Neuropeptides, Chemical Activity Profiling 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


Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cell-cell 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 define protease pathways involved in neuropeptide biosynthesis. Activity profiling of proteases, combined with mass spectrometry, has allowed identification 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 define 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 defined 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 Cell-Cell Communication in Nervous and Endocrine Systems

Neuropeptides mediate neurotransmission as peptide neurotransmitters and mediate cell-cell 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 3-40 residues. More than 100 different neuropeptides exist, and new neuropeptides are yet to be discovered.

The unique primary sequence of each neuropeptide defines 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 fi-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 (4-8).


Table 1. Neuropeptides in the Nervous and Endocrine Systems



Regulatory Function


steroid production


skin pigmentation, appetite


analgesia, pain relief


calcium regulation


learning, memory, and appetite

CRF (corticotropin releasing factor)

ACTH secretion


analgesia, pain relief




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 3-40 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), a-melanocyte stimulating hormone (a-MSH), neuropeptide Y (NPY), and pituitary adenylate cyclase-activating peptide (PACAP).



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.


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 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 (Fig. 2) (9-11). 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) (12-14). Proteolysis of these precursors, especially tissue-specific 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 residue sites that commonly flank the NH2 and COOH termini of neuropeptides within their precursors (Fig. 2). The dibasic residues Lys-Arg (KR) most often flank the neuropeptides; however, the dibasic sites Lys-Lys, Arg-Arg, and sometimes Arg-Lys 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.



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).


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 co- translationally 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 (15-18). 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 final neuropeptides.

Neuropeptides may also undergo post translational modification that modifies the biological activities of peptides. Activities of the neuropeptides may be altered by disulfide bond formation, glycosylation, COOH-terminal α-amidation, phosphorylaton, sulfation, and acetylation (6, 7). This article, however, will focus on protease mechanisms for neuropeptide biosynthesis.



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 identified 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.


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 identified for producing an active peptide, the neuropeptide field has used key criteria for successful elucidation of proteases that generate peptide neurotransmitters and hormones. These criteria 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 specificity 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 identified cysteine protease pathway and serine protease pathways, mediated by cathepsin L and proprotein convertases, respectively, for neuropeptide production (Fig. 3).


Chromaffin granules: model neurosecretory vesicles for proneuropeptide processing proteases

Elucidation of protease pathways for neuropeptide biosynthesis has been facilitated in the field with the use of isolated chromaffin secretory vesicles, a well-established model neurosecretory vesicle system used for investigation of proteases that synthesize neuropeptides and small-molecule neurotransmitters (19). Chromaffin 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 identified prohormone convertase processing enzymes (20-23).


Chemical biology for activity-based profiling and identification of proteases

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific 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 subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identification of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article.


Activity-based chemical profiling identifies the cathepsin L cysteine protease pathway in secretory vesicles that contributes to neuropeptide biosynthesis

Activity-based profiling of active cysteine proteases identified 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 specific affinity labeling of the 27 kDa protease enzyme of the “prohormone thiol protease” (PTP) complex (18, 22), which represents the major proenkephalin (PE) processing activity in chromaffin secretory vesicles (20, 21). The high molecular weight of the PTP complex of approximately 180-200 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 identification 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 27-29 kDa (Fig. 4), which was identified as cathepsin L by mass spectrometry of tryptic peptides. Confirmation of the localization of cathepsin L in secretory vesicles was demonstrated by immunofluorescence 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 findings suggested a new biological function for cathepsin L in secretory vesicles for producing the enkephalin and related neuropeptides.



Figure 4. Activity-based profiling for identification of proenkephalin cleaving activity as cathepsin L. Activity-based profiling (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 chromaffin secretory vesicles (also known as chromaffin granules) allowed affinity 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.


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

Cathepsin L knockout mice

Cathepsin L-deficient 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-deficient 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 deficiency 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.


Cellular gene expression of cathepsin L for enkephalin neuropeptide production in the regulated secretory pathway

Cellular routing and trafficking 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 trafficking to secretory 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, 18-19, 8-9, 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.


Cathepsin L cleavage specificity indicates the subsequent aminopeptidase B exopeptidase step for neuropeptide production

Studies of the cleavage specificity 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 specificity 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 chromaffin cells (33) and from pituitary (34) contain Arg/Lys aminopeptidase activity for neuropeptide production.

