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Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1&prime, position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

Peptides produced by the proteasome have been proposed to function as signaling molecules that regulate a number of biological processes. In the current study, we used quantitative peptidomics to test whether conditions that affect protein stability, synthesis, or turnover cause changes in the levels of peptides in Human Embryonic Kidney 293T (HEK293T) cells. Mild heat shock (42 &deg, C for 1 h) or treatment with the deubiquitinase inhibitor b-AP15 led to higher levels of ubiquitinated proteins but did not significantly increase the levels of intracellular peptides. Treatment with cycloheximide, an inhibitor of protein translation, did not substantially alter the levels of intracellular peptides identified herein. Cells treated with a combination of epoxomicin and bortezomib showed large increases in the levels of most peptides, relative to the levels in cells treated with either compound alone. Taken together with previous studies, these results support a mechanism in which the proteasome cleaves proteins into peptides that are readily detected in our assays (i.e., 6&ndash, 37 amino acids) and then further degrades many of these peptides into smaller fragments.

Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classical neurotransmitters, peptides undergo cell-type-specific post-translational modifications that affect their biological activity. To enable the identification of the peptide repertoire of a genetically-defined cell-type, we generated mice with a conditional disruption of the gene for carboxypeptidase E (Cpe), an essential neuropeptide-processing enzyme. The loss of Cpe leads to accumulation of neuropeptide precursors containing C-terminal basic residues which serve as tags for affinity-purification. The purified peptides are subsequently identified using quantitative peptidomics, thereby revealing the specific forms of neuropeptides in cells with the disrupted Cpe gene. To validate the method, we used mice expressing Cre recombinase under the proopiomelanocortin (Pomc) promoter and analyzed hypothalamic and pituitary extracts, detecting peptides derived from proopiomelanocortin (as expected) and also proSAAS in POMC neurons. This technique enables the analyses of specific forms of peptides in any Cre-expressing cell-type.

Mass spectrometry-based peptidomic approaches are powerful techniques to detect and identify the peptide content of biological samples. The present study investigated the limitations of peptidomic approaches using trimethylammonium butyrate isotopic tags to quantify relative peptide levels and Mascot searches to identify peptides. Data were combined from previous studies on human cell lines or mouse tissues. The combined databases contain 2155 unique peptides ranging in mass from 444 to 8765 Da, with the vast majority between 1 and 3 kDa. The amino acid composition of the identified peptides generally reflected the frequency in the Eukaryotic proteome with the exception of Cys, which was not present in any of the identified peptides in the free-SH form but was detected at low frequency as a disulfide with Cys residues, a disulfide with glutathione, or as S-cyanocysteine. To test if the low detection rate of peptides smaller than 500 Da, larger than 3 kDa, or containing Cys was a limitation of the peptidomics procedure, tryptic peptides of known proteins were processed for peptidomics using the same approach used for human cell lines and mouse tissues. The identified tryptic peptides ranged from 516 to 2418 Da, whereas the theoretical digest ranged from 217 to 7559 Da. Peptides with Cys were rarely detected and, if present, the Cys was usually modified S-cyanocysteine. Additionally, peptides with mono- and di-iodo Tyr and His were identified. Taken together, there are limitations of peptidomic techniques, and awareness of these limitations is important to properly use and interpret results. Graphical Abstract ᅟ.

Peptidomic techniques are powerful tools to identify peptides in a biological sample. This protocol describes a targeted peptidomic approach that uses affinity chromatography to purify peptides that are substrates of carboxypeptidase E (CPE), an enzyme present in the secretory pathway of neuroendocrine cells. Many CPE products function as neuropeptides and/or peptide hormones, and therefore represent an important subset of the peptidome. Because CPE removes C-terminal Lys and Arg residues from peptide-processing intermediates, organisms lacking CPE show a large decrease in the levels of the mature forms of most neuropeptides and peptide hormones, and a very large increase in the levels of the processing intermediates that contain C-terminal Lys and/or Arg (i.e., the CPE substrates). These CPE substrates can be purified on an anhydrotrypsin-agarose affinity resin, which specifically binds peptides with C-terminal basic residues. Not all peptides with basic C-terminal residues within a cell are CPE substrates, and these other peptides will also be purified on the anhydrotrypsin affinity column. However, a comparison of peptides purified from wild-type mice and from mice lacking CPE allows for the rapid identification of CPE substrates based on their large increase in the absence of CPE.

Peptides and small molecules that bind to peptide receptors are important classes of drugs that are used for a wide variety of different applications. The search for novel neuropeptides traditionally involved a time-consuming approach to purify each peptide to homogeneity and determine its amino acid sequence. The discovery in the 1980s of enkephalin convertase/carboxypeptidase E (CPE), and the observation that this enzyme was involved in the production of nearly every known neuropeptide led to the idea for a one-step affinity purification of CPE substrates. This approach was successfully used to isolate hundreds of known neuropeptides in mouse brain, as well as over a dozen novel peptides. Some of the novel peptides found using this approach are among the most abundant peptides present in brain, but had not been previously identified by traditional approaches. Recently, receptors for two of the novel peptides have been identified, confirming their role as neuropeptides that function in cell-cell signaling. Small molecules that bind to one of these receptors have been developed and found to significantly reduce food intake and anxiety-like behavior in an animal model. This review describes the entire project, from discovery of CPE to the novel peptides and their receptors.

Peptidomics techniques have identified hundreds of peptides that are derived from proteins present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular peptides to distinguish them from secretory pathway peptides that function primarily outside of the cell. The proteasome and thimet oligopeptidase participate in the production and metabolism of intracellular peptides. Many of the intracellular peptides are common among mouse tissues and human cell lines analyzed and likely to perform a variety of functions within cells. Demonstrated functions include the modulation of signal transduction, mitochondrial stress, and development; additional functions will likely be found for intracellular peptides.

Peptides have been studied for over 100 years, but for most of this time the focus was on a specific peptide or peptides, and not on the general peptidome of a biological sample. In the 1990s, mass spectrometry techniques were developed for the analysis of proteins, usually after digestion into peptides. The field of peptidomics started soon after proteomics and has grown to over 600 publications that use the word “peptidomic” or “peptidomics”. Although peptidomics is related to proteomics, there are fundamental differences. In this review, we discuss these differences along with the history of the field of peptidomics.

