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Review

Cite This: Chem. Rev. 2018, 118, 1137−1168 pubs.acs.org/CR

Proteolytic CleavageMechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational Modification Theo Klein,†,⊥ Ulrich Eckhard,†,§ Antoine Dufour,†,¶ Nestor Solis,† and Christopher M. Overall*,†,‡

† ‡ Sciences Institute, Department of Oral Biological and Medical Sciences, and Department of and Molecular , University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

ABSTRACT: enzymatically hydrolyze bonds in , resulting in a widespread, irreversible posttranslational modification of the ’s structure and biological function. Often regarded as a mere degradative mechanism in destruction of proteins or turnover in maintaining physiological homeostasis, recent research in the field of degradomics has led to the recognition of two main yet unexpected concepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire of proteases are pivotal regulators of most, if not all, physiological and pathological processes. Second, an unexpected in vivo abundance of stable cleaved proteins revealed pervasive, functionally relevant protein processing in normal and diseased tissuefrom 40 to 70% of proteins also occur in vivo as distinct stable proteoforms with undocumented N- or C- termini, meaning these proteoforms are stable functional cleavage products, most with unknown functional implications. In this Review, we discuss the aspects and mechanisms of by different classes. We also provide an overview of biological pathways that utilize specific proteolytic cleavage as a precision control mechanism in protein quality control, stability, localization, and maturation, as well as proteolytic cleavage as a mediator in signaling pathways. Lastly, we provide a comprehensive overview of analytical methods and approaches to study activity and substrates of proteolytic in relevant biological models, both historical and focusing on state of the art techniques in the field of degradomics research.

CONTENTS 4.3.2. C-terminal Proteomics 1156 5. Future Perspectives 1157 1. Introduction 1137 ff Author Information 1158 2. Di erent Classes of Proteases and Proteolytic Corresponding Author 1158 Mechanisms 1138 Present Addresses 1158 2.1. Metallopeptidases 1138 Notes 1158 2.2. Aspartic Peptidases 1140 Biographies 1158 2.3. Glutamate Peptidases 1141 Acknowledgments 1159 2.4. Peptide 1141 References 1159 2.5. Peptidases 1142 2.6. Peptidases 1143 2.7. Peptidases 1144 1. INTRODUCTION 3. Biological Processes Regulated by Proteolytic Cleavage 1145 Regulatory pathways interface to create a system capable of 3.1. Maturation of Translated Proteins 1145 selectively amplifying and integrating signals in a coordinated 3.2. Removal 1145 manner to achieve homeostasis. Defects in one pathway can 3.3. Proprotein Convertases 1145 destabilize the entire system, causing neoplastic, fibrotic, 3.4. Processing by 1146 inflammatory, autoimmune, and immunodeficiency diseases. 3.5. Ectodomain Shedding 1147 The biological function of proteins is regulated by an intricate 3.6. Modulation of Biochemical Pathways and network of posttranslational chemical and enzymatic mod- Signaling through Proteolytic Cleavage 1148 ifications such as , , ubiquitination, 3.7. Precision in Immunity 1150 and proteolysis. Proteolytic cleavage or proteolysis is the 4. Analytical Approaches for Proteases and Proteo- enzymatic of a in a peptide or protein lytic Processing 1151 substrate by a family of specialized enzymes termed proteases.1 4.1. Monitoring Substrate Cleavage 1151 This irreversible posttranslational modification is often 4.2. Substrate- and Activity-Based Probes 1152 4.3. Proteomics-Based Approaches for Substrate Special Issue: Posttranslational Protein Modifications Discovery 1152 4.3.1. N-terminal Proteomics 1153 Received: March 1, 2017 Published: December 21, 2017

© 2017 American Chemical Society 1137 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Figure 1. (a) Active-site view of the metzincin ADAM17 (pdb entry: 1bkc). The side chains of Glu406, His405, His409, His415, and Met435 are shown as stick models (sequence numbering following Uniprot entry P78536). Zn, . (b) Active-site view of the Clostridium tetani gluzincin T (pdb entry: 4ar9). The side chains of Glu466, Glu499, His465, and His469 are shown as stick models (sequence numbering following Uniprot entry Q899Y1). Zn, zinc. (c) Active-site view of E. coli (pdb entry: 2mat). The side chains of Glu204, Glu235, Asp97, Asp108, and His171 are shown as stick models (sequence numbering following Uniprot entry P0AE18). Co, . All figures were created within PyMOL with depicted in cyan, in red, in blue, and in yellow. regarded solely as a destructive mechanism, for example, in the (i) hydrophobic and electrostatic interactions with the substrate degradation of in inflammation and amino residue side chains in commonly well-defined intracellular proteins by the or in , but substrate-binding pockets and (ii) bonding with the recent decades of research in degradomics have highlighted peptide backbone of a substrate in an extended conformation.15 specific, limited precision proteolysis, termed processing, to be Whereas (EC 3.4.21−25) cleave substrates a crucial regulator of protein function in virtually all biological within the protein molecule, (EC 3.4.11−14) pathways.2 Thecompleterepertoireofproteases(the and carboxypeptidases (EC 3.4.15−18) remove up to three degradome) in man comprises 588 peptidases, which can be amino from the N- or C-terminus, respectively, and are grouped in 5 catalytic classes: 21 aspartyl-, 164 cysteine-, 192 jointly referred to as .16 A special case represents metallo-, 184 serine-, and 27 threonine peptidases,3 even the so-called omega peptidase family (EC 3.4.19), which outnumbering the large family (kinome) with its 518 releases terminal amino acids that are either chemically members.4 This means that nearly 3.1% of the 19 587 human modified (e.g., acetylated or formylated), cyclized (e.g., protein encoding genes5,6 encode for proteases, making these pyroglutamate), or linked by an . A more one of the most abundant classes of enzymes and systematic classification follows the of the and promoting them to center stage in a wide range of biological the or metal catalyzing the peptide-bond processes and diseases. cleavage. Thereby seven distinct classes of proteases can be Proteolytic processing is widespread, is pervasive, and identified: the aforementioned and well-established five major generates stable protein fragments as shown by targeted N- classes, the serine- (EC 3.4.21), cysteine- (EC 3.4.22), aspartic- terminal proteomics analysis in numerous biological samples. (EC3.4.23), metallo- (EC 3.4.24), and threonine peptidases Unexpectedly, the majority of identifiable protein N-termini are (EC 3.4.25), and two classes without known representatives in within the protein sequence, i.e., unannotated, neo N-termini : the glutamic peptidases (currently included in EC 77% in ,7 68% in erythrocytes,8 50−60% in normal and 3.4.23) and the asparagine peptide lyases (EC 4.3.2).17 The inflamed murine skin,9 and 78% in human dental pulp.10 The latter were established only in 2011 and represent true outliers interplay of interconnections between proteases and their in the world of proteolytic enzymes as their reaction endogenous inhibitor proteins, termed the protease web, mechanism does not include the addition of a molecule. remains understudied to date, leaving many unanswered Thus, they do not qualify as hydrolases or peptidases and have questions open for investigation.11 to be considered as lyases instead, as they utilize an elimination Although proteolytic degradation is vital for maintaining reaction where an internal asparagine residue first attacks its homeostasis, e.g., in protein turnover through the - own carbonyl carbon bond and cyclizes, followed by a proteasome system,12 this Review will focus on targeted, nucleophilic attack, leading to a succinimide-ring formation specific processing as a near-ubiquitous post-translational and peptide-.18 fi modi cation. Processing can profoundly alter the biological 2.1. Metallopeptidases properties of a protein, its localization, and stability. We discuss the repertoire of proteases, their different hydrolytic mecha- The metalloproteases (EC 3.4.24) represent a highly diverse group of endo- and exopeptidases. They all harness at least one nisms, the biological function of proteolytic cleavage, and 2+ approaches for the analysis of proteases and their substrates. divalent metal ion in their active site, typically Zn , which is coordinated by three amino acid side chains and a water molecule, where the latter plays a key role in substrate 2. DIFFERENT CLASSES OF PROTEASES AND 19,20 hydrolysis (Figure 1a and b). PROTEOLYTIC MECHANISMS The most common metal is , followed by In the absence of a protease, peptide bonds are highly stable at glutamate, aspartate, and . Additionally, an unpaired neutral pH and 25 °C with half- of >100 years as shown in cysteine is found in the propeptides of matrix metal- ancient human remains,13 whereas they are hydrolyzed within loproteinases (MMPs) and the families, which milliseconds in protease-assisted biochemical reactions.14 replaces the active site water in the form, rendering Proteases recognize and bind their substrates mainly through the protease inactive.21 Notably, in some families, two

1138 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Figure 2. Active-site view of the zinc metallo-deubiquitinase AMSH-LP (associated molecule with the SH3 domain of STAM-like protein) in complex with Lys63-linked diubiquitin (pdb code: 2znv; PMID: 18758443). (a) AMSH-LP is shown in semitransparent surface representation (middle), while the distal (left) and proximal (right) ubiquitin are depicted in cartoon rendering. The scissile isopeptide bond between the carboxyl group of the distal ubiquitin’s C-terminus (Gly76) and the ε-amino group of Lys63 of the proximal ubiquitin is positioned in the Zn2+ coordinating active site of AMSH-LP and is depicted as two spheres. Following the nomenclature of Schechter and Berger (PMID: 6035483), Arg72 to Gly76 of the distal ubiquitin and Lys63 and Glu64 of the proximal ubiquitin moiety represent the P5 to P1 and P1′ to P2′ positions of the deubiquitinating (DUB) cleavage site, respectively. Due to the special character of the isopeptide bond, Gln62 and Ile61 form an additional substrate− protease interface that we designate with a double prime symbol (″), and these are consequently designated as P2″ and P3″. (b) Schematic representation of the isopeptide bond of Lys63-linked diubiquitin, highlighting the T-shaped substrate-recognition motif of deubiquitinating enzymes. The sequence Gln62-Lys63-Glu64 forms together with the Lys63-linked C-terminal of the distal ubiquitin a unique interaction surface only found in Lys63-linked polyubiquitin. Similar but different structural arrangements are found in other polyubiquitin chains, rendering DUBs highly specific for individual ubiquitin linkages (PMID: 27012468). All structural figures were generated using PyMOL. cocatalytic metal are found, typically in the case of cobalt tidylinositol) anchors or a C-terminal transmembrane do- − (e.g., E. coli methionine aminopeptidase; Figure 1c) or main.29 31 The well-known and classic MMP substrate is (e.g., E. coli aminopeptidase A) but also sometimes ; MMPs 1, 8, 13, and especially MMP14, also known as for zinc (e.g., mammalian leucyl aminopeptidases). Importantly, membrane-type (MT) 1-MMP, cleave type-I collagen across all unlike serine and cysteine proteases, metallopeptidases do not three α-chains at a single point, namely, Gly775↓Ile776 for the form a covalent acyl−enzyme intermediate during substrate α1(I)-chain and Gly775↓Leu776 for the α2(I)-chain three- hydrolysis. Although irreversible inhibition of metalloprotease quarters of the length from the N-terminus of the ∼1000 amino activity has been described using cobalt-containing Schiff- acid residue-long collagen. However, not all MMPs are capable complexes as small-molecule inhibitors,22 no peptide-based of this cleavage, despite highly similar substrate specificity on covalent inhibitors exist for this class. the peptide level.32 Binding to the hemopexin domain is key to Upon substrate binding, the active-site water is displaced triple activity and scissile-bond access for cleavage.33 toward the general base glutamate whereas its oxygen remains Despite their undisputed role in extracellular matrix remodel- associated with the metal ion. Additionally, the carbonyl of the ing, MMPs are now well-established as signaling enzymes in scissile bond interacts now with the metal ion, leading to a many biological pathways and diseases such as arthritis, cancer, pentacoordinated transition state. The general base glutamate cardiovascular disease, and liver fibrosis.34,35 Of similar great then subtracts a from the water molecule, which interest are the , consisting of the ADAMs (a simultaneously performs a nucleophilic attack on the carbonyl and ; Figure 1a) and ADAMTS bond of the scissile bond, leading to cleavage and a gem-diol (ADAM with a thrombospondin type 1 motif) families. Like intermediate of the substrate. Subsequent protonation of the MMPs, the adamalysins are expressed as multidomain by the now general acid glutamate leads to the extracellular proteins, belong to the metzincin superfamily, release of the C-terminal substrate fragment, whereas an attack and employ a cysteine switch for zymogen inactivation. of a water molecule on the active site zinc is needed to displace Whereas the ADAMTS family comprises secreted proteins the -cleavage fragment.19,20,23 only, most ADAMs possess a C-terminal transmembrane Two major families of the metallopeptidases are distin- domain followed by a short cytoplasmic tail that is crucial for guished by their third zinc-binding ligand.24,25 Whereas intracellular signaling. Next to their emerging role in metzincins (Figure 1a) employ a total of three histidine , inflammation, and migration, ADAMTS residues within a HExxHxxGxxH motif, the gluzincin super- proteases are mostly known for their role in connective tissue family (Figure 1b) harnesses a glutamate further downstream of remodeling by proteolytic processing of, e.g., procollagens, α the central HExxH (HExxHxnE), often on an helix. , and versican. On the contrary, most ADAM Additionally, all metzincins possess a methionine-containing are involved in ectodomain shedding of 1,4- (Met-turn, hence the metzincin family name), which various plasma membrane-tethered growth factors, , forms a “hydrophobic basement” for the catalytic metal ion receptors, and/or adhesion molecules and thus play funda- (Figure 1a). One subgroup of the metzincin family, the MMPs, mental roles in controlling development and homeostasis, comprises 23 members in human. MMPs are encoded as thereby linking them to several pathological states such as − preproproteins with conserved active site features. The cancer, cardiovascular disease, and Alzheimer’s disease.36 38 presence or absence of accessory substrate-binding domains, Other prominent members of the metzincin superfamily are, − known as exosites,26 28 such as the hemopexin domain or the e.g., , and B, and ulilysin,39 now known as fibronectin type-II modules, distinguish individual family LysargiNase;40 the latter is used because of its -mirroring members, some of which also possess GPI (glycosylphospha- specificity in proteomics.40

