The Peptidase Zymogen Proregions - CiteSeerX

Current Pharmaceutical Design, 2002, 8, 125-133 1

The Peptidase Zymogen Proregions: Nature's Way of Preventing UndesiredActivation and Proteolysis.

Claude Lazure

Neuropeptides Structure and Metabolism Laboratory, Institut de recherches cliniques de Montréal, 110 Pine Avenue West,Montréal (Québec) Canada H2W 1R7, Tel: (514) 987-5593, Fax: (514) 987-5542, E-mail: [email protected]

Abstract: NOTE FOR AUTHOR: PLEAS WRITE THE ABSTRACT OF THIS MANUSCRIPTABOUT 250 WORDS. ABSTRACT IS VERY IMPORTANT PART OF OUR JOURNAL.

INTRODUCTION signaling/migration, immunological reactions, woundhealing and, ultimately, cell death through apoptosis. As aconsequence, by understanding their roles in normalprocesses, one can envision a plethora of potential benefitsarising from controlling the activities of peptidases involvedin various pathological states [2]. Along with the increasingnumber of peptidases being discovered, we are alsowitnessing the emergence of novel members and/or familiesfor which, apart from the cDNA sequences, very little isknown. One way, though peppered with caveats, to ascertainthe full extent of this issue is to examine the peptidasescontent of so far complete genomes. Indeed, the number ofproteases predicted from analyses of completetely sequencedgenomes tends to increase with the complexity of theorganism as indicated in Fig. (1). The percentage ofpeptidases derived from these computations is 2.05% ± 0.9of the total protein content. This number should beconsidered with caution with respect to higher organismssince full annotations of the human genome is not yetcompleted and most current information is derived from theanalyses of bacterial or archaea bacterial genomes. Forexample, according to the MEROPS databank, there arepresently 406 ORFs encoding peptidases in the humangenome. This is a low number when compared to muchsmaller genomes such as those from Drosophilamelanogaster, Caenorhabitis elegans and Saccharomycescerevisiae which encode 466, 334 and 107 peptidasesrespectively [5]. Based on the above figure of 2% of the totalprotein content, a conservative estimate of the number ofhuman peptidases would yield (if one assumes between40,000 to 60,000 genes) 700 to 1100 peptidases, a numbertwice as high as that already recognized [4]. Regardless ofthe actual number, however, it suffices to say that thisarsenal of useful but potentially destructive power must beharnessed and controlled in order for any organism tosurvive.

One of the great biochemists of our time, Dr HansNeurath, wrote in his 1984 review article entitled ''Evolutionof proteolytic enzymes'' that: ''Proteolytic enzymes are notonly a physiological necessity but also a potential hazard,since, if uncontrolled, they can destroy the proteincomponent of cells and tissues'' [1].

This far ranging observation was, at the time, basedmainly on knowledge gained through careful physiologicalstudies of peptidases1 involved in digestive processes, bloodcoagulation and fibrinolysis, the release of peptide hormonesfrom precursors, the transport of secretory proteins acrossmembranes, the organization of macromolecular structuresand proteolysis in general. Since then, not only have weadvanced toward a better understanding of the molecularbasis of peptidase function but also, by sequencing entiregenomes, we have achieved an appreciation of thetremendous diversity and unique specificities of peptidases.Most recently, the known function of peptidases have beenextended to include participation in other vital processessuch as fertilization, growth, differentiation, cell

*Address correspondence to this author at Neuropeptides Structure andMetabolism Laboratory, Institut de recherches cliniques de Montréal, 110Pine Avenue West, Montréal (Québec) Canada H2W 1R7, Tel: (514) 987-5593, Fax: (514) 987-5542, E-mail: [email protected]

1 The term peptidase (peptidyl bond hydrolase) will be used throughoutthis article in accordance to the classification suggested by Rawlings andBarrett [3,4] instead of the more familiar terms proteinase or protease.The nomenclature of peptidase families follows that of the InternationalUnion of Biochemists and Molecular Biologists (IUBMB).

1381-6128/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd.

2 Current Pharmaceutical Design, 2002, Vol. 8, No. 3 Claude Lazure

Fig. (1). Peptidases genome content.

The number of genes identified and/or predicted following complete genome sequencing is plotted against the number of peptidasesidentified and listed in the MEROPS databank [4] (accessible through http://www.Merops.co.uk website). At this stage, it can bereadily appreciated that the vast majority of completely sequenced genomes correspond to those from archaebacteria, bacteria andsimple eukaryotic organisms (see text).

There are a variety of ways in which peptidase activitycan be regulated in either a positive and/or negative mannerincluding (i) the synthesis of specific inhibitors that providea virtual fail-safe mechanisms terminating peptidase action;(ii) the influence of microenvironments which allowpeptidases to act in discrete and unique locations; (iii)substrate and/or product inhibition especially in the case ofmetabolic enzymes; (iv) the regulation of gene expressionenabling spatio-temporal regulation of activity; (v) cascaderegulation is well known in blood coagulation andfibrinolysis and; (vi) the activation of zymogens, the nascentinactive state of peptidases following their synthesis. None ofthese mechanisms is exclusive; more often than not,multiple mechanisms are used in order to control peptidaseactivities. In contrast to endocrine functions, for example,where the action of an hormone is restricted in time (i.eunder the control of appropriate stimuli) and space (i.ethrough interaction with a given receptor), a peptidase, onceexpressed and activated, will lead to irreversible modificationof its target substrates. Despite the built-in specificity ofnumerous peptidases, this in itself is not sufficient to preventcollateral damage from their actions in undesired locationson unitended susbtrates. Thus, highly diversified groups ofmolecules known as inhibitors have evolved in order toreversibly or irreversibly inactivate the peptidases before theycause deleterial effects. This group of molecules hasgenerated and continues to generate a lot of attention since

they can be seen as the final solution to stopping in aspecific and potent manner peptidase activity. As it isassumed that, in the human genome, these molecules arepresent at a 1:10 ratio compared to peptidases; hence 70 to110 genes encoding peptidase inhibitors should be present[5]. Understanding how these endogenous inhibitors functionwill hopefully pave the way to the development of specificand potent non-peptidic inhibitors (the use of peptides orproteins as therapeutic agents is often compromised by theirbioavailability, cost, instability and pharmacologicalproperties). It is the purpose of this review to illustrate that,within the structure of zymogens, lies information whichcould lead to the development of peptidase inhibitors.

There exist in the literature numerous reviews, some ofthem referred to in the following paragraphs, dealing withpeptidase inhibition and/or focused on each one of themechanisms described above. This review is not meant to beexhaustive and the author immediately apologizes for theomission of scientists and laboratories who have activelypursued research in this area. For an in depth exquisitemolecular detailing of zymogen activation, the reader mustconsult the review by Khan and James who have expertlyrecollected and documented data pertinent to zymogenactivation [6]. This review will survey novel zymogenstructures determined since 1997 and on zymogen or

The Peptidase Zymogen Proregions Current Pharmaceutical Design, 2002, Vol. 8, No. 3 3

peptidase families for which structural data is still verylimited or absent.

discuss below) preceding the catalytic unit. Some generalproperties and characteristics of the proregion can be defined.

I. PROREGION SEQUENCES: WHAT ARE THEYAND WHAT ARE THEIR FUNCTIONS?

I.1. Proregions are Localized at the N-Terminus ofZymogens

In order to prevent any undue activation of the zymogen,it is logical to consider that synthesis of the proregionprecedes that of the catalytic unit thus explaining its positionat the N-terminus often right after the signal peptide in thecase of secreted peptidases. However, in human protectiveprotein, a serine carboxypeptidase, an activation segment ispresent within the catalytic domain. Though not located toblock the already preformed active site, its removal isnecessary to confer activity though postulated conformationalchanges [16]. Similarly, proregions can be found at the C-terminus of Thermus aquaticus aqualysin I [17,18],Neisseria gonorrhoeae IgA protease [19] and Serrataimarcescens SSP protease [20] where they play an importantrole in extracellular protein secretion. Hence, for examplewith aqualysin I, the C-terminal proregion acts in concertwith the N-terminal proregion responsible for proper foldingand inhibition properties. A similarly located C-terminalproregion, though possessing none of the properties of the N-terminal proregion was located in a bacterial zincaminopeptidase but as yet its role remains undetermined[21]. Interestingly, as we shall see, the primary structure ofproregions even within endopeptidase families appears illconserved, though based on crystallographic studies thegeneral fold is.