Peptides represent a major class of cell-cell signaling molecules. Most peptidomic studies have focused on peptides present in brain or other tissues. For a peptide to function in intercellular signaling, it must be secreted. The present study was undertaken to identify the major peptides secreted from mouse brain slices that were cultured in oxygenated buffer for 3-4h. Approximately 75% of the peptides identified in extracts of cultured slices matched the previously reported peptide content of heat-inactivated mouse brain tissue, whereas only 2% matched the peptide content of unheated brain tissue; the latter showed a large number of postmortem changes. As found with extracts of heat-inactivated mouse brain, the extracts of cultured brain slices represented secretory pathway peptides as well as peptides derived from intracellular proteins such as those present in the cytosol and mitochondria. A subset of the peptides detected in the extracts of the cultured slices was detected in the culture media. The vast majority of secreted peptides arose from intracellular proteins and not secretory pathway proteins. The peptide RVD-hemopressin, a CB1 cannabinoid receptor agonist, was detected in culture media, which is consistent with a role for RVD-hemopressin as a non-classical neuropeptide. Taken together with previous studies, the present results show that short-term culture of mouse brain slices is an appropriate system to study peptide secretion, especially the non-conventional pathway(s) by which peptides produced from intracellular proteins are secreted. This article is part of a Special Issue entitled: An Updated Secretome.

The cytosolic carboxypeptidases (CCPs) are a subfamily of metalloenzymes within the larger M14 family of carboxypeptidases that have been implicated in the post-translational modification of tubulin. It has been suggested that at least four of the six mammalian CCPs function as tubulin deglutamylases. However, it is not yet clear whether these enzymes play redundant or unique roles within the cell. To address this question, genes encoding CCPs were identified in the zebrafish genome. Analysis by quantitative polymerase chain reaction indicated that CCP1, CCP2, CCP5, and CCP6 mRNAs were detectable between 2 h and 8 days postfertilization with highest levels 5-8 days postfertilization. CCP1, CCP2, and CCP5 mRNAs were predominantly expressed in tissues such as the brain, olfactory placodes, and pronephric ducts. Morpholino oligonucleotide-mediated knockdown of CCP1 and CCP5 mRNA resulted in a common phenotype including ventral body curvature and hydrocephalus. Confocal microscopy of morphant zebrafish revealed olfactory placodes with defective morphology as well as pronephric ducts with increased polyglutamylation. These data suggest that CCP1 and CCP5 play important roles in developmental processes, particularly the development and functioning of cilia. The robust and similar defects upon knockdown suggest that each CCP may have a function in microtubule modification and ciliary function and that other CCPs are not able to compensate for the loss of one.

Metallocarboxypeptidase D (CPD) is a membrane-bound component of the trans-Golgi network that cycles to the cell surface through exocytic and endocytic pathways. Unlike other members of the metallocarboxypeptidase family, CPD is a multicatalytic enzyme with three carboxypeptidase-like domains, although only the first two domains are predicted to be enzymatically active. To investigate the enzymatic properties of each domain in human CPD, a critical active site Glu in domain I and/or II was mutated to Gln and the protein expressed, purified, and assayed with a wide variety of peptide substrates. CPD with all three domains intact displays >50% activity from pH 5.0 to 7.5 with a maximum at pH 6.5, as does CPD with mutation of domain I. In contrast, the domain II mutant displayed >50% activity from pH 6.5–7.5. CPD with mutations in both domains I and II was completely inactive towards all substrates and at all pH values. A quantitative peptidomics approach was used to compare the activities of CPD domains I and II towards a large number of peptides. CPD cleaved C-terminal Lys or Arg from a subset of the peptides. Most of the identified substrates of domain I contained C-terminal Arg, whereas comparable numbers of Lys- and Arg-containing peptides were substrates of domain II. We also report that some peptides with C-terminal basic residues were not cleaved by either domain I or II, showing the importance of the P1 position for CPD activity. Finally, the preference of domain I for C-terminal Arg was validated through molecular docking experiments. Together with the differences in pH optima, the different substrate specificities of CPD domains I and II allow the enzyme to perform distinct functions in the various locations within the cell. This work was funded by the Spanish Ministry of Innovation and Competitiveness grants BIO2013-44973-R and BIO2016-78057-R (to FXA), by Plan Estatal grant number BIO2016-79960-R from the Spanish Ministry of Economy and Competitiveness (to JFR), and by grant R01-DA004494 from the United States’ National Institute of Health (to LDF).

PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide)-binding protein)-coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems-PEN-GPR83 and bigLEN-GPR171-may be functionally coupled in the regulation of feeding.

Carboxypeptidase A6 (CPA6) is a member of the A/B subfamily of M14 metallocarboxypeptidases that is expressed in brain and many other tissues during development. Recently, two mutations in human CPA6 were associated with febrile seizures and/or temporal lobe epilepsy. In this study we screened for additional CPA6 mutations in patients with febrile seizures and focal epilepsy, which encompasses the temporal lobe epilepsy subtype. Mutations found from this analysis as well as CPA6 mutations reported in databases of single nucleotide polymorphisms were further screened by analysis of the modeled proCPA6 protein structure and the functional role of the mutated amino acid. The point mutations predicted to affect activity and/or protein folding were tested by expression of the mutant in HEK293 cells and analysis of the resulting CPA6 protein. Common polymorphisms in CPA6 were also included in this analysis. Several mutations resulted in reduced enzyme activity or CPA6 protein levels in the extracellular matrix. The mutants with reduced extracellular CPA6 protein levels showed normal levels of 50-kDa proCPA6 in the cell, and this could be converted into 37-kDa CPA6 by trypsin, suggesting that protein folding was not greatly affected by the mutations. Interestingly, three of the mutations that reduced extracellular CPA6 protein levels were found in patients with epilepsy. Taken together, these results provide further evidence for the involvement of CPA6 mutations in human epilepsy and reveal additional rare mutations that inactivate CPA6 and could, therefore, also be associated with epileptic phenotypes.

