a non-peptidase APP is conserved in insect genomes is unclear. Almost all characterised aminopeptidases are metalloenzymes and therefore many are inhibited by chelators of divalent metal ions. More specific aminopeptidase inhibitors that are useful for characterising aminopeptidase activities in insects include amastatin, actinonin, bestatin and puromycin. Some insect neuropeptides are not readily hydrolysed by aminopeptidases (e.g., members of the AKH family), but others are and might also be susceptible to progressive removal of amino acids until the core sequence for signalling is under attack. Evidence for a role of an aminopeptidase in the inactivation of an insect neuropeptide comes from a study of the metabolism of proctolin (Arg-Tyr-LeuPro-Thr) in S. gregaria (Isaac, 1987). Locust neuronal membranes cleave proctolin to generate Tyr-Leu-Pro-Thr and Arg-Tyr as primary fragments, both of which are inactive in physiological assays (Starratt and Brown, 1979). Of these two inactivating reactions, only the aminopeptidase activity is enriched in synaptosomal membranes. Proctolin is also an important neuromodulator/transmitter in crustaceans and an extracellular aminopeptidase terminates proctolin activity in the stomatogastric ganglion (STG) of the crab Cancer borealis and the peptidase appears to be responsible for the differences in the pyloric rhythms elicited by two proctolinergic neurons (Wood and Nusbaum, 2002).

6. Dipeptidyl aminopeptidase III The removal of the N-terminal dipeptide of proctolin by cleavage of the Tyr-Leu peptide bond has been found in both nervous and peripheral tissues of several insect species (Isaac, 1987; Mazzocco et al., 2003, 2006; Quistad et al., 1984; Starratt and Steele, 1984, 1985). This activity is similar to that of mammalian dipeptidyl aminopeptidase III (EC 3.4.14.4, DPP-3), an enzyme implicated in the metabolism of enkephalin and angiotensin peptides (Smyth and O’Cuinn, 1994). Mazzocco and Puiroux purified two proteins with relative molecular masses of 80,000 and 76,000 from foregut membranes of the cockroach, Blaberus cranifer, which were identified as insect orthologues of mammalian DPP-3 (Mazzocco et al., 2001). The purified cockroach enzyme hydrolysed proctolin with a Km of 3.8 lM, a value which is an order of magnitude lower than the Km recorded for the proctolin-degrading aminopeptidase of locust synaptic membranes. Cockroach DPP-3 is a metalloenzyme, inhibited by EDTA and by tynorphin, a peptide inhibitor of mammalian DPP-3 (Mazzocco et al., 2001). Expression of the D. melanogaster DPP-3 gene (CG 7415) in S2 cells showed that a significant proportion of the protein was associated with a cell membrane fraction. Subsequently, soluble DPP-3, corresponding to CG7415 was purified from adult flies and was shown to exist as two isoforms of 89 and 82 kDa, which hydrolysed proctolin with a Km of 4.5 lM (Mazzocco et al., 2006). Strong anti-DPP-3 immuno-fluo-