Numerous proteins, apart from peptidases, aresynthesized as inactive zymogens that must be activatedprior to being able to express their full biological activities.The most frequent mechanism of activation, as it leads toirreversible action, is post-translational cleavage of peptidebond(s) within the zymogen molecule. In most cases, eitherthese cleavages which can be intramolecular orintermolecular, are catalysed either by peptidases acting onthemselves (autocatalytic) or by activating partners. In recentyears, four types of autocatalytic activation leading to N →O or N → S acyl rearrangements have been described(reviewed in [7,8]). Examples of these include proteinsplicing, the autoactivation of glycosylaspariginase,hedgehog proteins and pyruvoyl-dependant enzymes. It hasbeen further proposed that such acyl rearrangements might bemore common than hitherto recognized and can represent apathway for autoproteolysis to occur [8]. Actually, intein-mediated protein ligation represents a novel tool for proteinsynthesis and expression [9]. In the absence of these peptidebond rearrangements, the most common mechanism ofpeptide bond cleavages is the release of the active moietythrough the catalysed addition of a water molecule in a uniand/or bimolecular mode. Indeed, although aspartic and zinc-metallopeptidases lack covalent intermediate present in thecase of serine and thiol peptidases, all cleavages by thesepeptidases require the formation of a tetrahedral intermediate[10]. Hence, it was originally considered that proregions21

only function to suppress enzymatic activity by masking thecatalytic machinery and thus preventing formation of thecatalytic intermediate. Based on earlier studies oncrystallised forms of hydrolytic digestive enzymes such aspepsinogen, chymotrypsinogen and trypsinogen, it certainlyappeared to be the case. For example, Freer et al. [11] haveshown that, in the structure of chymotrypsinogen, which is107 times less active than chymotrypsin, all the catalyticmachinery is present but the specific binding site isdisordered and not accessible. Subsequent studies haveshown that the most common albeit not the only one, modeof action of proregions is to sterically occlude the bindingsites and this in all classes of peptidases. Indeed, aside fromsignal peptidases (reviewed in [12,13]) which cannot beclassified in any of the known peptidases families but whoseactivity is highly dependent on phospholipid moieties andfrom viral peptidases [14] though in the latter case, beinginscribed in a polyprotein precursor, part of which couldmaybe play a role of a proregion prior to self-cleavage, allknown cellular or bacterial peptidases are synthesized aszymogens. Hence, peptidases require prior to being activeremoval of the proregions which can range from as little as afew residues (two in the case of granzyme B [15] to up tomultidomain structures (in the case of caspases as we shall

I.2. Proregion Intramolecular Chaperone Properties canbe Independent of their Inhibition Properties

The role of proregions in assisting peptidases to gaintheir final active three-dimensional structure has been heavilydocumented as well as the way they can accomplish this feat.In a recent review article [22], Shinde and Inouye haveclassified proregions in two types namely type I which isdefined by the ability of proregions to elicit folding of theircognate peptidases (intramolecular chaperone or IMC) andtype II whereby the proregions are associated with otherfunctions. Here, it is easier to list the enzymes shown not torequire their proregions to fold properly. Hence, theseenzymes include, for example, those whose proregions aretoo short such as trypsin, chymotrypsin or elastase [23],viral peptidases such as HIV-1 [24] known to foldindependantly of their polyprotein precursor and the asparticpeptidase renin [25]. Furthermore, there exist peptidases forwhich no role of the proregion is known and hence thisprevents their classification as type I or II. For example,carboxypeptidase E (CPE) is first synthesized as a precursorcontaining a 14 residues extension at the N-terminus [26], asequence completely conserved between human, rat andmouse but conspicuously absent in anglerfish CPE [27].However, no role in inhibition as bovine proCPE is active[28], in expression of human and rat CPE [29,30] and/orintracellular routeing of the protein [31] could be preciselydefined. This is quite striking since the role of proregions incarboxypeptidases A and B, enzymes belonging to the same

2The term proregion will be used in this article and is equivalent to othernames such as propeptide, prosequence, actvation peptide, activationsequence and propart.


Fig. (2). Unimolecular mode of activation of prosubtilisin.

A scheme describing the activation of prosubtilisin based on both functional and structural studies is depicted [119]. Salient featuresof this model, many of them applicable to numerous peptidases, correspond to (i) initiation of the activation through an initialcleavage of the proregion in an autocatalytic manner, (ii) induced conformational change resulting in the movement of the peptidase N-terminal region towards a Ca++ -binding site resulting in enhanced stability of the peptidase, (iii) secondary cleavage within anaccessible site in the proregion resulting in the degadation of the proregion and in the loosening of the interactions between therporegion and the peptidase and (iv) complete dissociation of the proregion-peptidase complex yielding a fully stable and activeenzyme.

family as CPE, is well established and their removalnecessary for the enzymes to be active [31].

metalloprotease) families are well documented; this relies onthe conservation of a cysteine residue in the proregion whichis able to coordinate the active site Zinc ion [39,40].Regardless of the mechanism, it is therefore not surprisingthat numerous proregions acting as IMC are also potentendogenous inhibitors. Indeed, even after being cleaved,these proregions remain tightly associated with the enzymeforming a 1:1 inhibitor enzyme complex as illustrated for thesubtilisin-prosubtilisin complex in Fig (2) and (3). In thiscase, in order to fully activate the peptidase, furtherdegradation of the proregion may be mandatory. Here, it canbe seen readily that the proregion is able to interact directlywith at least two regions of the subtilisin namely the oneencompassing the active site and an exposed region of thecatlytic domain made up by two parallel helices of theenzyme interacting with a sheet structure of the proregion.Often, as we shall discuss later, this close fitting leads tostrong and exquisitely specific binding (experimentallydetermined in vitro Ki values are often in the nM to pMrange). This, in effect, also explains why following the firstcleavage of the proregion in the active site further cleavageselsewhere within the proregion are necessary if not obligatoryto release the active enzyme as shown in Fig (2). However,in the case of subtilisin E, the two functions are notabsolutely linked [41]. Even the mode of functioning in twoclosely related enzymes such as subtilisin E and aqualysin Ican also be different. The aqualysin I proregion is able byitself to fold spontaneously into a stable and a well definedconformation in the absence of the cognate enzyme, acharacteristic not shared with the subtilisin E proregionwhich does not exhibit a discernible conformation in theabsence of subtilisin E. Interestingly, the proaqualysin Iproregion can also support refolding of subtilisin E albeit ata much lower efficiency though its inhibiting potential is tentimes better than the cognate prosubtilisin E region [42].Finally, the introduction of point mutations in the peptidase-proregion subtilisin E interface do not alter the foldingcapacity of the proregion but seriously compromises its

Intramolecular chaperones can act directly to catalysefolding from a molten globule state to a fully folded state bylowering the energy barrier between the two states and/or bystabilizing of a rate-limiting folding intermediate. This hasbeen demonstrated through the study of refolding in vitro ofdenatured subtilisin upon addition of the proregion intrans32 [32,33]. In the case of α-lytic protease, the control ofrefolding is under kinetic control as the proregion helps instabilizing a folding intermediate between two lower energystates [34-36]. Furthermore, studies of IMC have led to theconcept of "protein memory" [37] whereby a mutation in theproregion is imprinted in the structure of the cognatepeptidase. This important role of the proregion implies thatthere exists an intimate structural relationships charcaterizedby numerous non-covalent interactions localized at thepeptidase-proregion interface. In essence, these interactionsare no different than those observed at the interface betweenthe catalytic domain and substrate or inhibitor binding.However, this is not an exclusive rule as contacts betweenproregion and peptidase can sometimes rely on covalentand/or coordination bonds. In the case of the former, a crystalstructure of procathepsin X has shown that there existsbetween the active site Cys residue and a Cys residue in theproregion a reversible covalent bond; the existence of such abond diminishes greatly the reliance of secondaryinteractions between the proregion and the cognate enzymepartly explaining why this proregion is so short (38residues) compared to those of other cathepsins [38]. In thecase of the latter, the existence of a cysteine switch in thecase of members of the ADAM (a disintegrin andmetalloproteinase domain) and MMP (matrix

3Cis activity is used to refer to the presence of the proregion within the

polypeptide section of the zymogen whereas trans activity refers to theactivity of the proregion added as a separate entity.


Fig. (3). The prosubtilisin-subtilisin complex.

The space-filling structure of the prosubtilisin-subtilisin complex based on the X-ray crystallographic data [119]. The maturesubtilisin is colored whereas the interacting prosubtilisin is uniformly labeled in green. Such a close interaction is seen to occur innumerous peptidases and this, irrespective of their classes though the molecular interactions as well as the inhibition mechanisms canbe widely different. In this model, Two interacting regions can be clearly defined namely (i) the proregion C-terminal chain is seen toenter deeply into the active site region thus enabling its interaction with residues in the substrate binding site and (ii) the sharedinterface between the proregion and the peptidase can be seen in the lower right of the figure. Data accessible through the BrookhavenDatabank (identifier 1SPB) were manipulated using the Hyperchem V4.0 software. For the sake of clirity, all hydrogen atoms andwater molecules were removed: carbon atoms are in light blue, nitrogen atoms in dark blue, sulfur atoms in yellow and oxygen atomsin red.

ability to inhibit the enzyme when added in trans [41].Based on these examples, it can be seen that potentinhibitors of peptidases cannot be taken as evidence that theywill function as intramolcular chaperones and vice versa.

delivery represent questionable issues. Hence, in thiscontext, it is not surprising that our increased understandingof proregions have led scientists to examine the usefulness ofproregions or fragments thereof in terms of inhibitors. It wasshown, for example, that synthetic propeptides could inhibitManduca sexta midgut trypsin [48] and hence could be usedas pest controls. Similarly, due to the implications of thecathepsin faimily in numerous pathological states, muchefforts have been devoted to using proregions as inhibitors.Indeed, it was shown that the proregions of cathepsin B, K,L and S are very potent (K is of 0.4, 2.6, 0.09 and 0.27 nMrespectively [49-52]). Furthermore, this inhibition is oftenpH dependant and sensitive to the presence ofpolysaccharides. In the case of cathepsins and more generallyof peptidases active in acidic environments, it is ofparamount importance that the interactions between theproregion and the peptidases be maintained from the site ofsynthesis in near neutral conditions up to where they areactivated in an acid media. Hence, a common feature ofproregions of these peptidases is to stabilize the cognateprotease structure by forming salt bridges. Comparison of thevarious recombinantly produced proregions from cathepsinK, L and S demonstrates that some selectivity can beachieved upon addition of proregions in trans to their parent

