Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

Vivian Hook, Shin-Rong Hwang, Jill Wegrzyn and Steven Bark, Skaggs School of Pharmacy, Pharmaceutical Sciences, University of California,
San Diego, California
doi: 10.1002/9780470048672.wecb381

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Neuropeptides for CellCell Communication in Nervous and Endocrine Systems Future Perspectives Chemical Approaches for Elucidating Neuropeptide Mechanisms for Translation into Therapeutic Applications

Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cellcell communication in neurotransmission and for regulation of endocrine functions. Neuropeptides are synthesized from protein precursors (termed proneuropeptides or prohormones) that require proteolytic processing within secretory vesicles that store and secrete active neuropeptides. This article describes the application of chemical biological approaches advantageously used to dene protease pathways involved in neuropeptide biosynthesis. Activity proling of proteases, combined with mass spectrometry, has allowed identication of the novel cathepsin L cysteine protease pathway for neuropeptide biosynthesis, which contributes to neuropeptide production with the subtilisin-like prohormone convertase pathway. Furthermore, proteomic approaches for identifying proteases and protein systems present in secretory vesicles dene the protease pathways and the functional protein systems that jointly operate in the secretory vesicle for production and secretion of active neuropeptides. Neuropeptidomic approaches allow dened primary structural analyses of neuropeptides. Future studies that gain understanding of protease mechanisms for generating active neuropeptides will be instrumental for translational research to develop therapeutic strategies for health and disease.

Neuropeptides for CellCell Communication in Nervous and Endocrine Systems

Neuropeptides mediate neurotransmission as peptide neurotransmitters and mediate cellcell communication as peptide hormones for endocrine regulation of target cellular systems. The term neuropeptides refers to this large, diverse class of peptide neurotransmitters and peptide hormones that typically consist of 340 residues. More than 100 different neuropeptides exist, and new neuropeptides are yet to be discovered. The unique primary sequence of each neuropeptide denes its selective and potent biological actions. The same neuropeptides often serve important functions in both the nervous system as neurotransmitters (Fig. 1) and as peptide hormones in peripheral endocrine systems. For example, enkephalins function as neurotransmitters in the brain and are involved in peripheral actions, including regulation of intestinal motility and immune

cell functions (1, 2). ACTH (adrenocorticotropin hormone) is present in the brain where it functions as a neuromodulator; furthermore, ACTH is a prominent peptide hormone released from the pituitary gland for control of glucocorticoid production in the adrenal cortex (3). Neuropeptides such as -endorphin, NPY (neuropeptide Y), galanin, CRF (corticotropin releasing factor), vasopressin, insulin, and numerous others (Table 1) mediate diverse physiological functions that include analgesia, feeding behavior and blood pressure regulation, cognition, stress, water balance, and glucose metabolism, respectively (48).

Proteolytic processing for neuropeptide biosynthesis

Proneuropeptide (prohormone) precursors
Neuropeptides are derived from larger protein precursors known as proneuropeptides or prohormones. Proneuropeptides refers to protein precursors of peptide neurotransmitters as well as peptide hormones, whereas prohormones refers primarily to 1

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

Figure 1 Peptide neurotransmitters in the brain. Neuropeptides in the brain function as peptide neurotransmitters to mediate chemical communications among neurons. Neuropeptides are synthesized within secretory vesicles that are transported from the neuronal cell body via the axon to nerve terminals. The proneuropeptide (or prohormone) is packaged with the newly formed secretory vesicle in the cell body, and proteolytic processing of the precursor protein occurs during axonal transport and maturation of the secretory vesicle. Mature processed neuropeptides are contained within secretory vesicles at the synapse where activity-dependent, regulated secretion of neuropeptides occurs to mediate neurotransmission via neuropeptide activation of peptidergic receptors.

Table 1 Neuropeptides in the Nervous and Endocrine Systems Neuropeptide ACTH -MSH beta-endorphin calcitonin cholecystokinin CRF (corticotropin releasing factor) enkephalin galanin glucagon insulin NPY PACAP somatostatin vasopressin Regulatory Function steroid production skin pigmentation, appetite analgesia, pain relief calcium regulation learning, memory, and appetite ACTH secretion analgesia, pain relief cognition glucose metabolism glucose metabolism obesity, blood pressure neuronal differentiation growth regulation water balance

Peptide neurotransmitters and hormones are collectively termed neuropeptides. Neuropeptides typically consist of small peptides of approximately 340 residues. Several neuropeptides and several of their regulatory functions are listed; these neuropeptides and others function in multiple roles as physiological regulators (too numerous to list in this short table). Abbreviations are adrenocorticotropin hormone (ACTH), -melanocyte stimulating hormone (-MSH), neuropeptide Y (NPY), and pituitary adenylate cyclase-activating peptide (PACAP).

