Carboxyterminal Protein Processing in Health and Disease - American

Sep 10, 2014 - are widely connected to various pathological processes such as carcinogenesis and neurodegenerative and cardiovascular diseases...
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Carboxyterminal Protein Processing in Health and Disease: Key Actors and Emerging Technologies Agnese Petrera,†,§ Zon Weng Lai,†,§ and Oliver Schilling*,†,‡ †

Institute of Molecular Medicine and Cell Research, ‡BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104 Freiburg, Germany ABSTRACT: Carboxypeptidases are important mediators of cellular behavior. Through C-terminal truncations, they alter protein functionality and participate in proteome turnover. Similarly, carboxypeptidases shape the human peptidome by targeting neuroendocrine and vasoactive peptides, thereby regulating signaling pathways in the nervous and cardiovascular systems as well as in embryonic development. Carboxypeptidases are widely connected to various pathological processes such as carcinogenesis and neurodegenerative and cardiovascular diseases. The repertoire of carboxypeptidase in vivo substrates still remains poorly defined, largely due to the lack of suitable experimental approaches. Understanding the precise role of carboxypeptidases is pivotal in the future development of diagnostic/prognostic markers in such diseases. To date, very little attention has been paid to the implication of carboxypeptidases in shaping the proteome as well as the peptidome. This review focuses on the patho-physiological function of carboxypeptidases and highlights the approaches by which proteomics-based technologies can be applied to characterize carboxypeptidases and to quantify the differential regulation of proteins by carboxypeptidases in a proteome-wide manner. KEYWORDS: Carboxypeptidase, degradomics, proteolysis, biomarkers, protease, peptidase

1. INTRODUCTION Carboxypeptidases represent a small group of the more than 550 genetically encoded proteases in humans. In Table 1, we list the human proteases annotated as carboxypeptidases or having carboxypeptidase activity. Carboxypeptidases perform C-terminal truncations by removing single residues or amino acid pairs from protein C-termini. There are multiple classification criteria to group carboxypeptidases. The first, based on the chemical nature of their catalytic site, defines three classes: serine carboxypeptidases, cysteine carboxypeptidases, and metallo-carboxypeptidases, with the latter group employing zinc as a catalytic cofactor (Figure 1). The second criteria classifies carboxypeptidases according to their substrate specificity: carboxypeptidases displaying preference for hydrophobic C-terminal amino acids (A-like metallocarboxypeptidases or C-type serine carboxypeptidases), carboxypeptidases cleaving C-terminal aspartate or glutamate residues, carboxypeptidases preferring C-terminal basic residues (B-like metallocarboxypeptidases or D-type serine carboxypeptidases), and other carboxypeptidases with broader substrate specificity.1 In addition, carboxypeptidases can be classified according to the physiological processes in which they are involved: the digestive carboxypeptidases, primarily involved in the degradation of intake proteins (such as the pancreatic CPA1), and the regulatory carboxypeptidases, which specifically truncate bioactive peptides, thereby participating in biological signaling pathways (such as CPN and CPU).2

physiologically relevant family M14.3 The M14 family is composed of four subfamilies, based on primary amino acid sequence similarities. The M14A subfamily (CPA/B) is named after the pancreatic digestive enzymes CPA and CPB. M14A carboxypeptidases are typically produced as zymogens, and once activated they display an optimal neutral pH.4 CPA members of the M14A subfamily preferentially process C-terminal aliphatic and aromatic residues, whereas the pancreatic carboxypeptidase B has a strong preference for basic residues.3 Although the primary function of pancreatic CPA and CPB is to break down peptides in the gastrointestinal system, other members of the M14A subfamily have a regulatory function, such as the plasmacirculating enzyme CPU (also named thrombin-activatable fibrinolysis inhibitor, TAFI), which displays an anti-fibrinolytic activity by removing C-terminal lysine residues of partially degraded fibrin.5 Mast cell-CPA (MC-CPA) has a substrate specificity similar to that of pancreatic CPA; however, its biological function, although not well-defined in vivo, has been correlated to the regulation of immune responses and the degradation of vasoactive peptides such as angiotensin II and endothelin I.6 Another noteworthy member of this subfamily is the non-digestive carboxypeptidase A4 (CPA4), which has been linked to prostate cancer aggressiveness, as discussed below. The M14B subfamily (CPN/E) is composed of metallocarboxypeptidases that are produced in an enzymatically active