Recent molecular cloning studies have identified aminopeptidase B as an appropriate Arg/Lys aminopeptidase (35). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that provided production of specific 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 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding 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 findings indicate differences in the cleavage specificity 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) (15-17,23). 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 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.


Chemical biology defines the novel cathepsin L cysteine protease pathway combined with the prohormone convertase subtilisin-like pathway for neuropeptide production

Significantly, the approach of activity profiling for cysteine proteases has established cathepsin L as a new protease pathway for neuropeptide biosynthesis. Together with current knowledge in the field, 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 (15-17) 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 (36-39) 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 first 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 a-helices, and the C-terminal domain contains a β-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.

The proteases that enter the secretory or lysosomal pathways are synthesized as precursors, with N-terminal propeptides and signal peptides (36-39). 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 specificity 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 deficient 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-deficient 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 identified the novel cathepsin L cysteine protease pathway as a candidate route for neuropeptide production.

More specifically, to integrate data from PC2- and PC1/3-deficient 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-deficient mice are summarized here. In PC2 knockout mice, many neuropeptides were partially reduced, with the exception of α-MSH that was nearly obliterated. PC2-deficient mice show increases in the POMC-derived peptide hormones ACTH and β-endorphin (1-31), which identifies them as substrates for PC2 (40). Among the POMC-derived peptide hormones, only α-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-deficient 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-deficient mice.

In PC1/3-deficient 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-deficient mice (47). Interestingly, no change in vasopressin occurred, and little change in POMC-derived peptide hormones were observed in the PC1/3-deficient mice compared with wild-type controls (48).

These findings demonstrate several features with regard to the selective roles of PC2 and PC1/3 in determining the production of neuropeptides. Firstly, PC-deficient mice may show changes in several neuropeptides but not all neuropeptides. Secondly, neuropeptides may show tissue-specific differences in PC-deficient mice. Thirdly, a particular tissue region may show selective alterations among different neuropeptides in each of the PC-deficient mice. These results demonstrate the roles of PC2 and PC1/3 in neuropeptide production. Notably, partial reductions of many neuropeptides in mice deficient in PC2 or PC1/3 are consistent with the presence of other processing enzymes such as the newly identified 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/at mice that contain mutant, inactive CPE, showed that these animals show altered neuropeptide production (49-55). Studies of CPE peptide substrates in the fat/fat mice have been facilitated with specific isolation of peptides with C-terminal basic residues by the anhydrotrypsin affinity column for enrichment of CPE substrates (49-53). Analyses of such CPE substrates have demonstrated that numerous neuropeptides use CPE for their biosynthesis (49-55).

Analogously, it will be important to evaluate cathepsin L-generated peptide products that serve as substrates for the subsequent aminopeptidase B (AP-B) step. Significantly, 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 affinity 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 structure-function features of neuropeptides. The development of mass spectrometry (MS) tools and approaches for identifying neurological peptides has become essential for defining neuropeptide structures that correspond to biological activities—this field 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 specific mass measurements by tandem mass spectrometry that defines neuropeptide structures. Neuropeptide analyses may be achieved by several types of mass spectrometers including MALDI-TOF (matrix-assisted laser desorption time-of-flight), ESI-Trap (electrospray ionization ion-trap), and ESI-qTOF (quadrupole-time-of-flight 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 identified directly by mass spectrometry approaches as specific 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 significant 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 fluids (61), and synaptosomes (62). In each of these studies, significant 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 defining 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.


Proteomics of Secretory Vesicles for Defining 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 chromaffin secretory vesicles revealed several functional protein categories that together support secretory vesicle production of neuropeptides and bioactive catecholamines for cell-cell 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 purified from neuroendocrine chromaffin cells. Proteins were separated by SDS-PAGE, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (pLC-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 identified 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 (15-17). 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 identified, which indicates a novel location for these previously known lysosomal proteases. The localization of cathepsin B in these secretory vesicles has been confirmed by immunoelectron microscopy (77). Cystatin C was identified 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 identified; furthermore, ubiquitin-binding protein S27A was also identified. These findings 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 identified 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 trafficking and exocytosis of secretory vesicle components (85, 86). Membranes also preferentially contained ATPases that regulate proton translocation (87). These results implicate membrane-specific 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 chromaffin 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.