endocrine peptide hormone precursors. To encompass peptide functions in both the nervous and endocrine systems, the terminology of neuropeptide and the respective proneuropeptides will be used in this article to refer to neuroendocrine functions of neuropeptides. Proneuropeptide precursors share distinct and common features. Notably, the small active form of each neuropeptide is a segment present within its full-length precursor protein. A proneuropeptide may contain one copy of the active neuropeptide, as represented by proNPY, progalanin, and provasopressin 2

(Fig. 2) (911). Alternatively, a precursor may contain multiple related copies of the active neuropeptide. For example, proenkephalin contains four copies of (Met)enkephalin, one copy of the related (Leu)enkephalin, and one copy each of the ME-Arg-Gly-Leu and ME-Arg-Phe (Fig. 2) (1214). Proteolysis of these precursors, especially tissue-specic proteolytic mechanisms, is required for biologically active neuropeptides to be generated. Although each proneuropeptide precursor possesses a distinct primary sequence, proteolytic processing occurs at dibasic

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

Figure 2 Proneuropeptides: structural features for proteolytic processing. Neuropeptides are synthesized as proneuropeptide precursors, also known as prohormones, that require proteolytic processing to liberate the active neuropeptide. Proteolytic processing occurs at dibasic and monobasic sites, as well as at multibasic sites. The precursor proteins may contain one copy of the active neuropeptide, such as the proneuropeptides for NPY, galanin, CRF, and vasopressin. Some proneuropeptides such as proenkephalin contain multiple copies of the active neuropeptide; proenkephalin contains four copies of (Met)enkephalin (ME), one copy of (Leu)enkephalin (LE), and the related opioid peptides ME-Arg-Phe (H) and ME-Arg-Gly-Leu (O).

residue sites that commonly ank the NH2 and COOH termini of neuropeptides within their precursors (Fig. 2). The dibasic residues LysArg (KR) most often ank the neuropeptides; however, the dibasic sites LysLys, ArgArg, and sometimes ArgLys also occur. Processing sometimes occurs at monobasic Arg sites as well as at multibasic residue sites. Processing at non-basic residues occurs occasionally. Overall, proteolytic processing is a key process required for the biosynthesis of numerous active neuropeptides from inactive precursors.

Proteolytic processing of precursors for neuropeptide biosynthesis

Biosynthesis of neuropeptides begins with the translation of the respective mRNAs to generate the preproneuropeptide or preprohormone precursors. Proteolytic processing begins cotranslationally at the rough endoplasmic reticulum (RER) where the NH2 -terminal signal peptide of the preproneuropeptide is cleaved by signal peptidase. The resulting proneuropeptide or prohormone is routed through the Golgi apparatus and is packaged into newly formed secretory vesicles together with processing proteases. As the secretory vesicle matures, proteolytic processing occurs so that the mature secretory vesicle contains processed, biologically active neuropeptide that awaits cellular stimuli for regulated secretion. Proteolytic processing at the dibasic or monobasic sites of proneuropeptides occurs primarily within regulated secretory vesicles (1518). Cleavage at the COOH-terminal side of the

paired basic residues results in peptide intermediates with basic residue extensions on their COOH-termini, which must then be removed by Lys/Arg carboxypeptidase to generate the mature neuropeptide (Fig. 3). Alternatively, cleavage of the precursor at the NH2 -terminal side of dibasic residue sites will generate peptide intermediates with basic residue extensions on their NH2 -termini, which then will be removed by aminopeptidase B to generate the active neuropeptide. Processing may also occur between the dibasic residues, which then will require both carboxypeptidase and aminopeptidase exopeptidase activities to generate the nal neuropeptides. Neuropeptides may also undergo post translational modication that modies the biological activities of peptides. Activities of the neuropeptides may be altered by disulde bond formation, glycosylation, COOH-terminal -amidation, phosphorylaton, sulfation, and acetylation (6, 7). This article, however, will focus on protease mechanisms for neuropeptide biosynthesis.