1139 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

One group of metalloproteases distinguishes itself by their substrates; instead of endo- or exoproteolytical cleavage, the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MNP+) domain proteases (JAMM) recognize the isopeptide bond formed by ubiquitin conjugated to the free ε-amino group of lysine in the protein sequence (Figure 2a and b). These deubiquitinating enzymes (DUBs) comprise a family of close to 100 metallo- and cysteine proteases in Mammalia41 that regulate the removal of ubiquitin from substrates, thereby forming an integral control mechanism in the ubiquitin system.12 Contrary to the mammalian (MMPs), bacterial collagenases such as collagenase G from Hathewaya histolytica (formerly known as Clostridium histolyticum42) belong to the gluzincin superfamily and digest native, triple-helical into a mixture of small under physiological Figure 3. (a) Active-site view of the porcine aspartic peptidase conditions.43 These collagenases cleave in the repeating Pro- (pdb code: 4pep). The side chains of Asp91 and Asp274 are shown as stick models (sequence numbering following Uniprot entry P00791). Hyp-↓-Gly sequence of collagen and select for a small ′ (b) Active-site view of the human aspartic peptidase pepsin in complex hydrophobic residue in P1 , whereas their mammalian with an active-site binding phosphonate inhibitor IVA-VAL-VAL- counterparts favor a large hydrophobic residue such as Leu LEU(P)-(O)PHE-ALA-ALA-OME (pdb code: 1qrp). The side chains 32,44 or Ile. In ColT from Clostridium tetani, the fourth zinc of Asp94 and Asp277 are shown as stick models (sequence numbering ligand, Glu499, lies 34 amino acids downstream of the following Uniprot entry P0DJD7). (c) Active-site view of the porcine H465ExxH motif (Figure 1b), in the center of the peptidase pepsin precursor pepsinogen (pdb code: 2psg) with the inhibitory domain resembling a -like peptidase (TLP) prodomain in place. The side chains of Asp91, Asp274, and Lys51 are 45,46 shown as stick models (sequence numbering following Uniprot entry fold. Like many proteases, thermolysin from Bacillus fi thermoproteolyticus is expressed as preproenzyme, where the P00791). All gures were created within PyMOL with carbon depicted in cyan, oxygen in red, nitrogen in blue, and sulfur in yellow. 200 amino acid prodomain acts as a molecular in the periplasm, leading to autoactivation and subsequent of the mature protease into the .47 The TLP The acidic residue in the second domain serves as the general fold imparts two roughly spherical subdomains that are base to abstract a proton from the active-site water, enabling it separated by a deep substrate-binding cleft where substrates to perform a nucleophilic attack on the carbonyl carbon of the bind from left to right when shown in the standard orientation bound substrate, whereas the first aspartate residue provides via of metallopeptidases.48 The upper subdomain is dominated by its protonated state electrophilic assistance to the carbonyl a five-pleated β-sheet with the active-site facing edge strand, oxygen. The tetrahedral oxyanion intermediate subsequently providing crucial contacts for substrate binding in antiparallel breaks down when the scissile amide bond acquires a proton fashion, whereas the lower C-terminal subdomain is built up from the solvent or through structural rearrangement of the predominantly by α-helices. The catalytic zinc ion is intermediate, releasing the cleavage fragments and regenerating − sandwiched in between by the HExxH-motif of the central the protonated state of the active site aspartate.57 59 helix and the glutamate from the gluzincin helix.49 Notably, a Pepsin represents the most-studied aspartic peptidase to similar five-pleated β-sheet is also found in MMPs and many date. It shows a bilobed, mostly β-sheet structure, with the other metallopeptidases.50 Another highly prominent gluzincin active site located at their interface. Every lobe harbors one of family member is the dipeptidyl angiotensin the aspartate residues of the catalytic dyad within a conserved I-converting enzyme (ACE), an unusual protease having two Asp-Xaa-Gly motif, in which Xaa is typically a serine or catalytic domains in the same protein encoded by the same threonine. Despite low sequence similarity, the high overall . As a central component of the -angiotensin system, suggests that one lobe most likely evolved from the ACE tightly regulates blood pressure by (i) removing a other by gene duplication.53,57 Approximately 45 residues dipeptide from the C-terminus of the decapeptide angiotensin downstream of the first aspartate is located a highly conserved I, generating the vasopressor angiotensin II, and (ii) residue, which separates and defines the S1 and S2′ inactivating the vasodilator by two sequential substrate-binding pockets. The tyrosine is also part of an 11- cleavages at the C-terminus, removing a total of four amino residue β-hairpin loop called flap, which caps the active site and acids.51 encloses substrates and inhibitors in the active groove upon binding. Whereas substrates are readily turned over, active-site 2.2. Aspartic Peptidases inhibitors bind in similar fashion, rendering the enzyme inactive Aspartic peptidases are the main proponents of the carboxyl by tight binding and displacing the active site water (Figure peptidase family, which is characterized by an acidic active site 3b). This structural feature is only present in the N-terminal residue (aspartate or glutamate), a low pH optimum, and an domain and thus constitutes the main asymmetric aspect of the − activated water molecule that attacks the scissile peptide bond overall structure.60 62 in a concerted general acid−base mechanism.52,53 In the The members of the retroviral retropepsin family constitute a majority of known aspartic peptidases, a pair of special case, as retropepsins consist only of one lobe and thus residues act together to bind and activate the catalytic water need to form a homodimer to build a functional catalytic site in molecule (Figure 3a), but in some, other amino acids replace between. Notably, these viral enzymes (e.g., HIV-1 and HIV-2 the second aspartate, e.g., Glu in the E. coli peptidase HybD54,55 protease) are not thought to be ancestral and are most likely or His in the histo- from Plasmodium derived from the N-terminal lobe of a pepsin homologue, as falciparum.56 they harbor the aforementioned active-site capping flap

1140 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review structure. Because of the presence of two flaps, the active-site Scytalidium lignicolum was determined, revealing that its grooves of retropepsins are shortened and accommodate only catalytic dyad consisted of a and a glutamine78 substrate residues from P3 to P3′ instead of P5 to P3′, with a (Figure 4a). Expressed as preproproteins, they are mostly found typical preference for hydrophobic residues around the scissile bond.63,64 Because of the inherent symmetry of the homodimer, the two aspartates cannot play predefined roles during substrate hydrolysis. Instead, they most likely engage in their individual roles only upon substrate binding, which breaks the initial symmetry.65,66 Importantly, many eukaryotic aspartic proteases are secreted as .62,67,68 Pepsinogen, for example, is synthesized in the and secreted into the , where a 44- residue N-terminal propeptide (Ile16-Leu62 in human ) displaces the first ∼10 residues of the mature pepsin sequence (Val63-Ala388) and blocks the active-site cleft with its α two -helices, whereas Lys51 displaces the active-site water Figure 4. (a) Active-site view of Scytalidium lignicolum glutamate (Figure 3c). Additionally, two tyrosine residues, namely, Tyr53 peptidase eqolisin (scytalidopepsin B, pdb code: 1s2b). The side and Tyr137, block the S1 and S1′ binding sites. A special role is chains of Glu190 and Gln107 are shown as stick models (sequence incumbent to the highly conserved Arg29 residue, which binds numbering following Uniprot entry P15369) (b) Active-site view of to Asp73, stabilizing the zymogen conformation and, together eqolisin with a bound N-terminal cleavage fragment Ala-Ile-His (pdb with other ionic interactions, being responsible for the acid- code: 1s2k). The side chains of Glu190 and Gln107 are shown as stick triggered conversion to pepsin in the stomach.69,70 models (sequence numbering following Uniprot entry P15369). All figures were created within PyMOL with carbon depicted in cyan, Other prominent members of the aspartic protease family oxygen in red, nitrogen in blue, and sulfur in yellow. include D and E, the β-secretases BACE1 and -2 (β- site amyloid precursor protein (APP)-cleaving enzyme-1 and γ in pathogenic fungi; several glutamic proteases also have been -2), and -secretase. D is a soluble lysosomal aspartic fi 79 peptidase with a 44-amino-acid long propeptide. After identi ed in and but not yet in . activation, further proteolytic processing yields the mature Because of their distinct fold, which is similar to concanavalin A fi but novel for a protease, eqolisin is insensitive to pepstatin and and active lysosomal protease in human, composed of disul de- 80 linked heavy and chains. Notably, in mice the single-chain several other inhibitors of aspartic proteases. A rather peculiar case of glutamate peptidases are the trimer-forming tail spike form dominates, whereas the bovine form shows equal amounts 68,71 and fiber proteins found in various . These of both variants. Additionally, has two proteins harbor a proteolytically active C-terminal domain asparagine-linked of high-mannose type, (CTD) as well as a domain structure associated with their which are not required for or enzyme activity primary function as, e.g., endosialidases or K5 lyases. The CTD but play an important role in lysosomal targeting of the protein initially acts as an intramolecular chaperone that is indis- via mannose-6-phosphate (M6P) receptors.72 is an pensable for correct folding of the full-length protein and the intracellular, nonlysosomal closely homologous to formation of a functional homotrimer. After assembly, the cathepsin D but mainly found in the . It autocatalytic release of the CTDs by the glutamate peptidase plays a vital role in protein degradation, as well as in the MHC 81,82 73 stabilizes the remaining trimer. class II processing pathway. Cathepsin E is rather The suggested hydrolytic mechanism for glutamic peptidases unusual among the pepsin-like , as it forms a fi is similar to that for their aspartic counterparts. A general base- disul de-bonded homodimer. Notably, monomeric and dimeric activated water molecule (Figure 4a and b), initially bound by cathepsin E share similar biochemical characteristics, but the and the general base glutamate of the catalytic dimerization evokes higher structural stability to alkaline pH dyad, acts as the , whereas the side-chain amide of and temperature changes, allowing the enzyme to thrive at the fi 74,75 the Gln provides electrophilic assistance rst by polarizing the close-to-neutral pH of the endoplasmic reticulum. BACE-1, carbonyl carbon of the scissile peptide bond and second by also known as Memapsin 2 (membrane aspartic protease of the stabilizing the tetrahedral intermediate. A proton transfer from pepsin family 2), is a membrane-bound aspartic protease, the active-site Glu to the leaving-group nitrogen then triggers mostly known for its activity on the APP, releasing the breakdown of the transition state and finalizes the the N-terminal ectodomain and initiating the subsequent proteolytic cleavage, followed by the regeneration of the cleavage of the C-terminal part by the y-secretase complex, active-site glutamate.78,83,84 ultimately forming a 30−51-amino-acid long isoform of the Aβ- 2.4. Asparagine Peptide Lyases peptide involved in the plaque formation in Alzheimer’s disease.68,76 The y-secretase complex itself consists of 5 Another family of proteolytic enzymes, which was initially individual proteins including the multipass thought to belong to the aspartic peptidases, is now known to -1, an aspartyl protease with an intramembranous be not even hydrolases but peptide lyases, as their cleavage active site enabling APP cleavage in its transmembrane mechanism does not involve a water molecule. Instead, an region.77 asparagine residue acts as a nucleophile and attacks its own main-chain carbonyl to create a five-membered succinimide 2.3. Glutamate Peptidases intermediate by intramolecular cyclization. In the right Historically misclassified as aspartic peptidases, glutamate physicochemical environment, this leads to the subsequent peptidases were only described in 2004, when the crystal cleavage of the peptide bond C-terminally of asparagine in an structure of scytalidoglutamic peptidase (eqolisin) from elimination reaction without the addition of a water molecule.

1141 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Furthermore, as every active site functions only once without of the first residue of the C-terminal extein any subsequent substrate turnover, the self-cleaving activity of (transesterification). The intein’s C-terminal asparagine then peptide lyases does not represent a typical enzymatic action attacks its own backbone carbonyl and releases the intein from after all.18,85 the polypeptide, similar to that described above for peptide Probably the most prominent members of the asparagine lyases in general. Finally, an acyl rearrangement of the peptide family are the self-splicing inteins (Figure 5). An bond linking the extein domains occurs to form a normal peptide bond between them. Importantly and name-giving, none of the peptide bonds in this process is broken by hydrolysis, and thus these enzymes have to be referred to as peptide lyases and not proteases.18,85,86 2.5. Serine Peptidases The serine peptidases hydrolyze peptide bonds very differently to the mechanisms described earlier for aspartate, glutamate, and metallopeptidases, as the initial nucleophilic attack is carried out by the side chain of a serine residue and not an activated water molecule, which leads to a covalent acyl− enzyme intermediate during peptide-bond cleavage. Thus, the catalytic mechanism is more similar to the asparagine peptide lyases, but with the important difference that the second tetrahedral intermediate is actually attacked by a water molecule, thus fully qualifying them as true hydrolases and peptidases.87,88 The most common (“classical”) of serine peptidases consists of histidine, aspartate, and serine (Figure 6a), not necessarily in that sequence order, but with highly similar three-dimensional arrangements. Whereas the serine acts as a nucleophile, the aspartate stabilizes the histidine side chain, which in turn acts as a proton donor and general base. It is noteworthy that glutamate and histidine replace the aspartate in bacterial LD-carboxypeptidases and herpes simplex , respectively, and lysine takes the role as general base in the Ser/Lys-dyad of Lon-A peptidase, which lacks the acidic residue. However, the probably most-studied serine peptidases, such as trypsin, , or , all have the classical catalytic triad.89 In chymotrypsin, the catalytic triad is built up by Ser214, His71, and Asp119 (Figure 6a and b). The latter orients His57 and reduces its pKa to enable the histidine to function as a general base in the reaction. The histidine then abstracts a proton from Ser195, which simultaneously performs a Figure 5. Schematic representation of the mechanism of intein- nucleophilic attack on the carbonyl of the substrate scissile mediated . Upon dimerization of N-intein and C-intein bond. A covalent link is formed between the serine side-chain (1) the first residue of the newly formed catalytic core (a serine or oxygen and the substrate, resulting in a tetrahedral intermediate cysteine) facilitates a nucleophilic N−O/S acyl shift (2) followed by (enzyme−acyl complex) and a negative charge on the peptide fi transthioesteri cation mediated by the last residue in the C-extein (3). carbonyl oxygen, which is stabilized by the protease backbone The C-terminal asparagine residue in the intein subsequently self- of Gly193 and Ser195, which form a positively charged cyclizes into a succinimidyl group, cleaving the peptide bond between intein and extein and allowing an S/O−N shift to link both exteins via pocket commonly referred to as an . Subsequent an amide bond (4). protonation of the substrate amide nitrogen by His57, which now acts as a general acid, collapses the complex and results in the release of the C-terminal part of the substrate ( intein is a of ∼135 residues inserted into a reaction), while the N-terminal part remains bound to the polypeptide that is responsible for catalyzing its own excision protease. Next, an activated water molecule, which displaced while simultaneously fusing the two flanking extein domains, the C-terminal fragment in the active site, attacks the similar to the splicing out of RNA from mRNA. To do bond of the acyl−enzyme intermediate (Figure 6c), resulting in so, the last residue of the intein domain must be asparagine, a second oxyanion hole-stabilized tetrahedral intermediate. whereas the first residues of both the intein and C-terminal Collapse of the latter releases the N-terminal substrate fragment extein domain have to be either cysteine, serine, or threonine. from the active site ( reaction) and regenerates the First, the side chain of the N-terminal amino acid of the intein serine side chain. Similar catalytic mechanisms are suggested for − attacks the preceding peptide bond, leading to an amide to the other nonclassical active sites of serine peptidases.87 89 ester (Ser, Thr) or thioester (Cys) rearrangement, i.e., the N- Another prototype of serine peptidase is the endopeptidase terminal extein domain is loaded onto the inteins N-terminal trypsin. It cleaves preferentially after or lysine, whereas side chain. Enabled by the close proximity of the inteins N- and the structurally nearly identical chymotrypsin selects for large C-terminus, the N-extein is subsequently transferred onto the hydrophobic residues. Trypsin is synthesized as preproprotein