I.3. Proregions can be a Source of Potent SmallerInhibitors Displaying Enhanced Selectivities

One of the central problems in designing potenttherapeutically active inhibitors is to confer selectivity tothese molecules. Indeed, in many cases, members of a givenfamily will display overlapping specificities especially whenthey are studied in vitro. Hence, the modeling of substratestructures can at times leads to great difficulties in designingselective inhibitors. Examples can be found in the recentproprotein convertases (PC, discussed below) and in therapidly expanding family of cathepsins. In the latter case,there exist some natural inhibitors such as cystatins [43,44]but these are too large to be of clinical or industrial value.Similarly in the case of the proprotein convertases,engineering of α1-antitrypsin [45], alpha-2 macroglobulin[46] or ovomucoid [47] inhibitors could potentially lead toincreasingly specific inhibitors but their size and way of


enzymes and that they show little inhibition of cathepsin Band papain [53]. Interestingly, it is worth mentioning thatproteins, completely unrelated to the previously mentionedendogenous inhibitor cystatins, isolated from Bombyx moridesignated as BCPI α and β [54,55] as well as from murineT-lymphocytes and mast cells designated CTLA-2 α and β[56] were shown to display significant sequence similaritiesto proregion sequences of members of the cathepsin family.In the case of the Bombyx proteins, these were shown toinhibit papain, cathepsin B and L. Synthetic fragmentsencompassing shorter sequences than full proregions werealso shown to display interesting properties in terms ofinhibition and selectivity. These studies demonstrated atleast for cathepsins B and D that the full proregion is notnecessary and that fragments thereof could be used either toinhibit cathepsin B [57] or purify cathepsin D [58].Recombinantly expressed papaya proteinase IV proregion (aplant enzyme) could not only inhibit cysteine peptidases ofpapaya but also those present in the digestive tract of insects,in this case the Colorado potato beetle, pointing to possibleuses of proregion as pest controls [59]. Finally, potent hexaand pentapeptides containing the Cys residue known to bepresent in the proregion of MMPs, stromelysin or MMP-3,have been shown to be potent inhibitors of the parentenzyme but also of procollagenase or MMP-1 [60].

procaspase 1, 2, 4, 5, 9 and 13 [67]. For example, caspase-8is interacting with the adaptor protein FADD through itsprodomain and ultimately to the Fas/APO-1/CD95 deathcomplex [68,69] whereas caspase-9 is interacting with Apaf-1in a similar manner [70]. In this context, the proregion isalso responsible for ultimately targeting caspase-2 to anuclear location [71]. The other procaspases namely 3,6 and7, termed executioner caspases, do not contain thesedomains. Another function of proregions is to favor pH-dependant association of propeptidases to membranes andthus provide sorting signals as in the case of procathepsins Dand L. Both of these procathepsins are known to bind tomembranes at pH 5.0 but not the active proteases [72]. Inthe case of procathepsin L, a nine residue sequence withinthe proregion was found responsible for this binding [73].However, in the case of procathepsin G, it was shown thatthe proform is not important for sorting to granules butserved mainly to maintain the enzyme in an inactive formduring intracellular transport [74]. It was also proposed inthe case of prorenin that the presence of protease cleavagesites in the proregion is enough to direct the enzyme in theregulated pathway [75]. Finally, the proregion of the ATP-dependant PIM1 protease is absolutely essential for targetingthis protease to its ultimate site of action, namely themitochondria [76]. Activation of this protease is actuallyrather unique since once located in the mitochondria, amithochondrial processing enzyme removes the N-terminal37 residues containing the targeting signal but leaving theprotease with a 61 residues N-terminal extension which isultimately cleaved off autocatalytically upon assembly of theprotease subunits into an homo-oligomeric complex.

I.4. Proregions can Exhibit Diverse Properties otherthan their Refolding or Inhibiting Properties

Though other roles of proregions have been described andin certain cases documented, this demonstration is far moredifficult to achieve. Indeed, considering that one of the mainand more frequently encountered roles is in helping its parentpeptidase to fold correctly and hence achieve a stablefunctional structure, one is frequently confronted by aseminal question as to whether the effect observed is due tofailure to fold properly or to exhibit a proregion specificproperty. Nevertheless, proregions have additional rolesother than folding and/or inhibition and many examples havebeen described and some already mentioned. Indeed, we havementioned the role of C-terminal located proregions insecretion into the yeast vacuolar space which was shown inthe case of IgA protease, aqualysin, SSP protease but alsoStreptomyces griseus proteinase B and carboxypeptidase Y[17-20,61,62]. Interestingly restoring this secretion can beaccomplished by supplying the proregion in trans.Furthermore proper secretion of aqualysin in an eukaryoteorganism, Escherichia coli, also requires the C-terminalproregion [63]. In the lesser known family of peptidasespresent in the eukaryotic 20S proteasome (see below), thepresence of the proregion are essential for the assembly i.e.oligomerization of the various subunits into the full particle[64,65] and addition of the proper proregion in trans can beas efficient as when present in cis [65] . However, assemblyand full activity of the 20S proteasome in Thermoplasmaacidophilum, an archaea-bacteria, do not require the presenceof the proregions [66]. Proregions of certain caspases(cysteine-dependant aspartate specific protease) containspecific regions involved in protein-protein interactions ofgreat importance in activation of the procaspases. These arethe death effector domain (DED) present in procaspase 8 and10 and the caspase recruitment domain (CARD) present in

As we have seen in this brief summary, it is ratherdifficult to generate a unifying hypothesis concerning thenature and the role of proregions as they exhibit inter or intrafamily diverse structures, diverse properties and finallydiverse roles. Furthermore, though it appeared that in certaincases they might be dispensable, it is more than likely that afunction has yet to be defined or ascertained. We shall brieflysee in the next section how proregions interact in molecularterms with their partners.

II. ZYMOGEN STRUCTURE DETERMINATION,WHAT MORE HAVE WE LEARNED?

More than any other experimental technique, X-raycrystallography has contributed to our present understandingof zymogen activation. Not only was this technique able todescribe in atomic terms the importance and role of activesite residues but also to describe how inhibitors, small andlarge, are able to interact with them. The contribution of thisapproach cannot be minimized and has proven essential inour understanding of the intimate relation of proregions withtheir cognate enzymes. As much information has alreadybeen described in a very detailed review article together withan in-depth description of molecular detailing [6], thissection will focus on structures determined since 1997 andespecially on novel information derived therein. Already, arepresentative of each recognized classes of peptidases wasavailable in the latter review, the recent years have led tonew members and/or members of new families (Table 1).These will be briefly described in the following paragraphs.


Table 1. Crystal Structures of Zymogens (1997-2000).

Zymogen Enzyme Family CLAN MEROPS ID Resolution(Å)

PDB accession Reference

human proCathepsin B

human proCathepsin Khuman proCathepsin X

Streptococcus proStreptopain1

Cysteine peptidase

Cysteine peptidaseCysteine peptidaseCysteinel peptidase

CA

CACACA

C01.060

C01.036C01.013C10.001

2.5

3.21.71.6

3PBH

7PCK/1BY81DEU1DKJ

[77]

[78,79][80][81]

Hordeum v. proPhytepsin

Plasmodium f. proPlasmepsin IIhuman proGastricsin2

Aspartic peptidase

Aspartic peptidaseAspartic peptidase

AA

AAAA

A01.020

A01.023A01.003

2.3

1.852.36

1QDM

1PFZ1AVF

[82]

[83][84]

human proPlasminogen3

Bacillus s. proSubtilisin E4

Lysobacter e.proαLytic Protease3

Human complement proFactor DHuman Factor VIIa5

Human single chain t-PA

Serine peptidase

Serine peptidaseSerine peptidaseSerine peptidaseSerine peptidaseSerine peptidase

PA

SBPAPAPAPA

S01.233

S08.001S01.268S01.191S01.215S01.232

2.0

2.03.02.12.83.25

1QRZ

1SCJ2PRO1FDP1QFK1BDA

[85]

[86][35}[87][88][89]

human proCarboxypeptidase A2

human proMMP-2 (gelatinase A)Clostridium b. Bontoxilysin AClostridium b. Bontoxilysin B

Zinc-CPase

Zinc matrix peptidaseZinc neurotoxin peptidase

Zinc neurotoxin peptidase

MC

MAMAMA

M14.002

M10.003M27.002M27.002

1.8

2.83.21.9

1AYE

1CK73BTA

1EPW/1F82

[90]

[91][92]

[93,94]

Bovine enzyme complex CPase A

Ser/Cys Chymotrypsin C Peptidase E

MC

PAPA

M14.001

S01.157S01.154

2.35 1PYT [95]

Yeast 20S proteasome Nth hydrolase PB T01.010 2.35 1RYP [96]

Bacillus m. P(46) Germination protease (GPR) UX U03.001 3.0 1C8B [97]

Notes:1: This structure corresponds to a C47S mutant.

2: This structure corresponds to a crystallized intermediate in the activation pathway of gastricsin.3. The proenzyme structure determined contains 3 nonessential mutations (M585Q, V673M and M788L) and represents the complex formed with streptokinase.4. The crystal structure of the Ser221Cys mutant corresponds to a non-covalent complex between the proregion and the cognate enzyme.5. Though this structure represents an activated form of the enzyme, this form exhibits a zymogen-like dormant state (see text).