Biochemical properties expected of proteases for neuropeptide production

Elucidation of proteases in brain and neuroendocrine tissues is complicated because of the many different cell types and the presence of proteases in many subcellular compartments of these cells. To ensure that authentic proteases are identied for producing an active peptide, the neuropeptide eld has used key criteria for successful elucidation of proteases that generate peptide neurotransmitters and hormones. These criteria 3

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

Figure 3 Cysteine protease and subtilisin-like protease pathways for proneuropeptide processing. Distinct cysteine protease and subtilisin-like protease pathways have been demonstrated for pro-neuropeptide processing. Recent studies have identied secretory vesicle cathepsin L as an important processing enzyme for the production of the endogenous enkephalin opioid peptide. Preference of cathepsin L to cleave at the NH2 -terminal side of dibasic residue processing sites yields peptide intermediates with NH2 -terminal residues, which are removed by Arg/Lys aminopeptidase. The well-established subtilisin-like protease pathway involves several prohormone convertases (PC). PC1/3 and PC2 have been characterized as neuroendocrine processing proteases. The PC enzymes preferentially cleave at the COOH-terminal side of dibasic processing sites, which results in peptide intermediates with basic residue extensions at their COOH-termini that are removed by carboxypeptidase E/H.

are as follows: 1) The processing protease must be present in the organelle site where production of the active peptide occurs, primarily in secretory vesicles; 2) the protease must possess the appropriate substrate cleavage specicity to generate the active peptide; and 3) the protease inhibition or gene knockdown should reduce production of the active peptide. Application of these criteria has led to elucidation of the recently identied cysteine protease pathway and serine protease pathways, mediated by cathepsin L and proprotein convertases, respectively, for neuropeptide production (Fig. 3).

Chemical biology for activity-based proling and identication of proteases

Recent achievements in the development of active-site directed afnity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (2427). These specic activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclassescysteine, serine, metallo, aspartic, and threoninecan provide advantageous chemical approaches for functional protease identication. Activity probe labeling of proteases allows direct identication of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identication of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article.

Chromafn granules: model neurosecretory vesicles for proneuropeptide processing proteases

Elucidation of protease pathways for neuropeptide biosynthesis has been facilitated in the eld with the use of isolated chromafn secretory vesicles, a well-established model neurosecretory vesicle system used for investigation of proteases that synthesize neuropeptides and small-molecule neurotransmitters (19). Chromafn secretory vesicles contain proneuropeptide precursors that undergo proteolytic processing to generate enkephalin, NPY, galanin, somatostatin, VIP, and other neuropeptides. These vesicles were used to identify the cathepsin L as a novel proneuropeptide processing enzyme using chemical biological approaches and have identied prohormone convertase processing enzymes (2023). 4

Activity-based chemical proling identies the cathepsin L cysteine protease pathway in secretory vesicles that contributes to neuropeptide biosynthesis
Activity-based proling of active cysteine proteases identied cathepsin L as a proenkephalin processing protease in secretory vesicles. The activity probe DCG-04, the biotinylated form of E64c that inhibits cysteine proteases, was used for specic afnity labeling of the 27 kDa protease enzyme of the prohormone thiol protease (PTP) complex (18, 22), which represents

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

the major proenkephalin (PE) processing activity in chromafn secretory vesicles (20, 21). The high molecular weight of the PTP complex of approximately 180200 kDa (21) suggested the presence of several protein subunits because proteases typically possess lower molecular masses than that of native PTP. Studies then were targeted to identify the catalytic subunit of PTP responsible for PE-cleaving activity. The activity probe DCG-04, combined with differential labeling in the presence of CA074, allowed identication of the 27 kDa protein as the active protease subunit of the PTP complex (18, 22). Two-dimensional gels resolved DCG-04-labeled proteins of 2729 kDa (Fig. 4), which was identied as cathepsin L by mass spectrometry of tryptic peptides. Conrmation of the localization of cathepsin L in secretory vesicles was demonstrated by immunouorescence confocal microscopy and immunoelectron microscopy, which illustrated the presence of cathepsin L in enkephalin and neuropeptide-containing secretory vesicles. The secretory vesicle function of cathepsin L contrasts with its well-known lysosomal function for degradation of proteins. These ndings suggested a new biological function for cathepsin L in secretory vesicles for producing the enkephalin and related neuropeptides.

Cathepsin L knockout mice

Cathepsin L-decient mice show decreased levels of enkephalin in the brain, with reduction by approximately one half (22). In addition, enkephalin brain levels are also reduced by about one half in PC2-decient mice (28). These results support dual roles for both cathepsin L and PC2 in enkephalin production. Ongoing studies indicate multiple neuropeptides that are substantially decreased by more than 50% in the brain and endocrine tissues of cathepsin L knockout mice (Funkelstein et al., submitted for publication). With the observed alterations in brain neuropeptides, it will be of interest in future studies to assess the behavioral effects of the loss of neuropeptides in cathepsin L knockout mice. Cathepsin L knockout mice are viable and show phenotypes of hair loss and cardiac myopathy (29, 30). The mechanism for these functional effects of cathepsin L deciency could possibly involve neuropeptides. New and continued investigations of neuropeptides in cathepsin L knockout mice will provide knowledge of the relative roles of cathepsin L in the production of particular neuropeptides.