1.1. Metallo-carboxypeptidases

Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment

One of the broader and better characterized classes is the metallo-carboxypeptidases, encoded by more than 26 genes in the human genome, most of them belonging to the © 2014 American Chemical Society

Received: June 10, 2014 Published: September 10, 2014 4497

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Table 1. Human Carboxypeptidases or Peptidases with Known Carboxypeptidase Activity Reported in MEROPSa

a

clan

family

MEROPS ID

peptidase or homologue name

MERNUM

gene

locus

CA MA MC MC MC MC MC MC MC MC MC MC MC MC MC MC MC MC MC MC MC MC MH MH MH MH SC SC SC SC

C1 M2 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M14 M20 M28 M28 M28 S10 S10 S10 S28

C01.013 M02.006 M14.001 M14.002 M14.003 M14.004 M14.005 M14.006 M14.009 M14.010 M14.011 M14.012 M14.017 M14.018 M14.020 M14.021 M14.025 M14.026 M14.027 M14.028 M14.029 M14.030 M20.011 M28.010 M28.012 M28.014 S10.002 S10.003 S10.013 S28.001

cathepsin Z angiotensin-converting enzyme-2 carboxypeptidase A1 carboxypeptidase A2 carboxypeptidase B carboxypeptidase N carboxypeptidase E carboxypeptidase M carboxypeptidase U carboxypeptidase A3 metallocarboxypeptidase D metallocarboxypeptidase Z carboxypeptidase A4 carboxypeptidase A6 carboxypeptidase A5 metallocarboxypeptidase O cytosolic carboxypeptidase 5 cytosolic carboxypeptidase 3 cytosolic carboxypeptidase 6 cytosolic carboxypeptidase 1 cytosolic carboxypeptidase 2 cytosolic carboxypeptidase 4 carboxypeptidase PM20D1 glutamate carboxypeptidase II glutamate carboxypeptidase III carboxypeptidase Q serine carboxypeptidase A vitellogenic carboxypeptidase-like protein RISC peptidase lysosomal Pro-Xaa carboxypeptidase

MER004508 MER011061 MER001190 MER001608 MER001194 MER001198 MER001199 MER001205 MER001193 MER001187 MER003781 MER003428 MER013421 MER013456 MER017121 MER016044 MER033174 MER033176 MER033178 MER033179 MER037713 MER034713 MER033182 MER002104 MER005238 MER005244 MER000430 MER005492 MER010960 MER000446

CTSZ ACE2 CPA1 CPA2 CPB1 CPN1 CPE CPM CPB2 CPA3 CPD CPZ CPA4 CPA6 CPA5 CPO AGBL5 AGBL3 AGBL4 AGTPBP1 AGBL2 AGBL1 PM20D1 FOLH1 NAALAD2 CPQ CTSA CPVL SCPEP1 PRCP

20q13 Xp22 7q32 7q32 3q24 10 4 12q15 13q14.11 3q21−q25 17p11.1−q11.2 4p16.1 7q32 8q12.3 7q32 2q34 2p23.3 7q33 1p33 9q22.1 11p11.2 15q25.3 1q32.1 11p11.2 11q14.3−q21 8q22.2 20q13.1 7p14−p15.3 17 11q14

Cathepsin B was omitted from the list due to its prominent endopeptidase activity.

Figure 1. Human carboxypeptidases classified according to chemical nature of their catalytic site. MEROPS family name and the representative peptidase are depicted in bold and italic, respectively. Where peptidase members are classified in subfamilies, the latter are indicated with a bold capital letter. Exemplary subfamily members are listed; Table 1 highlights the human subfamily members in greater detail.