II.1. Cysteine Peptidases activate themselves in an autocatalytic manner as shown forcathepsins B [101], procathepsin S [101] and procathepsin L[102] and require an acidic pH well in agreement with theircellular localization. However, in other cases, activationappears to be mediated by other peptidases such as pepsins,elastase, cathepsin D. Accordingly, it was recently shownthat procathepsin C (dipeptidyl peptidase I) is unable toautoactivate and absolutely requires for this process thepresence of cathepsin L and to a lesser extent cathepsin S[103]. The two novel structures recently determined bringabout two unprecedented findings namely (i) as abovementioned, in procathepsin X, the presence of a covalentbond between the proregion and the Cys in the active site[38] and (ii) disruption of the position of the catalyticessential His residue by a loop inserted in the active site inproStreptopain [81]. The latter observation is interesting asthis enzyme is clearly related to papain albeit in a remotemanner and should thus possess like members of this familyan already preformed active site. However, movement ofresidues elsewhere than in the active site but present in theoccluding loop following removal of the proregion had beenpreviously described in the case of procathepsin B forexample [104]. Finally crystal structures of Cys peptidaseshave permitted to define in addition to the substrate bindingsite, two other sites of major interactions which mighteventually be used to render synthetic inhibitors more

From the previous review, structures of procathepsins Band L as well as procaricain were known and this alreadyprovided a lot of information for this quickly expandingfamily which now comprises 11 members in the humannamely B, H, L, S, C, K, O, F, V, X and W [98].Proregion structural analyses provided further division of thisfamily into two namely (i) those resembling cathepsin Lwhich contain prodomain in excess of 100 residues to whichthe classical Cys peptidase papain belongs and (ii) thoseresembling cathepsin-B which contain a proregion of ca. 60residues. It can be readily appreciated that proregions fromthe various members exhibit very low sequence similaritythough two motifs can be identified, the first one labeledERF(W)NIN is present only in cathepsin-L like enzymesand the second one is GNFD. However, they are allstructurally similar sharing common fold and functions.Hence, part of the proregion is shown to cover the substratebinding cleft but in a reverse direction than a peptidylsubstrate. This places a normally scissile bond in anunfavorable position with respect to the active site residues[99]. Hence since positioning of the peptide chain preventsintramolecular cleavage, it is not surprising that activation ofcathepsin proceeds in an intermolecular fashion as wasshown for cathepsin-B [100]. In most cases, Cys peptidases


selective. These include the occluding-loop crevice and evenmore interestingly the proregion binding loop especiallysince as mentioned [104], it offers a large, exposed and partlyhydrophobic surface. An interesting proposition is thus touse this secondary site to improve binding and/or augmentthe specificity of an inhibitor in a manner reminiscent of theinteraction of thrombin and hirudin [105]. Two issues as yetunresolved appear to be whether proregions on their own canfold into a stable structure and how they help in Cyspeptidases folding.

This feature is not in line with the fact that in manypeptidases, and this irrespective of the class they belong to,active site are already formed but seen before in the case ofchymotrypsin-like enzymes. In this case, instead of shieldingthe active site, the role of the proregion is basically to keepthe active site Asp residues away from each other thuspreventing them from being operational [112]. Even moreintriguing is the case of prophytepsin which possess apreformed active site like the other Asp peptidases but here,it is shielded from the substrate not by the proregion but byresidues belonging to the catalytic chain. Activation byremoval of the proregion reverses the positioning of theresidues enabling the active site to be freely accessible [112].II.2. Aspartic Peptidases

Despite the varied functions and their distribution in allphyla from viruses to humans, much of our knowledgeconcerning this class of peptidases concern those found in thedigestive tract. Indeed, till 1997, crystal structures ofpepsinogen (probably the oldest zymogen known) fromhuman and porcine species as well as of progastricsin wereknown [6]. Further understanding came about by the successencountered in determining the structure of a processingintermediate corresponding to a non-covalent complex of apartially cleaved proregion [84,106]. By unraveling such astructure, it is now possible to look more closely at apeptidase on its way to become active as compared to theinitial and final pictures available so far with mostpropeptidases and peptidases. This ''frozen in time'' pictureallowed confirmation of the activation pathway proposed sofar for aspartic peptidases and revealed at what positions inthe sequence of the proregion the first cleavage occurs. Thus,the positively charged proregion by interacting with thenegatively charged catalytic region through a number of salt-bridges is able to shield the active site from a substrate hencekeeping in an inactive form. Following transfer to a low pHenvironment helping in loosening the salt bridges, theproregion is cleaved autocatalytically following a series ofcleavages through various intermediates. The activationpathway described for progastricsin contains features whichare proposed to be applicable to members of the Asppeptidases and has been detailed in numerous recent reviews[107-109]. However, some variations pertaining to the direct(removal of the proregion in a single cleavage) or sequentialpathway (removal of proregions through multiple cleavages)are known to occur in various species or depending upon theconcentration of enzymes [109]. Similarly, though a crystalstructure is not available, activation of procathepsin E isproposed to follow an identical route to pepsinogen orgastricsin. However, a different structural motif due to achanges in salt bridges being formed between the proregionand enzyme is proposed to interact with the active site [110].In a way unexpected, two novel structures are shedding newlights upon activation od Asp peptidases and thesecorrespond to proplasmepsin, an enzyme found in theparasite Plasmodium falciparum whose role is to degradehemoglobin [83], and a plant peptidase, prophytepsin [82].In the first case, proplasmepsin, the proregion is unusuallylong at 125 residues when compared to digestive tractproenzymes, contains a transmembrane segment instead of asignal peptide and requires for its removal the presence of anas yet unidentified acidic maturase [111]. Furthermore, itinactivates its cognate enzyme by preventing formation of theactive site though accessible but not yet assembled correctly.

II.3. Serine Peptidases

This family is composed of a variety of peptidases andthis diversity is also reflected in the manner used in terms oftheir activation. Indeed, numerous different mechanismsmore or less related are known so far for these familymembers. The first one and also one of the earliestdiscovered involves a significant conformational change fromthe zymogen into the active form which results in the properpositioning of the catalytic machinery (active site residuesand oxyanion hole). In this instance, the conformationalchange is brought about by cleavage of a peptide bond withensuing repositioning of the chains involving for examplethe N-terminal segment which will stabilize the now activepeptidase. This mode is used by chymotrypsin-likeenzymes; it is worth noting that, under certain circumstancessuch as with chymotrypsinogen and proelastase 2, theproregion is retained through disulfide bridges in thestructure of the active enzyme where it will impart a greaterchemical stability of the peptidase without affecting itskinetic or folding properties [113]. The inactive state of thepeptidase can be directly ascribed to the fact that thesubstrate binding cleft is not properly formed as seen in themany crystallographic structures available [6].

The second mechanism has already been described in thepreceding sections and includes the peptidases related to thebacterial subtilisin. As can be seen in Fig. (2,3), it stemsfrom the fact that the proregion interacts directly with apreformed active site thereby shielding it in a mannerreminiscent to proregion interactions in Cys peptidases. Inthis case, however, it is the residues occupying the C-terminus of the proregion which are localized in the substratebinding cleft in a product-like manner; as a general rule, itcan be said that activation will proceed throughautoactivation and that the nature of the residues occupyingthe C-terminus of the proregion will correspond closely tothe specificity of the cognate enzyme. This can be clearlyseen in the structure of the α-lytic protease complexed withits proregion [35]. We and others have shown in the case ofpairs of basic cleaving enzymes such as furin, PC1/3, PC2,PC7 and their yeast homologue kexin that the proregion C-terminal residues are responsible for the inhibition in transand that removing them prevents the proregion mediatedinhibition [114-118]. This is also clearly apparent in theprosubtilisin-BPN' structure (Fig. (3) [119]) and even moreso in the structure of the autoprocessed Ser221Cys-subtilisinE-propeptide complex [86] whereby the proregion C-terminal


Tyr residue is seen to interact through hydrogen-bonding tothe His64 and Ser221Cys which are part of the catalytictriad. However, the fact that in this Ser221Cys mutant(rendering the subtilisin E unable to further cleave theproregion), a stable complex can be isolated confirms twofurther properties of the subtilisin related proregions namely(i) they can behave as transient potent inhibitors and (ii) tofully activate the enzyme a secondary cleavage at anaccessible site and possibly more is necessary in order todegrade the proregion and prevent further inhibition. In thecase of subtilisin BPN', this secondary site was identified asa Glu residue occupying position 53 in the proregion and ledto the proposal that "it might be possible to make mutantproteins from the propeptide which exhibit permanentinhibition of subtilisin" [120].

Similarly, activation of human Factor VII is a veryinteresting refinement of this mode of inhibition as itrequires for full activation the presence of an accessoryprotein namely tissue factor (TF). Indeed, conversion of fullyinactive Factor VII into what one should expect would beactivated Factor VIIa following an internal cleavage as in thecase of trypsin-like enzymes, leads to generation of FactorVIIa which remains in a zymogen-like state i.e withoutcatalytic acitivity until efficient binding to its naturalregulator TF. The crystal structure of free Factor VIIa [88]following the one previously described of Factor VIIa incomplex with TF [122], allows one to better understand thedrastic effect of TF binding upon the enzyme. The presenceof a unique residue, Met306, as well as of an helix loop(307-312) are shown to exhibit widely different interactionsin the two structures with consequences on substratebinding. One of the role of TF binding would be to stabilizethis helix in a suitable position hence ensuring peptidaseactivity. It is our opinion that this type of refined regulationwill become more common as knowledge advances in theunraveling of numerous peptidases involved in manypathways.