Gene analyses of Cathepsin L in neuropeptide biosynthesis by protease gene knockout and gene expression approaches
S

Cellular gene expression of cathepsin L for enkephalin neuropeptide production in the regulated secretory pathway
Cellular routing and trafcking of cathepsin L to secretory vesicles for proneuropeptide processing was demonstrated by the coexpression of cathepsin L with proenkephalin in neuroendocrine PC12 cells (derived from rat adrenal medulla) (31). Expression of cathepsin L resulted in its trafcking to secretory

H NH

H HN O

HN HO O

O H2N O H N N H O N H

O H N

O O OEt

(a)

(b)

Figure 4 Activity-based proling for identication of proenkephalin cleaving activity as cathepsin L. Activity-based proling (APB) uses the strategy of labeling the active site of active proteases, often with an inhibitor-related probe, to identify proteolytic activity. Inhibition of proenkephalin cleaving activity by the cysteine protease inhibitor E64c in isolated chromafn secretory vesicles (also known as chromafn granules) allowed afnity labeling of the 27 kDa active protease enzyme proteins by a biotinylated form of E64 known as DCG-04 (Panel a). The inhibitor-labeled proteins were separated by two-dimensional gels (Panel b) and subjected to peptide sequencing by mass spectrometry, which revealed the identity of the proneuropeptide processing activity as cathepsin L. WILEY ENCYCLOPEDIA OF CHEMICAL BIOLOGY 2008, John Wiley & Sons, Inc.

Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

vesicles that contain enkephalin and chromogranin A. Furthermore, cathepsin L expression resulted in cellular processing of proenkephalin into (Met)enkephalin that undergoes regulated secretion from PC12 cells. Cathepsin L generated high molecular weight PE-derived intermediates (of about 23, 1819, 89, and 4.5 kDa) that represented PE-derived products in vivo (20). Such results demonstrated a cellular role for cathepsin L in the production of (Met)enkephalin in secretory vesicles for its regulated secretion.

containing C-terminal basic residue extensions that are removed by carboxypeptidase E. These dual protease pathways provide alternative routes for cellular processing of proneuropeptides into active peptide neurotransmitters and hormones.

Cathepsin L cleavage specicity indicates the subsequent aminopeptidase B exopeptidase step for neuropeptide production
Studies of the cleavage specicity of cathepsin L demonstrated that it prefers to cleave on the NH2 -terminal side of dibasic residue processing sites of enkephalin-containing peptide substrates BAM-22P and Peptide F (22) and to cleave at the N-terminal sides of dibasic residues within peptide-MCA substrates (32). The cleavage specicity of cathepsin L results in enkephalin intermediate peptides with NH2 -terminal basic residue extensions, which are then removed by Arg/Lys aminopeptidase. Secretory vesicles from adrenal medullary chromafn cells (33) and from pituitary (34) contain Arg/Lys aminopeptidase activity for neuropeptide production. Recent molecular cloning studies have identied aminopeptidase B as an appropriate Arg/Lys aminopeptidase (35). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA dened its primary sequence that provided production of specic antisera to demonstrate localization of AP-B in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and NPY. AP-B was also found in several neuroendocrine tissues by western blots. Recombinant bovine AP-B (35) and rat AP-B were compared. Recombinant bovine AP-B showed preference for Arg-MCA substrate compared with Lys-MCA. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin and Lys-(Met)enkephalin neuropeptide substrates to generate (Met)enkephalin, whereas rat AP-B preferred Arg-(Met)enkephalin. Furthermore, AP-B possesses an acidic pH optimum of 5.56.5 that is similar to the internal pH of secretory vesicles. The signicant nding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides. These ndings indicate differences in the cleavage specicity of cathepsin L for the N-terminal side of dibasic residues within proneuropeptides, compared with cleavage at the C-terminal side of dibasic residue processing sites by the prohormone convertases 1 and 2 (PC1/3 and PC2) (1517, 23). These differences result in the requirement for different exopeptidases following endoproteolytic processing by cathepsin L compared with PC1/3 or PC2. Although cathepsin L cleavage of neuropeptide precursors results in peptide intermediates extended with basic residues at their N-termini that can be removed by aminopeptidase B, PC1/3 and PC2 cleavage at the C-terminal side of dibasic residues of proneuropeptides results in peptide products 6

Chemical biology denes the novel cathepsin L cysteine protease pathway combined with the prohormone convertase subtilisin-like pathway for neuropeptide production
Signicantly, the approach of activity proling for cysteine proteases has established cathepsin L as a new protease pathway for neuropeptide biosynthesis. Together with current knowledge in the eld, these data demonstrate the existence of two distinct protease pathways for converting proneuropeptides into active peptide neurotransmitters and hormones. These dual pathways consist of the newly discovered cysteine protease pathway for proneuropeptide processing, which consists of cathepsin L followed by Arg/Lys aminopeptidase (aminopeptidase B), and the previously known proprotein convertase (PC) family of subtilisin-like proteases (1517) that process proneuropeptides with carboxypeptidase E (Fig. 3). Elucidation of these two protease pathways resulted from the application of the biochemical criteria required for processing proteases.