The first metallocarboxypeptidase (CP) was identified in pancreatic extracts more than 80 years ago and named carboxypeptidase A (CPA; now known as CPA1). Since that time, seven additional mammalian members of the CPA subfamily have been described, all of which are initially produced as proenzymes, are activated by endoproteases, and remove either C-terminal hydrophobic or basic amino acids from peptides. Here we describe the enzymatic and structural properties of carboxypeptidase O (CPO), a previously uncharacterized and unique member of the CPA subfamily. Whereas all other members of the CPA subfamily contain an N-terminal prodomain necessary for folding, bioinformatics and expression of both human and zebrafish CPO orthologs revealed that CPO does not require a prodomain. CPO was purified by affinity chromatography, and the purified enzyme was able to cleave proteins and synthetic peptides with greatest activity toward acidic C-terminal amino acids unlike other CPA-like enzymes. CPO displayed a neutral pH optimum and was inhibited by common metallocarboxypeptidase inhibitors as well as citrate. CPO was modified by attachment of a glycosylphosphatidylinositol membrane anchor to the C terminus of the protein. Immunocytochemistry of Madin-Darby canine kidney cells stably expressing CPO showed localization to vesicular membranes in subconfluent cells and to the plasma membrane in differentiated cells. CPO is highly expressed in intestinal epithelial cells in both zebrafish and human. These results suggest that CPO cleaves acidic amino acids from dietary proteins and peptides, thus complementing the actions of well known digestive carboxypeptidases CPA and CPB.

Carboxypeptidase A6 (CPA6) is an extracellular matrix-bound metallocarboxypeptidase (CP) that has been implicated in Duane syndrome, a neurodevelopmental disorder in which the lateral rectus extraocular muscle is not properly innervated. Consistent with a role in Duane syndrome, CPA6 is expressed in a number of chondrocytic and nervous tissues during embryogenesis. To better characterize the enzymatic function and specificity of CPA6 and to compare this with other CPs, CPA6 was expressed in HEK293 cells and purified. Kinetic parameters were determined using a panel of synthetic carboxypeptidase substrates, indicating a preference of CPA6 for large hydrophobic C-terminal amino acids and only very weak activity toward small amino acids and histidine. A quantitative peptidomics approach using a mixture of peptides representative of the neuropeptidome allowed the characterization of CPA6 preferences at the P1 substrate position and suggested that small and acidic P1 residues significantly inhibit CPA6 cleavage. Finally, a comparison of available kinetic data for CPA enzymes shows a gradient of specificity across the subfamily, from the very restricted specificity of CPA2 to the very broad activity of CPA4. Structural data and modeling for all CPA/B subfamily members suggests the structural basis for the unique specificities observed for each member of the CPA/B subfamily of metallocarboxypeptidases.

Metallocarboxypeptidase D (CPD) functions in protein and peptide processing. The Drosophila CPD svr gene undergoes alternative splicing, producing forms containing 1-3 active or inactive CP domains. To investigate the function of the various CP domains, we created transgenic flies expressing specific forms of CPD in the embryonic-lethal svr (PG33) mutant. All constructs containing an active CP domain rescued the lethality with varying degrees, and full viability required inactive CP domain-3. Transgenic flies overexpressing active CP domain-1 or -2 were similar to each other and to the viable svr mutants, with pointed wing shape, enhanced ethanol sensitivity, and decreased cold sensitivity. The transgenes fully compensated for a long-term memory deficit observed in the viable svr mutants. Overexpression of CP domain-1 or -2 reduced the levels of Lys/Arg-extended adipokinetic hormone intermediates. These findings suggest that CPD domains-1 and -2 have largely redundant functions in the processing of growth factors, hormones, and neuropeptides.

Prohormone convertase 2 (PC2) functions in the generation of neuropeptides from their precursors. A quantitative peptidomics approach was used to evaluate the role of PC2 in the processing of peptides in a variety of brain regions. Altogether, 115 neuropeptides or other peptides derived from secretory pathway proteins were identified. These peptides arise from 28 distinct secretory pathway proteins, including proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, and many others. Forty one of the peptides found in wild type mice were not detectable in any of the brain regions of PC2 knockout mice, and another twenty four peptides were present at levels ranging from 20–79% of wild type levels. Most of the other peptides were not substantially affected by the mutation, with levels ranging from 80–120% of wild type levels, and only three peptides were found to increase in one or more brain regions of PC2 knock-out mice. Taken together, these results are consistent with a broad role for PC2 in neuropeptide processing, but with functional redundancy for many of the cleavages. Comparison of the cleavage sites affected by the absence of PC2 confirms previous suggestions that sequences with a Trp, Tyr and/or Pro in the P1′ or P2′ position are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Carboxypeptidase A6 (CPA6) is an extracellular matrix metallocarboxypeptidase that modulates peptide and protein function by removal of hydrophobic C-terminal amino acids. Mutations in the human CPA6 gene that reduce enzymatic activity in the extracellular matrix are associated with febrile seizures, temporal lobe epilepsy, and juvenile myoclonic epilepsy. The characterization of these human mutations suggests a dominant mode of inheritance by haploinsufficiency through loss of function mutations, however the total number of humans with pathologic mutations in CPA6 identified to date remains small. To better understand the relationship between CPA6 and seizures we investigated the effects of morpholino knockdown of cpa6 mRNA in zebrafish (Danio rerio) larvae. Knockdown of cpa6 mRNA resulted in resistance to the effect of seizure-inducing drugs pentylenetetrazole and pilocarpine on swimming behaviors. Knockdown of cpa6 mRNA also reduced the levels of mRNAs encoding neuropeptide precursors (bdnf, npy, chga, pcsk1nl, tac1, nts, edn1), a neuropeptide processing enzyme (cpe), transcription factor (c-fos), and molecules implicated in glutamatergic signaling (grin1a and slc1a2b). Treatment of zebrafish embryos with 60 mM pilocarpine for 1 hour led to reductions in levels of many of the same mRNAs when measured 1 day after pilocarpine exposure, except for c-fos which was elevated 1 day after pilocarpine treatment. Pilocarpine treatment, like cpa6 knockdown, led to a reduced sensitivity to pentylenetetrazole when tested 1 day after pilocarpine treatment. Taken together, these results add to mounting evidence that peptidergic systems participate in the biological effects of seizure-inducing drugs, and are the first in vivo demonstration of the molecular and behavioral consequences of cpa6 insufficiency.