CPN and CPM.4 In particular, CPN is the largest regulatory carboxypeptidase described, circulating in the plasma as a 280 kDa protein complex. CPN represents the major blood inactivator of potent peptides such as kinins and anaphylotoxins.2 Generally, M14B metallo-carboxypeptidases favor substrates with a basic C-terminal residue (Arg or Lys). The subfamily M14C includes bacterial peptidoglycan hydrolyzing enzymes. Recently, a new subfamily has been

form and thus do not requiring zymogen activation. M14B members are primarily secreted or membrane-bound. With a broad neuroendocrine distribution and substrate specificity, CPE and CPD both play a relevant role in the biosynthesis of most neuroendocrine peptides within the late secretory pathway and the trans Golgi network of neuroendocrine cells, respectively.4 Two further carboxypeptidases participating in processing of peptide hormones, mainly vasoactive peptides, are 4498

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identified, M14D, also named cytosolic carboxypeptidases (CPPs). Novel findings, including direct studies on purified CCP1 and CCP5, suggest that CPPs function as deglutamylases, releasing glutamates from the C-terminus of tubulin and from polyglutamate side chains of tubulin.7,8 Carboxypeptidase A (CPA, also named CPA1) is considered to be the prototypical metallo-carboxypeptidase. Bovine pancreatic CPA was the third protein, after myoglobin and lysozyme, to have its structure solved at high resolution by Xray crystallography.9 Structural information from the highresolution structure of CPA has led to the elucidation of the catalytic mechanism of zinc-dependent proteases and to the development of specific inhibitors for zinc proteases. Over the past decade, the crystal structures of more metallo-carboxypeptidases were subsequently solved; among them are some important regulatory carboxypeptidases such as the human CPN, CPM, and CPU.10 In addition to the M14 family, other metalloprotease families comprise carboxypeptidases. In this context, angiotensinconverting enzyme-2 (ACE2) is of particular interest. Belonging to the M2 family, this secreted zinc metallocarboxypeptidase removes C-terminal hydrophobic or basic residues of several hormone peptides, such as the potent vasoconstrictor angiotensin II.11 Being a key player in the regulation of the renin−angiotensin system and blood pressure homeostasis, ACE inhibition has been clinically used in cardiovascular therapies for more than 20 years.12 Furthermore, ACE2 gained international attention when it was identified as being the functional receptor for the binding of the coronavirus that is responsible for severe acute respiratory syndrome during the epidemic outbreak in South East Asia in 2002.13 Further metalloprotease subfamilies with carboxypeptidase members include the M20 and M28 families. The M28 family contains glutamate carboxypeptidase II, a membrane-bound zinc metallo-carboxypeptidase with a relevant patho-physiological function in nutrient uptake, particularly related to glutamate or folate absorption.14

DPP7 (also named DPP2), lack major sequence similarity to any other protease.17 Structurally, all serine carboxypeptidases display an alpha/ beta hydrolase fold common to several hydrolytic enzymes with different phylogenetic origin and catalytic functions, such as acetylcholinesterases and lipases.18 However, the crystal structure of the human PRCP has revealed a novel helical structure domain that caps the active site, called the SKS domain.17 Serine carboxypeptidases are generally found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells, although some can also be secreted into the extracellular space. Their subcellular localization is attributed to their main biological function, which is intracellular protein turnover.19 However, some serine carboxypeptidases are major players in the regulation of hormone peptides, such as the human kidney prolyl CPD, which cleaves the C-terminal peptide bond of angiotensin II and III,20 and the yeast CPD coded by the KEX1 gene that proteolytically matures the αfactor mating pheromone.21 Serine carboxypeptidase A, commonly known as cathepsin A, is a serine carboxypeptidase belonging to the S10 family, sharing 30% sequence identity with the yeast carboxypeptidase Y and the wheat carboxypeptidase II (carboxypeptidase D-like). This enzyme is worth mentioning for its multifunctional roles in different subcellular localizations: in the lysosome, it plays a protective structural role in stabilizing the protein complex made up of βgalactosidase and neuroamidase-1; the same complex is found on the cell surface (having the 67 kDa splice variant of βgalactosidase) where it actively participates in the formation of elastic fibers;22 when secreted, it is involved in the processing of vascular peptides, such as endothelin-1, bradykinin, and angiotensin I.23 To highlight the physiological relevance of this serine carboxypeptidase, the impairment of the protective structural function of cathepsin A due to mutations or deficiency of the gene leads to the severe lysosomal storage disease galactosialidosis.24 1.3. Cysteine Carboxypeptidases