1142 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

side chains. As before, additional enzyme engineering is needed to fully convert, e.g., trypsin to .93 So, despite nearly identical three-dimensional structures and substrate binding in highly similar antiparallel β-sheet conformation, these three enzymes show very different substrate preferences, highlighting the structural fine-tuning during protein and the complexity of any structure−function analysis. 2.6. Threonine Peptidases Threonine peptidases exploit an N-terminal threonine as central catalytic residue and nucleophile for peptide-bond hydrolysis, whereas no other amino acids are strictly conserved in the active site.94 Instead, the N-terminal amino group, or a water molecule bound between the α- and the threonine side chain, acts as a general base in the reaction by providing the proton necessary for hydrolysis. Together with peptidases that harness Ser or Cys in the very same N-terminal position, threonine peptidases are colloquially grouped as N-terminal nucleophile (Ntn) hydrolases. Importantly, all Ntn hydrolases have to be activated first to liberate the N-terminal nucleophile, either by simple N-terminal methionine excision as seen, e.g., in bacterial (reviewed in ref 95)orbyan intramolecular autoactivation mechanism observed in the eukaryotic counterpart, probably involving a catalytic Thr/ − Asp/Lys triad.94,96 98 Undeniably, the proteasome (Figure 7a), which is a multisubunit complex comprising four stacked rings each Figure 6. (a) Catalytic triad of human chymotrypsin (pdb code: 4h4f). The side chains of Ser216, His74, and Asp121 are shown as stick models (sequence numbering following Uniprot entry Q99895). (b) Catalytic triad of porcine chymotrypsin (pdb code: 1esa). The side chains of Ser214, His71, and Asp119 are shown as stick models (sequence numbering following Uniprot entry P00772). (c) Acyl- enzyme complex of porcine chymotrypsin (pdb code: 1esa). The side chains of Ser214, His71, and Asp119 are shown as stick models (sequence numbering following Uniprot entry P00772). All figures were created within PyMOL with carbon depicted in cyan, oxygen in red, nitrogen in blue, and sulfur in yellow. including a 15- and 8-amino-acid long signal peptide and propeptide, respectively. Notably, whereas trypsin is rendered inactive yet highly stable at low pH, at its pH optimum of ∼8, Figure 7. (a) Active-site view of the threonine peptidase β7 subunit of binding ensures maximum activity and decreased the human proteasome (pdb code: 5le5). The side chains of Thr44 trypsin autodegradation. The propeptide harbors a conserved and Lys76 are shown as stick models (sequence numbering following recognition motif for the serine peptidase (also Uniprot entry Q99436). (b) Active-site view of the β7-subunit of the human proteasome complexed with inhibitor bortezomib (pdb code: called enterokinase), which cleaves after (Asp)4-Lys and thus unveils the N-terminus of mature trypsin, in which the catalytic 5lf3). The side chains of Thr44, Gly90, and Lys76 are shown as stick triad bridges two β-barrel domains. Whereas the guanidinium models (sequence numbering following Uniprot entry Q99436). All figures were created within PyMOL with carbon depicted in cyan, group of arginine-containing substrates is directly recognized by oxygen in red, nitrogen in blue, and sulfur in yellow. the carboxyl group of Asp189 at the bottom of the S1 binding pocket, the shorter lysine side chains are encompassed via a bridging water molecule, partly explaining the ∼6-fold containing six (in bacteria) or seven (in archaea and preference for arginine over lysine.90 In chymotrypsin-like eukaryotes) subunits as core particle, represents the most peptidases, the equivalent position is taken by serine, but prominent threonine peptidase. Although the subunits of the neither the of Asp to Ser in trypsin nor that from Ser outer rings (α-subunits) share the protein fold with the two to Asp in chymotrypsin fully converts the substrate specificity inner rings (β-subunits), only the latter can act as proteases despite the fact that nearly all other residues of the S1 subsite within the proteasome’s αββα-architecture. Additionally, in are identical. Additional surface loops that are not even part of eukaryotic proteasomes only 6 of the 14 homologues are the extended substrate have to be reciprocally proteolytically active, with always two sites showing chymo- engineered to render the respective enzyme specificity.91,92 tryptic, tryptic, and glutamyl endopeptidase activity, thus In elastase and elastase-like serine peptidases, the S1 cleaving preferentially after large hydrophobic, basic, and acidic substrate pocket represents only a shallow depression, as residues, respectively. On the contrary, archaeal proteasomes glycine residues that build the pocket in chymotrypsin and are composed of 14 identical active β-subunits. In each active trypsin are replaced by larger, hydrophobic residues that fill the subunit, a conserved lysine residue ∼30 amino acids down- pocket and instead provide interaction with small hydrophobic stream of the N-terminal threonine is suggested to assist

1143 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review substrate hydrolysis by decreasing the pKa of the amino terminus through electrostatic interactions (Figure 7a and b). Additionally, a conserved glutamate roughly halfway between the N-terminal threonine and the aforementioned lysine seems to be involved in the catalytic mechanism. After the nucleophilic attack, the main-chain amide of a conserved glycine residue stabilizes the tetrahedral transition state. After breakdown of the intermediate, the C-terminal part of the substrate is released, whereas the N-terminal part remains covalently bound to the threonine side chain as an acyl− enzyme complex. A subsequent nucleophilic attack by a water molecule closes the reaction circle by releasing the N-terminal 99−101 cleavage product and regenerating the free protease. Figure 8. (a) Catalytic triad of the Carica cysteine peptidase Notably, whereas the proteasome is highly conserved among (pdb code: 1ppn). The side chains of Cys158, His292, and eukaryotes, it seems sufficiently diverse to allow specific Asn308 are shown as stick models (sequence numbering following targeting of, e.g., disease-causing protozoan parasites, such as Uniprot entry P00784). (b) Catalytic triad of the Carica papaya Plasmodium, Trypanosoma, or , possibly opening a cysteine peptidase papain complexed with inhibitor E64 (pdb code: new road for, e.g., antimalarial agents.102,103 1pe6). The side chains of Cys158, His292, and Asn308 are shown as stick models (sequence numbering following Uniprot entry P00784). 2.7. Cysteine Peptidases All figures were created within PyMOL with carbon depicted in cyan, Cysteine peptidases are found through all kingdoms of life. oxygen in red, nitrogen in blue, and sulfur in yellow. Although evolutionary highly diverse, cysteine peptidases all share a common catalytic mechanism that involves a ization and deprotonation. It is still under debate whether the nucleophilic attack by a cysteine side chain, typically from a histidine also acts as proton acceptor or if the imidazole is per catalytic triad or dyad, for substrate hydrolysis. As the active site se protonated, as well as if the cysteine becomes ionized thiol group is highly prone to oxidation, cysteine peptidases are only upon substrate binding like in serine peptidases or if most active in a slightly reducing environment and/or at − cysteine proteases are a priori active. slightly acidic pH (4.0 6.5). In mammals, cytosolic Hydrolysis is initiated by the nucleophilic attack of the and lysosomal cathepsins are probably the most well-known activated thiolate anion on the carbonyl carbon of the substrate family members. Nevertheless, the most-studied cysteine scissile bond, resulting in the first tetrahedral transition state peptidase is papain from the tropical melon tree (Carica where the substrate is covalently attached to the cysteine, and papaya), which is also considered as the archetype of cysteine the reaction intermediate is stabilized by the oxyanion hole of proteinases. Papain is synthesized as a nonglycosylated the enzyme. Rotation of the imidazole ring enables a preproprotein of 345 amino acids with an 18- and 115- subsequent proton transfer to the amide nitrogen of the amino-acid long signal peptide and propeptide, respectively. attacked peptide bond, releasing the C-terminal fragment of the The mature protein folds into a globular domain stabilized by cleaved polypeptide, whereas the N-terminal part remains fi three disul de bridges, with two subdomains delimiting a covalently linked via its C-terminus. Next, a water molecule, surface-exposed active-site groove on the top, when shown in polarized by the active-site histidine, attacks the carbonyl standard orientation. Although the left subdomain is comprised carbon of the thioester-bonded acyl−enzyme complex, nearly entirely by the N-terminal part of the enzyme, the N- resulting in the second tetrahedral intermediate, which terminus is located on the distal right side, whereas the right ultimately decomposes and releases the N-terminal portion of − subdomain ends in a strand that extends proximal into the left the substrate, thereby regenerating the peptidase.106 109 domain. The catalytic residues Cys158 (left domain; corre- Lysosomal cysteine cathepsins represent a highly studied sponds to Cys25 in pepsin numbering starting after group of peptidases within the papain superfamily of cysteine prepropeptide removal) and His292 (right domain; His159) proteases. In , there are 11 representatives known, of the catalytic dyad are located on opposite sides of the namely, , C, F, H, K, L, O, S, V, X, and W. interface (Figure 8a and b). contain a number of , , , Several additional residues in papain are known to be and more than 50 different proteases, mainly aspartic, serine, important for substrate turnover but are considered as and cysteine peptidases. In addition to, e.g., the aspartic noncatalytic, as they are not essential for hydrolysis; e.g., protease cathepsin D, the , and the Asn308 (Asn175) resembles the structural equivalent of legumain, all kinds of cysteine cathepsins play − aspartate in the catalytic triad of serine peptidases (Ser/His/ a key role in lysosomes.110 113 One example is cathepsin B, Asp) but with much lower contribution to the catalytic which displays both endopeptidase and carboxydipeptidase efficiency. Mutation to glutamine only leads to a 3.4-fold activities; after initial cleavage within the substrate, the cleaved 104 drop in kcat/KM, whereas mutating the aspartate in the polypeptide chain is degraded in a processive manner by catalytic triad of trypsin results in a 104-fold decrease of removing dipeptides from the substrate C-terminus. Notably, enzymatic activity.105 In the case of papain-like proteinases, the compared to other papain-like enzymes, cathepsin B shows a catalytic center is complemented by the aforementioned rather broad specificity in the P2 position and accepts, e.g., even Asn308, but other residues such as aspartate or glutamate can arginine next to the more commonly found large hydrophobic be found in the equivalent position, especially in bacteria and residues, alleviating its degradative power.111,114 . The main role of the third residue during substrate In contrast, functions as either an endopeptidase hydrolysis is to orient the imidazole ring of the catalytic dyad, or aminopeptidase, depending on the presence of an 8-amino- ultimately allowing the histidine to assist in cysteine polar- acid long minichain originating from the propeptide. Cathepsin

1144 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

H lacking the minichain shows distinct endopeptidase activity. a process named N-terminal methionine excision (NME, However, when the minichain is bound, it occludes the reviewed in refs 125 and 126). and subsequent nonprimed subsides from S2 backward while providing a proper folding of proteins is an imperfect process. Although the negative charge through its C-terminus to bind the substrate N- role of the N-terminal methionine and the functional relevance terminus, abolishing endopeptidase activity and emanating in of its proteolytic excision is not fully understood, current an elevated aminopeptidase activity.115,116 Interestingly, in dogma dictates that these processes control protein stability , a unique 3-amino-acid long miniloop-insertion and half-life in the context of the N-end rule that determines (Ile-Pro-Gln) between the oxyanion hole glutamine and the protein fate.8,127,128 For cytosolic proteins, stability and quality active-site cysteine confers carboxypeptidase activity in a similar control is determined by the N-terminal residue after excision manner. The insertion is preceded by a histidine residue of the initiator methionine. Most residues (primary, secondary, (glycine in papain), which occupies the S2′ subsite and can and tertiary destabilizing residues) trigger ubiquitination and anchor the substrate C-terminus in two different side-chain proteasomal degradation if the protein is incorrectly folded with conformations, depending on carboxymonopeptidase or the N-terminal residue exposed. Acetylated N-terminal residues − carboxydipeptidase activity.117 119 can also destabilize proteins where the N-terminal residue is Overall, lysosomal cysteine cathepsins developed several either M, V, A, S, T, C, P, or G, termed secondary destabilizing modus operandi that reduce the number of substrate-binding residues. Lange et al.8 investigated protein stability to sites from either side and provide an electrostatic anchor for the determine if the neo-N-terminal residue exposed after protease respective substrate terminus, facilitating permanent or cleavage followed the N-end rule. While following the N-end temporary activity. Adjuvant to their special- rule, post translational neo-N-terminal unexpectedly ization as exo- or endopeptidases, localization and pH stabilized cleaved cytosolic proteins with primary destabilizing dependency are other unique features of individual cysteine residues (R, K, L, H, F, Y, W, and I). Thus, this differed from cathepsins; e.g., cathepsins B, C, H, L, and O are ubiquitously the destabilizing role for acetylated N-termini having secondary expressed in nearly all tissues and cells, whereas cathepsins S, K, destabilizing residues.8 V, F, X, and W show restricted organ distribution, suggesting For proteins moving through the ER, a quality-control housekeeping or specific functions, respectively. Additionally, system termed ER (endoplasmic reticulum)-associated protein cathepsin H is irreversibly inactivated above pH 7.0; its inactive degradation (ERAD) is in check. Misfolded proteins are precursor remains stable, and even shows activity recognized by the ERAD machinery, likely due to interactions and actually has its pH optimum somewhere between pH 6.0 of specific chaperones with exposed hydrophobic patches, and 7.5. incomplete disulfide bridges, and improper glycosylation, Importantly, like many other proteases, cysteine cathepsins leading to lysine-48 polyubiquitination of the misfolded protein are expressed with N-terminal signal-peptide and propeptide by ubiquitin E3 such as carboxy terminus of - sequences responsible for protein sorting into specific cellular interacting protein (CHIP), expulsion of the protein from the compartments or extracellular space and enzyme inhibition, ER lumen, and subsequent cytosolic proteolytic degradation of − respectively. In the inactive zymogen form, the prodomain the misfolded protein by the proteasome.129 131 fl forms a compact minidomain, where a stretched and exible 3.2. Signal Peptide Removal segment covers and tightly interacts with the active site cleft in backward orientation, occupying several substrate-binding Localization and transport of proteins in the secretory pathway pockets while also preventing hydrolysis. Activation occurs in is regulated by intrinsic sequences within the N-terminal region acidic milieu, at pH 5.0 or below, where the prodomain of these proteins termed signal peptides; short sequences are switches from the closed conformation favored at neutral or recognized during translation of the nascent protein by the signal-recognition particle in the ER that mediates translocation slightly acidic pH to an open conformation due to ionic 132 repulsion. The physical displacement yields a low-active into the ER lumen, allowing subsequent secretion of the ffi proteins via vesicular transport mechanisms. Alternatively, this cathepsin, which is su cient to activate another zymogen in 133 trans, thus triggering an autocatalytic activation cascade.109,120 process can occur by posttranslational processes. During Other prominent cysteine peptidases that are beyond the scope translocation, signal peptide sequences are cleaved from the of this Review are -1 and -2,121 the various ,122 preprotein by a family of serine proteases named signal the lysosomal protease legumain,123 and the peptidases (SPase), e.g., type-1 SPase in bacteria and the ER mucosa-associated lymphoid tissue translocation complex (SPC) in eukaryotes for proper gene 1 (MALT1),124 just to mention a few. localization (reviewed in ref 134). This process is further controlled by proteolytic cleavage of the signal peptide remnant 3. BIOLOGICAL PROCESSES REGULATED BY by a family of intramembrane cleaving aspartyl proteases called signal peptide peptidases (SPPs135) by regulated intramem- PROTEOLYTIC CLEAVAGE brane proteolysis (RIP, reviewed in ref 136). 3.1. Maturation of Translated Proteins Proteins targeted for the contain a variant signal peptide named peptide (unfortu- Various proteolytic cleavage events function as a control nately also designated as MMP), an 8−58-amino-acid long mechanism during translation and localization of proteins in the sequence rich in positively charged amino acids that targets the cell. In eukaryotic protein synthesis, mRNA generally starts preprotein to the mitochondrion and is subsequently cleaved with an AUG codon, and therefore the translated protein by the zinc metalloprotease mitochondrial processing peptidase sequence will almost exclusively start with a methionine (MPP).137 residue. Depending on the protein and the , this initiator methionine either can remain on the new protein or, in 3.3. Proprotein Convertases a large fraction of newly translated proteins, be rapidly removed Many proteins such as proteolytic enzymes, growth factors, and though proteolytic cleavage by methionine aminopeptidases in peptide are expressed and translated as inactive

