Proteolytic Cleavage—Mechanisms, Function, and “Omic ... committee...Proteolytic Cleavage Mechanisms, Function, and “Omic” Approaches for a Near-Ubiquitous Posttranslational

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

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

ABSTRACT: Proteases enzymatically hydrolyze peptide bonds in substrate proteins,resulting in a widespread, irreversible posttranslational modification of the protein’sstructure and biological function. Often regarded as a mere degradative mechanism indestruction of proteins or turnover in maintaining physiological homeostasis, recentresearch in the field of degradomics has led to the recognition of two main yet unexpectedconcepts. First, that targeted, limited proteolytic cleavage events by a wide repertoire ofproteases 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 tissuefrom 40 to 70% ofproteins also occur in vivo as distinct stable proteoforms with undocumented N- or C-termini, meaning these proteoforms are stable functional cleavage products, most withunknown functional implications. In this Review, we discuss the structural biology aspects and mechanisms of catalysis bydifferent protease classes. We also provide an overview of biological pathways that utilize specific proteolytic cleavage as aprecision control mechanism in protein quality control, stability, localization, and maturation, as well as proteolytic cleavage as amediator in signaling pathways. Lastly, we provide a comprehensive overview of analytical methods and approaches to studyactivity and substrates of proteolytic enzymes in relevant biological models, both historical and focusing on state of the artproteomics techniques in the field of degradomics research.

CONTENTS

1. Introduction 11372. Different Classes of Proteases and Proteolytic

Mechanisms 11382.1. Metallopeptidases 11382.2. Aspartic Peptidases 11402.3. Glutamate Peptidases 11412.4. Asparagine Peptide Lyases 11412.5. Serine Peptidases 11422.6. Threonine Peptidases 11432.7. Cysteine Peptidases 1144

3. Biological Processes Regulated by ProteolyticCleavage 11453.1. Maturation of Translated Proteins 11453.2. Signal Peptide Removal 11453.3. Proprotein Convertases 11453.4. Processing by Carboxypeptidases 11463.5. Ectodomain Shedding 11473.6. Modulation of Biochemical Pathways and

Signaling through Proteolytic Cleavage 11483.7. Precision Proteolysis in Immunity 1150

4. Analytical Approaches for Proteases and Proteo-lytic Processing 11514.1. Monitoring Substrate Cleavage 11514.2. Substrate- and Activity-Based Probes 11524.3. Proteomics-Based Approaches for Substrate

Discovery 11524.3.1. N-terminal Proteomics 1153

4.3.2. C-terminal Proteomics 11565. Future Perspectives 1157Author Information 1158

Corresponding Author 1158Present Addresses 1158Notes 1158Biographies 1158

Acknowledgments 1159References 1159

1. INTRODUCTION

Regulatory pathways interface to create a system capable ofselectively amplifying and integrating signals in a coordinatedmanner to achieve homeostasis. Defects in one pathway candestabilize the entire system, causing neoplastic, fibrotic,inflammatory, autoimmune, and immunodeficiency diseases.The biological function of proteins is regulated by an intricatenetwork of posttranslational chemical and enzymatic mod-ifications such as phosphorylation, glycosylation, ubiquitination,and proteolysis. Proteolytic cleavage or proteolysis is theenzymatic hydrolysis of a peptide bond in a peptide or proteinsubstrate by a family of specialized enzymes termed proteases.1

This irreversible posttranslational modification is often

Special Issue: Posttranslational Protein Modifications

Received: March 1, 2017Published: December 21, 2017

Review

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© 2017 American Chemical Society 1137 DOI: 10.1021/acs.chemrev.7b00120Chem. Rev. 2018, 118, 1137−1168

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regarded solely as a destructive mechanism, for example, in thedegradation of extracellular matrix in inflammation andintracellular proteins by the proteasome or in apoptosis, butrecent decades of research in degradomics have highlightedspecific, limited precision proteolysis, termed processing, to bea crucial regulator of protein function in virtually all biologicalpathways.2 The complete repertoire of proteases (thedegradome) in man comprises 588 peptidases, which can begrouped in 5 catalytic classes: 21 aspartyl-, 164 cysteine-, 192metallo-, 184 serine-, and 27 threonine peptidases,3 evenoutnumbering the large kinase family (kinome) with its 518members.4 This means that nearly 3.1% of the 19 587 humanprotein encoding genes5,6 encode for proteases, making thesehydrolases one of the most abundant classes of enzymes andpromoting them to center stage in a wide range of biologicalprocesses and diseases.Proteolytic processing is widespread, is pervasive, and

generates stable protein fragments as shown by targeted N-terminal proteomics analysis in numerous biological samples.Unexpectedly, the majority of identifiable protein N-termini arewithin the protein sequence, i.e., unannotated, neo N-termini77% in platelets,7 68% in erythrocytes,8 50−60% in normal andinflamed murine skin,9 and 78% in human dental pulp.10 Theinterplay of interconnections between proteases and theirendogenous inhibitor proteins, termed the protease web,remains understudied to date, leaving many unansweredquestions open for investigation.11

Although proteolytic degradation is vital for maintaininghomeostasis, e.g., in protein turnover through the ubiquitin-proteasome system,12 this Review will focus on targeted,specific processing as a near-ubiquitous post-translationalmodification. Processing can profoundly alter the biologicalproperties of a protein, its localization, and stability. We discussthe repertoire of proteases, their different hydrolytic mecha-nisms, the biological function of proteolytic cleavage, andapproaches for the analysis of proteases and their substrates.

2. DIFFERENT CLASSES OF PROTEASES ANDPROTEOLYTIC MECHANISMS

In the absence of a protease, peptide bonds are highly stable atneutral pH and 25 °C with half-lives of >100 years as shown inancient human remains,13 whereas they are hydrolyzed withinmilliseconds in protease-assisted biochemical reactions.14

Proteases recognize and bind their substrates mainly through

(i) hydrophobic and electrostatic interactions with the substrateamino acid residue side chains in commonly well-definedsubstrate-binding pockets and (ii) hydrogen bonding with thepeptide backbone of a substrate in an extended conformation.15

Whereas endopeptidases (EC 3.4.21−25) cleave substrateswithin the protein molecule, aminopeptidases (EC 3.4.11−14)and carboxypeptidases (EC 3.4.15−18) remove up to threeamino acids from the N- or C-terminus, respectively, and arejointly referred to as exopeptidases.16 A special case representsthe so-called omega peptidase family (EC 3.4.19), whichreleases terminal amino acids that are either chemicallymodified (e.g., acetylated or formylated), cyclized (e.g.,pyroglutamate), or linked by an isopeptide bond. A moresystematic classification follows the nature of the active site andthe amino acid or metal ion catalyzing the peptide-bondcleavage. Thereby seven distinct classes of proteases can beidentified: the aforementioned and well-established five majorclasses, the serine- (EC 3.4.21), cysteine- (EC 3.4.22), aspartic-(EC3.4.23), metallo- (EC 3.4.24), and threonine peptidases(EC 3.4.25), and two classes without known representatives inmammals: the glutamic peptidases (currently included in EC3.4.23) and the asparagine peptide lyases (EC 4.3.2).17 Thelatter were established only in 2011 and represent true outliersin the world of proteolytic enzymes as their reactionmechanism does not include the addition of a water molecule.Thus, they do not qualify as hydrolases or peptidases and haveto be considered as lyases instead, as they utilize an eliminationreaction where an internal asparagine residue first attacks itsown carbonyl carbon bond and cyclizes, followed by anucleophilic attack, leading to a succinimide-ring formationand peptide-bond cleavage.18

2.1. Metallopeptidases

The metalloproteases (EC 3.4.24) represent a highly diversegroup of endo- and exopeptidases. They all harness at least onedivalent metal ion in their active site, typically Zn2+, which iscoordinated by three amino acid side chains and a watermolecule, where the latter plays a key role in substratehydrolysis19,20 (Figure 1a and b).The most common metal ligand is histidine, followed by

glutamate, aspartate, and lysine. Additionally, an unpairedcysteine is found in the propeptides of matrix metal-loproteinases (MMPs) and the adamalysin families, whichreplaces the active site water in the zymogen form, renderingthe protease inactive.21 Notably, in some families, two

Figure 1. (a) Active-site view of the human metzincin ADAM17 (pdb entry: 1bkc). The side chains of Glu406, His405, His409, His415, and Met435are shown as stick models (sequence numbering following Uniprot entry P78536). Zn, zinc. (b) Active-site view of the Clostridium tetani gluzincincollagenase T (pdb entry: 4ar9). The side chains of Glu466, Glu499, His465, and His469 are shown as stick models (sequence numbering followingUniprot entry Q899Y1). Zn, zinc. (c) Active-site view of E. coli methionine aminopeptidase (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, Cobalt. All figures were createdwithin PyMOL with carbon depicted in cyan, oxygen in red, nitrogen in blue, and sulfur in yellow.

Chemical Reviews Review

DOI: 10.1021/acs.chemrev.7b00120Chem. Rev. 2018, 118, 1137−1168

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cocatalytic metal ions are found, typically in the case of cobalt(e.g., E. coli methionine aminopeptidase; Figure 1c) ormanganese (e.g., E. coli aminopeptidase A) but also sometimesfor zinc (e.g., mammalian leucyl aminopeptidases). Importantly,unlike serine and cysteine proteases, metallopeptidases do notform a covalent acyl−enzyme intermediate during substratehydrolysis. Although irreversible inhibition of metalloproteaseactivity has been described using cobalt-containing Schiff-basecomplexes as small-molecule inhibitors,22 no peptide-basedcovalent inhibitors exist for this class.Upon substrate binding, the active-site water is displaced

toward the general base glutamate whereas its oxygen remainsassociated with the metal ion. Additionally, the carbonyl of thescissile bond interacts now with the metal ion, leading to apentacoordinated transition state. The general base glutamatethen subtracts a proton from the water molecule, whichsimultaneously performs a nucleophilic attack on the carbonylbond of the scissile bond, leading to cleavage and a gem-diolintermediate of the substrate. Subsequent protonation of theamide product by the now general acid glutamate leads to therelease of the C-terminal substrate fragment, whereas an attackof a water molecule on the active site zinc is needed to displacethe carboxylate-cleavage fragment.19,20,23

Two major families of the metallopeptidases are distin-guished by their third zinc-binding ligand.24,25 Whereasmetzincins (Figure 1a) employ a total of three histidineresidues within a HExxHxxGxxH motif, the gluzincin super-family (Figure 1b) harnesses a glutamate further downstream ofthe central HExxH (HExxHxnE), often on an α helix.Additionally, all metzincins possess a methionine-containing1,4-turn (Met-turn, hence the metzincin family name), whichforms a “hydrophobic basement” for the catalytic metal ion(Figure 1a). One subgroup of the metzincin family, the MMPs,comprises 23 members in human. MMPs are encoded aspreproproteins with conserved active site features. Thepresence or absence of accessory substrate-binding domains,known as exosites,26−28 such as the hemopexin domain or thefibronectin type-II modules, distinguish individual familymembers, some of which also possess GPI (glycosylphospha-

tidylinositol) anchors or a C-terminal transmembrane do-main.29−31 The well-known and classic MMP substrate iscollagen; MMPs 1, 8, 13, and especially MMP14, also known asmembrane-type (MT) 1-MMP, cleave type-I collagen across allthree α-chains at a single point, namely, Gly775↓Ile776 for theα1(I)-chain and Gly775↓Leu776 for the α2(I)-chain three-quarters of the length from the N-terminus of the ∼1000 aminoacid residue-long collagen. However, not all MMPs are capableof this cleavage, despite highly similar substrate specificity onthe peptide level.32 Binding to the hemopexin domain is key totriple helicase activity and scissile-bond access for cleavage.33

Despite their undisputed role in extracellular matrix remodel-ing, MMPs are now well-established as signaling enzymes inmany biological pathways and diseases such as arthritis, cancer,cardiovascular disease, and liver fibrosis.34,35 Of similar greatinterest are the adamalysins, consisting of the ADAMs (adisintegrin and metalloproteinase; Figure 1a) and ADAMTS(ADAM with a thrombospondin type 1 motif) families. LikeMMPs, the adamalysins are expressed as multidomainextracellular proteins, belong to the metzincin superfamily,and employ a cysteine switch for zymogen inactivation.Whereas the ADAMTS family comprises secreted proteinsonly, most ADAMs possess a C-terminal transmembranedomain followed by a short cytoplasmic tail that is crucial forintracellular signaling. Next to their emerging role incoagulation, inflammation, and cell migration, ADAMTSproteases are mostly known for their role in connective tissueremodeling by proteolytic processing of, e.g., procollagens,aggrecan, and versican. On the contrary, most ADAMmetalloproteinases are involved in ectodomain shedding ofvarious plasma membrane-tethered growth factors, cytokines,receptors, and/or adhesion molecules and thus play funda-mental roles in controlling development and homeostasis,thereby linking them to several pathological states such ascancer, cardiovascular disease, and Alzheimer’s disease.36−38

Other prominent members of the metzincin superfamily are,e.g., astacin, meprin A and B, and ulilysin,39 now known asLysargiNase;40 the latter is used because of its trypsin-mirroringspecificity in proteomics.40

Figure 2. Active-site view of the zinc metallo-deubiquitinase AMSH-LP (associated molecule with the SH3 domain of STAM-like protein) incomplex 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 carboxylgroup of the distal ubiquitin’s C-terminus (Gly76) and the ε-amino group of Lys63 of the proximal ubiquitin is positioned in the Zn2+ coordinatingactive site of AMSH-LP and is depicted as two spheres. Following the nomenclature of Schechter and Berger (PMID: 6035483), Arg72 to Gly76 ofthe distal ubiquitin and Lys63 and Glu64 of the proximal ubiquitin moiety represent the P5 to P1 and P1′ to P2′ positions of the deubiquitinatingenzyme (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) Schematicrepresentation of the isopeptide bond of Lys63-linked diubiquitin, highlighting the T-shaped substrate-recognition motif of deubiquitinatingenzymes. The tripeptide sequence Gln62-Lys63-Glu64 forms together with the Lys63-linked C-terminal glycine of the distal ubiquitin a uniqueinteraction 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.