Sample preparation for neuropeptidomic studies is a critical issue since protein degradation can produce high levels of peptides that obscure the endogenous neuropeptides. We compared different extraction conditions for the recovery of neuropeptides and the formation of protein breakdown fragments from mouse hypothalami. Sonication and heating in water (70 degrees C for 20 min) followed by cold acid and centrifugation enabled the efficient extraction of many neuropeptides without the formation of protein degradation fragments seen with hot acid extractions. The hot water/cold acid extraction procedure resulted in the reproducible recovery of many hypothalamic peptides, including several novel peptides.

Neuropeptidomics is the analysis of the neuropeptides present in a tissue extract. Most neuropeptidomic studies use mass spectrometry to detect and identify the peptides, which provides information on the precise posttranslationally modified form of each peptide. Quantitative peptidomics uses isotopic labels to compare the levels of peptides in extracts from two different samples. This technique is ideal for examining neuropeptide levels in a variety of systems and is especially suited for studies of mice lacking peptide-processing enzymes. This review is focused on the neuropeptidomics technique and its application to the analysis of mice with a mutation that inactivates carboxypeptidase E, a critical enzyme in the biosynthesis of many neuroendocrine peptides. Mice without carboxypeptidase E activity are overweight, and a key question is the identification of the peptide or peptides responsible. The quantitative peptidomics approach has provided some insights toward the answer to this question.

Nna1 is a recently described gene product that has sequence similarity with metallocarboxypeptidases. In the present study, five additional Nna1-like genes were identified in the mouse genome and named cytosolic carboxypeptidase (CCP) 2 through 6. Modeling suggests that the carboxypeptidase domain folds into a structure that resembles metallocarboxypeptidases of the M14 family, with all necessary residues for catalytic activity and broad substrate specificity. All CCPs are abundant in testis and also expressed in brain, pituitary, eye, and other mouse tissues. In brain, Nna1/CCP1, CCP5, and CCP6 are broadly distributed, whereas CCP2 and 3 exhibit restricted patterns of expression. Nna1/CCP1, CCP2, CCP5, and CCP6 were found to exhibit a cytosolic distribution, with a slight accumulation of CCP5 in the nucleus. Based on the above results, we hypothesized that Nna1/CCP1 and CCP2-6 function in the processing of cytosolic proteins such as alpha-tubulin, which is known to be modified by the removal of a C-terminal tyrosine. Analysis of the forms of alpha tubulin in the olfactory bulb of mice lacking Nna1/CCP1 showed the absence of the detyrosinylated form in the mitral cells. Taken together, these results are consistent with a role for Nna1/CCP1 and the related CCPs in the processing of tubulin.

Nna1 has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal. Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.

Prohormone convertase (PC) 1/3 and 2 are involved in the generation of neuropeptides from their precursors. A quantitative peptidomic approach was used to explore the role PC2 plays in the processing of hypothalamic peptides. In this approach, extracts from mice lacking PC2 activity and from wild-type littermates were labeled with isotopic tags, combined, fractionated on a reverse phase HPLC column, and analyzed by electrospray ionization mass spectrometry. Altogether, 53 neuropeptides or other peptides derived from secretory pathway proteins were identified and sequenced using tandem mass spectrometry. These peptides arise from 21 distinct proteins: proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, promelanin-concentrating hormone, proneurotensin, proneuropeptide Y, provasopressin, pronociceptin/orphanin, prothyrotropin-releasing hormone, cocaine- and amphetamine-regulated transcript, chromogranin A and B, secretogranin II, prohormone convertase 1 and 2, propeptidyl-amidating monooxygenase, and proteins designated proSAAS and VGF. Approximately one third of the peptides found in wild-type mice were not detectable in PC2 knock-out mice, and another third were present at levels ranging from 25 to 75% of wild-type levels. Comparison of the cleavage sites suggests that sequences with a Trp, Tyr and/or Pro in the P1' or P2' position, or a basic residue in the P3 position, are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr PG33 mutants do not survive past the early larval stage. These mutants have a P-element insertion within exon 1B upstream of the initiation ATG, which greatly reduces mRNA levels of all forms of CPD. Both svr 1 and svr poi mutants are viable, with a silvery body color and pointed wings. The wing shape is generally similar between these two mutants, although svr poi mutants have smaller wings. The svr 1 gene has a three-nucleotide deletion in exon 6, removing a leucine in a region of the protein predicted to function as a folding domain for the second CP-like domain. svr poi has a 1072-bp duplication of the gene that introduces a stop codon into the open reading frame, causing the truncation of the protein in the middle of the second CP-like domain. Both deletions eliminate enzyme activity of the second CP-like domain and appear to cause the misfolding of the protein. This greatly reduces the levels of the long forms of CPD protein but do not affect the levels of the short forms. Taken together, these findings suggest that lethal and viable svr alleles differ in which protein forms are affected. Flies that retain the short form are viable, whereas flies that are missing all forms of CPD do not survive past the early larval stages.

Neuropeptides perform a large variety of functions as intercellular signaling molecules. While most proteomic studies involve digestion of the proteins with trypsin or other proteases, peptidomics studies usually analyze the native peptide forms. Neuropeptides can be studied by using mass spectrometry for identification and quantitation. In many cases, mass spectrometry provides an understanding of the precise molecular form of the native peptide, including post-translational cleavages and other modifications. Quantitative peptidomics studies generally use differential isotopic tags to label two sets of extracted peptides, as done with proteomic studies, except that the Cys-based reagents typically used for quantitation of proteins are not suitable because most peptides lack Cys residues. Instead, a number of amine-specific labels have been created and some of these are useful for peptide quantitation by mass spectrometry. In this review, peptidomics techniques are discussed along with the major findings of many recent studies and future directions for the field.