1145 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review precursor forms that require targeted, specific proteolytic processing of pro-opiomelanocortin (POMC) into adrenocor- cleavage events during their transition through the secretory ticotropic (ACTH) or alpha--stimulating pathway to become biologically active. In humans, this process hormone (αMSH).162 Furthermore, PC2 processes a wide is regulated by a family of seven (PC) variety of neuroendocrine precursor proteins such as enzymes (, PC1, -2, -4, -5, and -7, and paired basic amino , prosomatostatin, neurotensin, neuromedin N, acid cleaving enzyme 4 (PACE4)) that hydrolyze peptide prodynorphin, and nociceptin.163 PC1 knockout results in bonds at paired basic amino acid residues in substrate precursor severe growth defects that are likely caused by disrupted proteins, the subtilisin 1 (SKI-1) that cleaves processing of growth factor-1 and growth hormone- after nonbasic residues, and proprotein convertase subtilisin/ releasing hormone (GHRH).138 kexin 9 (PCSK9) that is only known to cleave itself in an PC4 is selectively expressed in germ cells in testes, ovaries, autocatalytic activation step.138,139 Depending on the substrate and placenta and plays an important role in maintaining fertility and its activating PC, cleavage and activation can occur at any and in reproduction, likely through cleavage of pituitary location in the secretory pathway from the trans-Golgi network, adenylate cyclase-activating polypeptide (PACAP) in the testes endosomes, and cell surface and, in the case of activation of and the activation of various ADAM proteases.138,164,165 SKI-1 polypeptide hormones, in secretory granules. PCs cleaves or S1P is a widely expressed protease with notably different precursor proteins by limited proteolysis within a sequence cleavage-motif preference than the other members of the PC ↓ ′ motif [R/K]-(X)0,2,4,6-[R/K] P 1, with a strong preference for family because it hydrolyzes substrates after any amino acid arginine in the P1 position.140 Because the cleavage-site other than cysteine and .166,167 SKI-1 is an activator of specificity of most PCs is so similar, and there is considerable membrane-bound factors in cholesterol metabo- redundancy in substrate cleavage in vitro, localization is likely lism and homeostasis ( regulatory element-binding the main determinant of substrate specificity.141,142 protein (SREBP)-1 and -2)168 and activating transcription In 1990, furin or PCSK3 was the first identified mammalian factor 6 (ATF6) in the endoplasmic reticulum (ER) stress PC as a homologue of the convertase kexin.143 Furin is a response.169 Besides this role in transcription-factor processing, membrane-bound protease that is subject to endosomal SKI-1 cleaves and activates viral proteins such as Lassa virus trafficking between the cell surface and the trans-Golgi network, glycoprotein, making the enzyme a potential target for antiviral where it processes numerous protein precursors by cleavage at therapeutics.138 dibasic motifs such as BXBB or BBXB (where B is a basic 3.4. Processing by Carboxypeptidases amino acid residue).141 Furin has been reported to be secreted into the extracellular space.144 Interestingly, some potent Proteases are divided into their ability to cleave proteins from human pathogens have evolved to contain furin-cleavage sites within a sequence (endoproteases) or the ends of a polypeptide in their virulence factors; for example, lethal factor and sequence (exopeptidases). Exopeptidases can be further avian influenza virus hemeagglutinin rely on host furin for classified if they cleave protein chains at their N terminus activation.141,145,146 Proteolytic cleavage by furin is essential to (aminopeptidases) or if they cleave from the C terminus 1 lifefurinknockoutmicearenotviableduetosevere (carboxypeptidases). In addition, peptidases and proteases can cardiovascular defects during embryogenesis that are likely a cleave single or two (or more) amino acids from the end of a result of insufficient activation of transforming growth factor protein or peptide such as in the case of peptidyl beta (TGFβ).147,148 Processing of TGFβ1 by furin is also and oligoendopeptidases, but this section will focus on single required in adaptive immunity; T cells lacking furin produce amino acid hydrolysis in the case strictly for carboxypeptidases. less mature TGFβ1 and are dysfunctional.149 Furin and PACE4 Peptidases and proteases can be classified by their mechanisms are capable of activation of multiple membrane-associated of peptide hydrolysis, namely, if the active-site residues essential metalloproteases in the metzincin superfamily such as MMPs, for hydrolysis are serine, threonine, cysteine, aspartate, and MT-MMPs and ADAMs, by selective proteolysis of motifs glutamate or if there is the necessity of a metal ion bound to the within the prodomain, leading to exposure of the catalytic cleft enzyme in the case of metalloproteases.170 The two largest − and accessibility for substrates.150 152 Cellular adhesion is groups for carboxypeptidases are serine carboxypeptidases and regulated by furin and furin-like (PC5) PCs through the metallocarboxypeptidases.171,172 Of these two groups, the activation of pro- that are required for interaction of metallocarboxypeptidase family has more members and is the cell with the extracellular matrix, and furin activity is high better characterized biochemically and structurally than serine during situations of tissue remodeling, such as in atherosclerotic carboxypeptidases. lesions.153 There is considerable apparent redundancy between Carboxypeptidases can recognize specific C-terminal residues furin and related PCs such as PC5, PC7, and PACE4, although that can be recognized by substrate-binding pockets and some substrates appear to be preferentially cleaved by one over subsequently cleaved. Serine carboxypeptidases often have pH the other enzymes.138 optima in the acidic range as opposed to most serine proteases PC1 and -2 are, as indicated by their original names pituitary and metallocarboxypeptidases, which are active in neutral to or prohormone convertase 1 and 2, respectively, important mildly basic ranges.172 While carboxypeptidases were originally activators of endocrine and neural polypeptide hor- discovered in the context of protein as in the case of − mones,154 157 which are localized in granules in hormone- bovine , the role of carboxypeptidases has producing cells and activate their substrates in the acidic now been shown to be more exquisite. There are 34 regulated secretory pathway.158,159 There is considerable carboxypeptidases in humans, which highlights the importance overlap in substrate specificity considering knockout mice of their biological role.173 show defects but are viable, and in unison these two proteases Metallocarboxypeptidases perform peptide hydrolysis regulate vital processes, such as blood sugar homeostasis through acid−base mechanisms mediated by a metal ion with through cleavage of pro-insulin and glucagon,160,161 and a solvent molecule to attack the bond.49 In modulate many hormonal signaling events through site-specific contrast, serine carboxypeptidases were originally characterized

1146 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review to be serine proteases due to being inhibited by diisopropyl- range of 5.0−5.5, which renders it inactive in the Golgi body fluorophosphate (DPF)172 and later confirmed by structural where it is synthesized, preventing it from acting on other studies to attack the scissile bond with a catalytic triad (Ser-His- cellular proteins.1,177 It is proposed that this pH range avails Asp).1 Metallocarboxypeptidases are structurally split into two activity in secretory vesicles, which are families that have been extensively reviewed by Gomis-Rüth:171 packed with peptide hormones that require processing.178 cowrins and funnelins. The cowrin subset of metallocarbox- Many peptide hormones require C-terminal trimming after ypeptidases is characterized by having a long and narrow endoprotease processing as well as in many cases peptide groove across the length of the protease with the active site amidation to render the hormone biologically active.179 A halfway through the groove. Such a groove allows for the widely known substrate of carboxypeptidase E is insulin,180 a insertion of unfolded sequences that can be key regulator of glucose intake in humans, which when cleaved at the last residue catalyzed by the metal ion (typically dysregulated causes . Insulin is synthesized as a prepro zinc). Cowrins have a consensus structural motif involved in form and upon endoprotease processing (prohormone catalysis, HExxH+ExxS/G+H+Y/R+Y, and their catalytic convertases 1 and 2) generates two pairs of C-terminal basic domains are 500−700 residues in size. Funnelins differ from residues: Lys-64/Arg-65 and Arg-31/Arg-32.181 These basic cowrins structurally in that funnelins have a shallow active cleft residues are substrates of carboxypeptidase E and when at the bottom of a funnel-like cavity and have the potential to removed result in a mature molecule of insulin. A mutation hydrolyze folded proteins. Funnelins also possess a consensus on Ser-202 to proline on carboxypeptidase E abrogates the motif important for their catalysis: HxxE+R+NR+H+Y+E with catalytic activity of the enzyme (also resulting in carboxypepti- a catalytic domain ∼300 residues large. Examples of cowrins dase E being degraded)182 and results in severe impairment of and funnelins are and carboxypeptidase A, maturation of insulin (10−50% of correct processing compared respectively. to wild-type), which in turn manifests as and diabetic As mentioned above, the role of carboxypeptidases was pathologies in mice models.1,183 It is clear that carboxypepti- initially described in the context of bovine pancreatic dases have profound biological roles across a variety of different carboxypeptidase A, one of the most studied funnelin-type , with functions that appear concerned with digestion carboxypeptidases and the first metalloprotease described of and precise maturation of peptide hormones. 174 (utilizing a zinc ion for catalysis). Carboxypeptidase A has 3.5. Ectodomain Shedding selectivity toward C-terminal residues that are aliphatic or aromatic (hence why it was termed carboxypeptidase Afor Proteins involved in intercellular signaling and interaction aromatic and aliphatic). was also described between cells are expressed and translated as membrane- in its role for digestive biology for its specificity toward basic anchored proteoforms that are sensitive to proteolytic cleavage residues at the C-termini of polypeptide chains. It is important by an array of mainly membrane-bound proteolytic enzymes. to note that carboxypeptidases A and B preferably act upon Such ectodomain shedding modulates the function of the peptides as opposed to large protein structures. Thus, they substrate protein, by activating precursor forms of, for example, necessitate the action of other endoproteases that can cleave cytokines and growth factors, directly and indirectly inhibiting proteins to generate smaller fragments with C-terminal residues signaling by release of membrane-bound receptors into the that are recognizable by the carboxypeptidase and release the extracellular space (negating signaling directly and also C-terminal amino acid, which then can be absorbed by the functioning as soluble dominant negative proteins indirectly), organism in the case of digestive enzymes.1 Trypsin is the most or by affecting cell motility and binding by cleavage of proteins notorious and well-studied endoprotease that acts concertedly involved in cell−cell and cell−matrix interactions. Following with carboxypeptidase B.1 Trypsin can activate procarbox- cleavage, the cell-associated usually C-terminal protein remnant ypeptidase B into its active form by releasing its propeptide,175 can be internalized by or further processing by allowing secreted carboxypeptidase B to act upon extracellular intramembrane proteases such as presenilin-1 in the γ-secretase proteins (ingested in the digestive system) as opposed to complex,184 leading to either degradation or intracellular intracellular proteins, where they can cause cellular damage if signaling events185,186 (Figure 9). not controlled. Carboxypeptidase B can later cleave C-terminal Although, technically, most extracellular and membrane- on tryptic peptides. Lysine is an essential amino acid in associated proteolytic enzymes could mediate ectodomain humans (namely, it cannot be synthesized by metabolic shedding and especially soluble187 and membrane-anchored processes), and so the activity of carboxypeptidase B is MMPs188,189 have been described to do so, the specialized essential for life. This role is mirrored by the activity of proteases that this function is most attributed to are ADAMs. carboxypeptidase A on C-terminal aliphatic amino acids that are As discussed above, the ADAMs190,191 are multidomain, important for nutrition in concerted action with pepsin and transmembrane (Figure 9) that are closely chymotrypsin endoprotease activity.1 related to the reprolysins. These binding In addition to carboxypeptidases A and B, which have been proteins were discovered as fertilization factors when it was described in great detail in the literature, carboxypeptidases reported that binding and fusion of guinea pig ovum and sperm have also evolved to have hormone-activating functions were partially mediated by PH-20 or -30 (fertilin α and − which consists of trimming small peptide sequences into β),192 195 proteins that were renamed ADAM1 and -2 after the biologically active forms. Of distinction is carboxypeptidase E, a discovery and cloning of more related proteins. Besides metallocarboxypeptidase that has specificity toward C-terminal mediating cell−cell and cell−matrix interaction through basic residues, much like carboxypeptidase B.176 Unlike selective integrin binding via their disintegrin domains carboxypeptidase B, however, the distribution of carboxypepti- (reviewed in ref 196), about half of mammalian ADAMs dase E is in tissues where many and peptide (12/22 in human) contain a consensus HExxHxxGxxH zinc- hormones are produced such as the brain and .176 binding motif in their metalloprotease domain, implicating Furthermore, the activity of carboxypeptidase E is in the pH them as potential active endopeptidases (reviewed in ref 197).