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One group of metalloproteases distinguishes itself by theirsubstrates; instead of endo- or exoproteolytical cleavage, theJab1/Mov34/Mpr1 Pad1 N-terminal+ (MNP+) domainproteases (JAMM) recognize the isopeptide bond formed byubiquitin conjugated to the free ε-amino group of lysine in theprotein sequence (Figure 2a and b). These deubiquitinatingenzymes (DUBs) comprise a family of close to 100 metallo-and cysteine proteases in Mammalia41 that regulate the removalof ubiquitin from substrates, thereby forming an integralcontrol mechanism in the ubiquitin system.12

Contrary to the mammalian collagenases (MMPs), bacterialcollagenases such as collagenase G from Hathewaya histolytica(formerly known as Clostridium histolyticum42) belong to thegluzincin superfamily and digest native, triple-helical collagensinto a mixture of small peptides under physiologicalconditions.43 These collagenases cleave in the repeating Pro-Hyp-↓-Gly sequence of collagen and select for a smallhydrophobic residue in P1′, whereas their mammaliancounterparts favor a large hydrophobic residue such as Leuor Ile.32,44 In ColT from Clostridium tetani, the fourth zincligand, Glu499, lies 34 amino acids downstream of theH465ExxH motif (Figure 1b), in the center of the peptidasedomain resembling a thermolysin-like peptidase (TLP)fold.45,46 Like many proteases, thermolysin from Bacillusthermoproteolyticus is expressed as preproenzyme, where the200 amino acid prodomain acts as a molecular chaperone in theperiplasm, leading to autoactivation and subsequent secretionof the mature protease into the extracellular space.47 The TLPfold imparts two roughly spherical subdomains that areseparated by a deep substrate-binding cleft where substratesbind from left to right when shown in the standard orientationof metallopeptidases.48 The upper subdomain is dominated bya five-pleated β-sheet with the active-site facing edge strand,providing crucial contacts for substrate binding in antiparallelfashion, whereas the lower C-terminal subdomain is built uppredominantly by α-helices. The catalytic zinc ion issandwiched in between by the HExxH-motif of the centralhelix and the glutamate from the gluzincin helix.49 Notably, asimilar five-pleated β-sheet is also found in MMPs and manyother metallopeptidases.50 Another highly prominent gluzincinfamily member is the dipeptidyl carboxypeptidase angiotensinI-converting enzyme (ACE), an unusual protease having twocatalytic domains in the same protein encoded by the samegene. As a central component of the renin-angiotensin system,ACE tightly regulates blood pressure by (i) removing adipeptide from the C-terminus of the decapeptide angiotensinI, generating the vasopressor angiotensin II, and (ii)inactivating the vasodilator bradykinin by two sequentialcleavages at the C-terminus, removing a total of four aminoacids.51

2.2. Aspartic Peptidases

Aspartic peptidases are the main proponents of the carboxylpeptidase family, which is characterized by an acidic active siteresidue (aspartate or glutamate), a low pH optimum, and anactivated water molecule that attacks the scissile peptide bondin a concerted general acid−base mechanism.52,53 In themajority of known aspartic peptidases, a pair of aspartic acidresidues act together to bind and activate the catalytic watermolecule (Figure 3a), but in some, other amino acids replacethe second aspartate, e.g., Glu in the E. coli peptidase HybD54,55

or His in the histo-aspartic protease from Plasmodiumfalciparum.56

The acidic residue in the second domain serves as the generalbase to abstract a proton from the active-site water, enabling itto perform a nucleophilic attack on the carbonyl carbon of thebound substrate, whereas the first aspartate residue provides viaits protonated state electrophilic assistance to the carbonyloxygen. The tetrahedral oxyanion intermediate subsequentlybreaks down when the scissile amide bond acquires a protonfrom the solvent or through structural rearrangement of theintermediate, releasing the cleavage fragments and regeneratingthe protonated state of the active site aspartate.57−59

Pepsin represents the most-studied aspartic peptidase todate. It shows a bilobed, mostly β-sheet structure, with theactive site located at their interface. Every lobe harbors one ofthe aspartate residues of the catalytic dyad within a conservedAsp-Xaa-Gly motif, in which Xaa is typically a serine orthreonine. Despite low sequence similarity, the high overallhomology suggests that one lobe most likely evolved from theother by gene duplication.53,57 Approximately 45 residuesdownstream of the first aspartate is located a highly conservedtyrosine residue, which separates and defines the S1 and S2′substrate-binding pockets. The tyrosine is also part of an 11-residue β-hairpin loop called flap, which caps the active site andencloses substrates and inhibitors in the active groove uponbinding. Whereas substrates are readily turned over, active-siteinhibitors bind in similar fashion, rendering the enzyme inactiveby tight binding and displacing the active site water (Figure3b). This structural feature is only present in the N-terminaldomain and thus constitutes the main asymmetric aspect of theoverall structure.60−62

The members of the retroviral retropepsin family constitute aspecial case, as retropepsins consist only of one lobe and thusneed to form a homodimer to build a functional catalytic site inbetween. Notably, these viral enzymes (e.g., HIV-1 and HIV-2protease) are not thought to be ancestral and are most likelyderived from the N-terminal lobe of a pepsin homologue, asthey harbor the aforementioned active-site capping flap

Figure 3. (a) Active-site view of the porcine aspartic peptidase pepsin(pdb code: 4pep). The side chains of Asp91 and Asp274 are shown asstick models (sequence numbering following Uniprot entry P00791).(b) Active-site view of the human aspartic peptidase pepsin in complexwith an active-site binding phosphonate inhibitor IVA-VAL-VAL-LEU(P)-(O)PHE-ALA-ALA-OME (pdb code: 1qrp). The side chainsof Asp94 and Asp277 are shown as stick models (sequence numberingfollowing Uniprot entry P0DJD7). (c) Active-site view of the porcinepepsin precursor pepsinogen (pdb code: 2psg) with the inhibitoryprodomain in place. The side chains of Asp91, Asp274, and Lys51 areshown as stick models (sequence numbering following Uniprot entryP00791). All figures were created within PyMOL with carbon depictedin cyan, oxygen in red, nitrogen in blue, and sulfur in yellow.



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structure. Because of the presence of two flaps, the active-sitegrooves of retropepsins are shortened and accommodate onlysubstrate residues from P3 to P3′ instead of P5 to P3′, with atypical preference for hydrophobic residues around the scissilebond.63,64 Because of the inherent symmetry of thehomodimer, the two aspartates cannot play predefined rolesduring substrate hydrolysis. Instead, they most likely engage intheir individual roles only upon substrate binding, which breaksthe initial symmetry.65,66

Importantly, many eukaryotic aspartic proteases are secretedas zymogens.62,67,68 Pepsinogen, for example, is synthesized inthe gastric mucosa and secreted into the stomach, where a 44-residue N-terminal propeptide (Ile16-Leu62 in human pepsinA) displaces the first ∼10 residues of the mature pepsinsequence (Val63-Ala388) and blocks the active-site cleft with itstwo α-helices, whereas Lys51 displaces the active-site water(Figure 3c). Additionally, two tyrosine residues, namely, Tyr53and Tyr137, block the S1 and S1′ binding sites. A special role isincumbent to the highly conserved Arg29 residue, which bindsto Asp73, stabilizing the zymogen conformation and, togetherwith other ionic interactions, being responsible for the acid-triggered conversion to pepsin in the stomach.69,70

Other prominent members of the aspartic protease familyinclude cathepsins D and E, the β-secretases BACE1 and -2 (β-site amyloid precursor protein (APP)-cleaving enzyme-1 and-2), and γ-secretase. Cathepsin D is a soluble lysosomal asparticpeptidase with a 44-amino-acid long propeptide. Afteractivation, further proteolytic processing yields the matureand active lysosomal protease in human, composed of disulfide-linked heavy and light chains. Notably, in mice the single-chainform dominates, whereas the bovine form shows equal amountsof both variants.68,71 Additionally, cathepsin D has twoasparagine-linked oligosaccharides of high-mannose type,which are not required for protein folding or enzyme activitybut play an important role in lysosomal targeting of the proteinvia mannose-6-phosphate (M6P) receptors.72 Cathepsin E is anintracellular, nonlysosomal glycoprotein closely homologous tocathepsin D but mainly found in the endoplasmic reticulum. Itplays a vital role in protein degradation, as well as in the MHCclass II antigen processing pathway.73 Cathepsin E is ratherunusual among the pepsin-like protein family, as it forms adisulfide-bonded homodimer. Notably, monomeric and dimericcathepsin E share similar biochemical characteristics, butdimerization evokes higher structural stability to alkaline pHand temperature changes, allowing the enzyme to thrive at theclose-to-neutral pH of the endoplasmic reticulum.74,75 BACE-1,also known as Memapsin 2 (membrane aspartic protease of thepepsin family 2), is a membrane-bound aspartic protease,mostly known for its sheddase activity on the APP, releasingthe N-terminal ectodomain and initiating the subsequentcleavage of the C-terminal part by the y-secretase complex,ultimately forming a 30−51-amino-acid long isoform of the Aβ-peptide involved in the plaque formation in Alzheimer’sdisease.68,76 The y-secretase complex itself consists of 5individual proteins including the multipass membrane proteinpresenilin-1, an aspartyl protease with an intramembranousactive site enabling APP cleavage in its transmembraneregion.77

2.3. Glutamate Peptidases

Historically misclassified as aspartic peptidases, glutamatepeptidases were only described in 2004, when the crystalstructure of scytalidoglutamic peptidase (eqolisin) from

Scytalidium lignicolum was determined, revealing that itscatalytic dyad consisted of a glutamic acid and a glutamine78

(Figure 4a). Expressed as preproproteins, they are mostly found

in pathogenic fungi; several glutamic proteases also have beenidentified in bacteria and archaea but not yet in eukaryotes.79

Because of their distinct fold, which is similar to concanavalin Abut novel for a protease, eqolisin is insensitive to pepstatin andseveral other inhibitors of aspartic proteases.80 A rather peculiarcase of glutamate peptidases are the trimer-forming tail spikeand fiber proteins found in various bacteriophages. Theseproteins harbor a proteolytically active C-terminal domain(CTD) as well as a domain structure associated with theirprimary function as, e.g., endosialidases or K5 lyases. The CTDinitially acts as an intramolecular chaperone that is indis-pensable for correct folding of the full-length protein and theformation of a functional homotrimer. After assembly, theautocatalytic release of the CTDs by the glutamate peptidasestabilizes the remaining trimer.81,82

The suggested hydrolytic mechanism for glutamic peptidasesis similar to that for their aspartic counterparts. A general base-activated water molecule (Figure 4a and b), initially bound bythe glutamine and the general base glutamate of the catalyticdyad, acts as the nucleophile, whereas the side-chain amide ofthe Gln provides electrophilic assistance first by polarizing thecarbonyl carbon of the scissile peptide bond and second bystabilizing the tetrahedral intermediate. A proton transfer fromthe active-site Glu to the leaving-group nitrogen then triggersthe breakdown of the transition state and finalizes theproteolytic cleavage, followed by the regeneration of theactive-site glutamate.78,83,84

2.4. Asparagine Peptide Lyases

Another family of proteolytic enzymes, which was initiallythought to belong to the aspartic peptidases, is now known tobe not even hydrolases but peptide lyases, as their cleavagemechanism does not involve a water molecule. Instead, anasparagine residue acts as a nucleophile and attacks its ownmain-chain carbonyl to create a five-membered succinimideintermediate by intramolecular cyclization. In the rightphysicochemical environment, this leads to the subsequentcleavage of the peptide bond C-terminally of asparagine in anelimination reaction without the addition of a water molecule.

Figure 4. (a) Active-site view of Scytalidium lignicolum glutamatepeptidase eqolisin (scytalidopepsin B, pdb code: 1s2b). The sidechains of Glu190 and Gln107 are shown as stick models (sequencenumbering following Uniprot entry P15369) (b) Active-site view ofeqolisin with a bound N-terminal cleavage fragment Ala-Ile-His (pdbcode: 1s2k). The side chains of Glu190 and Gln107 are shown as stickmodels (sequence numbering following Uniprot entry P15369). Allfigures were created within PyMOL with carbon depicted in cyan,oxygen in red, nitrogen in blue, and sulfur in yellow.



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Furthermore, as every active site functions only once withoutany subsequent substrate turnover, the self-cleaving activity ofpeptide lyases does not represent a typical enzymatic actionafter all.18,85

Probably the most prominent members of the asparaginepeptide lyase family are the self-splicing inteins (Figure 5). An

intein is a protein domain of ∼135 residues inserted into apolypeptide that is responsible for catalyzing its own excisionwhile simultaneously fusing the two flanking extein domains,similar to the splicing out of intron RNA from mRNA. To doso, the last residue of the intein domain must be asparagine,whereas the first residues of both the intein and C-terminalextein domain have to be either cysteine, serine, or threonine.First, the side chain of the N-terminal amino acid of the inteinattacks the preceding peptide bond, leading to an amide toester (Ser, Thr) or thioester (Cys) rearrangement, i.e., the N-terminal extein domain is loaded onto the inteins N-terminalside chain. Enabled by the close proximity of the inteins N- andC-terminus, the N-extein is subsequently transferred onto the

side chain of the first residue of the C-terminal extein(transesterification). The intein’s C-terminal asparagine thenattacks its own backbone carbonyl and releases the intein fromthe polypeptide, similar to that described above for peptidelyases in general. Finally, an acyl rearrangement of the thioesterbond linking the extein domains occurs to form a normalpeptide bond between them. Importantly and name-giving,none of the peptide bonds in this process is broken byhydrolysis, and thus these enzymes have to be referred to aspeptide lyases and not proteases.18,85,86

2.5. Serine Peptidases

The serine peptidases hydrolyze peptide bonds very differentlyto the mechanisms described earlier for aspartate, glutamate,and metallopeptidases, as the initial nucleophilic attack iscarried out by the side chain of a serine residue and not anactivated water molecule, which leads to a covalent acyl−enzyme intermediate during peptide-bond cleavage. Thus, thecatalytic mechanism is more similar to the asparagine peptidelyases, but with the important difference that the secondtetrahedral intermediate is actually attacked by a watermolecule, thus fully qualifying them as true hydrolases andpeptidases.87,88 The most common (“classical”) catalytic triadof serine peptidases consists of histidine, aspartate, and serine(Figure 6a), not necessarily in that sequence order, but withhighly similar three-dimensional arrangements.Whereas the serine acts as a nucleophile, the aspartate

stabilizes the histidine side chain, which in turn acts as a protondonor and general base. It is noteworthy that glutamate andhistidine replace the aspartate in bacterial LD-carboxypeptidasesand herpes simplex virus endopeptidase, respectively, and lysinetakes the role as general base in the Ser/Lys-dyad of Lon-Apeptidase, which lacks the acidic residue. However, theprobably most-studied serine peptidases, such as trypsin,chymotrypsin, or subtilisin, 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 His57and reduces its pKa to enable the histidine to function as ageneral base in the reaction. The histidine then abstracts aproton from Ser195, which simultaneously performs anucleophilic attack on the carbonyl of the substrate scissilebond. A covalent link is formed between the serine side-chainoxygen and the substrate, resulting in a tetrahedral intermediate(enzyme−acyl complex) and a negative charge on the peptidecarbonyl oxygen, which is stabilized by the protease backboneamides of Gly193 and Ser195, which form a positively chargedpocket commonly referred to as an oxyanion hole. Subsequentprotonation of the substrate amide nitrogen by His57, whichnow acts as a general acid, collapses the complex and results inthe release of the C-terminal part of the substrate (amidasereaction), while the N-terminal part remains bound to theprotease. Next, an activated water molecule, which displacedthe C-terminal fragment in the active site, attacks the esterbond of the acyl−enzyme intermediate (Figure 6c), resulting ina second oxyanion hole-stabilized tetrahedral intermediate.Collapse of the latter releases the N-terminal substrate fragmentfrom the active site (esterase reaction) and regenerates theserine side chain. Similar catalytic mechanisms are suggested forthe other nonclassical active sites of serine peptidases.87−89

Another prototype of serine peptidase is the endopeptidasetrypsin. It cleaves preferentially after arginine or lysine, whereasthe structurally nearly identical chymotrypsin selects for largehydrophobic residues. Trypsin is synthesized as preproprotein

Figure 5. Schematic representation of the mechanism of intein-mediated protein splicing. Upon dimerization of N-intein and C-intein(1) the first residue of the newly formed catalytic core (a serine orcysteine) facilitates a nucleophilic N−O/S acyl shift (2) followed bytransthioesterification mediated by the last residue in the C-extein (3).The C-terminal asparagine residue in the intein subsequently self-cyclizes into a succinimidyl group, cleaving the peptide bond betweenintein and extein and allowing an S/O−N shift to link both exteins viaan amide bond (4).