Cathepsin L: member of clan CA and the C1A papain subfamily

Properties of cathepsin L may be compared among papain-like cysteine cathepsins (3639) for understanding its role in producing neuropeptides. Cathepsin L belongs to the C1A subfamily of Clan CA (36). Clan CA was formed based on recognition of the rst cysteine protease papain. The crystal structure of papain shows two structural domains separated by an active-site cleft. The N-terminal domain is comprised of -helices, and the C-terminal domain contains a -barrel. Clan CA is composed of twenty families. Family C1 within clan CA is divided into two subfamilies that consist of C1A (papain subfamily) and C1B (bleomycin hydrolase subfamily) groups. The larger subfamily C1A consists of secreted and lysosomal proteases that include the animal cysteine cathepsin proteases cathepsins B, H, and L as well as the plant proteases papain, chymopapain, and actinidain. A division within the C1A subfamily exists between the papain-like proteases and the cathepsin B-like proteases. Included among the papain-like proteases are cathepsins O, H, L, K and S. Cathepsin O is divergent, whereas the others are more closely related. Among the cathepsin B-like proteases are dipeptidyl-peptidase I and the endopeptidases from Giardia . The distinction between exopeptidases and endopeptidases is merged for some members of the subfamily C1A. Dipeptidylpeptidase I acts principally as an exopeptidase, removing N-terminal dipeptides, but may have some endopeptidase activity. Cathepsins B and H both possess endopeptidase activity but also possess exopeptidase activities. Cathepsin B acts as a peptidyl-dipeptidase, releasing C-terminal dipeptides. Cathepsin X is a carboxypeptidase.

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

The proteases that enter the secretory or lysosomal pathways are synthesized as precursors, with N-terminal propeptides and signal peptides (3639). Most members of family C1 have propeptides similar to that of papain. The propeptides act by blocking the active site. Papain-like propeptides are indicated by the ERFNIN motif. Propeptide inserts relative to papain occur within the catalytic domain in other family members. Cathepsin B contains the occluding loop that carries the histidine residues important for peptidyl-dipeptidase activity. Most members of subfamily C1A are monomeric. In the case of cathepsin L, it exists as a single-chain form (28 kDa on SDS-PAGE) and as a heavy-chain and light-chain form (24 and 4 kDa, respectively, on SDS-PAGE). The specicity subsite that is dominant in most proteases of subfamily C1A is S2, which commonly displays a preference for occupation by a bulky hydrophobic side chain. Cathepsin L possesses such S2 subsite preference for aromatic residues in the P2 position. These properties contribute to the function of secretory vesicle cathepsin L as a processing enzyme that produces neuropeptides.

Neuropeptides in mice decient in PC2, PC1/3, and CPE: evidence for other processing enzymes
PC2 and PC1/3 prohormone convertases
A review of neuropeptide data from PC2 and PC1/3 knockout mice shows that the majority of neuropeptides studies are partially reduced in the knockout compared with wild-type controls. These results indicate roles for PC2 and PC1/3 in neuropeptide production. Importantly, the partial reduction of neuropeptides examined in PC2- and PC1/3-decient mice also indicates possible roles for other proteases for processing proneuropeptides, such as cathepsin L in secretory vesicles for neuropeptide production that has been discussed in this article. Thus, the chemical biology approach has identied the novel cathepsin L cysteine protease pathway as a candidate route for neuropeptide production. More specically, to integrate data from PC2- and PC1/3decient mouse studies with recent data that demonstrate a candidate role for cathepsin L in neuropeptide production, results of neuropeptides studied in PC2- and PC1/3-decient mice are summarized here. In PC2 knockout mice, many neuropeptides were partially reduced, with the exception of -MSH that was nearly obliterated. PC2-decient mice show increases in the POMC-derived peptide hormones ACTH and -endorphin (131), which identies them as substrates for PC2 (40). Among the POMC-derived peptide hormones, only -MSH was nearly completely absent in the PC2 knockout (40). NPY was unchanged in the brain but was decreased in ileum and was increased in adrenal (41). Somatostatin was increased in the brain and was unchanged in the intestine; (Met)enkephalin was partially decreased in the brain but was not altered in adrenal and intestinal tissues. (41). No changes in VIP, galanin, or CRF were observed in PC2-decient mice. Insulin in the pancreas was reduced by 75% compared with wild-type controls, and