1147 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Ectodomain shedding of growth factors involved in epidermal growth factor (EGFR) signaling is largely attributed to ADAMs. ADAM12, -10, and -17 process TGFα and -binding EGF (HB-EGF) factor to their soluble forms, leading to activation of EGF receptor signaling.185,213 Whereas physiological shedding of growth factors is required in processes such as , tissue repair, and angio- genesis, dysregulation of the shedding and subsequent aberrant EGFR activation can lead to unwanted tissue remodeling, such as in fibrosis,214 and uncontrolled cell proliferation and has therefore been extensively implicated in the pathogenesis of cancer.215,216 Figure 9. Schematic representation of ectodomain shedding mediated Several ADAMs can mediate cleavage and shedding of by ADAM proteases. Proteolytic cleavage mediated though the active amyloid precursor protein (APP) and may play a role in the ADAM metalloprotease domain in the membrane-bound substrate ’ proximal to the membrane releases the extracellular fragment, leading pathogenesis of Alzheimer s disease. APP can be processed by to activation of membrane-anchored proproteins, abrogated inter- three families of proteases with different biological outcomes: 217 action of the ADAM-expressing cell with neighboring cells or the α-secretases such as ADAM9, -10, and -17 and meprin β; β- extracellular matrix (ECM), or regulated intramembrane proteolysis of secretases BACE1 and -2; and in the γ-secretase the C-terminal remnant. C, C-terminal domain; TM, transmembrane complex.218,219 β-Secretase-mediated release of soluble APP domain; EGF, epidermal growth factor-like domain; CYS, cysteine-rich and subsequent regulated intramembrane processing of the domain; DIS, disintegrin domain; MP, metalloprotease domain. remnant by the γ-secretase complex yields an amyloidogenic Aβ fragment that is implicated in the formation of amyloid plaques causing Alzheimer’s disease, whereas shedding of APP by As with other metzincins such as MMPs and ADAMTS’s, ADAMs is mediated by a proteolytic cleavage within the Aβ ADAMs can target and degrade extracellular matrix proteins region itself and yields less toxic fragments.219 This protective such as collagens, , and fibronectin,198,199 but this role of a clan of metalloproteases illustrates an very real function is secondary to their ability to cleave membrane-bound dilemma in pharmaceutical intervention targeting proteases; proteins. many proteases have either protective or detrimental roles in The “sheddase” activity of ADAM proteases was first different diseases, making rational use of inhibitors challenging demonstrated when a disintegrin metalloprotease was described and open to side effects.220,221 as the main responsible protease for the release of active mature Besides cleaving APP, ADAM10 is known to be the tumor factor alpha (TNFα) from its membrane- responsible sheddase for multiple other substrates in the anchored precursor form.200,201 This process had already been central , implicating this protease as an attributed to metalloprotease activity earlier, although thought important regulator of neural development. ADAM10 was to be due to MMPs.202,203 The discovery of ADAM17 or TNFα found to be the mammalian homologue to the Drosophila converting enzyme (TACE) was the gateway to a multitude of protein kuzbanian (KUZ), which cleaves and activates the studies investigating ADAM proteases and their substrates in Notch receptor, mediates release of the Notch receptor ligand health and disease, with ADAM17 receiving the most academic delta, and has a crucial function in neurological development in interest, perhaps due to its potential therapeutic potential in the fruit fly.222 In mice, knockout of Adam10 leads to lethality inflammatory diseases. The last two decades of research have in an early embryonic stage due to major defects in neurological yielded dozens of putative substrates such as cytokines and development that bear a striking resemblance to Notch 204,205 growth factors, their receptors, and adhesion molecules, knockout models.223,224 Interestingly, Notch signaling itself although for many their physiological relevance remains unclear may act as a positive feedback loop via transcriptional activation as many have been discovered via in vitro experiments and of furin, which in turn leads to enhanced activation of ADAM10 significant redundancy appears to exist for substrate shedding (and others) by PC cleavage of the inhibitory prodomain.225 between individual ADAMs. Because academic efforts in this area have frequently focused on ADAM proteases play a pivotal role in regulation of select elucidating the role and substrates of ADAM10 and -17, some signaling, as exemplified by the well-documented other catalytically active ADAMs remain understudied to date, release of TNFα by ADAM17. Although multiple ADAMs (e.g., and further research is required to understand the intricate role α 206 -9, -10, and -19) are capable of processing proTNF in vitro, of ADAM-mediated shedding in health and disease. ADAM17 is the dominant factor in determining TNF response 3.6. Modulation of Biochemical Pathways and Signaling upon stimulation, as shown by a lack of soluble TNFα through Proteolytic Cleavage production by stimulation of ADAM17-deficient monocytes,200 whereas other ADAMs, like the phylogenetically most closely Targeted precision proteolysis is key in the initiation and related ADAM10, may regulate constitutive production of regulation of many biochemical processes and pathways, e.g., soluble TNFα.207 ADAM sheddase activity is involved in the well-documented limited-cleavage events in the coagulation interleukin-6 (IL6) signaling through release of membrane- cascade226 (Figure 10) and complement activation cascade227 bound IL6 receptor (IL6R) that is mediated by ADAM10 and (Figure 11), but also has important roles in many other − -17.208 210 Proteolytic release of the soluble IL6R, as opposed biological processes. to shedding of TNF superfamily receptors, is not inhibitory but Proteolysis by the family of cysteine endopeptidases rather an important agonistic mechanism in pro-inflammatory is a major regulator of cell death, through either activation of IL6 trans-signaling by binding to the membrane protein gp130 the immunologically silent apoptosis or necroptosis (reviewed on neighboring cells.211,212 in refs 228 and 229). Apoptosis can be initiated via two distinct

1148 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Figure 10. Schematic representation of the coagulation cascade where a series of sequential proteolytic cleavages of serine protease zymogens activates them, allowing the cascade to progress to the next tier. The intrinsic pathway is activated upon exposure of blood to negatively charged exposed surfaces and depends on proteolytic activation of Figure 11. Schematic overview of the complement activation cascade, factors XII, XI, and IX. The extrinsic pathway is activated when which is dependent on a series of sequential activations of, and by, vascular tissue damage leads to activation of factor VII. The pathways serine endopeptidases. Activation can occur via three distinct converge at proteolytic activation of the endopeptidase (also pathways: in the classical pathway via formation of a C1 complex known as Stuart-Prower factor), which is capable of converting upon −antigen ligation, in the mannose-binding lectin (MBL) prothrombin into its active enzyme form that subsequently activates pathway upon formation of an MBL-mannose-binding lectin factor XIII and forms a positive feedback loop into the intrinsic associated serine protease (MASP) complex, or in the alternative pathway. also cleaves and activates fibrinogen, allowing pathway by direct activation of factor C3 via an activating surface. All activated factor XIII to form a cross-linked polyfibrin clot. pathways ultimately lead to the formation of a membrane attach complex (MAC) that leads to lysis of the compromised cell and pathways: the intrinsic pathway is activated upon cellular stress removal of the pathogen. such as excessive formation or DNA damage, and the extrinsic pathway relies on activation of death receptor complex formation upon ligation of the appropriate receptor with, e.g., Fas or TNFα. In the intrinsic pathway, domain of a truncated form of c-FLIP (cellular FLICE-like proapoptotic effectors in the Bcl2 family permeabilize the inhibitory protein) upon ligation of the T cell receptor with an mitochondrial outer membrane, leading to release of antigen does not cause apoptosis yet is required for T cell proapoptotic factors such as and second survival and proliferation.231 mitochondrial activator of caspases (SMAC, or direct IAP- Caspases have a strong to even exclusive preference for an binding protein with low pI (DIABLO)), which subsequently aspartate residue in the P1 position yet seem to rely on other enable formation of a large complex that leads to additional features for substrate specificity as shown by limited dimerization and proteolytic activation of caspase-9. Caspase-9 proteolysis of substrates in vitro.232 Several large-scale profiling activates the effector proteases caspase-3, -6, and -7 that cleave a studies of substrates of caspase-mediated proteolysis during − large repertoire of substrates, leading to cell death. Under induced apoptosis have revealed a plethora of substrates,233 235 normal conditions, death receptors such as TNF-R are present but the question remains which of these cleavage events are in large signalosomes with antiapoptotic factors such as the critical in initiation and sustenance of the pathway and which inhibitors of apoptosis (IAP1 and -2) and linear ubiquitin are mere victims of bystander proteolysis. Interestingly, a very chains polymerized by linear ubiquitin assembly complex in-depth analysis of the apoptosis interactome and its (LUBAC) on RIP1 that keep apoptosis in check.230 In the relationship with proteolytic disassembly revealed that caspase absence of antiapoptotic mediators in the signalosome, ligation cleavage of substrates in early apoptosis occurs after with a death-inducing ligand leads to homodimerization and interactome disassembly and is not the cause of breakup of proteolytic activation of caspase-8 that in turn activates effector essential protein/protein interactions.233 This was unexpected caspases. Caspase-8 also cleaves Bcl-2 interacting domain as the dogma prevailing in the field has been that caspase (BID), leading to a cross-talk into the intrinsic pathway by activity disassembles protein complexes, leading to termination causing release of cytochrome c from mitochondria. Signaling of cellular functions. Thus, it appears that protein/protein through receptor pathways that can cause apoptosis is a interactions may mask caspase-cleavage sites or sterically complex biological system, since depending on cofactors and preclude caspase access and that caspase cleavage occurs stimuli many different outcomes are possible. Ironically, partial upon isolated proteins following dynamic protein exchange or activation of caspase-8 by dimerization with the caspase-like complex remodeling.

1149 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

3.7. Precision Proteolysis in Immunity deficient B cells from a human patient, we identified a specific Proteolytic activity is key in the regulation and execution of cleavage by MALT1 in ranBP-type and C3HC4-type zinc finger-containing protein 1 (RBCK1), better known as - several pathways in innate and adaptive immunity. Serine 257 proteases contained in secretory vesicles in immune cells, such oxidized IRP2 ubiquitin 1 (HOIL1). This cleavage was fi 258,259 as in cytotoxic or elastase in later con rmed by others. HOIL1 associates with , are involved in the to viral HOIL1-interacting protein (HOIP) and Shank-associated RH (reviewed in ref 236) and bacterial pathogens (reviewed in ref domain-interacting protein (SHARPIN) to form the linear 237). Although the exact details remain under debate,238 the ubiquitin chain assembly complex that mediates formation of a fi special head-to-tail, linear polyubiquitin linkage that has consensus emerging is that, upon speci c recognition of a viral 260−264 antigen presented through the major histocompatibility important roles in the . Upon cleavage complex on the surface of an infected cell by T cell receptors by MALT1, the C-terminal domain of HOIL-1 dissociates from of cytotoxic CD8+ T cells or activation of NK cells through LUBAC, which abrogates the formation of linear polyubiquitin, κ signals from infected cells, secretory vesicles containing thus terminating NF- B activation, and desensitizes cells from 257 fi proteases and the protein are mobilized reactivation for at least 2 h. This showed for the rst time and secreted into the extracellular space. This leads to that MALT1 proteolytic activity acts as a late negative regulator generation of perforin pores in the membrane of the infected of NF-κB at later time points, illustrating the complex and cell large enough to enable translocation of secreted granzyme pleiotropic nature of this unusual protease in immune cell into the of the target cell.239,240 Internalized granzyme activation and again highlighting the difficulties in drug B (GrzB) subsequently activates classical apoptosis by a specific development from incompletely validated drug targets. activating proteolytic cleavage of procaspase 8, which initiates MMPs are a class of protease that has revealed unpredicted the apoptotic cascade.241 GrzB further performs a specific and unexpected roles that span beyond their well-recognized cleavage in the protein BID, thereby directly activating matrix remodeling functions. As their names suggest, the first mitochondrial apoptosis.242,243 This seemingly elegant system efforts to identify substrates of MMPs were strictly focused on 265 does not tell the full story though, and knockout experiments extracellular matrix proteins such as the collagens. Recently, have demonstrated that other members of the granzyme family MMPs have been demonstrated to be immune regulators by such as GrzA and GrzM are involved in the process.244,245 The modifying, through the activation or inactivation of chemo- exact importance of each protease and their substrates may be kines, cytokines, cytokine-binding proteins, signaling pathways, 266−268 dependent on thus far unknown factors, is likely variable protease inhibitors, and cell-surface receptors. between species, and will require more study to elucidate the MMP2 was one of the first examples of an MMP modulating full mechanism. the function of a chemokine through the proteolytic removal of Activation of B and T lymphocytes after ligation of their the first four N-terminal amino acids (1QPVG4) of monocyte surface-expressed antigen receptor with the corresponding chemoattractant protein (MCP) 3 or CCL7 (chemokine (C−C antigen leads to a rapid mounting of a potent adaptive immune motif) ligand 7); this inactivation of MCP3 resulted in an response. After activation, a series of specific phosphorylation antagonistic effect for CC-receptors, leading to a loss of calcium 269 and ubiquitination events leads to activation of the transcription signaling and chemotactic activity in vivo. Similarly, the first factor nuclear factor kappa-B (NF-κB) and subsequent four amino acids (1KPVS4) of another chemokine, stromal cell- transcription and translation of a multitude of proteins involved derived factor 1 (SDF-1α and β/CXCL12), were cleaved by in inflammation, differentiation, and prolifera- several MMPs (MMP1, -2, -3, -9, -13, and -14), resulting in a − tion.246 248 One of the signaling nodes transducing the antigen loss of signaling by its cognate receptor CXCR (C-X-C signal down to NF-κB activation consists of a trimeric complex chemokine receptor type)-4.270 In fact, this cleavage switched of caspase recruitment domain family-11 (CARD11), B-cell receptor binding to the related CXCR3, again highlighting the lymphoma/leukemia-10 (BCL10), and MALT1, termed the importance of precision proteolysis as a potent posttranslational CBM.249 MALT1, the only paracaspase in humans, is a cysteine modification that profoundly regulates protein function.271 It is protease homologue of the found in yeast and now known that MMPs cleave most if not all human and plants124 that, in addition to the scaffolding function within the murine CXC and CC chemokines, thereby affecting virtually all fi aspects of leukocyte migration, chemotaxis, activation or CBM complex, cleaves a very speci c repertoire of substrates in − lymphocytes that regulates the NF-κB response after antigen inactivation, and signaling.272 274 receptor ligation (reviewed in ref 250). Although MALT1 Cytokines such as the interferons are critical during antiviral contains a consensus active site, it took until 2008 for the first immunity.275 During coxsackievirus type B3 or respiratory substrates to be discovered, when MALT1 was shown to cleave syncytial virus , metalloelastase BCL10 after Arg228 in the C-terminal region, a cleavage that is (MMP12) processes interferon-alpha (IFN-α)attheC- important during T cell activation although not required for terminus at two distinct sites (157leu↓Gln158 and 161Leu↓ NF-κB activation upon T cell receptor ligation.251 At the same Arg162) that are critical for binding IFN-α receptor-2 and time, a publication showed the negative regulator of NF-κB mediating downstream signaling.276 MMP12 was also demon- called A20 to be cleaved by MALT1 upon T cell activation, strated to have moonlighting roles by binding to the NFKBIA indicating MALT1 proteolytic activity enhances or fine-tunes (NF-kappa-B inhibitor alpha) promoter (encoding for inhibitor NF-κB activation in lymphocytes by removing negative of NF-kappa-B alpha or IκBα) to activate IκBα in order to checkpoints.252 Since these discoveries, MALT1 has been mediate the secretion of IFN-α from viral-infected cells.276 shown to cleave a handful of other negative regulators of Importantly, the intracellular and extracellular regulation of canonical NF-κB activation in activated lymphocytes such as IFN-α is specific as MMP12 is unable to cleave other type I the deubiquitinases A20252 and CYLD,253 as well as RelB,254 interferon cytokines such as IFN-β or bind its promoter.276 In Regnase-1,255 and Roquin-1 and -2.256 Recently, using TAILS, addition to being implicated in antiviral immunity, MMP12 was an unbiased N-terminomics proteomics analysis of MALT1- demonstrated to play a protective role in peritonitis and