Figure 5. Proteomics reveals functional secretory vesicle protein systems for neuropeptide biosynthesis, storage, and secretion. Chromaffin secretory vesicles (also known as chromaffin 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 trafficking 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 cell-cell communication in the control of neuroendocrine functions.


Future Perspectives—Chemical 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 specific 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 fluid) in neurological diseases can monitor brain nervous activity because neuropeptides represent active neurotransmission (93, 94).

Knowledge of specific 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 production—enkephalin, P-endorphin, and dynorphin—may lead to new drugs for analgesia and pain relief. Specific 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 (5-8).

Application of chemical biology and proteomic approaches for understanding protease mechanisms in the biosynthesis of neuropeptides in health and disease is an exciting area of research for neuropeptide regulation of neuroendocrine systems.



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



1. Akil H, Watson SJ, Young E, Lewis ME, Khachaturian H, Walker JM. Endogenous opioids: biology and function. Annu. Rev. Neurosci. 1984; 7:223-55.

2. Law PY, Loh HH. Regulation of opioid receptor activities. J. Pharmacol. Exp Ther. 1999; 289:607-24.

3. Norris D. In: Vertebrate Endocrinology. 1997. Academic Press, San Diego.

4. Brunton LL, Lazo JS, Parker KL. 2006. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics. 2001. McGraw-Hill, New York.

5. Steiner DF. New aspects of proinsulin physiology and pathophysiology. J. Pediatr. Endocrinol. Metab. 2000; 13:229-239.

6. Krieger D, Brownstein, MJ, Martin, JB. Brain Peptides. 1983. Wiley-Interscience, New York.

7. Siegel GJ AB, Albers RW, Fisher SK, Uhler MD. In: Basic Neurochemistry. 1999. Lippincott Williams and Wilkins, Philadelphia, PA.

8. Strand FL, Rose KJ, Zuccarelli LA, Kume J, Alves SE, Antonawich FJ, Garrett LY. Neuropeptide hormones as neurotrophic factors. Physiol. Rev. 1991; 71:1017-1046.

9. Higuchi H, Yang HY, Sabol SL. Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester. J. Biol. Chem. 1988; 263:6288-6295.

10. Rokaeus A, Brownstein MJ. Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones encoding a galanin precursor. Proc. Natl. Acad. Sci. U.S.A. 1986; 83:6287-6291.

11. Robinson BG, D’Angio LA, Jr., Pasieka KB, Majzoub JA. Preprocorticotropin releasing hormone: cDNA sequence and in vitro processing. Mol. Cell. Endocrinol. 1989; 61:175-180.

12. Yoshikawa K, Williams C, Sabol S. Rat brain preproenkephalin mRNA, cDNA cloning, primary structure, and distribution in the central nervous system. J. Biol. Chem. 1984; 259:14301-14308.

13. Comb M, Rosen H, Seeburg P, Adelman J, Herbert E. Primary structure of the human proenkephalin gene. DNA 1983; 2:213-229.

14. Roberts JL, Seeburg PH, Shine J, Herbert E, Baxter JD, Goodman HM. Corticotropin and beta-endorphin: construction and analysis of recombinant DNA complementary to mRNA for the common precursor. Proc. Natl. Acad. Sci. U.S.A. 1979; 76:2153-2157.

15. Zhou A, Webb G, Zhu X, Steiner DF. Proteolytic processing in the secretory pathway. J. Biol. Chem. 1999; 274:20745-20748.

16. Hook VY, Azaryan AV, Hwang SR, Tezapsidis N. Proteases and the emerging role of protease inhibitors in prohormone processing. FASEB J. 1994; 8:1269-1278.

17. Seidah NG, Prat A. Precursor convertases in the secretory pathway, cytosol and extracellular milieu. Essays Biochem. 2002; 38:79-94.

18. Hook V, Yasothornsrikul S, Greenbaum D, Medzihradszky KF, Troutner K, Toneff T, Bundey R, Logvinova A, Reinheckel T, Peters C, Bogyo M. Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones. Biol. Chem. 2004; 385:473-480.

19. Njus D, Kelley PM, Harnadek GJ. The chromaffin vesicle: a model secretory organelle. Physiologist 1985; 28:235-241.

20. Schiller MR, Mende-Mueller L, Moran K, Meng M, Miller KW, Hook VY. Prohormone thiol protease” (PTP) processing of recombinant proenkephalin. Biochemistry 1995; 34:7988-7995.