somatostatin-14 was reduced (42). Partial reductions in the neuropeptides CCK (43), nociceptin (44), and neurotensin (45) have been observed in PC2-decient mice. In PC1/3-decient mice, reduction of GnRH by about 80% was observed as well as decreases in GLP-1 and GLP-2 (46). Reduction in processing of proinsulin to insulin was observed in PC1/3-decient mice (47). Interestingly, no change in vasopressin occurred, and little change in POMC-derived peptide hormones were observed in the PC1/3-decient mice compared with wild-type controls (48). These ndings demonstrate several features with regard to the selective roles of PC2 and PC1/3 in determining the production of neuropeptides. Firstly, PC-decient mice may show changes in several neuropeptides but not all neuropeptides. Secondly, neuropeptides may show tissue-specic differences in PC-decient mice. Thirdly, a particular tissue region may show selective alterations among different neuropeptides in each of the PC-decient mice. These results demonstrate the roles of PC2 and PC1/3 in neuropeptide production. Notably, partial reductions of many neuropeptides in mice decient in PC2 or PC1/3 are consistent with the presence of other processing enzymes such as the newly identied cathepsin L protease pathway for neuropeptide production.

Carboxypeptidase E
After the actions of PC1/3 and PC2 for cleavage at the C-terminal side of paired basic residues (Fig. 3), the resulting peptide products contain basic residues at the C-termini that are then removed by carboxypeptidase E (CPE). Studies of CPEfat/fat mice that contain mutant, inactive CPE, showed that these animals show altered neuropeptide production (4955). Studies of CPE peptide substrates in the fat/fat mice have been facilitated with specic isolation of peptides with C-terminal basic residues by the anhydrotrypsin afnity column for enrichment of CPE substrates (4953). Analyses of such CPE substrates have demonstrated that numerous neuropeptides use CPE for their biosynthesis (4955). Analogously, it will be important to evaluate cathepsin L-generated peptide products that serve as substrates for the subsequent aminopeptidase B (AP-B) step. Signicantly, the AP-B substrates contain basic residues at their N-termini; these substrates do not contain basic residues at their C-terminal as CPE substrates do. Therefore, although the anhydrotrypsin afnity column can be used to isolated CPE substrates, selective analyses of AP-B peptide substrates will require other approaches for isolation and analyses. It will be of interest in future studies to characterize AP-B neuropeptide substrates generated by secretory vesicle cathepsin L.

Neuropeptidomics: LC-MS/MS tandem mass spectrometry analyses of neuropeptides

Primary structure analyses of neuropeptides are essential for understanding the structurefunction features of neuropeptides. The development of mass spectrometry (MS) tools and approaches for identifying neurological peptides has become essential for dening neuropeptide structures that correspond to 7

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biological activitiesthis eld has been termed neuropeptidomics. Combined LC-MS/MS (liquid chromatography tandem mass spectrometry) provides separation of neuropeptides by the high resolving power of chromatography for specic mass measurements by tandem mass spectrometry that denes neuropeptide structures. Neuropeptide analyses may be achieved by several types of mass spectrometers including MALDI-TOF (matrix-assisted laser desorption time-of-ight), ESI-Trap (electrospray ionization ion-trap), and ESI-qTOF (quadrupole-time-of-ight instruments). Neuropeptides may be directly analyzed by MS without proteolysis (by trypsin) to gain information about the endogenous biological peptide. Trypsinization and MS analyses can also enhance neuropeptide structural analyses, typically using standard shotgun proteomic approaches (56, 57). Furthermore, quantitative neuroproteomics advantageously provide information about how designated systems of neuropeptides may be regulated (48, 50, 58). Neuropeptidomic analyses have several advantages compared with traditional radioimmunoassay or antibody-based methods used for neuropeptide characterization. First, neuropeptides are identied directly by mass spectrometry approaches as specic molecular species rather than indirectly through binding to antibodies. Second, mass spectrometry is capable of rapid sequential analysis of multiple peptide species in a single experiment. Finally, the vast information content of simple experiments provides signicant knowledge about possible components and interactions among the neuropeptide processes in biological systems. Peptides have been investigated by neuropeptidomic approaches with considerable success in mammalian tissues including hypothalamus (48, 50, 58), pituitary (49), islets of Langerhans (60), brain extracellular uids (61), and synaptosomes (62). In each of these studies, signicant new information on potential neuropeptides and precursors was obtained. The extension of these studies to biological tissues has encompassed MALDI analysis of tissue sections (63) and even single mammalian cells (64). Studies have developed quantitative evaluation of neuropeptides using labeled and label-free methods. Because many proneuropeptides are processed by common protease processing pathways (i.e., Fig. 3), new neuropeptides and their precursor proteins may be predicted from genomic and protein databases (65). In addition to mammalian systems, neuropeptidomics analyzes nonmammalian model species including Aplysia (66), Apis (67), C. elegans (68), Drosophila (69), and Tribolium castaneum (70). Indeed, bioinformatics applications for neuropeptidomics have been most well developed in nonmammalian studies of neuropeptides. It is predicted that dening neuropeptide structures by neuropeptidomics will reveal novel peptide neurotransmitters and hormones that possess biological activities. With the numerous peptidergic receptors, largely G-protein coupled receptors, with unknown functions, it is clear that increased focus on endogenous neuropeptide ligands can facilitate knowledge of peptide mechanisms for neurotransmission and endocrine communication. 8