1150 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review by the inactivation of several complement 4.1. Monitoring Substrate Cleavage 277 components: C3, C3b, iC3b, C3a, C5a, C4, and C4b. The Perhaps the most straightforward approach to analysis of activation of C3 to C3b leads to the covalent binding and 278 proteolytic activity is in direct in vitro monitoring of hydrolysis opsonization of target cell surfaces, resulting in cell lysis. The of (putative) substrates by the protease of interest. Cleavage fl cleavage by MMP12 reduces the proin ammatory activation can be visualized by or , effect of complement, and C3b-tagged cells no longer induce fl 277 and synthetic quenched uorogenic peptide probes based on cell lysis. Also, MMP12 generates more potent fluorescence resonance energy transfer (FRET) or peptides enhancers through the processing of C3b and iC3b. Other C3 labeled with gold nanoparticles that emit a specific signal only or C5 cleavage products include C3a or C5a anaphylatoxins after hydrolysis of the peptide are now used for basic 277,279 serving to reduce their chemoattractant activities and are characterization of in vitro cleavage site specificity of the − implicated in the augmentation of vascular permeability. protease283 286 and are reviewed in ref 287. Several approaches Notably, MMP12 cleavage of C3a (740Arg↓Ala741) and C5a using extended synthetic peptide libraries combined with (746Asp↓Met747) results in the reduction of calcium flux, leading sequencing or mass spectrometric analysis and − to a reduction of neutrophil recruitment and macrophage analysis of the cleavage sites have been described.288 291 277 infiltration. Proteomic identification of protease cleavage sites (PICS)292,293 The protease-activated receptors (PARs) are an important utilizes peptide libraries generated though controlled proteol- class of -coupled receptors and have been implicated ysis of endogenous bacterial or human proteomes with in cancer and disorders of the cardiovascular, musculoskeletal, enzymes like trypsin, GluC, chymotrypsin, or LysargiNase. gastrointestinal, respiratory, and central nervous system.280 After chemical blocking of reactive thiol and amine groups, the PAR1 is required to enhance growth and invasion of breast peptide pool is incubated with the protease of interest, where carcinoma cells in a murine xenograft model, and MMP1, hydrolysis of the peptides reveals a free α-amine group on the secreted from proximal fibroblasts or platelets, is an agonist of C-terminal peptide remnant that is amendable to labeling and PAR1 by inducing Ca2+ signaling, Rho-GTP/MAPK (rho- enrichment. After LC-MS/MS (liquid chromatography-tandem guanosine triphosphate/mitogen-activated 1) mass spectrometry) identification of the enriched peptide pathways, and cell migration through the cleavage of a unique remnants, the nonprime peptide sequence can be identified tethered ligand sequence (41PRSFLLRN47).281,282 Therefore, bioinformatically, leading to in-depth profiling of the cleavage MMPs not only regulate the remodeling of the matrix by site specificity of the studied proteases.32,292,294 cleaving extracellular matrix (ECM) proteins but also influence Zymography is the umbrella term for methods visualizing (activation or inactivation) diverse immune responses through protein substrate conversion by proteases (reviewed in ref 295). the targeted processing of multiple chemokines, cytokines, and Zymography yields qualitative and, if performed within the cell-surface receptors. limited linear range of the assay, also quantitative information These specific examples highlight clearly the importance of about protease and proteolytic cleavage, and visualization can precision proteolysis in regulating signaling and other bioactive be performed in SDS-PAGE ( proteins as a critical posttranslational modification. We point polyacrylamide gel electrophoresis) gels on tissue sections out again that proteolysis is one of the few posttranslational and in intact organisms. Gel zymography was originally modifications regulated by cells after proteins are secreted from performed by submitting samples containing the putative cells, and thus it is critical to have knowledge of the N- and C- protease of interest to electrophoretic separation based on termini of proteins in order to correctly formulate hypotheses molecular weight using SDS-PAGE, followed by (partial) regarding the function of a protein and the system. reactivating of the protease by removal of SDS by nonionic detergents and overlay of the gel with an indicator gel containing a cleavable substrate (e.g., fibrin-agar for visual- 4. ANALYTICAL APPROACHES FOR PROTEASES AND 296 PROTEOLYTIC PROCESSING ization of active ). Improved methods directly incorporated a protein substrate such as in the SDS- Uncontrolled proteolytic cleavage has strong detrimental PAGE gel matrix, where (MMP2 and -9) activity physiological effects, and therefore protease activity is subject leads to clear bands on a bright gel background after Coomassie to multilayered posttranslational regulation. Proteases are often staining, yielding exquisite sensitivity and information about expressed and translated as inactive zymogen proforms that different catalytically active proteoforms in the sample, require an activation step before being able to process provided proteolysis leads to sufficient cleavage of the substrate substrates, either by allosteric or proteolytic removal of an to small enough peptides to allow diffusion from the gel.297 inhibitory prodomain or by other posttranslational modifica- Thus, one or two cleavages only will not be detected. Although tions such as phosphorylation or ubiquitination. Furthermore, several improvements and variations on the basic premise have colocalization of a protease and its substrate can be dependent been published, such as using in-gel zymography for testing on specific stimuli, and an arsenal of endogenous protease inhibitory incorporated in the gel, 2-dimensional inhibitors keeps proteolysis in check. This strong regulation of electrophoresis for enhanced resolution, and transfer zymog- proteolytic cleavage poses a challenge for analysis of proteases raphy for monitoring multiple substrates from the same gel and their targets because mere quantification of expression (reviewed extensively in ref 295), the main limitation of this levels or protein abundance does not necessarily reflect the technique lies in the fact that little quantitative and only limited biological impact of the protease. To overcome this short- qualitative information about the proteolytic potential in vivo is coming of traditional transcriptomic and proteomic techniques provided; noncovalent complexes of proteases and their in studying proteolysis, the development of targeted and more endogenous inhibitors (e.g., and tissue inhibitors functional analysis methods termed degradomics has been of metalloproteases (TIMPs)) are dissociated during electro- instrumental in enhancing our understanding of proteolytic phoresis so the zymogram will inevitably overestimate the cleavages in health and disease.2 proteolytic activity present. Finally, novices are often confused

1151 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review by the molecular weights of the protease forms in zymograms. As the analyses are performed and analyzed under nonreductive conditions, the presence of any disulfide bonds in the protease will cause the protease to electrophorese with a lower apparent molecular weight than that if reduced. Finally, protease prodomain electrophoretic bands will show activity on gels if SDS activates the enzyme or if the allosteric pressure exerted by the refolding procedure leads to protease autoactivation. However, this occurs in situ in the gel and so, despite a loss of the propeptide in gel, the proform is also revealed as an active protease, although it must be considered as an inactive Figure 12. Schematic overview of the activity-based probe (ABP) zymogen form. approach for selective analysis of protease activity and active proteases. 4.2. Substrate- and Activity-Based Probes A proteinaceous sample of interest (lysate, living cells, and tissue) is incubated with an ABP that consists of a peptide-like linker region Although in vitro cleavage monitoring of natural and synthetic specific for the protease (class) of interest, an electrophilic reactive substrates provides valuable kinetic information, visualization of group (warhead) for covalent linkage of the probe to the active protease activity under physiological conditions is required to protease, and a reporter moiety for visualization (e.g., a fluorophore) infer and validate biology. To selectively analyze active or affinity enrichment (e.g., biotin). In the case of serine and cysteine proteases, several research groups have dedicated their efforts peptidases, cleavage of the probe by the active protease leads to covalent derivatization of the catalytic residue with the probe, allowing to synthesize and utilize selective, substrate-based probes fi ff activity-speci c analysis after gel electrophoresis, or pull-down of the (SBPs) and activity-based probes (ABPs) for di erent protease labeled protein and analysis by mass spectrometry. classes (reviewed in refs 15, 298, and 299). SBPs are used in in situ and in vivo zymography to selectively visualize proteolytic diseases and cancer, where aberrant proteolytic activity cleavage in tissue sections or living intact small using − contributes to the pathology.303 305 either directly fluorescent or the more popular and sensitive Designing ABPs for protease classes that are not dependent internally quenched fluorescent probes that give a positive or on active-site nucleophile amino acids for substrate hydrolysis negative fluorescent signal upon cleavage of the probe by the 300,301 (metalloproteases and aspartyl proteases) has proven more protease of interest. Although SBPs are powerful tools to challenging. Covalent labeling is achieved by incorporating a study proteolytic cleavage in tissue and even in vivo, photoactivatable cross-linker group into the probe that forms a redundancy between enzymes, which is observed in many reactive species and covalently attaches to catalytic-site protease classes, makes unambiguous interpretation of the residues upon irradiation of the sample with UV light. Several ffi acquired data di cult. To overcome this issue, ABPs have been groups have employed this approach to profiling of metzincin developed that irreversibly modify the active protease and allow proteases such as MMPs and ADAMs, but low labeling yields fi postlabeling identi cation of the actual enzyme involved, e.g., and nonspecific labeling seem to have made this approach − by mass spectrometry. largely redundant.135,306 308 The use of nanomolar noncovalent Commonly, ABPs contain a substrate-like protease inhibitors affords one venue to visualize such proteases backbone sequence optimized for recognition by the protease that lack a covalent acyl intermediate in catalysis, but these can (class) of interest, an “warhead” that mediates a only be used under native conditions, e.g., in vivo imaging, and nucleophilic attack upon cleavage of the peptide backbone, not denaturing SDS-PAGE gels. The lower amounts of protein which leads to covalent labeling of the active site, and a reporter that can therefore be labeled necessitates highly sensitive group that can be a fluorophore for visualization of a group for detection systems, and this has driven the development of affinity enrichment of the labeled protease such as biotin or via noncovalent probes for positron emission tomography of click chemistry302 (Figure 12). Visualization of labeled protease metalloproteases using the potent, low-nanomolar inhibitor using SDS-PAGE, fluorescence microscopy, mass spectrometry, Marimastat as the label-accepting probe.309,310 and in vivo imaging provides qualitative and quantitative A variant method for enrichment of active proteases from information about the activity status that was present in vivo complex samples also utilizes reversible inhibitors containing under the experimental conditions, making these probes peptidomimetic recognition sequences for selectivity that are extremely powerful tools to study proteolysis. immobilized on a solid support or resin and can be applied as The ABP approach has been used extensively and an affinity matrix to selectively enrich proteases in their active, successfully to analyze activity of cysteine-, serine-, and noninhibited form. This method has been used to purify active threonine proteases. Because catalysis is mediated by an forms of collagenases using a weak metalloprotease inhibitor 311 amino acid residue in the catalytic site that functions as a with micromolar potency (Pro-Leu-Gly-hydroxamate), and nucleophile, probes containing a directly reactive electrophile optimization of the inhibitor backbone structure has yielded ffi label the catalytic residue in a true activity-dependent manner. a nity materials that have been used to enrich and analyze MMPs and ADAMs with limited success in biological Many chemical structures have been used as electrophile in 312−317 both irreversible inhibitors of these protease classes and ABPs, samples. e.g., activated ketones, phosphonylating and sulfonylating 4.3. Proteomics-Based Approaches for Substrate Discovery groups, , and Michael acceptors (reviewed in ref As the function of a protease is determined by the substrates it 298). Besides obviously valuable application in fundamental cleaves, obtaining a comprehensive picture of these substrates characterization of activity of a specific protease, a promising under specific conditions is required to interpret the biological application of ABPs lies in in vivo labeling strategies for in situ significance of proteolysis. Proteomics provides a toolkit for visualization of proteolytic activity in, e.g., inflammatory unbiased qualitative and quantitative profiling of proteins and

1152 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review proteoforms present in a biological sample, but typically these are minor and specific chemical labeling of the N-terminal proteolytic cleavages are elusive to regular “shotgun” α-amine without side reaction on ε-amino groups on lysine proteomics experiments. As with proteases themselves, mere residues in the protein is far from trivial, although several quantification of the level of a substrate in a sample provides groups have attempted this but with low fidelity (Figure 13). limited information; stable cleavage fragments will still be present, and the probability of identifying cleavage sites is One approach for positive selection after labeling of N- statistically unlikely. To overcome this problem, methods terminal utilizes a subtiligase enzyme to selectively label selectively analyzing cleaved substrates have emerged and broadened our knowledge on substrates of proteolysis (reviewed in refs 318−320). Initial efforts used the additional information on molecular weight of proteoforms in the samples from the once-popular method of 2-dimensional gel electrophoresis, which separates proteins based on isoelectric point and size,321 followed by excision of spots of interest, in-gel digestion with trypsin, and mass spectrometric identification using either MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) or LC- (ESI)-MS/MS.322 In a modified protocol termed 2-dimensional difference gel electrophoresis (2D-DIGE), protein samples corresponding to different experimental conditions (e.g., protease-treated and control) are differentially labeled with fluorescent dyes, combined, and resolved on a 2D polyacrylamide gel.323 Cleaved substrates are visualized as decreasing intensity of colored spots correspond- ing to the full-length proteins in the protease-treated sample and concurrent appearance of lower-molecular-weight frag- ments. Both spots can be excised and identified by mass spectrometry. The emergence of more powerful and faster mass spectrometers enabled in-depth coverage of proteins in excised slices of 1D gel electrophoresis of unfractionated samples, which made it possible to avoid the finicky 2D electrophoresis and laborious picking of (many) individual spots. Originally pioneered in 2002 by Guo et al.,324 this technique was greatly enhanced by developing easily accessible software and other technical advances leading to the development of PROTOMAP (protein topography and migration analysis platform). As before, this is a method that is based on standard SDS-PAGE resolution of protein samples harvested from cells where Figure 13. Schematic overview of degradomics approaches using proteolytic activity occurred or was induced (e.g., apoptotic fi positive selection of N-terminal peptides. A proteome sample is Jurkat cells) followed by LC-MS/MS identi cation and acquired after activation of the protease of interest (e.g., upon quantification by spectral counting of proteins extracted from induction of apoptosis). The sample consists of full-length, uncleaved 325,326 multiple gel slices. By bioinformatically combining mass proteins and fragments resulting from proteolysis containing neo-N- spectrometric identity information with molecular-size in- terminal protein ends. Positive selection of N-terminal peptides (neo- formation obtained from the electrophoresis data, putative and natural) can be achieved by enzymatic selective labeling of the N- substrates can be identified. Although this method gives an terminal α-amine, attaching a tobacco etch virus (TEV)-linked biotin group (method I). After digestion of the proteins with trypsin and indication of the approximate position in the protein sequence ffi fi where the cleavage occurred, the limited resolution of mass in biotin-(strept-)avidin a nity puri cation, the N-terminal peptides are collected by TEV protease cleavage and are amendable to mass gel electrophoresis combined with the low probability of spectrometric identification, allowing exact identification of the identifying the tryptic peptide of the cleavage site makes proteolytic cleavage sites on a large scale. A chemical approach for unambiguous identification of the exact point of cleavage ff α positive selection utilizes the di erence in pKa between N-terminal - unlikely. amines and lysine side-chain ε-amines. By performing a guanidination 4.3.1. N-terminal Proteomics. Selective analysis of at high pH, selective labeling of ε-amines is achieved (method II). This cleaved protein substrates and cleavage sites is the most allows for chemical derivatization with biotin of the N-terminal α- optimal scenario to elucidate the comprehensive degradome of amines with standard N-hydroxysuccinimide chemistry, followed by a certain protease, but this approach faces similar challenges as enrichment of the N-terminal peptides using (strept-)avidin and mass fi methods for analysis of other posttranslational modifications spectrometric identi cation of proteolytic cleavage sites. Method III uses N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) as a (PTMs) such as phosphorylation and ubiquitination, which α fi fi reagent for selective labeling of the N-terminal -amines, followed by require speci c enrichment of modi ed peptides or proteins for tryptic digestion and sample fractionation by reversed-phase successful profiling. At first glance, labeling of cleaved proteins α chromatography to enrich for TMPP-labeled peptides. A further at their N-terminal -amino group followed by positive enrichment of N-terminal peptides is achieved by affinity enrichment selection of the labeled fraction seems like an attractive option, using an α-TMPP antibody, allowing subsequent mass spectrometric but, although differences in acid-dissociation constant exist, identification of cleavage sites.