21. Yasothornsrikul S, Aaron W, Toneff T, Hook VY. Evidence for the proenkephalin processing enzyme prohormone thiol protease (PTP) as a multicatalytic cysteine protease complex: activation by glutathione localized to secretory vesicles. Biochemistry 1999; 8:7421-7430.

22. Yasothornsrikul S, Greenbaum D, Medzihradszky KF, Toneff T, Bundey R, et al. Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter. Proc. Natl. Acad. Sci. U.S.A. 2003; 100:9590-9595.

23. Azaryan AV, Krieger TJ, Hook VY. Purification and characteristics of the candidate prohormone processing proteases PC2 and PC1/3 from bovine adrenal medulla chromaffin granules. J. Biol. Chem. 1995; 270:8201-8208.

24. Barglow KT, Cravatt BF. Activity-based protein profiling for the functional annotation of enzymes. Nat. Methods 2007; 4:822-827.

25. Evans MJ, Cravatt BF. Mechanism-based profiling of enzyme families. Chem. Rev. 2006; 106:3279-3301.

26. Yuan F, Verhelst SH, Blum G, Coussens LM, Bogyo M. A selective activity-based probe for the papain family cysteine protease dipeptidyl peptidase I/cathepsin C. J. Am. Chem. Soc. 2006;128:5616-5617.

27. Kato D, Boatright KM, Berger AB, Nazif T, Blum G, Ryan C, Chehade KA, Salvesen GS, Bogyo M. Activity-based probes that target diverse cysteine protease families. Nat. Chem. Biol. 2005; 1:33-38.

28. Miller R, Toneff T, Vishnuvardhan D, Beinfeld M, Hook VY. Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice. Neuropeptides 2003;37:140-148.

29. Tobin DJ, Foitzik K, Reinheckel T, Mecklenburg L, Botchkarev VA, et al. The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling. Am. J. Pathol. 2002; 160:1807-1821.

30. Stypmann J, Glaser K, Roth W, Tobin DJ, Petermann I, et al. Dilated cardiomyopathy in mice deficient for the lysosomal cysteine peptidase cathepsin L. Proc. Natl. Acad. Sci. U.S.A. 2002;99:6234-6239.

31. Hwang SR, Garza C, Mosier C, Toneff T, Wunderlich E, et al. Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion. J. Biol. Chem. 2007; 282:9556-9563.

32. Azaryan AV, Hook VYH. (1994) Unique cleavage specificity of prohormone thiol protease related to proenkephalin processing. FEBS Lett. 1994; 341:197-202.

33. Yasothornsrikul S, Toneff T, Hwang SR, Hook VY. Arginine and lysine aminopeptidase activities in chromaffin granules of bovine adrenal medulla: relevance to prohormone processing. J. Neurochem. 1998; 70:153-163.

34. Gainer H, Russell JT, Loh YP. An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin 60-65. FEBS Lett. 1984; 175:135-139.

35. Hwang SR, O’Neill A, Bark S, Foulon T, Hook V. Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules. J. Neurochem. 2007; 100:1340-1350.

36. Barrett AJ, Rawlings ND, Woessner JF. Handbook of Proteolytic Enzymes. 2004. Elsevier Academic Press, Amsterdam, p. 1051-1057, 1097-1102.

37. Turk V, Turk B, Turk D. Lysosomal cysteine proteases: facts and opportunities. EMBO J. 2001; 20:4629-4633.

38. Turk D, Guncar G, Podobnik M, Turk B. Revised definition of substrate binding sites of papain-like cysteine proteases. Biol. Chem. 1998; 379:137-147.

39. Mohamed MM, Sloane BF. Cysteine cathepsins: multifunctional enzymes in cancer. Nat. Rev. 2006; 6:764-766.

40. Miller R, Aaron W, Toneff T, Vishnuvardhan D, Beinfeld, M, Hook VYH. Obliteration of a-melanocyte-stimulating hormone derived from POMC in pituitary and brains of PC2-deficient mice. J. Neurochem. 2003; 86:556-563.