Proteomics of Secretory Vesicles for Dening Proteases and Related Systems for Neuropeptide Biosynthesis
Direct proteomic approaches can be used to identify protease enzyme proteins, as well as protein categories in the biological system, that are present in secretory vesicles for neuropeptide production and secretion. Knowledge of the secretory vesicle proteome can advance our understanding of neuropeptide biosynthetic mechanisms that operate within this organelle. Recent examination of proteins in model chromafn secretory vesicles revealed several functional protein categories that together support secretory vesicle production of neuropeptides and bioactive catecholamines for cellcell communication (Fig. 5) (71). Protein systems involved in vesicular neuropeptide biosynthesis were examined in proteomic studies of soluble and membrane fractions of dense core secretory vesicles puried from neuroendocrine chromafn cells. Proteins were separated by SDS-PAGE, and proteins from systematically sectioned gel lanes were identied by microcapillary LC-MS/MS (LC-MS/MS) of tryptic peptides (71). Proteomic results revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several proteases of different mechanistic classes were identied by proteomics of secretory vesicles. These proteases included the subtilisin-like prohormone convertases 1 and 2 along with the metalloprotease carboxypeptidase E (CPE), which participate in prohormone processing (1517). CPE has also been proposed to function as a prohormone sorting receptor (72). Regulators of PC1 and PC2 were found to consist of proSAAS and 7B2 (73, 74), respectively. Interestingly, cathepsin B (cysteine protease) (75) and cathepsin D (aspartic protease) (76) were identied, which indicates a novel location for these previously known lysosomal proteases. The localization of cathepsin B in these secretory vesicles has been conrmed by immunoelectron microscopy (77). Cystatin C was identied in the membrane and soluble components, which may participate in the regulation of cathepsins L and S; cystatin C is a member of the cystatin superfamily of protease inhibitors (78, 79). Ubiquitin, a highly conserved 76 amino acid protein that is covalently linked to proteins targeted for degradation by the ubiquitin-proteosome system (80, 81), was identied; furthermore, ubiquitin-binding protein S27A was also identied. These ndings may possibly be interpreted to suggest that ubiquitin-targeted protein degradation by proteosomes may occur in secretory vesicles. TIMP, tissue inhibitor of metalloproteinase, was also present (82). These identied protease system components were mostly present in both soluble and membrane fractions. Furthermore, membrane-selective functions were implicated by proteomic data of these secretory vesicles. The membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules (83, 84), together with SNARE-related proteins and annexins that are involved in trafcking and exocytosis of secretory vesicle components (85, 86). Membranes also preferentially contained ATPases that regulate proton translocation

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

Production of Hormones/Transmitters/Factors: Prohormones, Proteases, Neurotransmitter Enzymes/Transporters

PRONEUROPEPTIDES

Internal Vesicular Environment: Redox, Proton Regulation, Protein Folding

SECRETORY VESICLES Production and Secretion of: Hormones, Neurotransmitters, Neuromodulatory Factors

Cell Adhesion

Vesicular Trafficking: Nucleotide-binding proteins, Ca2+ regulation, Exocytosis proteins

NEUROPEPTIDES, SECRETION

Figure 5 Proteomics reveals functional secretory vesicle protein systems for neuropeptide biosynthesis, storage, and secretion. Chromafn secretory vesicles (also known as chromafn granules) were isolated and subjected to proteomic analyses of proteins in the soluble and membrane components of the vesicles. Protein systems in secretory vesicle function consisted of those for 1) production of hormones, neurotransmitters, and neuromodulatory factors, 2) generating selected internal vesicular conditions for reducing condition, acidic pH conditions maintained by ATPases, and chaperones for protein folding, and 3) vesicular trafcking mechanisms to allow the mobilization of secretory vesicles for exocytosis, which uses proteins for nucleotide-binding, calcium regulation, and vesicle exocytosis. These protein systems are coordinated to allow the secretory vesicle to synthesize and release neuropeptides for cellcell communication in the control of neuroendocrine functions.