Cysteine proteases hydrolyze peptide bonds using the sulfhydryl group of a cysteine residue located at their active center, which acts as a nucleophile. Cathepsin Z is a lysosomal cysteine peptidase belonging to the family C1 (papain family). It is described as a carboxypeptidase with a very minor endopeptidase activity.25 Cathepsin B, another C1 family member, possesses both endopeptidase and exopeptidase activities, in the latter case acting as a peptidyl-dipeptidase on the C-termini of proteins or peptides.26 In addition to their involvement in lysosomal protein turnover, both cathepsin B and Z have been found to be secreted. They have been shown to be involved in several physiological processes, as reviewed elsewhere.27

1.2. Serine Carboxypeptidases

Serine carboxypeptidases share the conserved catalytic triad Ser-Asp-His characteristic of the serine proteases. Unlike prototypical serine proteases such as trypsin, serine carboxypeptidases are active only at acidic pH. This is attributed to a conserved pair of glutamic acid residues and an asparagine positioned near the catalytic triad that form a hydrogenbonding pattern stabilized at pH 5.5 or below.15 In addition to their peptidase activity, serine carboxypeptidases are able to release C-terminal amino acid amides or ammonia from peptide amides (amidase activity) and alcohols from peptide esters (esterase activity).16 Serine carboxypeptidases are categorized under the S10 and S28 families, with carboxypeptidase Y (Saccharomyces cerevisiae) and lysosomal Pro-Xaa carboxypeptidase (Homo sapiens) as prototypes, respectively. S10 family members are further classified according to substrate preferences: carboxypeptidase C-like, which prefers hydrophobic residues in positions P1 and P1′, and carboxypeptidase D-like, which favors basic amino acids on either side of the scissile bond, although it is still able to cleave peptides with hydrophobic residues in these positions. On the other hand, the S28 subfamily stands out from other protease subfamilies as its only two characterized members, human lysosomal Pro-Xaa carboxypeptidase (PRCP) and human

2. CARBOXYPEPTIDASES IN DISEASES The physiological roles of carboxypeptidases have long been associated primarily with food digestion. As described above, their biological role goes far beyond the protein turnover, as carboxypeptidases are important mediators of cellular behaviors. Importantly, they participate in shaping the human peptidome by maturation and processing of neuropeptides, peptide hormones, and chemokines. It is therefore not surprising that carboxypeptidases have important pathophysiological functions in tumorigenesis and neurodegenerative and cardiovascular diseases. 4499

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Figure 2. As essential players in regulatory mechanisms, carboxypeptidases are associated with several pathological conditions. Hence, the characterization of their substrate repertoire is the key for exploiting carboxypeptidases as drug targets. Proteomics-based approaches open new perspectives into substrate discovery for carboxypeptidases.

that lack a signal peptide and are localized intracellularly.35 An autosomal recessive mutation in the gene encoding CCP1 causes a neurodegenerative phenotype in mice with loss of Purkinje cells, retinal photoreceptor cells, and olfactory bulb mitral cells.36 The neurodegenerative phenotype is rescued by reconstituted expression of catalytically active CCP1 but not by a catalytically inactive counterpart.37 A neuroprotective function has also been established for the Drosophila CCP1 orthologue.38 During embryogenesis, CCP1 is expressed in brain, spinal cord, and peripheral nervous tissue.39 However, its expression in adult mouse tissue is considered to be controversial. While Harris et al. found CCP1 expression restricted to heart and brain,39 Kalinina et al. report a broad tissue distribution.35a There are diverging findings also with respect to its precise subcellular localization, with studies supporting cytoplasmic and nuclear localization in cultured mouse neurons,39 mitochondrial localization in Drosophila S2 cells and human embryonic kidney cells,37a or predominantly cytoplasmic localization in Neuro2A cells with only low nuclear levels.35a The role of CCP1 as a tubulin-processing enzyme is solidly confirmed in a study by Berezniuk and colleagues.8b The same group has recently contradicted a previously hypothesized role for CCP1 in peptide turnover by using a quantitative peptidomics approach on the Purkinje cell degeneration mouse model (pcd).40 In this work, the authors attributed the intracellular peptide accumulation observed in adult pcd mouse brain to secondary effects rather than to CCP1 activity.40 Cathepsin A (CathA), as discussed previously, plays a relevant role in the regulation of the vascular tone by processing several vasoactive peptides such as endothelin-1. Studies using transgenic mice that express catalytically inactive CathA further support this notion41 while simultaneously indicating a similar function for other lysosomal serine carboxypeptidase, such as Scpep1.42 These findings have shed light on the involvement of lysosomal serine carboxypeptidases in the pathophysiology of hypertension. Increasing interest in CathA has developed in recent years, primarily in the context of cardiac diseases. Its role in the regulation of the hemodynamics function, along with its poor expression in the vasculature and the acidic pH optimum, made CathA a potential and novel target in cardiovascular diseases.43 Inhibitors against CathA are currently in phase I clinical trials for cardiac hypertrophy and atrial fibrillation in heart failure.44 The general concept of cysteine cathepsins being primarily involved in intracellular protein turnover has dramatically changed over the past decade. Findings on the implication of cathepsin B and cathepsin Z in tumor invasion and metastasis represent indeed a clear mark. Sevenich et al. demonstrated that combined deficiency of cathepsin B and cathepsin Z results in a significantly reduced tumor and metastatic burden in a mouse model of breast cancer.45