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rescence was observed, using an antibody against the rat liver enzyme, in the larval central nervous system, suggesting a role in degradation of neuropeptides. The bulk of the immuno-fluorescence was, however, cytoplasmic. This supports the possibility that the membrane-associated activity observed in S2 cells might be mitochondrial rather than with the plasma membrane. It has previously been shown that the membrane DPP-3-like activity of the locust CNS is enriched in a mitochondrial subcellular fraction (Isaac, 1987). Nevertheless, the high affinity displayed by this enzyme for proctolin and its neuronal distribution suggest that DPP-3 might be physiologically important and worthy of further investigation. 7. Concluding remarks There is now strong evidence that cell-surface peptidases specialising in cleaving oligopeptides have an important role in regulating neuropeptide signalling in nervous systems of insects. These enzymes are evolutionarily conserved and the human homologues are established drug targets for treating human diseases. Of particular interest are the highly conserved NEP-like endopeptidases, not only because of their evolutionarily conserved role in terminating neuropeptide signalling, but also for their importance in peptide activation and receptor recycling, in the extracellular catabolism of the amyloid b-peptide (Ab) and in their roles in cancer progression (Dawson et al., 2006; Nalivaeva et al., 2008). Studying these peptidases in insects, especially in the genetically tractable D. melanogaster, will provide valuable information not only on fundamental mechanisms of signalling, but also in the development of model systems of practical use in studying the pathogenesis of diseases, such as Alzheimer’s, and development of new therapeutic interventions. Characterisation of the main neuropeptide-degrading enzymes in insects has also been of practical value in the design and synthesis of metabolically stable neuropeptide agonists with high resistance to degradation (Nachman et al., 1998, 2002). These synthetic compounds have potential as insect control agents as do inhibitors of some of the inactivating/processing peptidases (Isaac et al., 2007). Acknowledgments We thank the Biotechnology and Biological Sciences Research Council, UK, and the Wellcome Trust for supporting the work conducted in our laboratories on insect neuropeptidases. NDB was supported by a Ph.D. studentship from the Biotechnology and Biological Sciences Research Council, UK. References Ahn, K., Johnson, G.D., 2004. Endothelin-converting enzyme 1. In: Barret, A., Rawlings, N.D., Woessner, J. (Eds.), Handbook of Proteolytic Enzymes, vol. 1. Elsevier Academic Press, London, pp. 429–434. Antczak, C., De Meester, I., Bauvois, B., 2001. Ectopeptidases in pathophysiology. Bioessays 23, 251–260. Arulmozhi, D.K., Portha, B., 2006. GLP-1 based therapy for type 2 diabetes. Eur. J. Pharm. Sci. 28, 96–108. Auld, D.S., 2004. In: Barrett, A.J., Rawlings, N.D., Woessner, J.F. (Eds.), Handbook of Proteolytic Enzymes, vol. 1. Elsevier Academic Press, Amsterdam, pp. 812–821. Bendena, W.G., Donly, B.C., Tobe, S.S., 1999. Allatostatins: a growing family of neuropeptides with structural and functional diversity. Ann. NY Acad. Sci. 897, 311–329. Bland, N.D., Pinney, J.W., Thomas, J.E., Turner, A.J., Isaac, R.E., 2008. Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships. BMC Evol. Biol. 8, 16. Bland, N.D., Thomas, J.E., Audsley, N., Shirras, A.D., Turner, A.J., Isaac, R.E., 2007. Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster. Peptides 28, 127–135. Burnham, S., Smith, J.A., Lee, A.J., Isaac, R.E., Shirras, A.D., 2005. The angiotensin-converting enzyme (ACE) gene family of Anopheles gambiae. BMC Genomics 6, 172. Chihara, C.J., Song, C., LaMonte, G., Fetalvero, K., Hinchman, K., Phan, H., Pineda, M., Robinson, K., Schneider, G.P., 2005. Identification and partial characterization of

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Lamango, N.S., Nachman, R.J., Hayes, T.K., Strey, A., Isaac, R.E., 1997. Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica. Peptides 18, 47–52. Lamango, N.S., Sajid, M., Isaac, R.E., 1996. The endopeptidase activity and the activation by Cl- of angiotensin-converting enzyme is evolutionarily conserved: purification and properties of an an angiotensin-converting enzyme from the housefly, Musca domestica. Biochem. J. 314 (Pt 2), 639–646. Llorens-Cortes, C., Kordon, C., 2008. Jacques Benoit lecture: the neuroendocrine view of the angiotensin and apelin systems. J. Neuroendocrinol. 20, 279–289. Macours, N., Poels, J., Hens, K., Luciani, N., De Loof, A., Huybrechts, R., 2003. An endothelin-converting enzyme homologue in the locust, Locusta migratoria: functional activity, molecular cloning and tissue distribution. Insect Mol. Biol. 12, 233–240. Malfroy, B., Swerts, J.P., Guyon, A., Roques, B.P., Schwartz, J.C., 1978. 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