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including a 15- and 8-amino-acid long signal peptide andpropeptide, respectively. Notably, whereas trypsin is renderedinactive yet highly stable at low pH, at its pH optimum of ∼8,calcium binding ensures maximum activity and decreasedtrypsin autodegradation. The propeptide harbors a conservedrecognition motif for the serine peptidase enteropeptidase (alsocalled enterokinase), which cleaves after (Asp)4-Lys and thusunveils the N-terminus of mature trypsin, in which the catalytictriad bridges two β-barrel domains. Whereas the guanidiniumgroup of arginine-containing substrates is directly recognized bythe carboxyl group of Asp189 at the bottom of the S1 bindingpocket, the shorter lysine side chains are encompassed via abridging water molecule, partly explaining the ∼6-foldpreference for arginine over lysine.90 In chymotrypsin-likepeptidases, the equivalent position is taken by serine, butneither the mutation of Asp to Ser in trypsin nor that from Serto Asp in chymotrypsin fully converts the substrate specificitydespite the fact that nearly all other residues of the S1 subsiteare identical. Additional surface loops that are not even part ofthe extended substrate binding site have to be reciprocallyengineered to render the respective enzyme specificity.91,92

In elastase and elastase-like serine peptidases, the S1substrate pocket represents only a shallow depression, asglycine residues that build the pocket in chymotrypsin andtrypsin are replaced by larger, hydrophobic residues that fill thepocket and instead provide interaction with small hydrophobic

side chains. As before, additional enzyme engineering is neededto fully convert, e.g., trypsin to elastase.93 So, despite nearlyidentical three-dimensional structures and substrate binding inhighly similar antiparallel β-sheet conformation, these threeenzymes show very different substrate preferences, highlightingthe structural fine-tuning during protein evolution and thecomplexity of any structure−function analysis.2.6. Threonine Peptidases

Threonine peptidases exploit an N-terminal threonine ascentral catalytic residue and nucleophile for peptide-bondhydrolysis, whereas no other amino acids are strictly conservedin the active site.94 Instead, the N-terminal amino group, or awater molecule bound between the α-amine and the threonineside chain, acts as a general base in the reaction by providingthe proton necessary for hydrolysis. Together with peptidasesthat harness Ser or Cys in the very same N-terminal position,threonine peptidases are colloquially grouped as N-terminalnucleophile (Ntn) hydrolases. Importantly, all Ntn hydrolaseshave to be activated first to liberate the N-terminal nucleophile,either by simple N-terminal methionine excision as seen, e.g., inbacterial proteasomes (reviewed in ref 95) or by anintramolecular autoactivation mechanism observed in theeukaryotic counterpart, probably involving a catalytic Thr/Asp/Lys triad.94,96−98

Undeniably, the proteasome (Figure 7a), which is amultisubunit complex comprising four stacked rings each

containing six (in bacteria) or seven (in archaea andeukaryotes) subunits as core particle, represents the mostprominent threonine peptidase. Although the subunits of theouter rings (α-subunits) share the protein fold with the twoinner rings (β-subunits), only the latter can act as proteaseswithin the proteasome’s αββα-architecture. Additionally, ineukaryotic proteasomes only 6 of the 14 homologues areproteolytically active, with always two sites showing chymo-tryptic, tryptic, and glutamyl endopeptidase activity, thuscleaving preferentially after large hydrophobic, basic, and acidicresidues, respectively. On the contrary, archaeal proteasomesare composed of 14 identical active β-subunits. In each activesubunit, a conserved lysine residue ∼30 amino acids down-stream of the N-terminal threonine is suggested to assist

Figure 6. (a) Catalytic triad of human chymotrypsin (pdb code: 4h4f).The side chains of Ser216, His74, and Asp121 are shown as stickmodels (sequence numbering following Uniprot entry Q99895). (b)Catalytic triad of porcine chymotrypsin (pdb code: 1esa). The sidechains 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 sidechains of Ser214, His71, and Asp119 are shown as stick models(sequence numbering following Uniprot entry P00772). All figureswere created within PyMOL with carbon depicted in cyan, oxygen inred, nitrogen in blue, and sulfur in yellow.

Figure 7. (a) Active-site view of the threonine peptidase β7 subunit ofthe human proteasome (pdb code: 5le5). The side chains of Thr44and Lys76 are shown as stick models (sequence numbering followingUniprot entry Q99436). (b) Active-site view of the β7-subunit of thehuman proteasome complexed with inhibitor bortezomib (pdb code:5lf3). The side chains of Thr44, Gly90, and Lys76 are shown as stickmodels (sequence numbering following Uniprot entry Q99436). Allfigures were created within PyMOL with carbon depicted in cyan,oxygen in red, nitrogen in blue, and sulfur in yellow.



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substrate hydrolysis by decreasing the pKa of the aminoterminus through electrostatic interactions (Figure 7a and b).Additionally, a conserved glutamate roughly halfway betweenthe N-terminal threonine and the aforementioned lysine seemsto be involved in the catalytic mechanism. After thenucleophilic attack, the main-chain amide of a conservedglycine residue stabilizes the tetrahedral transition state. Afterbreakdown of the intermediate, the C-terminal part of thesubstrate is released, whereas the N-terminal part remainscovalently bound to the threonine side chain as an acyl−enzyme complex. A subsequent nucleophilic attack by a watermolecule closes the reaction circle by releasing the N-terminalcleavage product and regenerating the free protease.99−101

Notably, whereas the proteasome is highly conserved amongeukaryotes, it seems sufficiently diverse to allow specifictargeting of, e.g., disease-causing protozoan parasites, such asPlasmodium, Trypanosoma, or Leishmania, possibly opening anew road for, e.g., antimalarial agents.102,103

2.7. Cysteine Peptidases

Cysteine peptidases are found through all kingdoms of life.Although evolutionary highly diverse, cysteine peptidases allshare a common catalytic mechanism that involves anucleophilic attack by a cysteine side chain, typically from acatalytic triad or dyad, for substrate hydrolysis. As the active sitethiol group is highly prone to oxidation, cysteine peptidases aremost active in a slightly reducing environment and/or atslightly acidic pH (4.0−6.5). In mammals, cytosolic calpainsand lysosomal cathepsins are probably the most well-knownfamily members. Nevertheless, the most-studied cysteinepeptidase is papain from the tropical melon tree (Caricapapaya), which is also considered as the archetype of cysteineproteinases. Papain is synthesized as a nonglycosylatedpreproprotein of 345 amino acids with an 18- and 115-amino-acid long signal peptide and propeptide, respectively.The mature protein folds into a globular domain stabilized bythree disulfide bridges, with two subdomains delimiting asurface-exposed active-site groove on the top, when shown instandard orientation. Although the left subdomain is comprisednearly entirely by the N-terminal part of the enzyme, the N-terminus is located on the distal right side, whereas the rightsubdomain ends in a strand that extends proximal into the leftdomain. The catalytic residues Cys158 (left domain; corre-sponds to Cys25 in pepsin numbering starting afterprepropeptide removal) and His292 (right domain; His159)of the catalytic dyad are located on opposite sides of theinterface (Figure 8a and b).Several additional residues in papain are known to be

important for substrate turnover but are considered asnoncatalytic, as they are not essential for hydrolysis; e.g.,Asn308 (Asn175) resembles the structural equivalent ofaspartate in the catalytic triad of serine peptidases (Ser/His/Asp) but with much lower contribution to the catalyticefficiency. Mutation to glutamine only leads to a 3.4-folddrop in kcat/KM,

104 whereas mutating the aspartate in thecatalytic triad of trypsin results in a 104-fold decrease ofenzymatic activity.105 In the case of papain-like proteinases, thecatalytic center is complemented by the aforementionedAsn308, but other residues such as aspartate or glutamate canbe found in the equivalent position, especially in bacteria andviruses. The main role of the third residue during substratehydrolysis is to orient the imidazole ring of the catalytic dyad,ultimately allowing the histidine to assist in cysteine polar-

ization and deprotonation. It is still under debate whether thehistidine also acts as proton acceptor or if the imidazole is perse protonated, as well as if the cysteine thiol becomes ionizedonly upon substrate binding like in serine peptidases or ifcysteine proteases are a priori active.Hydrolysis is initiated by the nucleophilic attack of the

activated thiolate anion on the carbonyl carbon of the substratescissile bond, resulting in the first tetrahedral transition statewhere the substrate is covalently attached to the cysteine, andthe reaction intermediate is stabilized by the oxyanion hole ofthe enzyme. Rotation of the imidazole ring enables asubsequent proton transfer to the amide nitrogen of theattacked peptide bond, releasing the C-terminal fragment of thecleaved polypeptide, whereas the N-terminal part remainscovalently linked via its C-terminus. Next, a water molecule,polarized by the active-site histidine, attacks the carbonylcarbon of the thioester-bonded acyl−enzyme complex,resulting in the second tetrahedral intermediate, whichultimately decomposes and releases the N-terminal portion ofthe substrate, thereby regenerating the peptidase.106−109

Lysosomal cysteine cathepsins represent a highly studiedgroup of peptidases within the papain superfamily of cysteineproteases. In humans, there are 11 representatives known,namely, cathepsin B, C, F, H, K, L, O, S, V, X, and W.Lysosomes contain a number of lipases, amylases, nucleases,and more than 50 different proteases, mainly aspartic, serine,and cysteine peptidases. In addition to, e.g., the asparticprotease cathepsin D, the serine protease cathepsin A, and thecysteine protease legumain, all kinds of cysteine cathepsins playa key role in lysosomes.110−113 One example is cathepsin B,which displays both endopeptidase and carboxydipeptidaseactivities; after initial cleavage within the substrate, the cleavedpolypeptide chain is degraded in a processive manner byremoving dipeptides from the substrate C-terminus. Notably,compared to other papain-like enzymes, cathepsin B shows arather broad specificity in the P2 position and accepts, e.g., evenarginine next to the more commonly found large hydrophobicresidues, alleviating its degradative power.111,114

In contrast, cathepsin H functions as either an endopeptidaseor aminopeptidase, depending on the presence of an 8-amino-acid long minichain originating from the propeptide. Cathepsin

Figure 8. (a) Catalytic triad of the Carica papaya cysteine peptidasepapain (pdb code: 1ppn). The side chains of Cys158, His292, andAsn308 are shown as stick models (sequence numbering followingUniprot entry P00784). (b) Catalytic triad of the Carica papayacysteine peptidase papain complexed with inhibitor E64 (pdb code:1pe6). The side chains of Cys158, His292, and Asn308 are shown asstick models (sequence numbering following Uniprot entry P00784).All figures were created within PyMOL with carbon depicted in cyan,oxygen in red, nitrogen in blue, and sulfur in yellow.



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H lacking the minichain shows distinct endopeptidase activity.However, when the minichain is bound, it occludes thenonprimed subsides from S2 backward while providing anegative charge through its C-terminus to bind the substrate N-terminus, abolishing endopeptidase activity and emanating inan elevated aminopeptidase activity.115,116 Interestingly, incathepsin X, a unique 3-amino-acid long miniloop-insertion(Ile-Pro-Gln) between the oxyanion hole glutamine and theactive-site cysteine confers carboxypeptidase activity in a similarmanner. The insertion is preceded by a histidine residue(glycine in papain), which occupies the S2′ subsite and cananchor the substrate C-terminus in two different side-chainconformations, depending on carboxymonopeptidase orcarboxydipeptidase activity.117−119

Overall, lysosomal cysteine cathepsins developed severalmodus operandi that reduce the number of substrate-bindingsites from either side and provide an electrostatic anchor for therespective substrate terminus, facilitating permanent ortemporary exopeptidase activity. Adjuvant to their special-ization as exo- or endopeptidases, localization and pHdependency are other unique features of individual cysteinecathepsins; e.g., cathepsins B, C, H, L, and O are ubiquitouslyexpressed in nearly all tissues and cells, whereas cathepsins S, K,V, F, X, and W show restricted organ distribution, suggestinghousekeeping or specific functions, respectively. Additionally,cathepsin H is irreversibly inactivated above pH 7.0; its inactiveprecursor remains stable, and cathepsin S even shows activityand actually has its pH optimum somewhere between pH 6.0and 7.5.Importantly, like many other proteases, cysteine cathepsins

are expressed with N-terminal signal-peptide and propeptidesequences responsible for protein sorting into specific cellularcompartments or extracellular space and enzyme inhibition,respectively. In the inactive zymogen form, the prodomainforms a compact minidomain, where a stretched and flexiblesegment covers and tightly interacts with the active site cleft inbackward orientation, occupying several substrate-bindingpockets while also preventing hydrolysis. Activation occurs inacidic milieu, at pH 5.0 or below, where the prodomainswitches from the closed conformation favored at neutral orslightly acidic pH to an open conformation due to ionicrepulsion. The physical displacement yields a low-activecathepsin, which is sufficient to activate another zymogen intrans, thus triggering an autocatalytic activation cascade.109,120

Other prominent cysteine peptidases that are beyond the scopeof this Review are calpain-1 and -2,121 the various caspases,122

the lysosomal protease legumain,123 and the paracaspasemucosa-associated lymphoid tissue lymphoma translocationgene 1 (MALT1),124 just to mention a few.