A remarkable example of a carboxypeptidase with a double implication in carcinogenesis and neurological disorders is human glutamate carboxypeptidase II (GCPII, synonym is prostate specific membrane antigen, PSMA). Highly expressed in the nervous system, GCPII is the key regulator of glutamatedriven neurotransmissions. It targets the abundant brain peptide N-acetylaspartylglutamate (NAAG) and releases the carboxy-terminal glutamate. Since glutamate excitotoxicity is intimately connected to both acute and chronic neuronal disorders, including stroke, amyotrophic lateral sclerosis, traumatic brain injury, Alzheimer’s dementia, and Parkinson’s disease, a massive effort over the last 18 years was undertaken to achieve effective and specific inhibition of GCPII in the brain.28 The efficacy of GCPII inhibitors has been demonstrated in several animal models of neurological disease, thereby supporting their future clinical application. GCPII is strongly expressed in human prostatic tissue, and its expression correlates with disease progression. Given its restricted expression pattern and membrane localization, the enzyme is also an attractive target for antibody-based therapy and prostate cancer imaging.29 Presently, more than 260 entries in PubMed describe the usage of GCPII/PSMA for imaging purposes. Further pathologically relevant carboxypeptidases include carboxypeptidase A4 (CPA4), cytosolic carboxypeptidase 1 (CCP1), and cathepsin A (CathA). These enzymes represent three examples of carboxypeptidases profoundly connected to tumorigenesis and neurodegenerative and cardiovascular diseases. Carboxypeptidase A4 (CPA4) is an extracellular metallocarboxypeptidase belonging to the M14A subfamily. A cluster of four pancreatic and non-digestive human M14A metallocarboxypeptidases is located in a genomic region associated with prostate cancer aggressiveness on chromosome 7.30 Among these, the non-digestive carboxypeptidase A4 has received particular attention since its expression is upregulated by sodium butyrate in androgen-independent prostate cancer cells as well as upon treatment of human prostate cancer cell lines with the histone deacetylase inhibitor trichostatin A.31 While CPA4 is weakly expressed in human adult tissues, high levels of expression were found in fetal tissues as well as in adult benign hypertrophic prostate.32 Furthermore, the singlenucleotide polymorphism rs2171492 on the CPA4 gene, yielding a G303C amino acid substitution, is associated with an increased risk of aggressive disease in younger men.33 The substrate specificity of this enzyme resembles that of other M14A members, with a strong preference for hydrophobic and positively charged amino acids in the P1 position.34 Although CPA4 targets neuropeptides in vitro such as neurotensin, granins, and opioid peptides, the (patho-)physiological substrates remain unknown. Cytosolic carboxypeptidase I (CCP1) belongs to a recently identified subfamily of metallo-carboxypeptidases, i.e., M14D, 4500