41. Miller R, Toneff T, Vishnuvardhan D, Beinfeld M, Hook VYH. Selective roles for the PC2 processing enzyme in the regulation of peptide neurotransmitter levels in brain and peripheral neuroendocrine tissues of PC2 deficient mice. Neuropeptides 2003; 37:140-148.

42. Furuta M, Yano H, Zhou A, Rouille Y, Holst JJ, Carroll R, Ravazzola M, Orci L, Furuta H, Steiner DF. Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2. Proc. Natl. Acad. Sci. U.S.A. 1997; 94:6646-6651.

43. Rehfeld JF, Lindberg I, Friis-Hansen L. Increased synthesis but decreased processing of neuronal proCCK in prohormone convertase 2 and 7B2 knockout animals. J. Neurochem. 2002; 83:1329-1337.

44. Allen RG, Peng B, Pellegrino MJ, Miller ED, Grandy DK, Lundblad JR, Washburn CL, Pintar JE. Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2. J. Neurosci. 2001; 21:5864-5870.

45. Villeneuve P, Feliciangeli S, Croissandeau G, Seidah NG, Mbikay M, Kitabgi P, Beaudet A. Altered processing of the neurotensin/ neuromedin N precursor in PC2 knock down mice: a biochemical and immunohistochemical study. J. Neurochem. 2002; 82:783-793.

46. Zhu X, Zhou A, Dey A, Norrbom C, Carroll R, Zhang C, Laurent V, Lindberg I, Ugleholdt R, Holst JJ, Steiner DF. Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Proc. Natl. Acad. Sci. U.S.A. 2002; 99:10293-10298.

47. Zhu X, Orci L, Carroll R, Norrbom C, Ravazzola M, Steiner DF. Severe block in processing of proinsulin to insulin accompanied by elevation of des-64,65 proinsulin intermediates in islets of mice lacking prohormone convertase 1/3. Proc. Natl Acad. Sci. U.S.A. 2002; 99:10299-10304.

48. Pan H, Nanno D, Che FY, Zhu X, Salton SR, Steiner DF, Fricker LD, Devi LA. Neuropeptide processing profile in mice lacking prohormone convertase-1. Biochemistry 2005; 44:4939-4948.

49. Che FY, Yan L, Li H, Mzhavia N, Devi LA, Fricker LD. Identification of peptides from brain and pituitary of Cpe(fat)/Cpe(fat) mice. Proc. Natl. Acad. Sci. U.S.A. 2001; 98:9971-9976.

50. Che FY, Yuan Q, Kalinina E, Fricker LD. Peptidomics of Cpe fat/fat mouse hypothalamus: effect of food deprivation and exercise on peptide levels. J. Biol. Chem. 2005; 280:4451-4461.

51. Che FY, Fricker LD. Quantitative peptidomics of mouse pituitary: comparison of different stable isotopic tags. J. Mass Spectrom. 2005; 40:238-249.

52. Che FY, Biswas R, Fricker LD. Relative quantitation of peptides in wild-type and Cpe(fat/fat) mouse pituitary using stable isotopic tags and mass spectrometry. J. Mass Spectrom. 2005; 40:227-237.

53. Decaillot FM, Che FY, Fricker LD, Devi LA. Peptidomics of Cpefat/fat mouse hypothalamus and striatum: effect of chronic morphine administration. J. Mol. Neurosci. 2006; 28:277-284.

54. Naggert JK, Fricker LK, Varlamov O, Nishina PM, Rouille Y, Steiner DF, Carroll RJ, Paigen BJ, Leiter EH. Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity. Nat. Genet. 1995; 10:135-142.

55. Lim J, Berezniuk I, Che FY, Parikh R, Iswas R, Pan H, Fricker LD. Altered neuropeptide processing in prefrontal cortex of Cpe fat/fat mice: implications for neuropeptide discovery. J. Neurochem. 2006; 96:1169-1181.

56. McDonald WH, Yates JR III. Shotgun proteomics: integrating technologies to answer biological questions. Curr. Opin. Mol. Ther. 2003; 5:302-309.

57. Fournier MJ, Gilmore JM, Martin-Brown SA, Washburn MP. Multidimensional separations-based shotgun proteomics. Chem. Rev. 2007; 107:3654-3686.