The third mechanism is highlighted by the structure ofthe proenzyme domain of plasminogen corresponding to themicroplasminogen form which is devoid of the 5 kringleregion repeats. This mechanism is related to the immaturesubstrate binding cleft model observed in thechymotrypsinogen namely by the presence inmicroplasminogen of a distorted structure preventingformation of the oxyanion hole and leading to misplacementof the catalytic residues [85]. However, this inhibited state isfurther reinforced by the presence of a main chain residue(trp761) directly occupying the S143 subsite and hencetotally preventing binding of a possible substrate. Thus,activation of plasminogen must proceed though concomitantconformational movements of three peptide loops in order toform the proper catalytic triad, the oxyanion hole as well asthe S1 subsite.

II.4. Zinc-Metallopeptidases

Without going into details of exopeptidases activation,the newest addition of crystal structures in this group ofenzymes correspond on one hand to a member of the matrixmetallopeptidases, proMMP-2 or gelatinase A [91],following the one of stromelysin 1 (MMP-3) [123] and onthe other to the structure of zinc-dependent endopeptidase ofone of nature's deadliest poison, Botulinum neurotoxins [92-94,124]. The structure of proMMP-2 is revealing in thesense that it follows closely the one derived for MMP-3namely that the proregion is shielding adequately thesubstrate binding cleft, that one of its peptide chain interactsclosely with the active site Cys residue through coordinationof the catalytically important Zinc atom and finally that theproregion offers efficient baits for activating proteases [123].

However, it should be noted that the direction of thepeptide chain in contact with the active site is in the reversedirection of that seen in complex of stromelysin withsynthetic inhibitors [40]. Considering the high number (seebelow) of matrix metalloprotease family members,knowledge of the conservation of the activation mode ofthese two members should help in the understanding of theother members though, no doubt, more surprises areawaiting us.

A variation of this theme can be exemplified by the factthat in tissue-type plasminogen activator (t-PA), somecatalytic activity of the single chain form (zymogen) ispresent when compared with the fully activated two-chainsform; the reason for the difference in activity can beappreciated from the crystal structure of the single chain formwhere it can be seen that, unique to this molecule, a mainchain residue (Lys156) is able to form a salt bridge with theactive site Asp residue hence preventing but not totallyenzymatic activity [89]. The structure of complement factorD can also be seen as related to this mode of inhibition asFactor D is activated through the classical internal cleavagemode but remains inactive in circulation despite the fact thatit has lost its activation peptide and hence should be active.Full activation appears to be through the control of itsnatural substrate binding, its only one known so far the C3b-bound Factor B. In the crystal structure, it can be seen thatuntil binding, Factor D is in a auto-inhibited form throughthe positioning of a main chain Arg residue (Arg218) whichis able to form a salt bridge with an Asp residue (Asp189)leading to disordered conformations of the catalytic triad anda shielded S1 site [87]. Hence, conversion of profactor D isclearly a two steps process whereby removal of the activationpeptide and internal cleavage leads to a dormant enzyme(self-inhibited) and subsequent to substrate binding, convertsinto the suitable catalytic site and S1 conformations.

Botulinum neurotoxins are made up of two subunitslinked together through a disulfide bridge namely a heavychain carrying the target recognition properties and thetransfer properties of the toxic chain and a light chainrepresenting the toxic moiety identified as a Zinc-dependentpeptidase [124]. It has been shown that, though differentneurotoxins will target different substrates, all of the targetsbelong to synaptic vesicle docking and fusion complex andare cleaved by the Zinc-peptidase encoded by the light chain.In both structures determined, it can be seen that the N-terminal part of the heavy chain is wrapped around the lightchain and that the catalytic sites are deeply buried and hencenot accessible. However, whereas in BoNT/A, the heavy

4This nomenclature refers to the one introduced by Schecter and Berger

[121].


chain sterically obstruct the active site, it is not the case inBoNT/B where the active site is accessible. In both casesthough, the enzymes are rendered active through reduction ofthe disulfide bridge linking the heavy and light chain of thetoxin, an event which happens following internalization ofthe toxin by the target cells. In BoNT/A, it appears thatreduction of this disulfide bridge is enough to render theactive site accessible and hence relieve the light chain fromthe close interactions with the heavy chain. However, inBoNT/B, since the active site is already accessible, it seemsthat reduction of the disulfide bond allows conformationalchanges in certain loop structures in the light chain broughtabout through either substrate binding and/or heavy/lightchain separation.

The other novel structure listed relates to the germinationprotease (GPR) responsible for degrading small acid-solubleproteins which protect DNA prior to spore germination [97].This peptidase is first synthesized as an enzymaticallyinactive homotetramer P(46), following sporulation P(46) isprocessed by removal of a strech of 7 to 16 amino acids intoa smaller homotetrameric entity P(41) which isenzymatically active. The main problem presented by thisprotease is two-fold. Firstly the amino acid sequence lacksany relatedness to the sequences of other known peptidasesand not surprisingly define a new peptidase fold and theenzymatic activity cannot be inhibited by known inhibitors.Secondly, as seen in the structure of the enzymaticallyinactive P(46) the amino terminal extension cleaved off upongeneration of P(41) is barely, if at all, in contact with the restof the protein hence raising a lot of unresolved questionssurrounding their role in either stability and/or inactivity ofthe zymogen structure. Actually, before the crystal structurewas made available, it was shown that most of the proregionsequence can be deleted without effect and that the free regiondid not inhibit the enzyme [127]. It thus appears like in thepreviously mentioned Factor VIIa, careful comparison of theX-ray generated structure of both P(46) and P(41) formscould lead to resolving the issue of activation of thisenzyme. Thus, in a rare example, crystal structure hasyielded a beautiful and novel structure but also has raisedmore questions than it can solved.

II.5. Other Peptidases

Amongst the novel structures listed in Table 1, it isworth mentioning the fascinating image conveyed by thecrsytal structure of the yeast 20S proteosome which, morethan an enzymatic complex, represents truly an organelle.The 20S unit, itself part of the larger 26S proteasome, iscomposed of seven subunits which can be subdivided intotwo classes, α and β. Only three out of the 7 β-subunits (β1,β2 and β5 in the proteasome and interferon-γ induced β1i,β2i and βb5i in the immunoproteasome) are catalyticallyactive peptidases [125]. These peptidases are not related toany of the 4 classical families and actually belong to the N-terminal nucleophilic hydrolase family which also includepenicillin G amidase (PGA), glutamine PRPPamidotransferases, glycosylasparaginases (mentionedpreviously), conjugated bile acid hydrolases and penicillin Vamidase (PVA) [126]. Nevertheless, this diverse groupshares some features in common notably a unique proteinfold and the fact that they all need to be activated in order toexhibit their hydrolase properties. This family, and mostmembers therein, has been identified in the last few years.These hydrolases are characterized by the fact the active sitenucleophile, be it Thr, Cys or Ser, is located at the extremeN-terminal position and must be uncovered throughactivation in order to be active. We have already mentioned(see above) the important role of the proregion (up to 75residues) in targeting and in subunit oligomerization of theproteasome but this proregion is also crucial in theactivation process both in the proteasome and in theimmunoproteasome. This activation, once assembly is done,appears to take place in two steps, reminiscent of the stepsdescribed in the more classical peptidases, namely cleavageswithin the proregion sequences by neighboring active siteswhich are followed closely by autolytic uncovering of the N-terminal residue rendering the subunits active andcompetent. Finally, similarly to the role of the proregion insubtilisin-like peptidases, it has also been demonstrated thatthe proregions exhibit intramolecular chaperone properties inhelping the peptidase to fold, this effect being specific to eachsubunit and being carried out through non-covalentinteractions. Similarly to subtilisin-like peptidases, thechaperone function can also be carried out in trans and suchan addition can rescue folding and assembly of proregiondeleted β-subunits.

Crystallographic studies have contributed significantly toour understanding of peptidase activation, has provided inrecent years novel mechanisms or structures and this, even inwell established families, and no doubt will continue to be amajor tool in analyzing peptidase functioning. However, inrecent years as well as through our increasing knowledge oftotal genomes, there exist numerous emerging familiesand/or members which obviously do not havecrystallographic counterparts. In the next section, we willbriefly mention some of them.

III. EMERGING PEPTIDASE FAMILIES, NEWMEMBERS, NEW MODELS?

As mentioned in the introduction, there is no questionthat the present diversity in peptidases structure and functionwill expand considerably in future years. As a corollary, itcan be envisioned that the role of proregions as well as themodes of activation of these peptidases, not to mention thediscovery of new endogenous inhibitors, will follow suit.Already, there exist examples of emerging classes, like theaforementioned Nth-hydrolases, and new enzymes for whichwe do not as yet possess crystal structures to guide ourunderstanding. In the next few paragraphs, we will brieflydescribed some of them hoping that this presentation willconvey to the reader hints of what will come in followingyears.