3. BIOLOGICAL PROCESSES REGULATED BYPROTEOLYTIC CLEAVAGE

3.1. Maturation of Translated Proteins

Various proteolytic cleavage events function as a controlmechanism during translation and localization of proteins in thecell. In eukaryotic protein synthesis, mRNA generally startswith an AUG codon, and therefore the translated proteinsequence will almost exclusively start with a methionineresidue. Depending on the protein and the organism, thisinitiator methionine either can remain on the new protein or, ina large fraction of newly translated proteins, be rapidly removedthough proteolytic cleavage by methionine aminopeptidases in

a process named N-terminal methionine excision (NME,reviewed in refs 125 and 126). Translation and subsequentproper folding of proteins is an imperfect process. Although therole of the N-terminal methionine and the functional relevanceof its proteolytic excision is not fully understood, currentdogma dictates that these processes control protein stabilityand half-life in the context of the N-end rule that determinesprotein fate.8,127,128 For cytosolic proteins, stability and qualitycontrol is determined by the N-terminal residue after excisionof the initiator methionine. Most residues (primary, secondary,and tertiary destabilizing residues) trigger ubiquitination andproteasomal degradation if the protein is incorrectly folded withthe N-terminal residue exposed. Acetylated N-terminal residuescan also destabilize proteins where the N-terminal residue iseither M, V, A, S, T, C, P, or G, termed secondary destabilizingresidues. Lange et al.8 investigated protein stability todetermine if the neo-N-terminal residue exposed after proteasecleavage followed the N-end rule. While following the N-endrule, post translational neo-N-terminal acetylation unexpectedlystabilized cleaved cytosolic proteins with primary destabilizingresidues (R, K, L, H, F, Y, W, and I). Thus, this differed fromthe destabilizing role for acetylated N-termini having secondarydestabilizing residues.8

For proteins moving through the ER, a quality-controlsystem termed ER (endoplasmic reticulum)-associated proteindegradation (ERAD) is in check. Misfolded proteins arerecognized by the ERAD machinery, likely due to interactionsof specific chaperones with exposed hydrophobic patches,incomplete disulfide bridges, and improper glycosylation,leading to lysine-48 polyubiquitination of the misfolded proteinby ubiquitin E3 ligases such as carboxy terminus of Hsp70-interacting protein (CHIP), expulsion of the protein from theER lumen, and subsequent cytosolic proteolytic degradation ofthe misfolded protein by the proteasome.129−131

3.2. Signal Peptide Removal

Localization and transport of proteins in the secretory pathwayis regulated by intrinsic sequences within the N-terminal regionof these proteins termed signal peptides; short sequences arerecognized during translation of the nascent protein by thesignal-recognition particle in the ER that mediates translocationinto the ER lumen,132 allowing subsequent secretion of theproteins via vesicular transport mechanisms. Alternatively, thisprocess can occur by posttranslational processes.133 Duringtranslocation, signal peptide sequences are cleaved from thepreprotein by a family of serine proteases named signalpeptidases (SPase), e.g., type-1 SPase in bacteria and the ERsignal peptidase complex (SPC) in eukaryotes for properlocalization (reviewed in ref 134). This process is furthercontrolled by proteolytic cleavage of the signal peptide remnantby a family of intramembrane cleaving aspartyl proteases calledsignal peptide peptidases (SPPs135) by regulated intramem-brane proteolysis (RIP, reviewed in ref 136).Proteins targeted for the mitochondrion contain a variant

signal peptide named mitochondrial matrix peptide (unfortu-nately also designated as MMP), an 8−58-amino-acid longsequence rich in positively charged amino acids that targets thepreprotein to the mitochondrion and is subsequently cleavedby the zinc metalloprotease mitochondrial processing peptidase(MPP).137

3.3. Proprotein Convertases

Many proteins such as proteolytic enzymes, growth factors, andpeptide hormones are expressed and translated as inactive



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precursor forms that require targeted, specific proteolyticcleavage events during their transition through the secretorypathway to become biologically active. In humans, this processis regulated by a family of seven proprotein convertase (PC)enzymes (furin, PC1, -2, -4, -5, and -7, and paired basic aminoacid cleaving enzyme 4 (PACE4)) that hydrolyze peptidebonds at paired basic amino acid residues in substrate precursorproteins, the subtilisin kexin isozyme 1 (SKI-1) that cleavesafter nonbasic residues, and proprotein convertase subtilisin/kexin 9 (PCSK9) that is only known to cleave itself in anautocatalytic activation step.138,139 Depending on the substrateand its activating PC, cleavage and activation can occur at anylocation in the secretory pathway from the trans-Golgi network,endosomes, and cell surface and, in the case of activation ofpolypeptide hormones, in secretory granules. PCs cleavesprecursor proteins by limited proteolysis within a sequencemotif [R/K]-(X)0,2,4,6-[R/K]↓P′1, with a strong preference forarginine in the P1 position.140 Because the cleavage-sitespecificity of most PCs is so similar, and there is considerableredundancy in substrate cleavage in vitro, localization is likelythe main determinant of substrate specificity.141,142

In 1990, furin or PCSK3 was the first identified mammalianPC as a homologue of the yeast convertase kexin.143 Furin is amembrane-bound protease that is subject to endosomaltrafficking between the cell surface and the trans-Golgi network,where it processes numerous protein precursors by cleavage atdibasic motifs such as BXBB or BBXB (where B is a basicamino acid residue).141 Furin has been reported to be secretedinto the extracellular space.144 Interestingly, some potenthuman pathogens have evolved to contain furin-cleavage sitesin their virulence factors; for example, Anthrax lethal factor andavian influenza virus hemeagglutinin rely on host furin foractivation.141,145,146 Proteolytic cleavage by furin is essential tolifefurin knockout mice are not viable due to severecardiovascular defects during embryogenesis that are likely aresult of insufficient activation of transforming growth factorbeta (TGFβ).147,148 Processing of TGFβ1 by furin is alsorequired in adaptive immunity; T cells lacking furin produceless mature TGFβ1 and are dysfunctional.149 Furin and PACE4are capable of activation of multiple membrane-associatedmetalloproteases in the metzincin superfamily such as MMPs,MT-MMPs and ADAMs, by selective proteolysis of motifswithin the prodomain, leading to exposure of the catalytic cleftand accessibility for substrates.150−152 Cellular adhesion isregulated by furin and furin-like (PC5) PCs through theactivation of pro-integrins that are required for interaction ofthe cell with the extracellular matrix, and furin activity is highduring situations of tissue remodeling, such as in atheroscleroticlesions.153 There is considerable apparent redundancy betweenfurin and related PCs such as PC5, PC7, and PACE4, althoughsome substrates appear to be preferentially cleaved by one overthe other enzymes.138

PC1 and -2 are, as indicated by their original names pituitaryor prohormone convertase 1 and 2, respectively, importantactivators of endocrine and neural polypeptide hor-mones,154−157 which are localized in granules in hormone-producing cells and activate their substrates in the acidicregulated secretory pathway.158,159 There is considerableoverlap in substrate specificity considering knockout miceshow defects but are viable, and in unison these two proteasesregulate vital processes, such as blood sugar homeostasisthrough cleavage of pro-insulin and glucagon,160,161 andmodulate many hormonal signaling events through site-specific

processing of pro-opiomelanocortin (POMC) into adrenocor-ticotropic hormone (ACTH) or alpha-melanocyte-stimulatinghormone (αMSH).162 Furthermore, PC2 processes a widevariety of neuroendocrine precursor proteins such asproenkephalin, prosomatostatin, neurotensin, neuromedin N,prodynorphin, and nociceptin.163 PC1 knockout results insevere growth defects that are likely caused by disruptedprocessing of insulin growth factor-1 and growth hormone-releasing hormone (GHRH).138

PC4 is selectively expressed in germ cells in testes, ovaries,and placenta and plays an important role in maintaining fertilityand in reproduction, likely through cleavage of pituitaryadenylate cyclase-activating polypeptide (PACAP) in the testesand the activation of various ADAM proteases.138,164,165 SKI-1or S1P is a widely expressed protease with notably differentcleavage-motif preference than the other members of the PCfamily because it hydrolyzes substrates after any amino acidother than cysteine and proline.166,167 SKI-1 is an activator ofmembrane-bound transcription factors in cholesterol metabo-lism and homeostasis (sterol regulatory element-bindingprotein (SREBP)-1 and -2)168 and activating transcriptionfactor 6 (ATF6) in the endoplasmic reticulum (ER) stressresponse.169 Besides this role in transcription-factor processing,SKI-1 cleaves and activates viral proteins such as Lassa virusglycoprotein, making the enzyme a potential target for antiviraltherapeutics.138

3.4. Processing by Carboxypeptidases

Proteases are divided into their ability to cleave proteins fromwithin a sequence (endoproteases) or the ends of a polypeptidesequence (exopeptidases). Exopeptidases can be furtherclassified if they cleave protein chains at their N terminus(aminopeptidases) or if they cleave from the C terminus(carboxypeptidases).1 In addition, peptidases and proteases cancleave single or two (or more) amino acids from the end of aprotein or peptide such as in the case of peptidyl dipeptidasesand oligoendopeptidases, but this section will focus on singleamino acid hydrolysis in the case strictly for carboxypeptidases.Peptidases and proteases can be classified by their mechanismsof peptide hydrolysis, namely, if the active-site residues essentialfor hydrolysis are serine, threonine, cysteine, aspartate, andglutamate or if there is the necessity of a metal ion bound to theenzyme in the case of metalloproteases.170 The two largestgroups for carboxypeptidases are serine carboxypeptidases andmetallocarboxypeptidases.171,172 Of these two groups, themetallocarboxypeptidase family has more members and isbetter characterized biochemically and structurally than serinecarboxypeptidases.Carboxypeptidases can recognize specific C-terminal residues

that can be recognized by substrate-binding pockets andsubsequently cleaved. Serine carboxypeptidases often have pHoptima in the acidic range as opposed to most serine proteasesand metallocarboxypeptidases, which are active in neutral tomildly basic ranges.172 While carboxypeptidases were originallydiscovered in the context of protein digestion as in the case ofbovine carboxypeptidase A, the role of carboxypeptidases hasnow been shown to be more exquisite. There are 34carboxypeptidases in humans, which highlights the importanceof their biological role.173

Metallocarboxypeptidases perform peptide hydrolysisthrough acid−base mechanisms mediated by a metal ion witha solvent molecule to attack the target peptide bond.49 Incontrast, serine carboxypeptidases were originally characterized



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to be serine proteases due to being inhibited by diisopropyl-fluorophosphate (DPF)172 and later confirmed by structuralstudies to attack the scissile bond with a catalytic triad (Ser-His-Asp).1 Metallocarboxypeptidases are structurally split into twofamilies that have been extensively reviewed by Gomis-Ruth:171

cowrins and funnelins. The cowrin subset of metallocarbox-ypeptidases is characterized by having a long and narrowgroove across the length of the protease with the active sitehalfway through the groove. Such a groove allows for theinsertion of unfolded oligopeptide sequences that can becleaved at the last residue catalyzed by the metal ion (typicallyzinc). Cowrins have a consensus structural motif involved incatalysis, HExxH+ExxS/G+H+Y/R+Y, and their catalyticdomains are 500−700 residues in size. Funnelins differ fromcowrins structurally in that funnelins have a shallow active cleftat the bottom of a funnel-like cavity and have the potential tohydrolyze folded proteins. Funnelins also possess a consensusmotif important for their catalysis: HxxE+R+NR+H+Y+E witha catalytic domain ∼300 residues large. Examples of cowrinsand funnelins are neurolysin and carboxypeptidase A,respectively.As mentioned above, the role of carboxypeptidases was

initially described in the context of bovine pancreaticcarboxypeptidase A, one of the most studied funnelin-typecarboxypeptidases and the first metalloprotease described(utilizing a zinc ion for catalysis).174 Carboxypeptidase A hasselectivity toward C-terminal residues that are aliphatic oraromatic (hence why it was termed carboxypeptidase Aforaromatic and aliphatic). Carboxypeptidase B was also describedin its role for digestive biology for its specificity toward basicresidues at the C-termini of polypeptide chains. It is importantto note that carboxypeptidases A and B preferably act uponpeptides as opposed to large protein structures. Thus, theynecessitate the action of other endoproteases that can cleaveproteins to generate smaller fragments with C-terminal residuesthat are recognizable by the carboxypeptidase and release theC-terminal amino acid, which then can be absorbed by theorganism in the case of digestive enzymes.1 Trypsin is the mostnotorious and well-studied endoprotease that acts concertedlywith carboxypeptidase B.1 Trypsin can activate procarbox-ypeptidase B into its active form by releasing its propeptide,175

allowing secreted carboxypeptidase B to act upon extracellularproteins (ingested in the digestive system) as opposed tointracellular proteins, where they can cause cellular damage ifnot controlled. Carboxypeptidase B can later cleave C-terminallysines on tryptic peptides. Lysine is an essential amino acid inhumans (namely, it cannot be synthesized by metabolicprocesses), and so the activity of carboxypeptidase B isessential for life. This role is mirrored by the activity ofcarboxypeptidase A on C-terminal aliphatic amino acids that areimportant for nutrition in concerted action with pepsin andchymotrypsin endoprotease activity.1

In addition to carboxypeptidases A and B, which have beendescribed in great detail in the literature, carboxypeptidaseshave also evolved to have hormone-activating functionswhich consists of trimming small peptide sequences intobiologically active forms. Of distinction is carboxypeptidase E, ametallocarboxypeptidase that has specificity toward C-terminalbasic residues, much like carboxypeptidase B.176 Unlikecarboxypeptidase B, however, the distribution of carboxypepti-dase E is in tissues where many neurotransmitters and peptidehormones are produced such as the brain and pancreas.176

Furthermore, the activity of carboxypeptidase E is in the pH

range of 5.0−5.5, which renders it inactive in the Golgi bodywhere it is synthesized, preventing it from acting on othercellular proteins.1,177 It is proposed that this pH range availscarboxypeptidase E activity in secretory vesicles, which arepacked with peptide hormones that require processing.178

Many peptide hormones require C-terminal trimming afterendoprotease processing as well as in many cases peptideamidation to render the hormone biologically active.179 Awidely known substrate of carboxypeptidase E is insulin,180 akey regulator of glucose intake in humans, which whendysregulated causes diabetes. Insulin is synthesized as a preproform and upon endoprotease processing (prohormoneconvertases 1 and 2) generates two pairs of C-terminal basicresidues: Lys-64/Arg-65 and Arg-31/Arg-32.181 These basicresidues are substrates of carboxypeptidase E and whenremoved result in a mature molecule of insulin. A mutationon Ser-202 to proline on carboxypeptidase E abrogates thecatalytic activity of the enzyme (also resulting in carboxypepti-dase E being degraded)182 and results in severe impairment ofmaturation of insulin (10−50% of correct processing comparedto wild-type), which in turn manifests as obesity and diabeticpathologies in mice models.1,183 It is clear that carboxypepti-dases have profound biological roles across a variety of differentorganisms, with functions that appear concerned with digestionof food and precise maturation of peptide hormones.