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Figure 3. C-TAILS work-flow. To analyze protein C-termini in complex proteomes, −SH, −NH2, and −COOH groups of full-length proteins are chemically protected. After trypsination, internal, but not C-terminal, peptides possess neo-COOH groups and are coupled to the polymer polyallylamine. C-terminal peptides remain uncoupled and are separated by ultrafiltration, followed by LC−MS/MS.55

3. PROTEOMICS-BASED APPROACHES TO STUDY CARBOXYPEPTIDASES AND THEIR SUBSTRATES While the field of carboxypeptidases is gaining significant research interest, technological advancements in mass spectrometry-based proteome analysis have improved throughput and sensitivity. MS-based proteomics is now readily applicable in most proteome and peptidome studies, especially in biomarker discovery, personalized medicine, and clinical validation. When combined with bioinformatics pipelines with comprehensive database mining, accurate qualitative and quantitative analyses can be performed with ease. Accordingly, increasing numbers of proteomics-related carboxypeptidase studies have surfaced in recent years (Figure 2). Classical shotgun proteomics-based studies occasionally lead to the identification of novel carboxypeptidases. This is exemplified by a recent study in two dermatophyte fungi species, Arthroderma benhamiae and Microsporum canis, using Orbitrap MS analyzers46 that highlighted the involvement of novel acidic carboxypeptidases of the S10 family in dermatophyte virulence.46 Mass spectrometry-based proteomics is also used to gain deeper insight into the biology of individual carboxypeptidases. For example, a matrix assisted laser desorption ionization (MALDI tandem time-of-flight (TOF/TOF)) study revealed that the human carboxypeptidase angiotensin-converting enzyme 2 is a substrate of the tumor necrosis factor alpha converting enzyme (TACE).47

targeting protein C-termini together with acetylated N-termini was reported by Dormeyer et al.49 Selective enrichment of these termini is achieved on the basis of their reduced basicity in comparison to that of non-N-acetylated N-termini and internal tryptic peptides.50 Strong cation exchange (SCX) based fractionation separates protein C-termini together with acetylated N-termini from internal tryptic peptides. MS analysis of a crude membrane fraction from a human embryonic carcinoma cell line using combined SCX prefractionation and an Orbitrap analyzer identified 168 IPI annotated and 193 unpredicted C-termini.49 Interestingly, a number of strategies have looked at the implication of carboxypeptidases on the processing of bioactive peptides. Peptidomics is particularly advantageous in studying endogenous peptides that are often not amenable to conventional antibody-based approaches. A peptidomic study led by Villanueva et al. described disease-specific exoproteolytic (including carboxypeptidase activities) processing of serum peptides.51 Serum peptides were extracted using an automated format based on ionic strength and hydrophobicity. Eluted peptides were mixed with matrix directly spotted onto MALDI target.52 Tandem MS-based sequence identification of 61 signature peptides from 106 serum samples showed both cancer-related and cancer-type-specific serum peptides with characteristic N- and C-terminal truncations. These may serve as potential candidates for diagnostic and prognosis biomarkers.51 Peptidomics was also used to characterize putative carboxypeptidase E substrates. Che et al. looked at the changes in peptidomic levels in mice lacking the enzyme upon starvation or increased exercise.53 Comparative profiling of the extracted peptides was performed by isotopic labeling followed by MS analysis using quadrupole TOF (Q-TOF) platform. More than 50 peptides were identified and quantified.53 A subsequent study from the same laboratory investigated differential expression levels of native neuropeptides in mice expressing inactive carboxypeptidase E.54 MS analyses using the LC−MS/MS workflow described above in conjunction with a stable isotope labeling strategy identified and quantified 158 peptides stemming from 32 known neuropeptides precursors.54 In the absence of carboxypeptidase

3.1. Mass Spectrometry-Based Strategies for Proteome-Wide Analysis of Carboxy Termini and Identification of Carboxypeptidase Substrates

In recent years, several proteomic-based approaches have been established to investigate protein C-termini and their proteolytic processing. Sechi and Chait isolated the C-termini of Lys-C digested peptides using agarose-immobilized anhydrotrypsin,48 which captures peptides with a C-terminal lysine or arginine residue. Lys-C digestion yields internal peptides with C-terminal lysine residues. These are bound to the agarose and removed, while native C-termini can be subsequently analyzed by MS.48 Although this is a straightforward approach, the effectiveness is limited to peptides that lack arginine or lysine residues at the C-termini.48 Another earlier study 4501