The biosynthesis of most neuropeptides and peptide hormones requires a carboxypeptidase such as carboxypeptidase E, which is inactive in Cpe(fat/fat) mice due to a naturally occurring point mutation. To assess the role of carboxypeptidase E in the processing of peptides in the prefrontal cortex, we used a quantitative peptidomics approach to examine the relative levels of peptides in Cpe(fat/fat) versus wild-type mice. Peptides representing internal fragments of prohormones and other secretory pathway proteins were decreased two- to 10-fold in the Cpe(fat/fat) mouse prefrontal cortex compared with wild-type tissue. Degradation fragments of cytosolic proteins showed no major differences between Cpe(fat/fat) and wild-type mice. Based on this observation, a search strategy for neuropeptides was performed by screening for peptides that decreased in the Cpe(fat/fat) mouse. Altogether, 32 peptides were identified, of which seven have not been previously reported. The novel peptides include fragments of VGF, procholecystokinin and prohormone convertase 2. Interestingly, several of the peptides do not fit with the consensus sites for prohormone convertase 1 and 2, raising the possibility that another endopeptidase is involved with their biosynthesis. Taken together, these findings support the proposal that carboxypeptidase E is the major, but not the only, peptide-processing carboxypeptidase and also demonstrate the feasibility of searching for novel peptides based on their decrease in Cpe(fat/fat) mice.

We recently developed a quantitative peptidomics method using stable isotopic labels and mass spectrometry to both quantify and identify a large number of peptides. To test this approach and screen for peptides regulated by cocaine administration, 32 Cpefat/fat mice and 16 wild-type mice were treated twice daily for 5 d either with saline or 10 mg/kg cocaine. Peptides were extracted from striatum, hypothalamus, hippocampus, and prefrontal cortex, and extracts from groups of eight mice were labeled with the N-hydroxysuccinimide ester of trimethylammonium butyrate containing either nine deuterium or nine hydrogen atoms. Pools of heavy- and light-labeled peptides were combined, purified on an anhydrotrypsin affinity column, and analyzed on a reversephase column coupled to an electrospray ionization quadrapole time-of-flight mass spectrometer. Changes in peptide levels upon cocaine treatment were determined from the relative peak intensities of the cocaine versus saline peaks, and peptides were identified from collision-induced dissociation spectra. Ten peptides were found to increase or decrease in each of two separate analyses from distinct groups of mice. Peptides found to increase corresponded to fragments of proenkephalin, prothyrotropin-releasing hormone, provasopressin, proSAAS, secretogranin II, chromogranin B, and peptidyl-glycine-alpha-amidating mono-oxygenase in the hypothalamus. The same peptidyl-glycine-alpha-amidating mono-oxygenase peptide decreased in the prefrontal cortex, along with striatal neurokinin B and two unidentified peptides. Thirty other peptides were not substantially affected by cocaine treatment in both replicates. Taken together, the quantitative peptidomics approach provides an efficient method to screen for changes in a large number of peptides.

In neuropeptidomics, the degradation of a small fraction of abundant proteins overwhelms the low signals from neuropeptides, and many neuropeptides cannot be detected by mass spectrometry without extensive purification. Protein degradation was prevented when mice were sacrificed with focused microwave irradiation, permitting the detection of hypothalamic neuropeptides by mass spectrometry. Here we report an alternative and very simple method utilizing an ordinary microwave oven to inhibit enzymatic degradation. We used this technique to identify brain and pituitary neuropeptides. Quantitative analysis using mass spectrometry in combination with stable isotopic labeling was performed to determine the effect of microwave irradiation on relative levels of neuropeptides and protein degradation fragments. Microwave irradiation greatly reduced the levels of degradation fragments of proteins. In contrast, neuropeptide levels were increased about 2-3 times in hypothalamus by the microwave irradiation but not increased in pituitary. In a second experiment, three brain regions (hypothalamus, hippocampus, and striatum) from microwave-irradiated mice were analyzed. Altogether 41 neuropeptides or fragments of secretory pathway proteins were identified after microwave treatment; some of these are novel. These peptides were derived from 15 proteins: proopiomelanocortin, proSAAS, proenkephalin, preprotachykinins A and B, provasopressin, prooxytocin, melanin-concentrating hormone, proneurotensin, chromogranins A and B, secretogranin II, prohormone convertases 1 and 2, and peptidyl amidating monooxygenase. Although some protein degradation fragments were still found after microwave irradiation, these appear to result from protein breakdown during the extraction and not to an enzymatic reaction during the postmortem period. Two of the protein fragments corresponded to novel protein forms: VAP-33 with a 7-residue N-terminal extension and beta tubulin with a glutathione on the Cys near the N terminus. In conclusion, microwave irradiation with an ordinary microwave oven effectively inhibits enzymatic postmortem protein degradation, increases the recovery of neuropeptides, and makes it possible to conduct neuropeptidomic studies with mouse brain tissues.

Prohormone convertase 1 (PC1; also known as PC3) is believed to be responsible for the processing of many neuropeptide precursors. To look at the role PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined changes in the levels of multiple neuropeptide products in PC1 knockout (KO) mice. The processing of proenkephalin was impaired in PC1 KO mouse brains with a decrease in the level of Met-Enkephalin immunoreactivity (ir-Met-Enk) and an accumulation of higher molecular weight processing intermediates containing ir-Met-Enk. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains with a decrease in the level of an endogenous 3 kDa C-terminal peptide. In contrast, the processing of proSAAS into PEN was not altered in PC1 KO mouse brains. Quantitative mass spectrometry was used to analyze a number of peptides derived from proopiomelanocortin (POMC), provasopressin, prooxytocin, chromogranin A, chromogranin B, and secretogranin II. Among them, the levels of oxytocin and peptides derived from chromogranin A and B dramatically decreased in the PC 1 KO mouse pituitaries, while the levels of peptides derived from proopiomelanocortin and provasopressin did not show substantial changes. In conclusion, these results support the notion that PC1 plays a key role in the processing of multiple neuroendocrine peptide precursors and also reveal the presence of a redundant system in the processing of a number of physiologically important bioactive peptides.