(87). These results implicate membrane-specic functions for signaling and exocytosis that allow secretory vesicles to produce, store, and secrete active neuropeptides for the control of physiological functions. The protein systems used in these chromafn vesicles, which represent dense core secretory vesicles (71), resemble those of brain synaptic vesicles (88) and secretory vesicles in the liver (89). Proteomic studies provide inference for secretory vesicle protein systems used for functions of these vesicles, including their biogenesis, that are required for production of enkephalin and related neuropeptides in brain and endocrine tissues. Secretory vesicles at synaptic nerve terminals in the brain are essential for chemical neurotransmission among neurons. Proteomic studies of synaptic proteins have revealed their regulation by brain injury (90), brain-derived neurotrophic factor (BDNF) (91), and drug regulation by morphine (92). The protein systems that support secretory vesicle exocytosis of peptide neurotransmitters and receptor activation at synaptic junctions of neurons function in concert to achieve neuropeptide-mediated communication in neural circuits.

Future PerspectivesChemical Approaches for Elucidating Neuropeptide Mechanisms for Translation into Therapeutic Applications
It is extremely important to apply knowledge of protease mechanisms for neuropeptide biosynthesis to small-molecule strategies for the development of therapeutic agents that can modulate the production of specic peptide neurotransmitters or hormones.

Current and future research using new approaches and tools, as discussed in this article, can provide insight into selective pharmacological approaches for exogenous therapeutic regulation of neuropeptide actions. Numerous health and disease conditions are regulated by neuropeptides. Proteases are essential for the conversion of inactive proprotein precursors into the active neuropeptides. Two main protease pathways have been elucidated for processing proneuropeptides and hormones: the recently discovered cysteine protease cathepsin L with aminopeptidase B and the well-established subtilisin-like serine proteases that consist of prohormone convertases 1 and 2 followed by carboxypeptidase E/H. Endogenous regulators modulate these two protease pathways as endogenous peptide inhibitors, activators, and in vivo secretory vesicle proteins. Neuropeptides in CSF (cerebrospinal uid) in neurological diseases can monitor brain nervous activity because neuropeptides represent active neurotransmission (93, 94). Knowledge of specic regulators for particular neuropeptides can lead to future translational research for small-molecule regulation of prohormone convertases and cathepsin L pathways in the control of physiological functions. For example, regulation of opioid peptide productionenkephalin, -endorphin, and dynorphinmay lead to new drugs for analgesia and pain relief. Specic small-molecule control of hypothalamic NPY in the control of feeding behavior may lead to improvement in obese conditions. Regulation of hypothalamic CRF and pituitary ACTH production is important for the control of steroid biosynthesis in the adrenal cortex for metabolic regulation. PC-related proteases have been implicated in sterol and lipid metabolism, tumor progression, atherosclerosis, and other physiological and disease conditions (58). Application of chemical biology and proteomic approaches for understanding protease mechanisms in the biosynthesis of 9

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Neuropeptides, Chemical Activity Proling and Proteomic Approaches for

neuropeptides in health and disease is an exciting area of research for neuropeptide regulation of neuroendocrine systems.
19.

Acknowledgments
Support from the National Institutes of Health for this research is appreciated.

20.

21.

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Further Reading
Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease proling and discovery tools. Chem Biol. 2000;7:569581. Pan Z, Jeffery DA, Chehade K, Beltman J, Clark JM, Grothaus P, Bogyo M, Baruch A. Development of activity-based probes for trypsin-family serine proteases. Bioorg. Med. Chem. Lett. 2006;16: 28822885. Sadaghiani AM, Verhelst SH, Gocheva V, Hill K, Majerova E, Stinson S, Joyce JA, Bogyo M. Design, synthesis, and evaluation of in vivo potency and selectivity of epoxysuccinyl-based inhibitors of papain-family cysteine proteases. Chem Biol. 2007;14:499511. Sieber SA, Niessen S, Hoover HS, Cravatt BF. Proteomic proling of metalloprotease activities with cocktails of active-site probes. Nat. Chem. Biol. 2006; 2: 274281. Speers AE, Cravatt BF. Chemical strategies for activity-based proteomics. ChemBioChem 2004;5:4147. Verhelst SH, Witte MD, Arastu-Kapur S, Fonovic M, Bogyo M. Novel aza peptide inhibitors and active-site probes of papain-family cysteine proteases. ChemBioChem 2006;7:943950.

See Also
Neurotransmitter: Production and Storage Protease Pathways, Small Molecules to Elucidate Neuropeptidomics Systems Biology

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