58. Mann M. Functional and quantitative proteomics using SILAC. Nat. Rev. 2006;7:952-958.

59. Pan H, Che FY, Peng B, Steiner DF, Pintar JE, Fricker LD. The role or prohormone convertase-2 in hypothalamic neuropeptide processing: a quantitative neuropeptidomic study. J. Neurochem. 2006; 98:1763-1777.

60. Boonen K, Baggerman G, Hertog WD, Husson SJ, Overbergh L, Mathieu C, Schoofs L. Neuropeptides of the islets of Langerhans: A peptidomics study. Gen. Comp. Endocrinol. 2007; 152:231-241.

61. Haskins WE, Watson CJ, Cellar NA, Powell DH, Kennedy RT. Discovery and Neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples. Anal. Chem. 2004; 76:5523-5533.

62. Parkin MC, Wei H, O’Callaghan JP. Kennedy RT Sample dependent effects on the neuropeptidome eetected in rat brain tissue preparations by capillary liquid chromatography with tandem mass spectrometry. Anal. Chem. 2005; 77:6331-6338.

63. Cornett DS, Reyzer ML, Chaurand P, Caprioli RM. MALDI imaging mass spectrometry: molecular snapshots of biochemical systems. Nat. Methods 2007; 4:828-833.

64. Rubakhin SS, Churchill JD, Greenough WT, Sweedler JV. Profiling signaling peptides in single mammalian cells using mass spectrometry. Anal. Chem. 2006; 78:7267-7272.

65. Amare A, Hummon AB, Southey BR, Zimmerman TA, Rodriguez-Zas SL, Sweedler JV. Bridging neuropeptidomics and genomics with bioinformatics: prediction of mammalian neuropeptide prohormone processing. J. Proteome Res. 2006; 5:1162-1167.

66. Proekt A, Vilim FS, Alexeeva V, Brezina V, Friedman A, Jing J, Li L, Zhurov Y, Sweedler JV, Weiss KR. Identification of a new neuropeptide precursor reveals novel source of extrinsic modulation in the feeding system of Aplysia. J. Neurosci. 2005; 25:9637-9648.

67. Hummon AB, Richmond TA, Verleyen P, Baggerman G, Huybrechts J, Ewing MA, Vierstraete E, Rodriguez-Zas SL, Schoofs L, Robinson GE, Sweedler JV. From the genome to the proteome: uncovering peptides in the Apis brain. Science 2006; 314:647-649.

68. Husson SJ, Schoofs L. Altered neuropeptide profile of Caenorhabditis elegans lacking the chaperone protein 7B2 as analyzed by mass spectrometry. FEBS Lett. 2007; 81:4288-4292.

69. Baggerman G, Liu F, Wets G, Schoofs L. Bioinformatic analysis of peptide precursor proteins. Ann. N.Y. Acad. Sci. 2005; 1040:59-65.

70. Amare A, Sweedler JV. Neuropeptide precursors in tribolium castaneum. Peptides 2007;28:1282-1291.

71. Wegrzyn J, Lee J, Neveu JM, Lane WS, Hook V. Proteomics of neuroendocrine secretory vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters. J. Proteome Res. 2007; 6:1652-1665.

72. Cool DR, Normant E, Shen F, Chen HC, Pannell L, Zhang Y, Loh YP. Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice. Cell 1997; 88:73-83.

73. Basak A, Koch P, Dupelle M, Fricker LD, Devi LA, Chretien M, Seidah NG. Inhibitory specificity and potency of proSAAS-derived peptides toward proprotein convertase 1. J. Biol. Chem. 2001; 276:32720-32728.

74. Van Horssen AM, Van den Hurk WH, Bailyes EM, Hutton JC, Martens GJM, Lindberg I. Identification of the region within the neuroendocrine polypeptide 7B2 responsible for the inhibition of prohormone convertase PC2. J. Biol. Chem. 1995; 270:14292-14296.

75. Bechet DM, Ferrara MJ, Mordier SB, Roux MP, Deval CD, Obled A. Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation, characterization of a cDNA encoding bovine cathepsin B. J Biol Chem. 1991; 266:14104-14112.

76. Higuchi, M, Miyashita N, Nagamine Y, Watanabe A, Awata T. The complementary DNA sequence and polymorphisms of bovine procathepsin-D (CTSD). J. Anim. Breed. Genet. 2003; 120:322- 330.