Carboxypeptidase E is a major enzyme in the biosynthesis of numerous neuroendocrine peptides. Previously, we developed a technique for the isolation of neuropeptide-processing intermediates from mice that lack carboxypeptidase E activity (Cpefat/fat mice) due to a naturally occurring point mutation. In the present study, we used a differential labeling procedure with stable isotopic tags and mass spectrometry to quantitate the relative changes in a number of hypothalamic peptides in Cpefat/fat mice in two different paradigms that each cause an ∼10% decrease in body mass. One paradigm involved a 2-day fast under normal sedentary conditions (i.e. standard mouse cages); the other involved giving mice access to an exercise wheel for 4 weeks with free access to food. Approximately 50 peptides were detected in both studies, and over 80 peptides were detected in at least one of the two studies. Twenty-eight peptides were increased >50% by food deprivation, and some of these were increased by 2- to 3-fold. In contrast, only three peptides were increased >50% in the group with exercise wheels, and many peptides showed a slight 15–30% decrease upon exercise. Approximately one-half of the peptides detected in both studies were identified by tandem mass spectrometry. Peptides found to be elevated by food deprivation but not exercise included a number of fragments of proenkephalin, prothyrotropin-releasing hormone, secretogranin II, chromogranin B, and pro-SAAS. Taken together, the differential regulation of these peptides in the two paradigms suggests that the regulation is not due to the lower body weight but to the manner in which the paradigms achieved this lower body weight.

Neuropeptides are generally produced from precursor proteins by selective cleavage at specific sites, usually involving basic amino acids. Enzymes such as the prohormone convertases and carboxypeptidase E are highly specific for these basic amino acid-containing sites. In addition to this "traditional" pathway, several neuropeptides are known to be cleaved at non-basic sites, and the enzymes responsible for these cleavages have not been conclusively identified. In a recent search for novel members of the metallocarboxypeptidase family, we found three human genes. One of these, named "CPA-5," has a specificity for C-terminal hydrophobic amino acids and mRNA expression in brain, pituitary, and testis. To test whether CPA-5 protein has a distribution pattern in pituitary that is consistent with a role for this enzyme in the non-basic processing of proopiomelanocortin-derived peptides such as beta-endorphin and adrenocorticotropin, we examined the distribution of CPA-5 using immunocytochemistry. In the pituitary, CPA-5 is detected in the neurointermediate lobe and in scattered cells in the anterior lobe. In the AtT-20 corticotroph cell line, CPA-5 has a perinuclear distribution. Taken together, these results are consistent with a role for CPA-5 in the intracellular processing of proopiomelanocortin-derived peptides at non-basic sites.

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.

Metallocarboxypeptidase D (CPD), is a 180-kDa protein that contains three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail and which functions in the processing of proteins that transit the secretory pathway. An initial report on the Drosophila melanogaster silver gene indicated a CPD-like protein with only two and a half carboxypeptidase-like domains with no transmembrane region (Settle, S. H., Jr., Green, M. M., and Burtis, K. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9470-9474). A variety of bioinformatics and experimental approaches were used to determine that the Drosophila silver gene corresponds to a CPD-like protein with three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. In addition, two alternative exons were found, which result in proteins with different carboxypeptidase-like domains, termed domains 1A and 1B. Northern blot, reverse transcriptase PCR, and sequence analysis were used to confirm the presence of the various mRNA forms. Individual domains of Drosophila CPD were expressed in insect Sf9 cells using the baculovirus expression system. Media from domain 1B- and domain 2-expressing cells showed substantial enzymatic activity, whereas medium from domain 1A-expressing cells was no different from cells infected with wild-type virus. Domains 1B and 2 were purified, and the enzymatic properties were examined. Both enzymes cleaved substrates with C-terminal Arg or Lys, but not Leu, and were inhibited by conventional metallopeptidase inhibitors and some divalent cations. Drosophila domain 1B is more active at neutral pH and greatly prefers C-terminal Arg over Lys, whereas domain 2 is more active at pH 5-6 and slightly prefers C-terminal Lys over Arg. The differences in pH optima and substrate specificity between Drosophila domains 1B and 2 are similar to the differences between duck CPD domains 1 and 2, suggesting that these properties are essential to CPD function.

Neuroendocrine peptides play important roles as intercellular messengers. We previously developed a technique to isolate and identify a large number of neuroendocrine peptides from Cpe(fat)/Cpe(fat) mice (Che, F.; et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9971-6); these mice lack carboxypeptidase E activity and this defect causes an accumulation of neuropeptide intermediates that contain C-terminal Lys or Arg residues (Naggert, J. K.; et al. Nat. Genet. 1995, 10, 135-42). In the present study, we have developed a differential isotopic-labeling technique that can be used to quantitate changes in neuropeptide levels in Cpe(fat)/Cpe(fat) mouse tissues. Samples are treated with either the H6 or the D6 form of acetic anhydride, peptides that contain C-terminal basic amino acids are isolated by affinity chromatography on anhydrotrypsin agarose, and the isolated peptides are analyzed by mass spectrometry. Measurement of the regulation of pituitary peptides in response to dehydration showed a decrease in vasopressin. The general method described in this report should be widely applicable to a large number of neuroendocrine peptides, known and novel, in a variety of regulatory paradigms.

ProSAAS (encoded by mouse gene Pcsk1n) is a recently described neuroendocrine secretory pathway protein that is cleaved into smaller peptides that may function in cell-cell signalling. ProSAAS and its processing intermediates are also potent inhibitors of prohormone convertase 1 (PC1), which is encoded by mouse gene Pcsk1. In order to gain insight into the function of proSAAS, we have examined the distribution of several proSAAS-derived peptides and PC1 by immunohistochemistry throughout mouse development. The distribution patterns of both SAAS and PC1 are broad from E9 to E11, with some enrichment in neural tube-derived tissues. By E15, the expression of SAAS is largely restricted to neuroendocrine tissues known to produce bioactive peptides. In general, the expression pattern of PC1 overlaps with that of SAAS and other proSAAS-derived peptides, consistent with the hypothesis that proSAAS functions as an endogenous PC1 inhibitor.

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT-20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C-terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C-terminal region functions in TGN-retention; removal of 10 amino acids from the C-terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half-life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10-residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half-life than either the wild type sequence or the EDE mutant. Adaptor protein-1 and -2 bind to most of the deletion mutants, the EDE point mutant, and the CK2-phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C-terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins.

Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy.