III.1. The Caspases: Funambulists on the Rope of CellProliferation and Cell Death

Since the discovery of capase-1, also known asinterleukin-1β-converting enzyme [128,129], 10 new


members have been identified and numbered accordingly inhumans (reviewed in [67,130]) though this number mightnot be final. These enzymes belong to the Cys-peptidasesfamily and demonstrate a strong preference for cleaving afterAsp residues in a variety of substrates, indeed a uniquepreference in eukaryotic enzymes. As a group, they can befurther divided in three namely, cytokine activators (caspase-1, 4, 5, 13), initiator caspases (caspase-2, 8, 9, 10) andexecutioner caspases (caspase-3, 6, 7) depending on theirinteractions with various proteins and each others and theirlocalization in the pathway leading to cell death byapopotosis. Interestingly, to a certain extent thisclassification also defines the type of proregions within eachas well as the mechanism by which they are activated.Indeed, as above mentioned, some of these possessunusually long proregions in excess of 200 residues (forexample caspase-8) while others have relatively shortsequences (for example, 22 residues for caspase-6). Thelonger proregions are now known to contain structuresimportant for protein-protein interactions pivotal fortransmitting signals through the presence of DED or CARDdomains. Since it is not the purpose of this section todiscuss the events leading to or upstream of the activation ofcaspases, we will refer the reader to numerous reviews[67,130-133], suffice to say that fortunately, there exist manyregulatory steps aiming at preventing undue activation ofthese entities. However, once a signal has been transduced,for exemple by binding of Fas at its receptor, then caspaseswill be activated through a two steps process identical for allnamely removal of the proregions and internal cleavages ofan activation sequence linking in the zymogen the heavywith the light subunits resulting in an heterodimericenzyme. This process, in the case of the initiator caspases, iscarried out by the enzymes themselves in a characteristicautocatalytic manner once they have formed the appropriatecomplexes. In contrast, executioner caspase such as caspase-3, 6 and 7 are activated through cleavages by initiatorcaspases and/or other enzymes in an hetero-activationmanner. So far, though complex, activation mechanism donot seem to detract from what was presented in the othersections. However, there are some surprises with certaincaspases rendering their activation quite a unique process.The first one concerns the fact that some caspases, even intheir zymogen form, possess or exhibit considerable intrinsicenzyme activity as shown in the case of caspase-8 and -9.Evidently, such an activity would be detrimental to the lifeof cells and hence must be taken care of by endogenouscaspase inhibitors [134]. The second one, resembling whatwas observed in the case of Factor VIIa, concerns caspase-9where it was shown that removal of the activation sequencelinking the heavy and light subunit is not necessary norsufficient for activation as both the zymogen and the cleavedcaspase-9 possess catalytic activities [135]. Furthermore, in acase rarely encountered, removal of the proregion is notaccomplished in the activated caspase-9 molecule; both ofthese characteristics point to the fact that caspase-9 activationis highly dependant upon co-factor or activator binding. Thethird one concerns the role of the proregions of theexecutioner caspases (caspase-3,9 and 7) as it was shown thatneither the activity nor the rate of activation of caspase-3 isinfluenced by the short proregion raising the question ofwhat is its role [136]. Lastly, it has been recently reportedthat a unique and rare post-translational protein modification,

namely nitrosylation, could play a role in the activation ofcaspase-3. Indeed, Mannick et al [137] have shown that theactive site cys in zymogen caspase-3 is nitrosylated and thatin addition to cleavage into active subunits, this residueneeds to be denitrosylated for caspase-3 functioning.

III.2. ADAMS Family: an Array of Diverse Functionswith a Commensurate High Number of Members

An ADAM protein can be simplistically described as aprotein incorporating a series of domain each having itsdistinct structure and properties, Thus, one can find insuccession starting from the N-terminus and in a protein oftotal length of ca. 750 residues, a proregion, ametalloprotease, a disintegrin, cysteine-rich, epidermalgrowth factor-like and, if membrane-bound, a transmembraneand a cytosolic domain. Furthermore, each one of thesedomains is involved in a specific function pertaining toproteolysis, adhesion, signaling and fusion events. Thepeptidase domain falls in the adamalysin/reprolysinsubfamily of metzincin peptidase family [138-140]. At thetime of writing, 29 cDNAs encoding ADAM have beensequenced but, of this number, only 17 would appear toencode fully functional peptidases. Hence, only this numberis proposed to possess the necessary attributes forfunctioning as Zinc-metallopeptidases, in particular, bypossessing the family signature of HEXGHXXGXXHD ofthe active site. These enzymes, globally, are involved in therelease of numerous membrane-bound factors such as, forexample, tumor necrosis factor by ADAM-17, Delta, a Notchligand. by ADAM-10 and heparin-binding epidermal-growthfactor-like growth factor by ADAM-9 [139]. In most casesthough, the actual substrates recognized and cleaved byADAMs remain to be identified. In addition of sharing anactive site sequence, these enzymes also appear to sharesimilar mechanism of activation which requires removal ofthe proregion. [6]. As previously mentioned, members ofthis family share with those belonging to the MMPs (seebelow) a protective mechanism namely a cysteine switch[39] as shown in the case of ADAM-12. In the latter, it wasconclusively shown that chemical modification and/ormutagenesis of the proregion reactive Cys will result in itsfailure to interact with the Zinc in the active site andultimately will lead to enzymatic activity even in thepresence of the proregion [39]. Furthermore, removal of theproregion does not appear to be autocatalytic as an active sitemutant is still correctly processed [141] hence pointing topossible activation by other peptidases [141,142].Furthermore, this proregion cleavage, contrary to MMPs,appears to be an early event occurring well before the enzymeis secreted and/or is presented at the surface. Interestingly, inkeeping with functions already ascribed to proregions inother families, the ADAM-12 proregion is absolutelyessential for proper secretion as a direct consequence of theproregion role in folding of active enzyme. A certainlyworthy question raised by the proregion roles so far definedfor ADAM-12 is why is this proregion so long (179residues) when compared to the 82 residues of MMP-3. Thelatter acts in a similar manner whereby the proregion Cysbinds to the Zinc in the active site, the cysteine switch, andthe whole proregion renders the active site unaccessiblethough shielding [123].


Not included in the ADAM members above described,there also exists a further group of rapidly expanding numberand importance namely these related peptidases in which adisintegrin-like domain is linked to 1 to manythrombospondin type-1 modules, collectively known asADAM-TS [143]. Following the first successful cloning ofADAM-TS1 in 1997 [144], the number of ADAM-TS hasincreased to 11 members even though most be considered byanalogy to receptors as orphan peptidases (10 sequences havebeen published at this date); the last two ones being ADAM-TS9 [145] and ADAM-TS12 [146]. In keeping with theirsequence relatedness to ADAM, these enzymes possess aproregion which contains the Cys involved in the switchmechanism as well as cleavage sites necessary for removal ofthe proregion by activating peptidases such as furin. Indeed,furin involvement had been proposed in activating someADAMs and might be very well involved in activatingADAM-TS1 [147]. Furthermore, ADAM-TS8 has also beenshown to be processed by removal of the proregion but alsoto even lower molecular weight forms [148]. Due to therecent discovery of these peptidases, little is knownconcerning their activation and the role of prodomain, butgiven their tissue distribution as well as their selectiveexpression in development and their involvement in variouspathologies, this aspect surely represents an active field.

proelastase, the proregion remains attached to the catalyticdomain through a disulfide bridge with no obviousinterference in the peptidase activity. In this case, theproregion can be seen as entirely responsible of retaining theactive peptidase to the membrane as it is acting as a tether. Itis worth mentioning that a recent review article on hepsinhas been published [157] and also review the perplexingresults obtained during studies with an hepsin knock-outmurine model [158].

III.4. Matrix Metalloproteases (MMPs)

Matrix metalloproteases (MMPs) or matrixins aresynthesized as zymogens which, in the case of solublematrixins, are secreted (8 members) while other members ofthe family remains bound to the cell surface through atransmembrane anchor (9 members often named as MT-MMPs) [159,160]. In addition, members of the matrixinfamily are often further classified according to the substraterecognized as being of the collagenase, gelatinase,stromelysin or membrane-bound. It can be argued that theyshould not be in the context of the present review discussedherein since two crystal structures, those of proMMP-2 (72kDa gelatinase A) and proMMP-3 (stromelysin 1) have beenreported (Table 1, [91, 123]. However, there exist goodreasons to do so as with the rapid increase of the number offamily members as well as the composite nature of theirstructure, a feature shared with ADAMs and ADAMs-TS,will surely yield novel information concerning theiractivation. In terms of molecular organization, matrilysin(MMP-7) is surely the simplest structure as it containssolely a typical Zinc-peptidase domain preceded by aproregion and a signal peptide. Many members (MMP-1, 3,8, 10, 12,13, 18, 19, 20 and 22) contain a C-terminalhemopexin-like domain whereas other members containfibronectin-like, vitronectin-like Cys and Pro-rich domainand HL-1 receptor like domains. Of particular interest is thefact that membrane-bound MMPs, and also MMP-3(stromelysin), all possess a sequence RX(K/R)R at theextreme C-terminus of the proregion, a sequence whichrepresents a hallmark for cleavage by the ubiquitouslydistributed convertase furin (see last section). Actually, it hasbeen shown that indeed stromelysin [161,162] as well asMT1-MMP [163,164] can be cleaved intracellularly by furin,a result well in line with the fact that MT1-MMP isexpressed on the cell surface as an active entity [164].However, there still exists considerable controversysurrounding this possible activation step as a furin-independant pathway MT1-MMP has been described [165]and further the zymogen form of MT1-MMP has beenreported to possess intrinsic enzymatic activity [166,167].To complicate the matter further, progelatinase A (MMP-2)which does not possess a typical furin cleavage site but dopossess further upstream of this site a RXXR sequence incommon with all MMPs except MMP-12 and MMP-7, wasrecently shown to be processed by furin [168]. Similarly,depending on the identity of the matrixins, the role of theproregion as an intramolecular chaperone could be widelydifferent. Indeed, with stromelysin, the proregion does notappear to play any significant role in protein folding, a roleimparted to a second Zinc-binding site [169]. On the otherhand, a proregion mediated folding of the peptidase, as well