3.5. Ectodomain Shedding

Proteins involved in intercellular signaling and interactionbetween cells are expressed and translated as membrane-anchored proteoforms that are sensitive to proteolytic cleavageby an array of mainly membrane-bound proteolytic enzymes.Such ectodomain shedding modulates the function of thesubstrate protein, by activating precursor forms of, for example,cytokines and growth factors, directly and indirectly inhibitingsignaling by release of membrane-bound receptors into theextracellular space (negating signaling directly and alsofunctioning as soluble dominant negative proteins indirectly),or by affecting cell motility and binding by cleavage of proteinsinvolved in cell−cell and cell−matrix interactions. Followingcleavage, the cell-associated usually C-terminal protein remnantcan be internalized by endocytosis or further processing byintramembrane proteases such as presenilin-1 in the γ-secretasecomplex,184 leading to either degradation or intracellularsignaling events185,186 (Figure 9).Although, technically, most extracellular and membrane-

associated proteolytic enzymes could mediate ectodomainshedding and especially soluble187 and membrane-anchoredMMPs188,189 have been described to do so, the specializedproteases that this function is most attributed to are ADAMs.As discussed above, the ADAMs190,191 are multidomain,transmembrane glycoproteins (Figure 9) that are closelyrelated to the reprolysins. These snake venom integrin bindingproteins were discovered as fertilization factors when it wasreported that binding and fusion of guinea pig ovum and spermwere partially mediated by PH-20 or -30 (fertilin α andβ),192−195 proteins that were renamed ADAM1 and -2 after thediscovery and cloning of more related proteins. Besidesmediating cell−cell and cell−matrix interaction throughselective integrin binding via their disintegrin domains(reviewed in ref 196), about half of mammalian ADAMs(12/22 in human) contain a consensus HExxHxxGxxH zinc-binding motif in their metalloprotease domain, implicatingthem as potential active endopeptidases (reviewed in ref 197).



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As with other metzincins such as MMPs and ADAMTS’s,ADAMs can target and degrade extracellular matrix proteinssuch as collagens, laminin, and fibronectin,198,199 but thisfunction is secondary to their ability to cleave membrane-boundproteins.The “sheddase” activity of ADAM proteases was first

demonstrated when a disintegrin metalloprotease was describedas the main responsible protease for the release of active maturetumor necrosis factor alpha (TNFα) from its membrane-anchored precursor form.200,201 This process had already beenattributed to metalloprotease activity earlier, although thoughtto be due to MMPs.202,203 The discovery of ADAM17 or TNFαconverting enzyme (TACE) was the gateway to a multitude ofstudies investigating ADAM proteases and their substrates inhealth and disease, with ADAM17 receiving the most academicinterest, perhaps due to its potential therapeutic potential ininflammatory diseases. The last two decades of research haveyielded dozens of putative substrates such as cytokines andgrowth factors, their receptors, and adhesion molecules,204,205

although for many their physiological relevance remains unclearas many have been discovered via in vitro experiments andsignificant redundancy appears to exist for substrate sheddingbetween individual ADAMs.ADAM proteases play a pivotal role in regulation of select

cytokine signaling, as exemplified by the well-documentedrelease of TNFα by ADAM17. Although multiple ADAMs (e.g.,-9, -10, and -19) are capable of processing proTNFα in vitro,206

ADAM17 is the dominant factor in determining TNF responseupon stimulation, as shown by a lack of soluble TNFαproduction by stimulation of ADAM17-deficient monocytes,200

whereas other ADAMs, like the phylogenetically most closelyrelated ADAM10, may regulate constitutive production ofsoluble TNFα.207 ADAM sheddase activity is involved ininterleukin-6 (IL6) signaling through release of membrane-bound IL6 receptor (IL6R) that is mediated by ADAM10 and-17.208−210 Proteolytic release of the soluble IL6R, as opposedto shedding of TNF superfamily receptors, is not inhibitory butrather an important agonistic mechanism in pro-inflammatoryIL6 trans-signaling by binding to the membrane protein gp130on neighboring cells.211,212

Ectodomain shedding of growth factors involved inepidermal growth factor receptor (EGFR) signaling is largelyattributed to ADAMs. ADAM12, -10, and -17 process TGFαand heparin-binding EGF (HB-EGF) factor to their solubleforms, leading to activation of EGF receptor signaling.185,213

Whereas physiological shedding of growth factors is required inprocesses such as wound healing, tissue repair, and angio-genesis, dysregulation of the shedding and subsequent aberrantEGFR activation can lead to unwanted tissue remodeling, suchas in fibrosis,214 and uncontrolled cell proliferation and hastherefore been extensively implicated in the pathogenesis ofcancer.215,216

Several ADAMs can mediate cleavage and shedding ofamyloid precursor protein (APP) and may play a role in thepathogenesis of Alzheimer’s disease. APP can be processed bythree families of proteases with different biological outcomes:α-secretases such as ADAM9, -10, and -17 and meprin β;217 β-secretases BACE1 and -2; and presenilins in the γ-secretasecomplex.218,219 β-Secretase-mediated release of soluble APPand subsequent regulated intramembrane processing of theremnant by the γ-secretase complex yields an amyloidogenic Aβfragment that is implicated in the formation of amyloid plaquescausing Alzheimer’s disease, whereas shedding of APP byADAMs is mediated by a proteolytic cleavage within the Aβregion itself and yields less toxic fragments.219 This protectiverole of a clan of metalloproteases illustrates an very realdilemma in pharmaceutical intervention targeting proteases;many proteases have either protective or detrimental roles indifferent diseases, making rational use of inhibitors challengingand open to side effects.220,221

Besides cleaving APP, ADAM10 is known to be theresponsible sheddase for multiple other substrates in thecentral nervous system, implicating this protease as animportant regulator of neural development. ADAM10 wasfound to be the mammalian homologue to the Drosophilaprotein kuzbanian (KUZ), which cleaves and activates theNotch receptor, mediates release of the Notch receptor liganddelta, and has a crucial function in neurological development inthe fruit fly.222 In mice, knockout of Adam10 leads to lethalityin an early embryonic stage due to major defects in neurologicaldevelopment that bear a striking resemblance to Notchknockout models.223,224 Interestingly, Notch signaling itselfmay act as a positive feedback loop via transcriptional activationof furin, which in turn leads to enhanced activation of ADAM10(and others) by PC cleavage of the inhibitory prodomain.225

Because academic efforts in this area have frequently focused onelucidating the role and substrates of ADAM10 and -17, someother catalytically active ADAMs remain understudied to date,and further research is required to understand the intricate roleof ADAM-mediated shedding in health and disease.

3.6. Modulation of Biochemical Pathways and Signalingthrough Proteolytic Cleavage

Targeted precision proteolysis is key in the initiation andregulation of many biochemical processes and pathways, e.g.,the well-documented limited-cleavage events in the coagulationcascade226 (Figure 10) and complement activation cascade227

(Figure 11), but also has important roles in many otherbiological processes.Proteolysis by the caspase family of cysteine endopeptidases

is a major regulator of cell death, through either activation ofthe immunologically silent apoptosis or necroptosis (reviewedin refs 228 and 229). Apoptosis can be initiated via two distinct

Figure 9. Schematic representation of ectodomain shedding mediatedby ADAM proteases. Proteolytic cleavage mediated though the activeADAM metalloprotease domain in the membrane-bound substrateproximal to the membrane releases the extracellular fragment, leadingto activation of membrane-anchored proproteins, abrogated inter-action of the ADAM-expressing cell with neighboring cells or theextracellular matrix (ECM), or regulated intramembrane proteolysis ofthe C-terminal remnant. C, C-terminal domain; TM, transmembranedomain; EGF, epidermal growth factor-like domain; CYS, cysteine-richdomain; DIS, disintegrin domain; MP, metalloprotease domain.



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pathways: the intrinsic pathway is activated upon cellular stresssuch as excessive reactive oxygen species formation or DNAdamage, and the extrinsic pathway relies on activation of deathreceptor complex formation upon ligation of the appropriatereceptor with, e.g., Fas or TNFα. In the intrinsic pathway,proapoptotic effectors in the Bcl2 family permeabilize themitochondrial outer membrane, leading to release ofproapoptotic factors such as cytochrome c and secondmitochondrial activator of caspases (SMAC, or direct IAP-binding protein with low pI (DIABLO)), which subsequentlyenable formation of a large apoptosome complex that leads todimerization and proteolytic activation of caspase-9. Caspase-9activates the effector proteases caspase-3, -6, and -7 that cleave alarge repertoire of substrates, leading to cell death. Undernormal conditions, death receptors such as TNF-R are presentin large signalosomes with antiapoptotic factors such as theinhibitors of apoptosis (IAP1 and -2) and linear ubiquitinchains polymerized by linear ubiquitin assembly complex(LUBAC) on RIP1 that keep apoptosis in check.230 In theabsence of antiapoptotic mediators in the signalosome, ligationwith a death-inducing ligand leads to homodimerization andproteolytic activation of caspase-8 that in turn activates effectorcaspases. Caspase-8 also cleaves Bcl-2 interacting domain(BID), leading to a cross-talk into the intrinsic pathway bycausing release of cytochrome c from mitochondria. Signalingthrough receptor pathways that can cause apoptosis is acomplex biological system, since depending on cofactors andstimuli many different outcomes are possible. Ironically, partialactivation of caspase-8 by dimerization with the caspase-like

domain of a truncated form of c-FLIP (cellular FLICE-likeinhibitory protein) upon ligation of the T cell receptor with anantigen does not cause apoptosis yet is required for T cellsurvival and proliferation.231

Caspases have a strong to even exclusive preference for anaspartate residue in the P1 position yet seem to rely on otheradditional features for substrate specificity as shown by limitedproteolysis of substrates in vitro.232 Several large-scale profilingstudies of substrates of caspase-mediated proteolysis duringinduced apoptosis have revealed a plethora of substrates,233−235

but the question remains which of these cleavage events arecritical in initiation and sustenance of the pathway and whichare mere victims of bystander proteolysis. Interestingly, a veryin-depth analysis of the apoptosis interactome and itsrelationship with proteolytic disassembly revealed that caspasecleavage of substrates in early apoptosis occurs afterinteractome disassembly and is not the cause of breakup ofessential protein/protein interactions.233 This was unexpectedas the dogma prevailing in the field has been that caspaseactivity disassembles protein complexes, leading to terminationof cellular functions. Thus, it appears that protein/proteininteractions may mask caspase-cleavage sites or stericallypreclude caspase access and that caspase cleavage occursupon isolated proteins following dynamic protein exchange orcomplex remodeling.

Figure 10. Schematic representation of the coagulation cascade wherea series of sequential proteolytic cleavages of serine protease zymogensactivates them, allowing the cascade to progress to the next tier. Theintrinsic pathway is activated upon exposure of blood to negativelycharged exposed surfaces and depends on proteolytic activation offactors XII, XI, and IX. The extrinsic pathway is activated whenvascular tissue damage leads to activation of factor VII. The pathwaysconverge at proteolytic activation of the endopeptidase factor X (alsoknown as Stuart-Prower factor), which is capable of convertingprothrombin into its active enzyme form that subsequently activatesfactor XIII and forms a positive feedback loop into the intrinsicpathway. Thrombin also cleaves and activates fibrinogen, allowingactivated factor XIII to form a cross-linked polyfibrin clot.

Figure 11. Schematic overview of the complement activation cascade,which is dependent on a series of sequential activations of, and by,serine endopeptidases. Activation can occur via three distinctpathways: in the classical pathway via formation of a C1 complexupon antibody−antigen ligation, in the mannose-binding lectin (MBL)pathway upon formation of an MBL-mannose-binding lectinassociated serine protease (MASP) complex, or in the alternativepathway by direct activation of factor C3 via an activating surface. Allpathways ultimately lead to the formation of a membrane attachcomplex (MAC) that leads to lysis of the compromised cell andremoval of the pathogen.



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3.7. Precision Proteolysis in Immunity

Proteolytic activity is key in the regulation and execution ofseveral pathways in innate and adaptive immunity. Serineproteases contained in secretory vesicles in immune cells, suchas granzymes in cytotoxic lymphocytes or neutrophil elastase inneutrophils, are involved in the immune response to viral(reviewed in ref 236) and bacterial pathogens (reviewed in ref237). Although the exact details remain under debate,238 theconsensus emerging is that, upon specific recognition of a viralantigen presented through the major histocompatibilitycomplex on the surface of an infected cell by T cell receptorsof cytotoxic CD8+ T cells or activation of NK cells throughsignals from infected cells, secretory vesicles containinggranzyme proteases and the protein perforin are mobilizedand secreted into the extracellular space. This leads togeneration of perforin pores in the membrane of the infectedcell large enough to enable translocation of secreted granzymeinto the cytosol of the target cell.239,240 Internalized granzymeB (GrzB) subsequently activates classical apoptosis by a specificactivating proteolytic cleavage of procaspase 8, which initiatesthe apoptotic cascade.241 GrzB further performs a specificcleavage in the protein BID, thereby directly activatingmitochondrial apoptosis.242,243 This seemingly elegant systemdoes not tell the full story though, and knockout experimentshave demonstrated that other members of the granzyme familysuch as GrzA and GrzM are involved in the process.244,245 Theexact importance of each protease and their substrates may bedependent on thus far unknown factors, is likely variablebetween species, and will require more study to elucidate thefull mechanism.Activation of B and T lymphocytes after ligation of their

surface-expressed antigen receptor with the correspondingantigen leads to a rapid mounting of a potent adaptive immuneresponse. After activation, a series of specific phosphorylationand ubiquitination events leads to activation of the transcriptionfactor nuclear factor kappa-B (NF-κB) and subsequenttranscription and translation of a multitude of proteins involvedin inflammation, lymphocyte differentiation, and prolifera-tion.246−248 One of the signaling nodes transducing the antigensignal down to NF-κB activation consists of a trimeric complexof caspase recruitment domain family-11 (CARD11), B-celllymphoma/leukemia-10 (BCL10), and MALT1, termed theCBM.249 MALT1, the only paracaspase in humans, is a cysteineprotease homologue of the metacaspases found in yeast andplants124 that, in addition to the scaffolding function within theCBM complex, cleaves a very specific repertoire of substrates inlymphocytes that regulates the NF-κB response after antigenreceptor ligation (reviewed in ref 250). Although MALT1contains a consensus active site, it took until 2008 for the firstsubstrates to be discovered, when MALT1 was shown to cleaveBCL10 after Arg228 in the C-terminal region, a cleavage that isimportant during T cell activation although not required forNF-κB activation upon T cell receptor ligation.251 At the sametime, a publication showed the negative regulator of NF-κBcalled A20 to be cleaved by MALT1 upon T cell activation,indicating MALT1 proteolytic activity enhances or fine-tunesNF-κB activation in lymphocytes by removing negativecheckpoints.252 Since these discoveries, MALT1 has beenshown to cleave a handful of other negative regulators ofcanonical NF-κB activation in activated lymphocytes such asthe deubiquitinases A20252 and CYLD,253 as well as RelB,254