77. Hook V, Toneff T, Bogyo M, Greenbaum D, Medzihradszky KF, Neveu J, Lane W, Hook G, Reisine T. Inhibition of cathepsin B reduces β-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate β-secretase of Alzheimer’s disease. Biol. Chem. 2005; 386:931-940.

78. Abrahamson M, Alvarez-Fernandez M, Nathanson CM. Cystatins. Biochem. Soc. Symp. 2003; 70:179-199.

79. Mussap M, Plebani M. Biochemistry and clinical role of human cystatin C. Crit. Rev. Clin. Lab. Sci. 2004; 41:467-550.

80. Welchman RL, Gordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat. Rev. Mol. Cell Biol. 2005; 6:599-609.

81. Ye Y. The role of the ubiquitin-proteasome system in ER quality control. Essays Biochem. 2005; 41:99-112.

82. Wurtz S0, Schrohl AS, Sprensen NM, Lademann U, Christensen IJ, Mouridsen H, Brunner N. Tissue inhibitor of metalloproteinases-1 in breast cancer. Endocr. Relat. Cancer 2005; 12:215-227.

83. Pfeffer S, Aivazian D. Targeting RAB GTPases to distinct membrane compartments. Nat. Rev. Mol. Cell Biol. 2004; 5:886-896.

84. Colicelli, J. Human RAS superfamily proteins and related GT- Pases. Sci. STKE 2004; 250:13.

85. Ungar D, Hughson FM. SNARE protein structure and function. Annu. Rev. Cell Dev. Biol. 2003; 19:493-517.

86. Gerke V, Moss SE. Annexins: from structure to function. Physiol. Rev. 2002; 82:331-371.

87. Taupenot L, Harper KL, O’Connor DT. Role of H+-ATPase-mediated acidification in sorting and release of the regulated secretory protein chromogranin A: Evidence for a vesiculogenic function. J. Biol. Chem. 2005; 280:3885-3897.

88. Takamori S, Holt M, Stenius K, Lemke EA, Gronborg M, et al. Molecular anatomy of a trafficking organelle. Cell 2006; 127:831- 846.

89. Gilchrist A, Au CE, Hiding J, Bell, AW, Fernandez-Rodriquez J, Lesimple S, Nagaya H, Roy L, Gosline SJC, Hallett M, Paiement J, Kearney RE, Nilsson T, Bergeron JJM. Quantitative proteomic analysis of the secretory pathway. Cell 2006; 127:1265-1281.

90. Kobeissy FH, Ottens AK, Zhang Z, Liu MC, Denslow ND, et al. Novel differential neuroproteomics analysis of traumatic brain injury in rats. Mol. Cell. Proteomics 2006; 5:1887-1898.

91. Liao L, Pilotte J, Xu T, Wong CC, Edelman GM, et al. BDNF induces widespread changes in synaptic protein content and up-regulates components of the translation machinery: an analysis using high-throughput proteomics. J. Proteome Res. 2007; 6:1059-1071.

92. Moron JA, Abul-Husn NS, Rozenfeld R, Dolios G, Wang R, Devi LA. Morphine administration alters the profile of hippocampal postsynaptic density-associated proteins: a proteomics study focusing on endocytic proteins. Mol. Cell. Proteomics 2007; 6:29-42.

93. Noben JP, Dumont D, Kwasnikowska N, Verhaert P, Somers V, et al. Lumbar cerebrospinal fluid proteome in multiple sclerosis: characterization by ultrafiltration, liquid chromatography, and mass spectrometry. J. Proteome Res. 2006; 5:1647-1657.

94. Svensson M, Skold K, Nilsson A, Falth M, Nydahl K, et al. Neuropeptidomics: MS applied to the discovery of novel peptides from the brain. Anal. Chem. 2007; 79:15-6, 8-21.


Further Reading

Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem Biol. 2000; 7:569-581.

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; 16:2882-2885.

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;14:499-511.

Sieber SA, Niessen S, Hoover HS, Cravatt BF. Proteomic profiling of metalloprotease activities with cocktails of active-site probes. Nat. Chem. Biol. 2006; 2: 274-281.

Speers AE, Cravatt BF. Chemical strategies for activity-based proteomics. ChemBioChem 2004; 5:41-47.

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; 7:943-950.


See Also

Neurotransmitter: Production and Storage

Protease Pathways, Small Molecules to Elucidate


Systems Biology