1153 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

α-amino groups in a proteome sample with a biotinylated short peptide sequence containing a tobacco etch virus (TEV) protease cleavage site. After tryptic digestion of the sample, labeled peptides are enriched using avidin affinity extraction, and peptides are eluted from the avidin matrix by cleavage with TEV protease, followed by LC-MS/MS identification of the labeled peptides from which putative substrates can be 234,327 ff inferred. Another approach uses the di erence in pKa between α- and ε-amino groups to selectively block lysine side- chain amines by guanidination in the presence of o- methylthiourea at high pH, followed by labeling of free N- terminal amines with a disulfide-containing biotinylation reagent, extraction of the biotinylated peptides with immobi- lized avidin, and elution of the peptides under reductive conditions.328 In a comparable approach using selective labeling of N-terminal α-amines at high pH with N-tris(2,4,6- trimethoxyphenyl)phosphonium acetyl (TMPP), N-terminal peptides can be enriched using an antibody that recognizes TMPP-labeled peptides that can be extracted with magnetic beads prior to mass spectrometric analysis.329,330 Because of the technical difficulties of specifically labeling α- amines and the inherent lower high-accuracy coverage obtained, the majority of methods for targeted analysis of protein N-termini now rely on the opposite principal: depletion of tryptic, internal peptides to effectively lead to negative Figure 14. Schematic overview of the most common biologically enrichment of N-terminal peptides. Although several methods fi have been published in the past decade, the basic premise is occurring chemical modi cation of the N-terminus (a) and C-terminus (b) of proteoforms. always to chemically block all primary amines in the sample of interest at the protein level, either by N-hydroxysuccinimide (NHS)-activated reagents or reductive amination (also known both the protease-containing and control samples as a high- as reductive alkylation) with an aldehyde using cyanoborohy- ratio peptide. dride as a catalyst.331 By labeling at the whole-protein level by Approaches that have been used to deplete the sample of definition then, any N-terminal labeled peptide had to have internal peptides include immobilization of free amine containing peptides with functionalized been present and exposed in the sample prior to labeling. If the 332 sample was handled with appropriate care using protease polystyrene beads or aldehyde-functionalized POROS beads using reductive amination,333 biotinylation of free α- inhibitors and taking precautions to avoid proteolysis on amines with NHS biotin and depletion with avidin beads,334,335 sample preparation, then such labeled N-termini on peptides and phospho-tagging (PTAG) internal peptides via reductive represent either mature protein N-termini or protease- amination with glyceraldehyde-3-phosphate, followed by generated neo-N-termini. After incubation with trypsin to chromatographic separation of phosphorylated internal pep- digest the proteins into LC-MS amendable peptides, the newly tides from the N-termini of interest using a titanium dioxide generated internal (i.e., nonterminal), tryptic peptides that are affinity column.336 However, an inherent problem with bead- not the N-terminome are removed from the sample, leading to based approaches is nonspecific binding of peptides to these negative enrichment of the blocked N-termini. media, leading to false positives or saturation of MS analyses. One major advantage of this approach over the positive- Although many methods have been published and have been selection methods lies not so much in the discovery of cleaved shown to function with varying degrees of reliability and substrates but in that it allows for comprehensive analysis of the validation, two distinct workflows have become more widely N-terminome in the sample, including naturally occurring N- used than othersterminal amine isotopic labeling of fi terminal modi cations such as , acetylation, and substrates (TAILS337,338), combined fractional diagonal fatty acid derivatization (Figure 14) that also have important 339,340 179 chromatography (COFRADIC ), and the variant charge- biological implications on protein function. The chemical based fractional diagonal chromatography (ChaFRA- − blocking step provides an ideal opportunity for isotopic DIC341 343)(Figure 15). COFRADIC relies on chemical ff di erential labeling and multiplexing of samples (e.g., modification of the hydrophobicity of internal peptides and protease-treated versus control, or different patients, or removal by reversed-phase chromatography, leading to negative different stimulation conditions) by using stable isotope-labeled enrichment of N-terminal peptides. All amines are initially reagents such as 13C- or deuterium-labeled or blocked by acetylation, followed by digestion of the sample formaldehyde, or isobaric proteomics tags such as iTRAQ with trypsin. The sample is subsequently prefractionated by (isobaric tags for relative and absolute quantitation) or TMTs strong cation-exchange (SCX) chromatography, which neg- (tandem mass tags). Another advantage is that the natural N- atively enriches for the acetylated N-termini (both natural as terminal peptides can be used to statistically determine the chemically modified) due to their low affinity for the SCX variation within and between experiments and hence used to material caused by the difference in dissociation constant statistically discriminate the neo-N-termini derived from the compared to unblocked, internal peptides. The enriched protease of interest from background proteolysis present in peptides are fractionated further by C18 chromatography, and

1154 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Figure 15. Schematic overview of the most commonly used degradomics approaches using negative selection of N-terminal peptides. Proteome samples under different conditions (e.g., control cells versus protease-deficient cells, or protease-overexpressing cells) are acquired that contain uncleaved, intact proteins and cleavage fragments resulting from proteolytic activity. All primary amines in the proteins are blocked by dimethylation or acetylation, using isotopically labeled reagents to differentially label proteins from different conditions, or in TAILS by isobaric mass spectrometry reagents such as iTRAQ or TMT. After combining the samples and digestion with trypsin, TAILS (left column) uses a soluble hyperbranched

1155 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Figure 15. continued polyglycerol polymer conjugated with aldehyde groups (HPG-ALD) for depletion of internal tryptic peptides that bind via their unblocked α-amino groups by reductive amination. The polymer and bound internal peptides are subsequently removed via molecular weight cutofffilter centrifugation, allowing for negative enrichment of blocked N-terminal peptides and mass spectrometric analysis. The isotopic or isobaric tagging of the individual samples allows for relative quantification of abundance of the N-terminal peptide, enabling the analysis of more subtle differences in proteolytic cleavage compared to only “on/off” approaches. COFRADIC (center column) relies on a series of chromatographic fractionations and chromatographic depletion of internal (i.e., unlabeled) tryptic peptides after derivatization with a hydrophobic reagent (TNBS). The extensive fractionation yields samples with a high enrichment for N-terminal peptides but with very large fraction numbers to be analyzed by mass spectrometry. ChaFRADIC (right column) is a COFRADIC variant that relies on the charge shift of nonterminal, tryptic peptides upon chemical acetylation that allows removal by SCX chromatography and negative enrichment of N-terminal peptides. each fraction is labeled with 2,4,6-trinitrobenzenesulfonic acid relies on the digestion of entire lysates with a site-specific (TNBS), which increases the hydrophobicity of the remaining enzyme, commonly trypsin, and then fractionating the resulting internal peptides, allowing their efficient removal through peptides prior to injection into the mass spectrometer and reversed-phase fractionation. In general, COFRADIC leads to subsequent identification. While there are significant advantages high numbers of samples due to the extensive sequential to fractionation at the peptide level and the identification of fractionation steps. A recent variant of COFRADIC termed those peptides, there is loss of protein context.350 Isoforms and ChaFRADIC simplifies the approach and massively reduces the proteolytically generated protein fragments are usually lost in amount of sample required for analysis to the low-microgram the vast numbers of internal tryptic peptides due to the fact that range. ChaFRADIC utilizes an optimized SCX fractionation to the peptides that can differentiate isoforms or protein fragments separate peptide fractions based on their charge state, followed are fewer in abundance and often have less amenable by acetylation of internal, tryptic peptides, which reduces the ionization/fragmentation properties. Trypsin is commonly charge state. During a second SCX fractionation on each of the used due to its strict substrate specificity, which generates C- original samples, nonterminal peptides will display a shifted terminal and lysines, both basic residues. The retention profile, and by only selecting the fraction that did not presence of these amino acids confers tryptic peptides excellent exhibit charge shift, enrichment of blocked N-terminal peptides fragmentation patterns, as it will preferentially generate stable y- is achieved.342 ions as opposed to lesser stable b-ions when using collision- On the other hand, TAILS is much simpler and faster and induced dissociation (CID).351 Peptides derived from C- circumvents the high peptide losses inherent to chromatog- terminal regions of proteins are not always fully tryptic. Over raphy-based approaches by the development of a unique ∼100 80% of proteins annotated in the human proteome (Swiss- kDa aldehyde-functionalized soluble hyperbranched polyglycer- Prot) do not have a C-terminal basic residue, making them ol polymer (HPG-ALD) that is commercially available (http:// semitryptic if digested with trypsin. Consequently, identifica- flintbox.com/public/project/1948). TAILS relies on blocking tion of C-terminal peptides is a particular challenge as C- all primary amines at the protein level using either reductive terminal peptides, while perhaps being the most specific amination with isotopically labeled formaldehyde or labeling sequences to a protein,352 (a) are in significantly less abundance with isobaric NHS tags such as iTRAQ or TMT. After mixing than internally generated fully tryptic peptides and (b) have of differentially labeled samples and digestion of the proteins impaired fragmentation properties relative to fully tryptic with trypsin, GluC, or LysargiNase, the internal tryptic peptides peptides. To overcome the second issue listed here, new are depleted by binding to HPG-ALD via reductive amination. enzymes have been introduced to proteomic workflows in The polymer with bound internal peptides can be conveniently order to identify C-terminal peptides. Namely, the proteases removed by filtration through a molecular-weight cutofffilter, Lys-N353 and LysargiNase40 are now available to digest leading to enriched N-termini (generally >95%) in an proteomes and enhance C-terminal peptide identification. unfractionated sample that can be analyzed by LC-MS/MS. A LysargiNase was demonstrated to improve the identification bioinformatics suite containing CLIPPER344 and tools of C-terminal peptides in a human breast cancer model. As TopFIND345 and TopFINDer346 is available for further analysis opposed to trypsin, Lys-N and LysargiNase both generate a of the obtained data. TAILS has been successfully employed to positively charged N-terminus, which generates b- and a-ion study the proteolytic landscape in several cell-based models and ladders, which greatly assist in peptide identification by to date remains the only N-terminomics approach reported to database search algorithms.351 Despite the advances provided be capable of in vivo tissue N-terminomics, including inflamed by Lys-N and lysargiNase as proteome-digestion alternatives, skin9 and arthritic ankle joints,277 antiviral responses,276 a the caveat of abundant internal peptides still remains, and model,347 and lymphocytes from an immune- similarly to other post-translationally modified peptides, C- deficient patient.257 One of the major mysteries in regards to terminal peptides require enrichment for in-depth profiling. proteolytic cleavage is the extent and genesis of neo-N-termini As opposed to other post-translational modifications where a in any given tissue.348 N-terminal proteomics studies regularly specific chemical group is added at functional sites on peptides find that the majority of protein termini are the result of and proteins, proteolytic processing does not have a single nonannotated proteolytic cleavage, and so questions arise chemical group that allows for affinity purification. While there including which proteases are responsible for this and what are are methods to enrich N-terminal peptides, the chemistry for the biological implications of these cleavages.179,349 isolating C-terminal peptides is not as straightforward. 4.3.2. C-terminal Proteomics. Detection of the natural C COFRADIC, discussed earlier, can be adapted for the isolation terminus of a protein or the proteolytically processed end of a of C-terminal peptides using a combination of metabolic protein is not a trivial matter when employing mass- labeling, acetylation, oxidation, and strong cation exchange.340 spectrometric approaches. Conventional shotgun proteomics For cell cultures and biological systems that are not amenable