Regnase-1,255 and Roquin-1 and -2.256 Recently, using TAILS,an unbiased N-terminomics proteomics analysis of MALT1-

deficient B cells from a human patient, we identified a specificcleavage by MALT1 in ranBP-type and C3HC4-type zincfinger-containing protein 1 (RBCK1), better known as heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1).257 This cleavage waslater confirmed by others.258,259 HOIL1 associates withHOIL1-interacting protein (HOIP) and Shank-associated RHdomain-interacting protein (SHARPIN) to form the linearubiquitin chain assembly complex that mediates formation of aspecial head-to-tail, linear polyubiquitin linkage that hasimportant roles in the immune system.260−264 Upon cleavageby MALT1, the C-terminal domain of HOIL-1 dissociates fromLUBAC, which abrogates the formation of linear polyubiquitin,thus terminating NF-κB activation, and desensitizes cells fromreactivation for at least 2 h.257 This showed for the first timethat MALT1 proteolytic activity acts as a late negative regulatorof NF-κB at later time points, illustrating the complex andpleiotropic nature of this unusual protease in immune cellactivation and again highlighting the difficulties in drugdevelopment from incompletely validated drug targets.MMPs are a class of protease that has revealed unpredicted

and unexpected roles that span beyond their well-recognizedmatrix remodeling functions. As their names suggest, the firstefforts to identify substrates of MMPs were strictly focused onextracellular matrix proteins such as the collagens.265 Recently,MMPs have been demonstrated to be immune regulators bymodifying, through the activation or inactivation of chemo-kines, cytokines, cytokine-binding proteins, signaling pathways,protease inhibitors, and cell-surface receptors.266−268

MMP2 was one of the first examples of an MMP modulatingthe function of a chemokine through the proteolytic removal ofthe first four N-terminal amino acids (1QPVG4) of monocytechemoattractant protein (MCP) 3 or CCL7 (chemokine (C−Cmotif) ligand 7); this inactivation of MCP3 resulted in anantagonistic effect for CC-receptors, leading to a loss of calciumsignaling and chemotactic activity in vivo.269 Similarly, the firstfour amino acids (1KPVS4) of another chemokine, stromal cell-derived factor 1 (SDF-1α and β/CXCL12), were cleaved byseveral MMPs (MMP1, -2, -3, -9, -13, and -14), resulting in aloss of signaling by its cognate receptor CXCR (C-X-Cchemokine receptor type)-4.270 In fact, this cleavage switchedreceptor binding to the related CXCR3, again highlighting theimportance of precision proteolysis as a potent posttranslationalmodification that profoundly regulates protein function.271 It isnow known that MMPs cleave most if not all human andmurine CXC and CC chemokines, thereby affecting virtually allaspects of leukocyte migration, chemotaxis, activation orinactivation, and signaling.272−274

Cytokines such as the interferons are critical during antiviralimmunity.275 During coxsackievirus type B3 or respiratorysyncytial virus infections, macrophage metalloelastase(MMP12) processes interferon-alpha (IFN-α) at the C-terminus at two distinct sites (157leu↓Gln158 and 161Leu↓Arg162) that are critical for binding IFN-α receptor-2 andmediating downstream signaling.276 MMP12 was also demon-strated to have moonlighting roles by binding to the NFKBIA(NF-kappa-B inhibitor alpha) promoter (encoding for inhibitorof NF-kappa-B alpha or IκBα) to activate IκBα in order tomediate the secretion of IFN-α from viral-infected cells.276

Importantly, the intracellular and extracellular regulation ofIFN-α is specific as MMP12 is unable to cleave other type Iinterferon cytokines such as IFN-β or bind its promoter.276 Inaddition to being implicated in antiviral immunity, MMP12 wasdemonstrated to play a protective role in peritonitis and



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rheumatoid arthritis by the inactivation of several complementcomponents: C3, C3b, iC3b, C3a, C5a, C4, and C4b.277 Theactivation of C3 to C3b leads to the covalent binding andopsonization of target cell surfaces, resulting in cell lysis.278 Thecleavage by MMP12 reduces the proinflammatory activationeffect of complement, and C3b-tagged cells no longer inducecell lysis.277 Also, MMP12 generates more potent phagocytosisenhancers through the processing of C3b and iC3b. Other C3or C5 cleavage products include C3a or C5a anaphylatoxinsserving to reduce their chemoattractant activities277,279 and areimplicated in the augmentation of vascular permeability.Notably, MMP12 cleavage of C3a (740Arg↓Ala741) and C5a(746Asp↓Met747) results in the reduction of calcium flux, leadingto a reduction of neutrophil recruitment and macrophageinfiltration.277

The protease-activated receptors (PARs) are an importantclass of G protein-coupled receptors and have been implicatedin cancer and disorders of the cardiovascular, musculoskeletal,gastrointestinal, respiratory, and central nervous system.280

PAR1 is required to enhance growth and invasion of breastcarcinoma cells in a murine xenograft model, and MMP1,secreted from proximal fibroblasts or platelets, is an agonist ofPAR1 by inducing Ca2+ signaling, Rho-GTP/MAPK (rho-guanosine triphosphate/mitogen-activated protein kinase 1)pathways, and cell migration through the cleavage of a uniquetethered ligand sequence (41PRSFLLRN47).281,282 Therefore,MMPs not only regulate the remodeling of the matrix bycleaving extracellular matrix (ECM) proteins but also influence(activation or inactivation) diverse immune responses throughthe targeted processing of multiple chemokines, cytokines, andcell-surface receptors.These specific examples highlight clearly the importance of

precision proteolysis in regulating signaling and other bioactiveproteins as a critical posttranslational modification. We pointout again that proteolysis is one of the few posttranslationalmodifications regulated by cells after proteins are secreted fromcells, and thus it is critical to have knowledge of the N- and C-termini of proteins in order to correctly formulate hypothesesregarding the function of a protein and the system.

4. ANALYTICAL APPROACHES FOR PROTEASES ANDPROTEOLYTIC PROCESSING

Uncontrolled proteolytic cleavage has strong detrimentalphysiological effects, and therefore protease activity is subjectto multilayered posttranslational regulation. Proteases are oftenexpressed and translated as inactive zymogen proforms thatrequire an activation step before being able to processsubstrates, either by allosteric or proteolytic removal of aninhibitory prodomain or by other posttranslational modifica-tions such as phosphorylation or ubiquitination. Furthermore,colocalization of a protease and its substrate can be dependenton specific stimuli, and an arsenal of endogenous proteaseinhibitors keeps proteolysis in check. This strong regulation ofproteolytic cleavage poses a challenge for analysis of proteasesand their targets because mere quantification of expressionlevels or protein abundance does not necessarily reflect thebiological impact of the protease. To overcome this short-coming of traditional transcriptomic and proteomic techniquesin studying proteolysis, the development of targeted and morefunctional analysis methods termed degradomics has beeninstrumental in enhancing our understanding of proteolyticcleavages in health and disease.2

4.1. Monitoring Substrate Cleavage

Perhaps the most straightforward approach to analysis ofproteolytic activity is in direct in vitro monitoring of hydrolysisof (putative) substrates by the protease of interest. Cleavagecan be visualized by gel electrophoresis or mass spectrometry,and synthetic quenched fluorogenic peptide probes based onfluorescence resonance energy transfer (FRET) or peptideslabeled with gold nanoparticles that emit a specific signal onlyafter hydrolysis of the peptide are now used for basiccharacterization of in vitro cleavage site specificity of theprotease283−286 and are reviewed in ref 287. Several approachesusing extended synthetic peptide libraries combined withsequencing or mass spectrometric analysis and bioinformaticsanalysis of the cleavage sites have been described.288−291

Proteomic identification of protease cleavage sites (PICS)292,293

utilizes peptide libraries generated though controlled proteol-ysis of endogenous bacterial or human proteomes withenzymes like trypsin, GluC, chymotrypsin, or LysargiNase.After chemical blocking of reactive thiol and amine groups, thepeptide pool is incubated with the protease of interest, wherehydrolysis of the peptides reveals a free α-amine group on theC-terminal peptide remnant that is amendable to labeling andenrichment. After LC-MS/MS (liquid chromatography-tandemmass spectrometry) identification of the enriched peptideremnants, the nonprime peptide sequence can be identifiedbioinformatically, leading to in-depth profiling of the cleavagesite specificity of the studied proteases.32,292,294

Zymography is the umbrella term for methods visualizingprotein substrate conversion by proteases (reviewed in ref 295).Zymography yields qualitative and, if performed within thelimited linear range of the assay, also quantitative informationabout protease and proteolytic cleavage, and visualization canbe performed in SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) gels on tissue sectionsand in intact organisms. Gel zymography was originallyperformed by submitting samples containing the putativeprotease of interest to electrophoretic separation based onmolecular weight using SDS-PAGE, followed by (partial)reactivating of the protease by removal of SDS by nonionicdetergents and overlay of the gel with an indicator gelcontaining a cleavable substrate (e.g., fibrin-agar for visual-ization of active plasmin).296 Improved methods directlyincorporated a protein substrate such as gelatin in the SDS-PAGE gel matrix, where gelatinase (MMP2 and -9) activityleads to clear bands on a bright gel background after Coomassiestaining, yielding exquisite sensitivity and information aboutdifferent catalytically active proteoforms in the sample,provided proteolysis leads to sufficient cleavage of the substrateto small enough peptides to allow diffusion from the gel.297

Thus, one or two cleavages only will not be detected. Althoughseveral improvements and variations on the basic premise havebeen published, such as using in-gel zymography for testinginhibitory antibodies incorporated in the gel, 2-dimensionalelectrophoresis for enhanced resolution, and transfer zymog-raphy for monitoring multiple substrates from the same gel(reviewed extensively in ref 295), the main limitation of thistechnique lies in the fact that little quantitative and only limitedqualitative information about the proteolytic potential in vivo isprovided; noncovalent complexes of proteases and theirendogenous inhibitors (e.g., gelatinases and tissue inhibitorsof metalloproteases (TIMPs)) are dissociated during electro-phoresis so the zymogram will inevitably overestimate theproteolytic activity present. Finally, novices are often confused



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by the molecular weights of the protease forms in zymograms.As the analyses are performed and analyzed under nonreductiveconditions, the presence of any disulfide bonds in the proteasewill cause the protease to electrophorese with a lower apparentmolecular weight than that if reduced. Finally, proteaseprodomain electrophoretic bands will show activity on gels ifSDS activates the enzyme or if the allosteric pressure exerted bythe refolding procedure leads to protease autoactivation.However, this occurs in situ in the gel and so, despite a lossof the propeptide in gel, the proform is also revealed as anactive protease, although it must be considered as an inactivezymogen form.

4.2. Substrate- and Activity-Based Probes

Although in vitro cleavage monitoring of natural and syntheticsubstrates provides valuable kinetic information, visualization ofprotease activity under physiological conditions is required toinfer and validate biology. To selectively analyze activeproteases, several research groups have dedicated their effortsto synthesize and utilize selective, substrate-based probes(SBPs) and activity-based probes (ABPs) for different proteaseclasses (reviewed in refs 15, 298, and 299). SBPs are used in insitu and in vivo zymography to selectively visualize proteolyticcleavage in tissue sections or living intact small animals usingeither directly fluorescent or the more popular and sensitiveinternally quenched fluorescent probes that give a positive ornegative fluorescent signal upon cleavage of the probe by theprotease of interest.300,301 Although SBPs are powerful tools tostudy proteolytic cleavage in tissue and even in vivo,redundancy between enzymes, which is observed in manyprotease classes, makes unambiguous interpretation of theacquired data difficult. To overcome this issue, ABPs have beendeveloped that irreversibly modify the active protease and allowpostlabeling identification of the actual enzyme involved, e.g.,by mass spectrometry.Commonly, ABPs contain a substrate-like peptidomimetic

backbone sequence optimized for recognition by the protease(class) of interest, an electrophile “warhead” that mediates anucleophilic attack upon cleavage of the peptide backbone,which leads to covalent labeling of the active site, and a reportergroup that can be a fluorophore for visualization of a group foraffinity enrichment of the labeled protease such as biotin or viaclick chemistry302 (Figure 12). Visualization of labeled proteaseusing SDS-PAGE, fluorescence microscopy, mass spectrometry,and in vivo imaging provides qualitative and quantitativeinformation about the activity status that was present in vivounder the experimental conditions, making these probesextremely powerful tools to study proteolysis.The ABP approach has been used extensively and

successfully to analyze activity of cysteine-, serine-, andthreonine proteases. Because catalysis is mediated by anamino acid residue in the catalytic site that functions as anucleophile, probes containing a directly reactive electrophilelabel the catalytic residue in a true activity-dependent manner.Many chemical structures have been used as electrophile inboth irreversible inhibitors of these protease classes and ABPs,e.g., activated ketones, phosphonylating and sulfonylatinggroups, epoxides, and Michael acceptors (reviewed in ref298). Besides obviously valuable application in fundamentalcharacterization of activity of a specific protease, a promisingapplication of ABPs lies in in vivo labeling strategies for in situvisualization of proteolytic activity in, e.g., inflammatory

diseases and cancer, where aberrant proteolytic activitycontributes to the pathology.303−305

Designing ABPs for protease classes that are not dependenton active-site nucleophile amino acids for substrate hydrolysis(metalloproteases and aspartyl proteases) has proven morechallenging. Covalent labeling is achieved by incorporating aphotoactivatable cross-linker group into the probe that forms areactive radical species and covalently attaches to catalytic-siteresidues upon irradiation of the sample with UV light. Severalgroups have employed this approach to profiling of metzincinproteases such as MMPs and ADAMs, but low labeling yieldsand nonspecific labeling seem to have made this approachlargely redundant.135,306−308 The use of nanomolar noncovalentprotease inhibitors affords one venue to visualize such proteasesthat lack a covalent acyl intermediate in catalysis, but these canonly be used under native conditions, e.g., in vivo imaging, andnot denaturing SDS-PAGE gels. The lower amounts of proteinthat can therefore be labeled necessitates highly sensitivedetection systems, and this has driven the development ofnoncovalent probes for positron emission tomography ofmetalloproteases using the potent, low-nanomolar inhibitorMarimastat as the label-accepting probe.309,310

A variant method for enrichment of active proteases fromcomplex samples also utilizes reversible inhibitors containingpeptidomimetic recognition sequences for selectivity that areimmobilized on a solid support or resin and can be applied asan affinity matrix to selectively enrich proteases in their active,noninhibited form. This method has been used to purify activeforms of collagenases using a weak metalloprotease inhibitorwith micromolar potency (Pro-Leu-Gly-hydroxamate),311 andoptimization of the inhibitor backbone structure has yieldedaffinity materials that have been used to enrich and analyzeMMPs and ADAMs with limited success in biologicalsamples.312−317

4.3. Proteomics-Based Approaches for Substrate Discovery

As the function of a protease is determined by the substrates itcleaves, obtaining a comprehensive picture of these substratesunder specific conditions is required to interpret the biologicalsignificance of proteolysis. Proteomics provides a toolkit forunbiased qualitative and quantitative profiling of proteins and

Figure 12. Schematic overview of the activity-based probe (ABP)approach for selective analysis of protease activity and active proteases.A proteinaceous sample of interest (lysate, living cells, and tissue) isincubated with an ABP that consists of a peptide-like linker regionspecific for the protease (class) of interest, an electrophilic reactivegroup (warhead) for covalent linkage of the probe to the activeprotease, and a reporter moiety for visualization (e.g., a fluorophore)or affinity enrichment (e.g., biotin). In the case of serine and cysteinepeptidases, cleavage of the probe by the active protease leads tocovalent derivatization of the catalytic residue with the probe, allowingactivity-specific analysis after gel electrophoresis, or pull-down of thelabeled protein and analysis by mass spectrometry.