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E, many of these peptides were found to be upregulated.54 In another study, Tanco et al. looked at the ability of carboxypeptidase A4 to cleave naturally occurring peptides.34 Peptides were first extracted from murine brain, followed by incubation with human recombinant carboxypeptidase A4. The samples were isotopically labeled and analyzed by LC−MS/ MS.34 Substrate specificity analyses identified 16 potential substrates with high preference for cleavage of Leu and Phe at position P1′, whereas 24 other peptides were identified as less efficient substrates with Leu, Phe, Tyr, Val, Ile, Met, Ala, Thr, and His as C-terminal residues.34 In 2010, Schilling et al. introduced a strategy for the enrichment of C-termini, termed C-terminal amine isotope labeling of substrates (C-TAILS)55 (Figure 3). In C-TAILS, the primary amines of proteins are protected by reductive dimethylation using formaldehyde, while all carboxyl groups are protected by carbodiimide-mediated, N-hydroxy succinimide-assisted condensation with ethanolamine.55 These modified proteins are trypsinized, and the resulting tryptic peptides, which contain newly generated N-termini and internal tryptic peptides, are dimethylated and then removed based on their unprotected C-termini by coupling to a linear polyallylamine polymer with high molecular weight.55 Ultrafiltration using spin columns separates the uncoupled, blocked Cterminal peptides from polymer-bound peptides, which are to be used for LC−MS/MS analysis. 55 This approach is particularly useful when combined with quantitative strategies such as isotopic dimethyl labeling. In Escherichia coli, C-TAILS identified hundreds of native or proteolytically generated Ctermini.56 C-TAILS includes chemical tagging of protein Ctermini, which might facilitate the identification of true Ctermini in downstream analysis. Different chemical compounds can be potentially used for the C-terminal protection, including aniline, ammonium, and glycinamide. Combined fractional diagonal chromatrography (COFRADIC) represents another strategy that highlights the use of MSbased isolation of C-termini.57 COFRADIC is used to enrich for C- as well as N-termini based on the strong cation exchange separation of peptides. Proteins are first S-alkylated, and primary amines are blocked by (trideutero-)acetylation.57 Following the modification of proteins, trypsin is used to generate α-amino-blocked N-terminal peptides, α-amino-free internal peptides, and α-amino-free C-terminal peptides that all end with an arginine residue, except for the C-terminal peptides.57 In subsequent steps, digested peptides are separated by strong cation exchange chromatography under acidic conditions.57 Given the low pH, internal peptides are positively charged and thus adhere to the stationary phase, while αamino-blocked N-terminal peptides and α-amino-free-Cterminal peptides, lacking a net positive charge, pass through the column.57 Enriched N- and C-terminal peptides are then separated by reversed-phase high-performance liquid chromatography (RP-HPLC).57 Additional steps were used to isolate C-termini from N-termini. As such, the primary α-amines of Cterminal peptides are butyrylated, leading to increased hydrophobicity and capturing by a second RP-HPLC.57 Following elution, these enriched C-terminal peptides are analyzed by LC−MS/MS. This approach identified 965 database-annotated C-termini, 334 neo-C-termini generated by granzyme B, and 16 neo-C-termini generated by carboxypeptidase A4 in human cell lysates.57 Through the COFRADIC-based approach, Tanco et al. recently profiled substrate specificities of carboxypeptidases.1 Peptide libraries