III.3. Type II Transmembrane Serine Proteases, SerinePeptidases with Novel Features

When we thought that chymotrypsin and/or trypsin-likeenzymes were confined to roles in digestive processes and incirculation, a completely novel family emerged characterizedby the presence of a transmembrane segment as well as otherdomains now recognized as type II transmembrane serineproteases or TTSPs for short (reviewed in [149]). Allmolecules share the same basic architecture namely a shortcytosolic segment, a classical transmembrane segment(except maybe in the case of enterokinase, a classicalmisnomer, more appropriately known as enteropeptidase[150]), a segment of variable length encoding or not variousstructural domains such as frizzled, LDL receptor class A,CUB, SEA or MAM domain, a proregion and at the extremeC-terminus a catalytic Ser peptidase. Following the earlycloning of the first member, hepsin, present in the liver andhepatic cells [151], the total number of recognizedmammalian members now totaled 17 including the longsought after pro-atrial natriuretic factor (ANF) activatingprotease, corin, [152,153]. In addition to sharing thecatalytic triad of the chymotrypsin-like enzymes, all TTSPsare synthesized as inactive zymogen which in all likelihoodare activated once embedded in the membrane by activatingproteases which remain in most cases to be identified.However, one can not dismiss the fact that some or allmembers of this family could also autoactivate. Indeed, uponexpression, mutant forms of mouse hepsin as well as humanMT-SP1 (matriplase or epithin) are able to autoactivate[154,155]. However, whether this observation can begeneralized remains to be established as, for example, theproregion of enteropeptidase, in addition to playing a role incellular targeting, is poorly cleaved by autocatalysis [156].Another unique aspect of TTSP activation resides in the factthat, as mentioned before for chymotrypsinogen and


as successful trafficking to the cell surface, has beenadvocated for MT1-MMP wherein it was demonstrated muchin common with subtilisin family members that addition intrans of the proregion leads to expression of proregion-deleted peptidase [170]. Nevertheless, activation of MMPsall required proteolytic cleavages within the bait regions ofthe proregion and subsequent release of the Cys-Zn switch[171]; this activation can proceed through other peptidases oreven through various agents such as thiol-reactivecompounds, reactive oxygen and denaturants. However, thispicture appears to be far from realistic as activation of MMPscan require other participants as aptly described in the case ofthe activation of gelatinase A which needs in addition toactivated MT1-MMP the presence of a natural endogenousinhibitor, a tissue-derived inhibitor of matrix peptidases(TIMP-2). This results in the formation of a ternary complexwhere TIMP-2 is bound to the hemopexin domain ofcollagenase-2 which can be subsequently activated by MT1-MMP [172-175]. Hence, upon formation of the complex,MT1-MMP is able to cleave the gelatinase A proregion inthe bait region and this event is quickly followed by asecond cleavage through autoproteolysis in order to releasethe functional enzyme [176]. Finally, it is worh noting thatMMPs are part of the larger metzincsin family which alsocomprises reprolysins, serralysins and astacins which alsoinclude Meprin [177]. However, while they share the Cys-Znswitch with reprolysins (ADAM family), this feature isabsent in meprin and astacins (for example, a Cys residue iscompletely absent in meprin α-subunit). Hence the basis forpeptidase latency in these peptidases is clearly different thanfor MMPs and ADAMs. It has been proposed that, in thepresence of the proregion, some critically importanthydrogen bonds found in the active enzyme specially thosemediated by the first two amino acids in the mature chain,cannot form hence keeping the enzymes in a quiescent state[178]. Thus, as already mentioned, even within familiesthere exists a diversity in proregion roles and functioning.

residues within the structure of the catalytic domain [184].This observation is based on sequence comparison betweenthe aligned sequence of memapsin 2 and its close relativepepsinogen. As in the case of aspartyl protease, the proregionblocks the entrance to the active site (see section 2.2).However, it has been reported that the expressed recombinantpro-memapsin 2 is enzymatically active as this enzyme doesnot appear to autoactivate contrary to the acid-promotedautoactivation of digestive aspartyl peptidases [185,186].However, similarly to what is observed in the case of thegastrincsin 2 intermediate [84], the active site could exist inthree states namely fully closed (the proregion preventingaccess to the active site), partially open (in analogy to pHexposure of the pepsinogen active site) and fully open (byproteolytic removal of the proregion) following full removalof the proregion. According to this proposal [184], the N-terminal Glu-33 would truly represent an intermediate on itsway to full activation hence resembling the gastrincsin2structure; however, it must be said that cleavages prior theGlu33 and the one prior to Gly45 were obtained in studiesusing various trypsin-like enzymes such as clostripain,kallikrein and trypsin. Nevertheless, the most often observedform of recombinantly produced enzyme clearly correspondsto a cleavage at a sequence characterized by an RXXRsequence just preceding Glu-33. It has been shown that thiscleavage happens very early on upon the transit of the proteinfrom the endoplasmic reticulum to the plasma membrane andthat this cleavage could very well be mediated by a knownconvertase namely furin [185-187]. Hence such a cleavagewould account for the recognized N-terminal sequencestarting at Glu33. Finally, it must also be said that theproregion, contrary to what is observed in the case of, forexample, cathepsin D, has minimal inhibitory activity but,on the other hand, it appears to be playing an important rolein terms of protein folding [186]. Indeed, similarly to whatis observed for subtilisin and α-lytic protease, it is able topromote correct refolding when added in trans. However, tocomplicate the issue of β-APP processing, an homologue ofmemapsin 2, named memapsin 1, has been cloned [190-192]and it has been proposed that it could very well be a secondβ-secretase. The peptidase is produced as a zymogen and theproregion removal is accomplished by cleavage at a Leu-Alajunction [193]. Contrary to memapsin 2, this proteolyticcleavage is accomplished in the endoplasmic reticulum/earlyGolgi in an entirely autocatalytic fashion [194] whereas asabove discussed, activation of memapsin 2 is accomplishedin the trans-Golgi by another enzyme. Similarly to theprevious, it appears that the proregion does not haveinhibitory action but might be necessary for proper folding.Hence, this is yet another example of proregions where rolesother than inhibition of enzymatic activity are important forexpression of an active peptidase and that, even in closelyrelated peptidases, the activation mechanism can bealtogether different.

III.5. An Interesting Aspartyl Peptidase which isMembrane-Bound (Memapsin 2)

Many of the recently emerging members and families aremembrane-bound as was mentioned in previous sections.There are surely numerous other enzymes which rightfullydeserve to be discussed herein but, at least, one merits aparticular attention on two accounts. Firstly, memapsin 2(membrane-anchored aspartic protease of the pepsin family,also known as β-secretase or BACE) is an aspartyl proteasewhich is located in cellular membranes. Secondly thisenzyme has been implicated in the pathogenesis ofAlzheimer's disease by being responsible for the cleavage ofthe β-amyloid precursor. Indicative of the importance of thispeptidase is the fact that 5 separate groups were almostsimultaneously able to decipher the nucleotide sequence[179-183]. The 501 residues long memapsin 2 contains a 21residues signal peptide immediately followed by a short 24residues prodomain preceding the pepsin-related catalyticdomain followed by a transmembrane region and a very shortcytosolic tail. It is very interesting to note that, though theN-terminal sequence of active memapsin 2 was firmlyestablished as starting at Glu occupying position 33, it hasbeen proposed that the proregion might further extend by 16

IV. A CASE-STUDY: PROPROTEIN CONVERTASEPROREGIONS

After more than 20 years following the hypothesisaccording to which polypeptide homones and biologicallyactive peptides were synthesized as larger molecular weight


precursors which are subsequently cleaved at pairs of basicamino acids, the nature and identity of the peptidasesresponsible were finally obtained in the early 1990s.Actually, this unrelented search for converting enzymesyielded an entirely new family of eukaryotic enzymes knownas the proprotein convertases (PC) or a family of subtilisin-like calcium dependent serine peptidases. Thus, sevenmembers are known so far and include PC1 (also calledPC3), PC2, PC4, PC5 (also called PC6), PC7 (also calledSPC7, PC8 or LPC), furin and PACE4 and all prefer tocleave at basic amino acids in various sequence context(reviewed in [195-198]. They are functionally andstructurally related to a yeast enzyme Kex2p or kexin andtogether they belong to the subtilase family (reviewed in[199]). Furthermore, another enzyme not cleaving at basicresidues but much more so at Leu residues in the context ofan R/KXXL sequence has been identified as SKI-1(subtilisin-kexin-isozyme) or S1P [200,201]. All theseenzymes are first synthesized as zymogen with a N-terminalproregion varying in length from 81 residues for furin to 104for PC7. They differ mostly by the composition of C-terminal tail which contains various domains or regions,altogether absent in the prototypical subtilisin, such as ahomo-B or P-domain whose role is of paramount importancefor activity [202], Ser/Thr-rich, Cys-rich, amphipatic andtransmembrane regions. They also differ by theirlocalizations and even by their sites of action, for examplePC1/3 and PC2 are secretory granules resident peptidaseswith acidic pH optimum within the regulated pathway ofsecretion whereas furin is active thoughout the constitutivesecretory pathway. Furthermore, furin can process precursorsand protein in a variety of compartments such as the trans-Golgi network, the endosomes, the cell surface and evenpossibly, upon shedding, extracellular [203].

solution structure in the absence of the cognate enzymesexcept, very recently, in the case of PC1/3 proregion [205].This solution structure determined by NMR closely followsthe one described for subtilisin-BPN' notably in terms ofloops, β-sheets and α-helices but was notably determined inthe absence of the peptidase. This property is not sharedwith the proregion of subtilisin BPN' which adopts itsstructure upon interacting with the enzyme. In this contextthough, it must be said that a C-terminal helix (α2)structure exists in solution for the synthetic 24 residuespeptide corresponding to the PC7 proregion C-terminus[206]. The other important conserved aspect of PCsproregion concern the preservation of another basic aminoacids containing sequence in the center of the proregion(though this region is not readily apparent in PC7). Again,in a similar fashion to subtilases proregions, this sequenceoffers a suitable cleavage site termed secondary for the PCs toget rid of the proregions.