Using a technique to identify substrates of the peptide processing enzyme carboxypeptidase E (CPE), several novel peptides were detected in the brain and pituitary of Cpe(fat)/Cpe(fat) mice and found to be derived from a single precursor, termed proSAAS. In order to gain further information regarding the potential physiological roles of these peptides, we have examined the distribution of two proSAAS-derived peptides, ARPVKEPRSLSAASAPLAETSTPLRL (SAAS) and LENSSPQAPARRLLPP (LEN), in rat neuroendocrine tissues using immunohistochemistry. Both peptides are detected throughout the brain, with the highest concentrations of SAAS peptide in the hypothalamus. In the hippocampus, both peptides are co-localized with prohormone convertase 1 in the dentate gyrus and CA1-3 region. In cerebellum, SAAS peptide is co-localized with prohormone convertase 1 in Purkinje and granular cells, whereas LEN is much more abundant in the Purkinje cells relative to the granular cells. Similarly, SAAS and prohormone convertase 1 are co-localized in the dorsal horn of the spinal cord, while LEN is mainly restricted to fibers of the white matter. In the pituitary, SAAS, LEN, and prohormone convertase 1 are detected in all three lobes. In the pancreas, SAAS, LEN, and prohormone convertase 1 are only detected in the islets, although the peptides are enriched in the peripheral cells (alpha and/or delta) while prohormone convertase 1 is only expressed in the inner cells (beta). Both SAAS and LEN are present in the adrenal medulla along with prohormone convertase 1. Taken together, these data are consistent with the proposed role for proSAAS as an endogenous inhibitor of prohormone convertase 1 in many, but not all cell types. However, the broader localization of the peptides allows for the possibility that they perform additional functions.

ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide-processing endopeptidase. Several proSAAS-derived peptides were previously identified in the brain and pituitary of the Cpe(fat)/Cpe(fat) mouse based on the accumulation of C-terminally extended peptides due to the absence of enzymatically active carboxypeptidase E, a peptide-processing exopeptidase. In the present study, antisera against different regions of proSAAS were used to develop radioimmunoassays and examine the processing profile of proSAAS in wild type and Cpe(fat)/Cpe(fat) mouse tissues following gel filtration and reverse phase high performance liquid chromatography. In wild type mouse brain and pituitary, the majority of proSAAS is processed into smaller peptides. These proSAAS-derived peptides elute from the reverse-phase column in the same positions as synthetic peptides that correspond to little SAAS, PEN, and big LEN. Mass spectrometry revealed the presence of peptides with the expected molecular masses of little SAAS and big LEN in the fractions containing immunoreactive peptides. The processing of proSAAS is slightly impaired in Cpe(fat)/Cpe(fat) mice, relative to wild-type mice, leading to the accumulation of partially processed peptides. One of these peptides, the C-terminally extended form of PEN, is known to inhibit PC1 activity and this could account for the reduction in enzymatically active PC1 seen in Cpe(fat)/Cpe(fat) mice. The observation that little SAAS and big LEN are the major forms of these peptides produced in mouse brain and pituitary raises the possibility that these peptides function as neurotransmitters or hormones.

Carboxypeptidase D (CPD) is a transmembrane protein that processes proteins in the trans-Golgi network (TGN). A 20-residue region within the cytoplasmic tail of CPD binds protein phosphatase 2A (PP2A). PP2A also binds to the cytoplasmic tails of other secretory pathway proteins: peptidylglycine-(amino)-amidating mono-oxygenase, the cation-independent mannose-6-phosphate receptor and TGN38. The CPD tail is phosphorylated on Thr residues in the AtT-20 cell line. The CPD tail can also be phosphorylated by purified protein kinase A, protein kinase C and casein kinase II. Both the in vitro and the in vivo phosphorylated CPD tail can be dephosphorylated by purified PP2A. The binding of CPD tail peptide to PP2A does not influence phosphatase activity. The rate of transport of CPD from the TGN to the cell surface of AtT-20 cells is decreased 45% by okadaic acid, a PP2A inhibitor. Microinjection of the CPD tail into AtT-20 cells inhibits the transition of CPD from endosomal compartments to the TGN. However, okadaic acid does not affect the rate of budding of CPD from the TGN into nascent vesicles or the rate of uptake from the cell surface into endosomal compartments. These results are consistent with the model that PP2A is involved in the trafficking of proteins between a TGN recycling loop and a cell-surface recycling loop, but is not involved in the individual recycling loops.

Carboxypeptidase E (CPE) is involved in the biosynthesis of peptide hormones and neurotransmitters, including insulin. One of the features of type 2 diabetes mellitus (T2DM) is an elevation in the proinsulin level and/or proinsulin/insulin molar ratio, suggesting that mutations in proinsulin processing enzymes may contribute to the development of T2DM. We scanned CPE for mutations in a collection of Ashkenazi T2DM families and identified five novel single nucleotide polymorphisms (SNPs). An SNP in the 283rd codon, c.847C>T, changes arginine to tryptophan (R283W). The residue Arg283 is conserved among CPE orthologs as well as most enzymatically active metallocarboxypeptidases. Of the 272 Ashkenazi T2DM pedigrees screened, we found four families segregating R283W. Within these four families, patients who inherited one copy of this variant had much earlier age of onset for T2DM. The R283W CPE protein cleaves peptide substrates with substantially lower efficiencies and is less stable at elevated temperature. In addition, the R283W CPE variant has a narrower pH optimum and is much less active at pH 6.0–6.5, indicating that the R283W CPE variant would be substantially less active than wild type CPE in the trans-Golgi network and immature secretory vesicles where the enzyme functions in vivo. To summarize, we uncovered a rare non-conservative missense mutation in CPE and demonstrated that the mutant protein has altered enzymatic properties. We predict that this mutant could cause hyperproinsulinism and diabetes in the homozygous state. Hum Mutat 18:120–131, 2001. © 2001 Wiley-Liss, Inc.

ProSAAS is a recently discovered 26-kDa neuroendocrine protein that was previously found to inhibit prohormone convertase (PC) 1 and not PC2. In the present study, the specificity of proSAAS toward other members of the prohormone convertase family was determined. Two microm proSAAS selectively inhibits PC1 but not furin, PACE4, PC5A, or PC7. The PC1 inhibitory region of proSAAS was mapped to an 8-12-residue region near the C terminus that includes a critical Lys-Arg sequence. Synthetic peptides corresponding to this region are competitive inhibitors of PC1 with apparent K(i) values of 14-40 nm. The inhibition becomes more effective with incubation time, indicating that the inhibitor is slow binding. A fusion protein containing the inhibitory region of proSAAS linked to the C terminus of glutathione S-transferase binds the 71-kDa form but not the 85-kDa form of PC1. This binding, which occurs at pH 5.5 and not at pH 7.4, is stable to incubation at room temperature for 1 h in the presence or absence of 0.5% Triton X-100 and/or 0.5 m NaCl. The removal of Ca(2+) with chelating agents partially releases the bound PC1. High concentrations of the inhibitory peptide quantitatively release the bound PC1. Taken together, these data support the proposal that proSAAS functions as an endogenous inhibitor of PC1.

Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in thetrans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.

Post-translational processing is essential for the biological activation of many proteins and peptides. After precursor cleavage at specific single residues or pairs of basic residues by the proprotein convertases, the C-terminal basic residues are removed. Carboxypeptidase E was thought to be the only enzyme responsible. Recent studies with carboxypeptidase E-deficient mice, Cpe fat / Cpe fat , indicated the existence of carboxypeptidase E-like carboxypeptidases, such as carboxypeptidase D. In order to define potential redundant functions in vivo , we compared the distributions of both carboxypeptidases in the rat central nervous system and selected endocrine tissues. Carboxypeptidase D messenger RNA was abundantly expressed in glial cells in the gray and white matter, while neurons in several brain regions, such as the piriform cortex, basolateral amygdala and hippocampus, also expressed high levels of carboxypeptidase D messenger RNA. Co-localization of carboxypeptidases E and D messenger RNAs was observed in many brain regions, the spinal cord and endocrine tissues. Immunohistochemistry showed the intracellular distribution of carboxypeptidase D with a perinuclear pattern. The extensive distribution of carboxypeptidase D in both glial and neuronal cells indicates the important role of carboxypeptidase D in peptide processing, possibly working together with furin, a ubiquitously expressed proprotein convertase. The co-localization of carboxypeptidases D and E suggests that carboxypeptidase D may, at least partially, compensate for carboxypeptidase E processing functions in Cpe fat / Cpe fat mice.

Duck gp180 was previously identified by its ability to bind to the preS envelope protein of duck hepatitis B virus particles (Kuroki, K., Cheung, R., Marion, P. L., and Ganem, D. (1994) J. Virol. 68, 2091–2096). Cloning and sequencing of gp180 cDNA revealed that it is a polyprotein with three carboxypeptidase-like domains (Kuroki, K., Eng, F., Ishikawa, T., Turck, C., Harada, F., and Ganem, D. (1995)J. Biol. Chem. 270, 15022–15028). To evaluate enzymatic properties of this protein, a soluble 170-kDa form of the protein (gp170) lacking the C-terminal transmembrane domain and cytoplasmic tail was expressed in a baculovirus system. The purified 170-kDa protein cleaved 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg with a pH optimum of 5.5–6.5. With this substrate at pH 5.5, the 170-kDa protein displayed a K m of 12 μm and a K cat of 57 s−1. Dansyl-Pro-Ala-Arg and dansyl-Phe-Phe-Arg were cleaved with K m values of 17 and 21 μm, and K cat values of 57 and 17 s−1, respectively. Constructs containing only the first or second carboxypeptidase domains also showed enzymatic activity. The effects of inhibitors and ions on enzyme activity of gp170 were generally similar to the effects of these compounds on purified bovine carboxypeptidase D. To evaluate the regions within gp180 necessary for binding preS, a series of deletion mutants were expressed in the 293T human kidney cell line. Deletions of the first and second domains, leaving the third domain intact, eliminated carboxypeptidase activity but retained preS binding. Deletion of the third domain eliminated preS binding but not carboxypeptidase activity. These results indicate that the third domain is responsible for preS binding, and this binding does not require carboxypeptidase activity.

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.

A spontaneous point mutation in the coding region of the carboxypeptidase E (CPE) gene in Cpe(fat)/Cpe(fat) mice affects proinsulin processing. Cell lines derived from the pancreatic beta-cells of Cpe(fat)/Cpe(fat) mice were generated by crossing C57BLKS/J-Cpe(fat)/+ mice with NOD mice expressing the simian virus 40 large T oncogene under the control of the rat insulin II promoter. Two cell lines, designated NIT-2 and NIT-3, were cultured from adenomatous islets obtained from F2 littermates and were compared with the NIT-1 cell line previously developed from mice with wild-type CPE. Electron microscopy of the cultured NIT-2 and -3 cells showed increased numbers of enlarged and electron-lucent granules compared with NIT-1 cells. Pro-CPE, but not the mature form of CPE, is present in NIT-2 and -3 cells, and neither pro-CPE nor CPE are secreted into the medium. Immunocytochemistry shows the pro-CPE to be localized to an endoplasmic reticulum-like structure in NIT-3 cells. Proinsulin is less extensively processed in NIT-2 and -3 cells than in NIT-1 cells, indicating that the Cpe(fat) mutation affects both the endopeptidase and carboxypeptidase reactions. The secretion of insulin/proinsulin from NIT-2 and -3 cells is significantly elevated by secretagogues, indicating that CPE is not required for sorting proinsulin into the regulated pathway.

A novel cDNA, designated carboxypeptidase Z (CPZ), was identified based on its homology to known metallocarboxypeptidases. Northern blot analysis shows bands of 2.1 and/or 2.6 kilobases in all tissues examined. The major form of CPZ mRNA in human salivary gland encodes a protein with an open reading frame of 641 amino acids. In addition, three variants were found that presumably arise due to alternative intron splicing. The 641-amino acid protein contains an 18-residue signal peptide-like sequence, a 120-residue region that shows 23–29% amino acid identity with a Cys-rich domain found in frizzled proteins and in type XVIII collagen, and then a 390-residue carboxypeptidase domain with 49% amino acid identity to carboxypeptidases E and N. The 641-amino acid form of CPZ expressed in the baculovirus system cleaves 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Phe-Ala-Arg, although the level of enzyme activity was approximately 10-fold lower than either carboxypeptidase E or D expressed using the same viral system. The CPZ activity is more active at neutral pH than at pH 5.5 and is inhibited by active site-directed inhibitors of metallocarboxypeptidases. In summary, CPZ is a novel metallocarboxypeptidase that is active toward substrates with C-terminal basic amino acids.