were generated by the digestion of human cell line derived proteome, using chymotrypsin and Lys-N.1 These peptide libraries were initially prefractionated on a reversed-phase HPLC and were subsequently hydrolyzed by a carboxypeptidase of interest in the presence of 18O-labeled water.1 The resulting neo-C-termini are distinguished from the parental Cterminal counterparts using LC−MS/MS analyses and database searching.1 At present, both C-TAILS and C-terminal COFRADIC approaches identify lower numbers of protein termini when compared to their counterpart strategies that target N-terminal peptides. While the above approaches are particularly advantageous in allowing the identification of protein C-termini on a proteomewide scale, a range of complementary techniques focuses on the C-terminal analysis of less complex protein mixtures. In 1995, Patterson et al. demonstrated the capability of carboxypeptidase-mediated C-terminal ladder sequencing using MALDITOF MS.58 Synthetic peptides are first aliquoted onto a MALDI target, followed by the addition of a carboxypeptidase at varying concentrations.58 Peptide digestion proceeds at room temperature until the liquid mixture on MALDI plate is dry. MALDI matrices (e.g., α-cyano-4-hydroxycinnamic acid) are aliquoted onto each well containing digested peptides. MALDITOF analysis of 22 peptide mimetics of various charges and polarity, cleaved by carboxypeptidase Y (an enzyme that nonspecifically cleaves all residues from the C-terminus), demonstrated significant sequence coverage.58 Samyn et al. highlighted the utilization of chemical cleavage of proteins using cyanogen bromide (CNBr).59 Protein mixtures are first separated on a 1D- or a 2D-PAGE, followed by gel staining. Gel bands containing proteins of interest are excised and destained. In-gel digestion using CNBr is performed, following reduction, alkylation, and subsequent washes to remove excess salt.59 Alternatively, this approach is adaptable to protein mixtures in solution without the use of SDS-/2D-PAGE.59 The cleavage of proteins using CNBr generates internal peptides that have homoserine lactone derivative at the C-termini. When these peptide mixtures are subsequently processed using carboxypeptides, only unmodified and natural occurring C-termini are hydrolyzed, forming peptide ladders with shortened Ctermini.59 This progressive truncation of C-termini can be analyzed by MS. A detailed protocol was described in ref 60. Alternatively, Miyazaki et al. introduced another gel-based, carboxypeptidase-free approach to perform C-terminal sequencing, based on the truncation of proteins using acetic anhydride.61 In addition, Kuyama et al. described an effective method for C-terminal sequencing of proteins, where proteins are first cleaved by Lys-C, generating fragments with two amino groups, each at one end of the peptide.62 However, native Cterminal peptides (that lack a C-terminal basic residue) contain only one α-amino group. Enrichment of C-termini peptide begins with the protection of α-amino groups using succinimidyloxycarbonylmethyl tris (2.4.6-trimethoxyphenyl) phosphonium bromide, followed by separating internal peptides from C-terminal peptides using p-phenyllenediisothiocyanate resin, which binds all internal peptides. The C-terminal fragments can thus be recovered and are subjected to de novo sequencing analysis by MS. Taken together, there exists now a range of proteomic techniques to study protein C-terminal processing by carboxypeptidases. Nevertheless, the majority of degradomic studies have so far focused on amino- and endopeptidases.63 As 4502

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a result, our knowledge on protein C-terminal processing remains comparably limited.

4. CONCLUSIONS Carboxypeptidases are involved in many important biological functions in the eukaryotic system. From digestive enzymes, carboxypeptidases turn into main players in a broad spectrum of patho-physiological conditions. Echoing the findings on carboxypeptidase roles in diseases, the development of inhibitors was undertaken, and some brilliant examples are offered by ACE inhibitors, which decrease mortality by up to one-fifth in cardiovascular patients, and GCPII inhibitors in the fight against neuropathy and neuropathic pain. The overall characterization of carboxypeptidases through proteomicsbased studies is gaining significant research interest. Perhaps the absence of highly sensitive and accurate MS analyzers in previous years would explain the lack of the above-mentioned approaches to be used as widely and as routinely. We expect that more studies, together with innovative approaches, will be brought forward in the future. Generally, little is known about post-translational processing of the C-termini, including modifications such as such as C-termininal amidation of proteins/peptides, as well as proteolytic truncations.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +49 761 203 9615. Fax: +49 761 203 9602. E-mail: [email protected]. Author Contributions §

A.P. and Z.W.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS O.S. is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SCHI 871/2 and SCHI 871/5) and the SFB850 (Project B8), a starting grant from the European Research Council (Programme “Ideas”; call identifier: ERC2011-StG 282111-ProteaSys), and the Excellence Initiative of the German Federal and State Governments (EXC 294, BIOSS). W.L. acknowledges a Marie Curie IIF fellowship (FP7-PEOPLE-2012-IIF).

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