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proteoforms present in a biological sample, but typicallyproteolytic cleavages are elusive to regular “shotgun”proteomics experiments. As with proteases themselves, merequantification of the level of a substrate in a sample provideslimited information; stable cleavage fragments will still bepresent, and the probability of identifying cleavage sites isstatistically unlikely. To overcome this problem, methodsselectively analyzing cleaved substrates have emerged andbroadened 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-popularmethod of 2-dimensional gel electrophoresis, which separatesproteins based on isoelectric point and size,321 followed byexcision of spots of interest, in-gel digestion with trypsin, andmass spectrometric identification using either MALDI-TOF(matrix-assisted laser desorption/ionization time-of-flight) orLC-electrospray ionization (ESI)-MS/MS.322 In a modifiedprotocol termed 2-dimensional difference gel electrophoresis(2D-DIGE), protein samples corresponding to differentexperimental conditions (e.g., protease-treated and control)are differentially labeled with fluorescent dyes, combined, andresolved on a 2D polyacrylamide gel.323 Cleaved substrates arevisualized as decreasing intensity of colored spots correspond-ing to the full-length proteins in the protease-treated sampleand concurrent appearance of lower-molecular-weight frag-ments. Both spots can be excised and identified by massspectrometry.The emergence of more powerful and faster mass

spectrometers enabled in-depth coverage of proteins in excisedslices of 1D gel electrophoresis of unfractionated samples,which made it possible to avoid the finicky 2D electrophoresisand laborious picking of (many) individual spots. Originallypioneered in 2002 by Guo et al.,324 this technique was greatlyenhanced by developing easily accessible software and othertechnical advances leading to the development of PROTOMAP(protein topography and migration analysis platform). Asbefore, this is a method that is based on standard SDS-PAGEresolution of protein samples harvested from cells whereproteolytic activity occurred or was induced (e.g., apoptoticJurkat cells) followed by LC-MS/MS identification andquantification by spectral counting of proteins extracted frommultiple gel slices.325,326 By bioinformatically combining massspectrometric identity information with molecular-size in-formation obtained from the electrophoresis data, putativesubstrates can be identified. Although this method gives anindication of the approximate position in the protein sequencewhere the cleavage occurred, the limited resolution of mass ingel electrophoresis combined with the low probability ofidentifying the tryptic peptide of the cleavage site makesunambiguous identification of the exact point of cleavageunlikely.4.3.1. N-terminal Proteomics. Selective analysis of

cleaved protein substrates and cleavage sites is the mostoptimal scenario to elucidate the comprehensive degradome ofa certain protease, but this approach faces similar challenges asmethods for analysis of other posttranslational modifications(PTMs) such as phosphorylation and ubiquitination, whichrequire specific enrichment of modified peptides or proteins forsuccessful profiling. At first glance, labeling of cleaved proteinsat their N-terminal α-amino group followed by positiveselection of the labeled fraction seems like an attractive option,but, although differences in acid-dissociation constant exist,

these are minor and specific chemical labeling of the N-terminalα-amine without side reaction on ε-amino groups on lysineresidues in the protein is far from trivial, although severalgroups have attempted this but with low fidelity (Figure 13).One approach for positive selection after labeling of N-

terminal amines utilizes a subtiligase enzyme to selectively label

Figure 13. Schematic overview of degradomics approaches usingpositive selection of N-terminal peptides. A proteome sample isacquired after activation of the protease of interest (e.g., uponinduction of apoptosis). The sample consists of full-length, uncleavedproteins and fragments resulting from proteolysis containing neo-N-terminal protein ends. Positive selection of N-terminal peptides (neo-and natural) can be achieved by enzymatic selective labeling of the N-terminal α-amine, attaching a tobacco etch virus (TEV)-linked biotingroup (method I). After digestion of the proteins with trypsin andbiotin-(strept-)avidin affinity purification, the N-terminal peptides arecollected by TEV protease cleavage and are amendable to massspectrometric identification, allowing exact identification of theproteolytic cleavage sites on a large scale. A chemical approach forpositive selection utilizes the difference in pKa between N-terminal α-amines and lysine side-chain ε-amines. By performing a guanidinationat high pH, selective labeling of ε-amines is achieved (method II). Thisallows for chemical derivatization with biotin of the N-terminal α-amines with standard N-hydroxysuccinimide chemistry, followed byenrichment of the N-terminal peptides using (strept-)avidin and massspectrometric identification of proteolytic cleavage sites. Method IIIuses N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) as areagent for selective labeling of the N-terminal α-amines, followed bytryptic digestion and sample fractionation by reversed-phasechromatography to enrich for TMPP-labeled peptides. A furtherenrichment of N-terminal peptides is achieved by affinity enrichmentusing an α-TMPP antibody, allowing subsequent mass spectrometricidentification of cleavage sites.



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α-amino groups in a proteome sample with a biotinylated shortpeptide 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 withTEV protease, followed by LC-MS/MS identification of thelabeled peptides from which putative substrates can beinferred.234,327 Another approach uses the difference in pKabetween 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 biotinylationreagent, extraction of the biotinylated peptides with immobi-lized avidin, and elution of the peptides under reductiveconditions.328 In a comparable approach using selective labelingof N-terminal α-amines at high pH with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), N-terminalpeptides can be enriched using an antibody that recognizesTMPP-labeled peptides that can be extracted with magneticbeads prior to mass spectrometric analysis.329,330

Because of the technical difficulties of specifically labeling α-amines and the inherent lower high-accuracy coverageobtained, the majority of methods for targeted analysis ofprotein N-termini now rely on the opposite principal: depletionof tryptic, internal peptides to effectively lead to negativeenrichment of N-terminal peptides. Although several methodshave been published in the past decade, the basic premise isalways to chemically block all primary amines in the sample ofinterest at the protein level, either by N-hydroxysuccinimide(NHS)-activated reagents or reductive amination (also knownas reductive alkylation) with an aldehyde using cyanoborohy-dride as a catalyst.331 By labeling at the whole-protein level bydefinition then, any N-terminal labeled peptide had to havebeen present and exposed in the sample prior to labeling. If thesample was handled with appropriate care using proteaseinhibitors and taking precautions to avoid proteolysis onsample preparation, then such labeled N-termini on peptidesrepresent either mature protein N-termini or protease-generated neo-N-termini. After incubation with trypsin todigest the proteins into LC-MS amendable peptides, the newlygenerated internal (i.e., nonterminal), tryptic peptides that arenot the N-terminome are removed from the sample, leading tonegative enrichment of the blocked N-termini.One major advantage of this approach over the positive-

selection methods lies not so much in the discovery of cleavedsubstrates but in that it allows for comprehensive analysis of theN-terminome in the sample, including naturally occurring N-terminal modifications such as methylation, acetylation, andfatty acid derivatization (Figure 14) that also have importantbiological implications on protein function.179 The chemicalblocking step provides an ideal opportunity for isotopicdifferential labeling and multiplexing of samples (e.g.,protease-treated versus control, or different patients, ordifferent stimulation conditions) by using stable isotope-labeledreagents such as 13C- or deuterium-labeled acetate orformaldehyde, or isobaric proteomics tags such as iTRAQ(isobaric tags for relative and absolute quantitation) or TMTs(tandem mass tags). Another advantage is that the natural N-terminal peptides can be used to statistically determine thevariation within and between experiments and hence used tostatistically discriminate the neo-N-termini derived from theprotease of interest from background proteolysis present in

both the protease-containing and control samples as a high-ratio peptide.Approaches that have been used to deplete the sample of

internal peptides include immobilization of free aminecontaining peptides with carboxylic acid functionalizedpolystyrene beads332 or aldehyde-functionalized POROSbeads using reductive amination,333 biotinylation of free α-amines with NHS biotin and depletion with avidin beads,334,335

and phospho-tagging (PTAG) internal peptides via reductiveamination with glyceraldehyde-3-phosphate, followed bychromatographic separation of phosphorylated internal pep-tides from the N-termini of interest using a titanium dioxideaffinity column.336 However, an inherent problem with bead-based approaches is nonspecific binding of peptides to thesemedia, leading to false positives or saturation of MS analyses.Although many methods have been published and have been

shown to function with varying degrees of reliability andvalidation, two distinct workflows have become more widelyused than othersterminal amine isotopic labeling ofsubstrates (TAILS337,338), combined fractional diagonalchromatography (COFRADIC339,340), and the variant charge-based fractional diagonal chromatography (ChaFRA-DIC341−343) (Figure 15). COFRADIC relies on chemicalmodification of the hydrophobicity of internal peptides andremoval by reversed-phase chromatography, leading to negativeenrichment of N-terminal peptides. All amines are initiallyblocked by acetylation, followed by digestion of the samplewith trypsin. The sample is subsequently prefractionated bystrong cation-exchange (SCX) chromatography, which neg-atively enriches for the acetylated N-termini (both natural aschemically modified) due to their low affinity for the SCXmaterial caused by the difference in dissociation constantcompared to unblocked, internal peptides. The enrichedpeptides are fractionated further by C18 chromatography, and

Figure 14. Schematic overview of the most common biologicallyoccurring chemical modification of the N-terminus (a) and C-terminus(b) of proteoforms.



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



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each fraction is labeled with 2,4,6-trinitrobenzenesulfonic acid(TNBS), which increases the hydrophobicity of the remaininginternal peptides, allowing their efficient removal throughreversed-phase fractionation. In general, COFRADIC leads tohigh numbers of samples due to the extensive sequentialfractionation steps. A recent variant of COFRADIC termedChaFRADIC simplifies the approach and massively reduces theamount of sample required for analysis to the low-microgramrange. ChaFRADIC utilizes an optimized SCX fractionation toseparate peptide fractions based on their charge state, followedby acetylation of internal, tryptic peptides, which reduces thecharge state. During a second SCX fractionation on each of theoriginal samples, nonterminal peptides will display a shiftedretention profile, and by only selecting the fraction that did notexhibit charge shift, enrichment of blocked N-terminal peptidesis achieved.342

On the other hand, TAILS is much simpler and faster andcircumvents the high peptide losses inherent to chromatog-raphy-based approaches by the development of a unique ∼100kDa aldehyde-functionalized soluble hyperbranched polyglycer-ol polymer (HPG-ALD) that is commercially available (http://flintbox.com/public/project/1948). TAILS relies on blockingall primary amines at the protein level using either reductiveamination with isotopically labeled formaldehyde or labelingwith isobaric NHS tags such as iTRAQ or TMT. After mixingof differentially labeled samples and digestion of the proteinswith trypsin, GluC, or LysargiNase, the internal tryptic peptidesare depleted by binding to HPG-ALD via reductive amination.The polymer with bound internal peptides can be convenientlyremoved by filtration through a molecular-weight cutoff filter,leading to enriched N-termini (generally >95%) in anunfractionated sample that can be analyzed by LC-MS/MS. Abioinformatics suite containing CLIPPER344 and database toolsTopFIND345 and TopFINDer346 is available for further analysisof the obtained data. TAILS has been successfully employed tostudy the proteolytic landscape in several cell-based models andto date remains the only N-terminomics approach reported tobe capable of in vivo tissue N-terminomics, including inflamedskin9 and arthritic ankle joints,277 antiviral responses,276 apancreatic cancer model,347 and lymphocytes from an immune-deficient patient.257 One of the major mysteries in regards toproteolytic cleavage is the extent and genesis of neo-N-terminiin any given tissue.348 N-terminal proteomics studies regularlyfind that the majority of protein termini are the result ofnonannotated proteolytic cleavage, and so questions ariseincluding which proteases are responsible for this and what arethe biological implications of these cleavages.179,349

4.3.2. C-terminal Proteomics. Detection of the natural Cterminus of a protein or the proteolytically processed end of aprotein is not a trivial matter when employing mass-spectrometric approaches. Conventional shotgun proteomics

relies on the digestion of entire lysates with a site-specificenzyme, commonly trypsin, and then fractionating the resultingpeptides prior to injection into the mass spectrometer andsubsequent identification. While there are significant advantagesto fractionation at the peptide level and the identification ofthose peptides, there is loss of protein context.350 Isoforms andproteolytically generated protein fragments are usually lost inthe vast numbers of internal tryptic peptides due to the fact thatthe peptides that can differentiate isoforms or protein fragmentsare fewer in abundance and often have less amenableionization/fragmentation properties. Trypsin is commonlyused due to its strict substrate specificity, which generates C-terminal arginines and lysines, both basic residues. Thepresence of these amino acids confers tryptic peptides excellentfragmentation patterns, as it will preferentially generate stable y-ions as opposed to lesser stable b-ions when using collision-induced dissociation (CID).351 Peptides derived from C-terminal regions of proteins are not always fully tryptic. Over80% of proteins annotated in the human proteome (Swiss-Prot) do not have a C-terminal basic residue, making themsemitryptic if digested with trypsin. Consequently, identifica-tion of C-terminal peptides is a particular challenge as C-terminal peptides, while perhaps being the most specificsequences to a protein,352 (a) are in significantly less abundancethan internally generated fully tryptic peptides and (b) haveimpaired fragmentation properties relative to fully trypticpeptides. To overcome the second issue listed here, newenzymes have been introduced to proteomic workflows inorder to identify C-terminal peptides. Namely, the proteasesLys-N353 and LysargiNase40 are now available to digestproteomes and enhance C-terminal peptide identification.LysargiNase was demonstrated to improve the identificationof C-terminal peptides in a human breast cancer model. Asopposed to trypsin, Lys-N and LysargiNase both generate apositively charged N-terminus, which generates b- and a-ionladders, which greatly assist in peptide identification bydatabase search algorithms.351 Despite the advances providedby Lys-N and lysargiNase as proteome-digestion alternatives,the caveat of abundant internal peptides still remains, andsimilarly to other post-translationally modified peptides, C-terminal peptides require enrichment for in-depth profiling.As opposed to other post-translational modifications where a

specific chemical group is added at functional sites on peptidesand proteins, proteolytic processing does not have a singlechemical group that allows for affinity purification. While thereare methods to enrich N-terminal peptides, the chemistry forisolating C-terminal peptides is not as straightforward.COFRADIC, discussed earlier, can be adapted for the isolationof C-terminal peptides using a combination of metaboliclabeling, acetylation, oxidation, and strong cation exchange.340

For cell cultures and biological systems that are not amenable

Figure 15. continued

polyglycerol polymer conjugated with aldehyde groups (HPG-ALD) for depletion of internal tryptic peptides that bind via their unblocked α-aminogroups by reductive amination. The polymer and bound internal peptides are subsequently removed via molecular weight cutoff filter centrifugation,allowing for negative enrichment of blocked N-terminal peptides and mass spectrometric analysis. The isotopic or isobaric tagging of the individualsamples allows for relative quantification of abundance of the N-terminal peptide, enabling the analysis of more subtle differences in proteolyticcleavage compared to only “on/off” approaches. COFRADIC (center column) relies on a series of chromatographic fractionations andchromatographic depletion of internal (i.e., unlabeled) tryptic peptides after derivatization with a hydrophobic reagent (TNBS). The extensivefractionation yields samples with a high enrichment for N-terminal peptides but with very large fraction numbers to be analyzed by massspectrometry. ChaFRADIC (right column) is a COFRADIC variant that relies on the charge shift of nonterminal, tryptic peptides upon chemicalacetylation that allows removal by SCX chromatography and negative enrichment of N-terminal peptides.