1156 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review to metabolic labeling, an alternate method can be utilizedC- changes resulted in a total 481 C-terminal peptides from 369 TAILS. Alternative C-terminomic strategies can also be found proteins in E. coli. described in the review by Tanco and colleagues.173 Contrasting to the inclusion of singly charged species in the Schilling and colleagues presented a negative selection mass spectrometer, Somasundaram and colleagues performed strategy to study C-terminal peptides as a modified method “charge-reversal derivatization” to convert C-terminal peptides of N-terminal amine isotopic labeling of substrates (N- into species that behave more like fully tryptic peptides.357 TAILS).354,355 The original TAILS method reported utilizes a Utilizing similar principles to reduce water availability during water-soluble polymer to deplete internal peptides using EDC coupling, Somasundaram and colleagues used the basic reductive alkylation chemistry following amine blocking at the amines N,N-dimethylethylenediamine and (4-aminobutyl)- protein level. C-TAILS follows a similar principle. A polymer is instead of ethanolamine. In doing so, it increased utilized to deplete internal tryptic peptides after the protein is the charge of peptides and provided a basic group at the C- blocked at carboxylic acids. Contrary to the original TAILS terminus, which increased the yield of y-ions, allowing for more fi fi method where primary amines react only with aldehydes to con dent identi cation using database search engines. How- form covalent bonds, C-TAILS chemistry reacts carboxylic ever, the increase in charge of trypsin-generated peptides acids with primary amines. As such, C-TAILS requires blocking requires the utilization of alternate fragmentation methods, such as electron-transfer dissociation (ETD), which perform at multiple levels. First, thiol groups are alkylated at the protein 358 level and then primary amines (N-termini and lysines) are best at higher charge states. Thus, they utilized a reductively dimethylated, rendering those groups unavailable combination of chymotrypsin, trypsin, and ETD on a LTQ for later reaction. This prevents cyclization and aggregation of Orbitrap Velos. This report found 424 C-termini from using proximal primary amines to carboxylic acids in the amidation two digestion enzymes and two fragmentation methods. ff The number of identified peptides across all these studies reaction. Following bu er exchange to 4-morpholinoethane- fi sulfonic acid (MES) buffer pH 5, proteins are then reacted with shows that the number of identi ed C-terminal peptides is significantly lower compared not only to N-terminomic a primary amine, originally published with ethanolamine, and a methodologies available but to the predicted number of combination of 1-ethyl-3-(3-(dimethylamino)propyl)- proteins in E. coli. Thus, C-TAILS and other carboxy-oriented carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (s- methods are still in their infancy and hold much promise for the NHS), both in excess quantities. Carboxylic acids and primary future of sequencing the ends of proteins. amines do not react in isolation; it requires the activation of the carboxylic acid. EDC activates carboxylic acids into an unstable 5. FUTURE PERSPECTIVES O-acylisourea intermediate. This intermediate can (1) be Regulated proteolytic processing is dysfunctional in diseases stabilized by s-NHS to form the amine-reactive compound, ffl fi  which then forms an amide bond with ethanolamine, or (2) that a ict us all yet is still ill-de ned only 340 of 565 human hydrolyze back to the carboxylic acid. Because of the back- proteases have known substrates. Deciphering cleavage events conversion back to carboxylic acid, addition of EDC and s-NHS at decision points of cell and tissue function will reveal an unexplored layer of complexity in the hierarchy of cell is repeated three times in C-TAILS over 18 h to maximize ff carboxylic acid blocking, which is critical for the success of the regulation. The past decade of increasing research e ort and attention in the field of degradomics has transformed our view method. of proteolysis from a mere destructive mechanism, such as in Proteins are then precipitated to eliminate excess reagent and MMP-mediated degradation of extracellular matrix, to an digested with trypsin to generate peptides that contain newly intricate and selective posttranslational modification with formed primary amines and carboxylic acids. For quantification important regulatory functions in many biological pathways. purposes and to prevent aggregation and cyclization, peptides With increasing technological capabilities (e.g., more powerful are labeled at their newly formed N-termini with isotopic or ff mass spectrometers and improved bioinformatics), our ability isobaric labels. Following bu er exchange, the freely available to probe substrate proteolysis deeper and more specifically in C-termini from internal tryptic peptides are reacted in a similar  various processes in health and disease has provided more EDC/s-NHS scheme with a water-soluble linear polymer insight into how proteolysis changes protein function, yet many polyallylamine (PAA). This polymer captures internal tryptic questions remain to be answered. peptides and can be separated from the unbound C-terminally fi fi An unexpected general nding of such unbiased terminomics blocked peptides utilizing a spin- lter membrane with a high- approaches is the very high percentage of protein populations ff molecular-weight cuto . C-TAILS was originally utilized to that are present with undocumented N- or C-termini present study the E. coli C-terminome, and it achieved >70% of concurrent with their mature parent protein as translated and peptides being C-terminally blocked, identifying 460 peptides 348 354 matured in the classical biochemical pathways. Thus, in from 196 proteins. normal skin ∼50% of proteins have a different N-terminus to Since the originally published protocol in 2010, there have that annotated in Uniprot;9 in platelets and erythrocytes this been novel developments to address some caveats in C-TAILS. percentage rises to 77%7 and 68%,8 respectively, ∼4% of which While the original method utilized dimethylation of primary are internal alternate start sites.8 Although these two cell types amines for blocking, a report in 2015 by Zhang and colleagues are unusual, these numbers seem representative, and indeed we showed there are potential benefits for utilization of acetylation found in human dental pulp that 78% of proteins occurred as to improve Mascot scores.356 In addition, they prepurified PAA different cleaved proteoforms.10 As has been discussed polymer to remove monomers and allowed for the inclusion of throughout this Review, the position and chemistry of the N- singly charged species to be fragmented by the mass terminal amino acid residues and their specific modifications spectrometer. Most notable was the inclusion of acetonitrile can profoundly alter the function of the protein. Thus, these in the EDC reactions in order to minimize water concen- proteoforms not only increase protein diversity in vivo but they trations while allowing proteins to remain soluble. Overall these alter our conceptual models of protein, organelle, cell, and

1157 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review tissue functionin dynamic ongoing physiological processes phosphorylation, ubiquitination, and proteolytic cleavage will that maintain or restore homeostasis and go astray, leading to integrate into a true systems analysis platform to study the role pathology or occurring to switch function as a protective host of specific proteolytic cleavages in biology and pathology. This response to pathology. The genesis of these neo-termini needs nuanced new level of biological control heralds a bright new era to be deciphered, and for that vast majority generated other and realm of biology and protein and cell regulation. than by alternate start sites and splicing, the cognate proteases leading to these proteoforms need to be identified. In AUTHOR INFORMATION pathology, these proteases or proteoforms may form valuable Corresponding Author new drug targets or biomarkers of disease. In any case, both * need to be recognized and the biological implications need to E-mail: [email protected]. be understood for more accurate understanding of the function Present Addresses ⊥ and response networks of a proteome, at rest and when (T.K.) Department of Clinical Chemistry; Erasmus Medical stressed. Center, Rotterdam, The Netherlands. § Current proteomics techniques aimed at unbiased study of (U.E.) Division of Structural Biology, Department of proteolysis of substrates provide temporal snapshots of the , University of Salzburg, Salzburg, Austria. state of cleavage in a tissue and give invaluable insights into ¶ fi (A.D.) Department of Physiology and Pharmacology; what substrates are cleaved, e.g., in response to a speci c University of Calgary, Calgary, Canada. stimulus or disease state, but interpreting these pictures can prove challenging. Most useful proteomics techniques are based Notes on relative quantification between samples, so the stoichiom- The authors declare no competing financial interest. etry between cleaved and intact substrate is often overlooked, Biographies and it is unknown how this affects biology. Many of the background cleavages may occur at low stoichiometry, and for Theo Klein obtained his Ph.D. in Analytic Biochemistry in 2008 from these, are they relevant? For each protein, what is the tipping the University of Groningen, The Netherlands, where he worked on point for physiological relevance in altering protein function, development of novel and proteomics methods for particularly when the cleavage products are antagonists or qualitative and quantitative analysis of protease activity. After a brief dominant negatives of the parent protein. How stable are the segue into biotech at Brains Online in Groningen, he joined Chris parent and neoproteins? A related question is how many of Overall’s lab in Vancouver in 2011 to study the role of proteolytic these cleavages are also silent? Thus, perhaps the generation of cleavage in adaptive immunity, focusing on the role of the paracaspase neotermini in homeostatic conditions is just the price the MALT1 in antigen-receptor signaling. system pays for maintaining a responsive pathway and network Ulrich Eckhard obtained his Ph.D. degree in 2011 from the University that can rapidly change the levels of a protein or its activity on of Salzburg, Austria, where he focused on the biochemical and demand. Similarly, deciphering protease redundancy for these structural elucidation of bacterial collagenases. He then joined Prof. cleavages is important to understand. Redundancy is often too Chris Overall at the University of British Columbia, Vancouver, easy an excuse for lack of deeper understanding. That is, a Canada, where he secured a postdoctoral fellowship from the Michael system may not have redundant capacity but rather the Smith Foundation for Health Research and expanded his structural organism does, and in different cells, tissue, or organs, under ff ff biochemical methodological repertoire to state-of-the-art proteomics. di erent stresses or nutritional and hormonal statuses, di erent Since September 2016, he has worked as a researcher at Uppsala proteases cut in and out. This is important to really understand within a multidisciplinary project trying to unravel how protein in order to formulate good hypotheses and insightful structure and function are intertwined in the evolution of new protein experiments. Localization of a given protease and its substrate variants. greatly affects the outcome and effect of cleavage, and more sensitive analytical techniques will enable more detailed analysis Antoine Dufour obtained his Ph.D. degree in 2010 from Stony Brook of specific tissues and subcellular fractions. University, NY, U.S.A. He developed a patent on the composition and Another challenge in analyzing proteolytic cleavage in methods for the inhibition of MMP9-mediated cell migration. He then complex, biologically relevant samples is the interplay that began a postdoctoral fellowship with Prof. Chris Overall at the exists between different proteases and their inhibitors. The University of British Columbia, focused on the investigation of fl direct source of an observed proteolytic cleavage is often proteases in in ammatory diseases such as cancer, arthritis, and intractable and can be due to indirect effects, e.g., deactivation systemic lupus erythematosus using proteomics. In 2012 he was of a protease inhibitor by another protease, often of a different awarded a fellowship from the Canadian Institutes of Health Research class,11 or activation of another protease. Ongoing advances in to continue his work on the roles of matrix metalloproteinases in bioinformatics are the key to give insights into the intricacies of breast cancer. the protease web and interpreting the genesis of proteolytic Nestor Solis obtained his Ph.D. in microbial proteomics in 2014 from proteoforms in cells and tissues. Finally, proteolytic cleavage The University of Sydney, Australia, where he explored the can be regulated or affected by other posttranslational protein identification of cell-surface proteins from Staphylococcus species modifications. Activity of proteases may rely on specific using cell-shaving proteomics. He joined the laboratory of Professor phosphorylation or ubiquitination events, and glycosylation or Christopher Overall in Vancouver, Canada, in 2014 to further expand phosphorylation or other posttranslational modifications of on proteomic methods to study N- and C-terminomes in macro- substrates may enable, or prevent, proteolysis. So, do cleavage phages. He was awarded two fellowships in 2016 to study macrophage sites function combinatorially or together with other post- terminomes: a Michael Smith Foundation for Health Research translational modifications? Integrating ap- postdoctoral fellowship from British Columbia, Canada, and a CJ proaches that profile posttranslational modifications will further Martin Early Career Fellowship from the National Health and Medical our insight into this, and current targeted analysis platforms for Research Council from Australia.

1158 DOI: 10.1021/acs.chemrev.7b00120 Chem. Rev. 2018, 118, 1137−1168 Chemical Reviews Review

Chris Overall is a Professor and Tier 1 Canada Research Chair in (10) Eckhard, U.; Marino, G.; Abbey, S. R.; Tharmarajah, G.; Protease Proteomics and Systems Biology at the University of British Matthew, I.; Overall, C. M. The Human Dental Pulp Proteome and N- Columbia (UBC), Life Sciences Institute. He is a pioneer of Terminome: Levering the Unexplored Potential of Semitryptic degradomics, a term he coined. He has had several sabbaticals in the Peptides Enriched by TAILS to Identify Missing Proteins in the and Pharmaceutical industries and is an Honorary in Underexplored Tissues. J. Proteome Res. 2015, 14, 3568−3582. Professor at the Freiburg Institute for Advanced Studies, Albert- ̈ (11) Fortelny, N.; Cox, J. H.; Kappelhoff, R.; Starr, A. E.; Lange, P. Ludwigs Universitat Freiburg, Germany. Dr. Overall was awarded the F.; Pavlidis, P.; Overall, C. M. Network Analyses Reveal Pervasive 2002 CIHR Scientist of the Year, the UBC Killam Senior Researcher Functional Regulation between Proteases in the Human Protease Award 2005, the Tony Pawson Canadian National Proteomics Web. PLoS Biol. 2014, 12, e1001869. Network Award for Outstanding Contribution and Leadership to (12) Hershko, A.; Ciechanover, A. The Ubiquitin System. Annu. Rev. the Canadian Proteomics Community 2014, and the HUPO Biochem. 1998, 67, 425−479. Proteomics Discovery Award in 2018. He is an Associate Editor of (13) Janko, M.; Zink, A.; Gigler, A. M.; Heckl, W. M.; Stark, R. W. the Journal of Proteome Research, Editor of mSystems, and on the Nanostructure and Mechanics of Mummified Type I Collagen from Editorial Boards of the Journal of Molecular Cellular Proteomics, Matrix the 5300-Year-Old Tyrolean Iceman. Proc. R. Soc. London, Ser. B 2010, − Biology, and Biological Chemistry and the Advisory Committee of the 277, 2301 2309. International Union of Basic and Clinical Pharmacology. (14) Radzicka, A.; Wolfenden, R. Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases. J. Am. Chem. Soc. 1996, 118, 6105−6109. ACKNOWLEDGMENTS (15) Deu, E.; Verdoes, M.; Bogyo, M. New Tools for Dissecting C.M.O. is funded by a Foundation Grant from the Canadian Protease Function: Implications for Inhibitor Design, and Probe Development. Nat. Struct. Mol. Biol. 2012, 19,9−16. Institute for Health Research and a Canada Research Chair in (16) Hooper, N. M. Proteases: A Primer. Essays Biochem. 2002, 38, Protease Proteomics and Systems Biology. A.D. was supported 1−8. by a Michael Smith Foundation for Health Research (17) Rawlings, N. D.; Barrett, A. J.; Finn, R. Twenty Years of the postdoctoral fellowship. U.E. acknowledges a postdoctoral MEROPS Database of Proteolytic Enzymes, Their Substrates and fellowship from the Michael Smith Foundation for Health Inhibitors. Nucleic Acids Res. 2016, 44 (D1), D343−D350. Research. N.S. is funded through a Canadian MSFHR Trainee (18) Rawlings, N. D.; Barrett, A. J.; Bateman, A. Asparagine Peptide Fellowship (Grant ID 16642) and an Australian NHMRC Early Lyases: A Seventh Catalytic Type of Proteolytic Enzymes. J. Biol. Career Fellowship (GNT1126842). Additional funding inclues Chem. 2011, 286, 38321−38328. C.I.H.R. Foundation Grant (FDN: 148408) (C.M.O.); Canada (19) Vallee, B. L.; Auld, D. S. Active-Site Zinc Ligands and Activated − Research Chair (Tier I) in Metalloproteinase Proteomics and H2O of Zinc Enzymes. Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 220 Systems Biology (950-01-126; 950-20-3877) (C.M.O.); 224. (20) Auld, D. S. Zinc Coordination Sphere in Biochemical Zinc Sites. C.I.H.R. Operating Grant (MOP-133632) (C.M.O.). BioMetals 2001, 14, 271−313. (21) Springman, E. B.; Angleton, E. L.; Birkedal-Hansen, H.; Van REFERENCES Wart, H. E. 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