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http://flintbox.com/public/project/1948

http://flintbox.com/public/project/1948


to metabolic labeling, an alternate method can be utilizedC-TAILS. Alternative C-terminomic strategies can also be founddescribed in the review by Tanco and colleagues.173

Schilling and colleagues presented a negative selectionstrategy to study C-terminal peptides as a modified methodof N-terminal amine isotopic labeling of substrates (N-TAILS).354,355 The original TAILS method reported utilizes awater-soluble polymer to deplete internal peptides usingreductive alkylation chemistry following amine blocking at theprotein level. C-TAILS follows a similar principle. A polymer isutilized to deplete internal tryptic peptides after the protein isblocked at carboxylic acids. Contrary to the original TAILSmethod where primary amines react only with aldehydes toform covalent bonds, C-TAILS chemistry reacts carboxylicacids with primary amines. As such, C-TAILS requires blockingat multiple levels. First, thiol groups are alkylated at the proteinlevel and then primary amines (N-termini and lysines) arereductively dimethylated, rendering those groups unavailablefor later reaction. This prevents cyclization and aggregation ofproximal primary amines to carboxylic acids in the amidationreaction. Following buffer exchange to 4-morpholinoethane-sulfonic acid (MES) buffer pH 5, proteins are then reacted witha primary amine, originally published with ethanolamine, and acombination of 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (s-NHS), both in excess quantities. Carboxylic acids and primaryamines do not react in isolation; it requires the activation of thecarboxylic acid. EDC activates carboxylic acids into an unstableO-acylisourea intermediate. This intermediate can (1) bestabilized by s-NHS to form the amine-reactive compound,which then forms an amide bond with ethanolamine, or (2)hydrolyze back to the carboxylic acid. Because of the back-conversion back to carboxylic acid, addition of EDC and s-NHSis repeated three times in C-TAILS over 18 h to maximizecarboxylic acid blocking, which is critical for the success of themethod.Proteins are then precipitated to eliminate excess reagent and

digested with trypsin to generate peptides that contain newlyformed primary amines and carboxylic acids. For quantificationpurposes and to prevent aggregation and cyclization, peptidesare labeled at their newly formed N-termini with isotopic orisobaric labels. Following buffer exchange, the freely availableC-termini from internal tryptic peptides are reacted in a similarEDC/s-NHS scheme with a water-soluble linear polymerpolyallylamine (PAA). This polymer captures internal trypticpeptides and can be separated from the unbound C-terminallyblocked peptides utilizing a spin-filter membrane with a high-molecular-weight cutoff. C-TAILS was originally utilized tostudy the E. coli C-terminome, and it achieved >70% ofpeptides being C-terminally blocked, identifying 460 peptidesfrom 196 proteins.354

Since the originally published protocol in 2010, there havebeen novel developments to address some caveats in C-TAILS.While the original method utilized dimethylation of primaryamines for blocking, a report in 2015 by Zhang and colleaguesshowed there are potential benefits for utilization of acetylationto improve Mascot scores.356 In addition, they prepurified PAApolymer to remove monomers and allowed for the inclusion ofsingly charged species to be fragmented by the massspectrometer. Most notable was the inclusion of acetonitrilein the EDC reactions in order to minimize water concen-trations while allowing proteins to remain soluble. Overall these

changes resulted in a total 481 C-terminal peptides from 369proteins in E. coli.Contrasting to the inclusion of singly charged species in the

mass spectrometer, Somasundaram and colleagues performed“charge-reversal derivatization” to convert C-terminal peptidesinto species that behave more like fully tryptic peptides.357

Utilizing similar principles to reduce water availability duringEDC coupling, Somasundaram and colleagues used the basicamines N,N-dimethylethylenediamine and (4-aminobutyl)-guanidine instead of ethanolamine. In doing so, it increasedthe charge of peptides and provided a basic group at the C-terminus, which increased the yield of y-ions, allowing for moreconfident identification using database search engines. How-ever, the increase in charge of trypsin-generated peptidesrequires the utilization of alternate fragmentation methods,such as electron-transfer dissociation (ETD), which performbest at higher charge states.358 Thus, they utilized acombination of chymotrypsin, trypsin, and ETD on a LTQOrbitrap Velos. This report found 424 C-termini from usingtwo digestion enzymes and two fragmentation methods.The number of identified peptides across all these studies

shows that the number of identified C-terminal peptides issignificantly lower compared not only to N-terminomicmethodologies available but to the predicted number ofproteins in E. coli. Thus, C-TAILS and other carboxy-orientedmethods are still in their infancy and hold much promise for thefuture of sequencing the ends of proteins.

5. FUTURE PERSPECTIVESRegulated proteolytic processing is dysfunctional in diseasesthat afflict us all yet is still ill-definedonly 340 of 565 humanproteases have known substrates. Deciphering cleavage eventsat decision points of cell and tissue function will reveal anunexplored layer of complexity in the hierarchy of cellregulation. The past decade of increasing research effort andattention in the field of degradomics has transformed our viewof proteolysis from a mere destructive mechanism, such as inMMP-mediated degradation of extracellular matrix, to anintricate and selective posttranslational modification withimportant regulatory functions in many biological pathways.With increasing technological capabilities (e.g., more powerfulmass spectrometers and improved bioinformatics), our abilityto probe substrate proteolysis deeper and more specifically invarious processes in health and disease has provided moreinsight into how proteolysis changes protein function, yet manyquestions remain to be answered.An unexpected general finding of such unbiased terminomics

approaches is the very high percentage of protein populationsthat are present with undocumented N- or C-termini presentconcurrent with their mature parent protein as translated andmatured in the classical biochemical pathways.348 Thus, innormal skin ∼50% of proteins have a different N-terminus tothat annotated in Uniprot;9 in platelets and erythrocytes thispercentage rises to 77%7 and 68%,8 respectively, ∼4% of whichare internal alternate start sites.8 Although these two cell typesare unusual, these numbers seem representative, and indeed wefound in human dental pulp that 78% of proteins occurred asdifferent cleaved proteoforms.10 As has been discussedthroughout this Review, the position and chemistry of the N-terminal amino acid residues and their specific modificationscan profoundly alter the function of the protein. Thus, theseproteoforms not only increase protein diversity in vivo but theyalter our conceptual models of protein, organelle, cell, and



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tissue functionin dynamic ongoing physiological processesthat maintain or restore homeostasis and go astray, leading topathology or occurring to switch function as a protective hostresponse to pathology. The genesis of these neo-termini needsto be deciphered, and for that vast majority generated otherthan by alternate start sites and splicing, the cognate proteasesleading to these proteoforms need to be identified. Inpathology, these proteases or proteoforms may form valuablenew drug targets or biomarkers of disease. In any case, bothneed to be recognized and the biological implications need tobe understood for more accurate understanding of the functionand response networks of a proteome, at rest and whenstressed.Current proteomics techniques aimed at unbiased study of

proteolysis of substrates provide temporal snapshots of thestate of cleavage in a tissue and give invaluable insights intowhat substrates are cleaved, e.g., in response to a specificstimulus or disease state, but interpreting these pictures canprove challenging. Most useful proteomics techniques are basedon relative quantification between samples, so the stoichiom-etry between cleaved and intact substrate is often overlooked,and it is unknown how this affects biology. Many of thebackground cleavages may occur at low stoichiometry, and forthese, are they relevant? For each protein, what is the tippingpoint for physiological relevance in altering protein function,particularly when the cleavage products are antagonists ordominant negatives of the parent protein. How stable are theparent and neoproteins? A related question is how many ofthese cleavages are also silent? Thus, perhaps the generation ofneotermini in homeostatic conditions is just the price thesystem pays for maintaining a responsive pathway and networkthat can rapidly change the levels of a protein or its activity ondemand. Similarly, deciphering protease redundancy for thesecleavages is important to understand. Redundancy is often tooeasy an excuse for lack of deeper understanding. That is, asystem may not have redundant capacity but rather theorganism does, and in different cells, tissue, or organs, underdifferent stresses or nutritional and hormonal statuses, differentproteases cut in and out. This is important to really understandin order to formulate good hypotheses and insightfulexperiments. Localization of a given protease and its substrategreatly affects the outcome and effect of cleavage, and moresensitive analytical techniques will enable more detailed analysisof specific tissues and subcellular fractions.Another challenge in analyzing proteolytic cleavage in

complex, biologically relevant samples is the interplay thatexists between different proteases and their inhibitors. Thedirect source of an observed proteolytic cleavage is oftenintractable and can be due to indirect effects, e.g., deactivationof a protease inhibitor by another protease, often of a differentclass,11 or activation of another protease. Ongoing advances inbioinformatics are the key to give insights into the intricacies ofthe protease web and interpreting the genesis of proteolyticproteoforms in cells and tissues. Finally, proteolytic cleavagecan be regulated or affected by other posttranslational proteinmodifications. Activity of proteases may rely on specificphosphorylation or ubiquitination events, and glycosylation orphosphorylation or other posttranslational modifications ofsubstrates may enable, or prevent, proteolysis. So, do cleavagesites function combinatorially or together with other post-translational modifications? Integrating systems biology ap-proaches that profile posttranslational modifications will furtherour insight into this, and current targeted analysis platforms for

phosphorylation, ubiquitination, and proteolytic cleavage willintegrate into a true systems analysis platform to study the roleof specific proteolytic cleavages in biology and pathology. Thisnuanced new level of biological control heralds a bright new eraand realm of biology and protein and cell regulation.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Present Addresses⊥(T.K.) Department of Clinical Chemistry; Erasmus MedicalCenter, Rotterdam, The Netherlands.§(U.E.) Division of Structural Biology, Department ofMolecular Biology, University of Salzburg, Salzburg, Austria.¶(A.D.) Department of Physiology and Pharmacology;University of Calgary, Calgary, Canada.

Notes

The authors declare no competing financial interest.

Biographies

Theo Klein obtained his Ph.D. in Analytic Biochemistry in 2008 fromthe University of Groningen, The Netherlands, where he worked ondevelopment of novel chemical biology and proteomics methods forqualitative and quantitative analysis of protease activity. After a briefsegue into biotech at Brains Online in Groningen, he joined ChrisOverall’s lab in Vancouver in 2011 to study the role of proteolyticcleavage in adaptive immunity, focusing on the role of the paracaspaseMALT1 in antigen-receptor signaling.

Ulrich Eckhard obtained his Ph.D. degree in 2011 from the Universityof Salzburg, Austria, where he focused on the biochemical andstructural elucidation of bacterial collagenases. He then joined Prof.Chris Overall at the University of British Columbia, Vancouver,Canada, where he secured a postdoctoral fellowship from the MichaelSmith Foundation for Health Research and expanded his structuralbiochemical methodological repertoire to state-of-the-art proteomics.Since September 2016, he has worked as a researcher at Uppsalawithin a multidisciplinary project trying to unravel how proteinstructure and function are intertwined in the evolution of new proteinvariants.

Antoine Dufour obtained his Ph.D. degree in 2010 from Stony BrookUniversity, NY, U.S.A. He developed a patent on the composition andmethods for the inhibition of MMP9-mediated cell migration. He thenbegan a postdoctoral fellowship with Prof. Chris Overall at theUniversity of British Columbia, focused on the investigation ofproteases in inflammatory diseases such as cancer, arthritis, andsystemic lupus erythematosus using proteomics. In 2012 he wasawarded a fellowship from the Canadian Institutes of Health Researchto continue his work on the roles of matrix metalloproteinases inbreast cancer.

Nestor Solis obtained his Ph.D. in microbial proteomics in 2014 fromThe University of Sydney, Australia, where he explored theidentification of cell-surface proteins from Staphylococcus speciesusing cell-shaving proteomics. He joined the laboratory of ProfessorChristopher Overall in Vancouver, Canada, in 2014 to further expandon proteomic methods to study N- and C-terminomes in macro-phages. He was awarded two fellowships in 2016 to study macrophageterminomes: a Michael Smith Foundation for Health Researchpostdoctoral fellowship from British Columbia, Canada, and a CJMartin Early Career Fellowship from the National Health and MedicalResearch Council from Australia.



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mailto:[email protected]


Chris Overall is a Professor and Tier 1 Canada Research Chair inProtease Proteomics and Systems Biology at the University of BritishColumbia (UBC), Life Sciences Institute. He is a pioneer ofdegradomics, a term he coined. He has had several sabbaticals in theBiotechnology and Pharmaceutical industries and is an HonoraryProfessor at the Freiburg Institute for Advanced Studies, Albert-Ludwigs Universitat Freiburg, Germany. Dr. Overall was awarded the2002 CIHR Scientist of the Year, the UBC Killam Senior ResearcherAward 2005, the Tony Pawson Canadian National ProteomicsNetwork Award for Outstanding Contribution and Leadership tothe Canadian Proteomics Community 2014, and the HUPOProteomics Discovery Award in 2018. He is an Associate Editor ofthe Journal of Proteome Research, Editor of mSystems, and on theEditorial Boards of the Journal of Molecular Cellular Proteomics, MatrixBiology, and Biological Chemistry and the Advisory Committee of theInternational Union of Basic and Clinical Pharmacology.

ACKNOWLEDGMENTS

C.M.O. is funded by a Foundation Grant from the CanadianInstitute for Health Research and a Canada Research Chair inProtease Proteomics and Systems Biology. A.D. was supportedby a Michael Smith Foundation for Health Researchpostdoctoral fellowship. U.E. acknowledges a postdoctoralfellowship from the Michael Smith Foundation for HealthResearch. N.S. is funded through a Canadian MSFHR TraineeFellowship (Grant ID 16642) and an Australian NHMRC EarlyCareer Fellowship (GNT1126842). Additional funding incluesC.I.H.R. Foundation Grant (FDN: 148408) (C.M.O.); CanadaResearch Chair (Tier I) in Metalloproteinase Proteomics andSystems Biology (950-01-126; 950-20-3877) (C.M.O.);C.I.H.R. Operating Grant (MOP-133632) (C.M.O.).

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