Following the study of furin activation [114], it wasreadily apparent that activation of PCs followed very closelythe mechanism illustrated in Fig. (2) namely that it involvedan initial cleavage at the primary site followed by cleavage atthe secondary site. This is required due to the potentinhibition brought about by the proregion which after theinitial cleavage, remains attached through non-covalentinteractions with the enzyme. This is known also for theprototypical kexin [118] but also for theSchizosaccharomyces pombe kexin homolog [207]. Indeed,we have shown that the proregion of PC1/3 is both a potent(Ki = 6 nM) and tight-binding inhibitor [115]. Using asimilar approach, with iodosobenzoic acid cleaved proPC2,we obtained a fragment encompassing positions 1 to 105hence containing the whole proregion. Upon incubation withCHO-produced PC2 (a gift from Iris Lindberg, LSU, [208]),this segment is able to inhibit the enzyme in an identicalfashion to what we observed with the PC1/3 proregion whenassayed with PC1/3 (Fig. (4)). Similarly, PC7 was alsopotently inhibited in trans by its recombinant bacterialproregion which also displayed selectivity against otherconvertases [117]. Furthermore, we have shown that theproPC1/3 region also potently inhibits furin but not so PC2;in the last case it is behaving as a competitive inhibitor withmuch diminished Ki. This result agrees entirely with theability of PC1/3 and furin proregions to interchange witheach other [209] but not with the proPC2 region. Indeed,PC2 not only requires its own proregion [210] but also thepresence of another protein namely 7B2 [211] which wasshown to interact closely with proPC2 and is mandatory forefficient expression [212]. As can be seen it appears that.actually, proPC2 activation mostly occuring in secretorygranules is quite unique as it requires low pHs and thepresence of 7B2. In addition, contrary to other PCs, itappears that cleavage at the secondary site is not required foreffective removal of the proregion from PC2 [116]. This issurely an unexpected result considering the observed potent(nM range) inhibiton of PC2 activity when the proPC2fragment is added in trans (Fig (4)). Finally, similarly to theuse of papaya proteinase IV proregion [59], it was shownthat vaccinia virus encoded proregions of PC7 and furin caninhibit through transient expression the ex vivo processing ofbiologically active peptides through inhibition ofintracellular PCs [117].

IV.1. Proregions as Inhibitor of Proprotein Convertases

At the same time that PC cDNAs structure wereestablished and due to their obvious relatedness bothfunctionally and structurally to the subtilase familymembers, it was reasoned that PC proregions could functionas intramolecular chaperone as well as endogenousinhibitors. Based on these predictions, it was hypothesizedthat similarly to already mentioned examples such assubtilisin BPN', subtilisin E, α-lytic protease, aqualysin orcathepsin D, these proregions would exhibit inhibitionconstants in the order of 10-7to 10-11. However, sequence-wise, when one compared PC proregions to each other,similarly to what was observed with procathepsins, onecannot fail to notice the low degree of sequence conservation.Indeed, they share 30-67% residues identity between eachother [196,197]. However, one can see some conservedregions especially the C-terminal segment always endingwith PCs preferred substrate specificity namely pairs of basicamino acids as well as an absolute conservation of 8residues. Moreover, similar comparison at the secondarystructure levels, allows one to predict that they would sharea very closely related fold with a structure consisting of threeβ sheets and two α-helices in the order β1-β2-α1-β3-α2[204]. On the other hand, numerous efforts using for examplebacterially expressed proregions have failed so far todemonstrate that PC proregions are able to adopt a stable


Fig. (4). In vitro inhibition of recombinant PC2 by a proPC2 containing peptide.

Progress curves of enzymatic activity of recombinant CHO-mPC2 (kindly made available by Dr Iris Lindberg, Louisiana StateUniversity, New Orleans, USA) as measured using a fluorogenic substrate, pGlu-Arg-Thr-Lys-Arg-MCA (100µM), in the presencefrom top to bottom increasing amounts (pmole level) of purified proPC2 fragment encompassing positions 1 to 105 of the proPC2 inthe presence of 5mM Ca++ and at pH 5.5. The proregion fragment was obtained following isolation of the complete proPC2 moleculefrom inclusion bodies of baculovirus infected insect cells, chemically cleaved following Trp residues by iodosobenzoic acid andpurified chromatographically as done similarly in the case of proPC1/3 [115].

Similarly to studies done on cathepsin proregions, it wasalso demonstrated that synthetic fragments encompassing theC-terminus of the proregion, as long as they possess the C-terminal basic residues [114-118], were potent inhibitors.Indeed, studies done on synthetic peptides representing C-terminal segments of proPC1/3 proregion, especially region50-83, yielded potent in vitro inhibitors [212]. Furthermore,comparison of peptides representing proPC1/3 proregion 50-83 and 74-83 indicates that they can inhibit PC1/3 activitywith Ki in the nM range but they nevertheless differ by theirmode of inhibition. Indeed, proPC1/3 74-83 is a competitiveinhibitor whereas the proPC1/3 50-83 behaves as a non-competitive inhibitor hinting that indeed this segment couldinteract at another site in addition to the active site (Basak,A. and Lazure, C., unpublished results). In the same vein,furin and PC7 proregions derived peptides were shown to bepotent and to exhibit selectivity when assayed with differentPCs [116], In particular the 24 C-terminal amino acidsproPC7 peptide inhibits PC7 enzymatic activity in purecompetitive manner with a Ki of 7 nM [206]. Similar resultswere also described in the case of convertase SKI-1 or S1P.Whereas it appears that the S1P proregion is cleaved withinthe endoplasmic reticulum at multiple sites, syntheticproregion derived peptides are able to efficiently inhibit S1Pactivity in vitro [214]. Similarly, peptidic substrates derivedfrom the proregion were also used in order to study S1Pproregion cleavages [214, 215]. On the other hand, as furtherevidence that activation of proPC2 is different than the one of

other PCs, synthetic peptides even including onerepresenting proPC2 positions 54-84 are micromolarinhibitors of PC2 [116]. In conclusion, it appears thatcomplete proregions and/or fragments thereof can be used toselectively and potently inhibit PCs enzymatic activities andthis even intracellularly.

IV.2. Proregions: Intramolecular Chaperone ofProprotein Convertases and other Roles

The evidence surrounding the role of PC proregions is atthe moment circumstancial in the sense that nocrystallographic structure of either the catalytic domainand/or its complex with the proregion is available nor wasthere direct studies like those above described withsubtilisins, aimed at addressing the issue. Actually, the onlydirect study with PC2, contrary to much expectation, provedrather inconclusive as following extensive or partialdenaturation, it was not possible to detect, upon renaturationin the presence of proPC2 and even in the presence of 7B2,any recovery of enzymatic activity [116]. Possibleexplanations have been put forward namely that PC2 foldingis a complex process that might require other as yetunidentified factors or that the inability to refold in vitro maybe due to the large size of the convertase when compared tosubtilisins. On the other hand, due to the high resemblancein terms of inhibition and relatedness to subtilisin as well as


the absolute necessity for the proregion for efficientexpression of PCs, it can readily be envisioned thatproregions play a significant role in PC folding.Furthermore, this involvement was demonstrated in the pastthrough site-directed mutagenesis of certain key proregionresidues; these mutants in general never made it out of theendoplasmic reticulum [216,217].

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Interestingly, as observed with other subtilases,proregions can often exhibit other roles than as an inhibitorand an intramolecular chaperone, the proregion of PC2 hasbeen proposed to play an important role in targeting thisenzyme in the regulated pathway of secretion. Indeed, it wasshown that the proregion contains a transferable aggregationand membrane binding sequence [206, 218]. This result wasrecently further documented by the use of a synthetic peptidecorresponding to residues 45-84 of proPC2. This peptidewas able, as the complete proPC2 molecule, to associatewith membranes in a pH and Ca++ manner. Further, thispeptide was able to prevent interactions of proPC2 in a dose-dependant manner to membranes [219]. This associationwould implicate a lipid component of the membrane which,ultimately would be responsible for its proper sorting tosecretory granules. It would surely be of interest to determinewhether the same role can be ascribed to the proregion ofPC1/3 as these two are the main secretory granulesconvertases.

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V. CONCLUSIONS[14] Babé, L.M., Craik, C.S., Cell 1997, 91 , 427-430.

In the introduction, it was mentioned that a plethora ofpeptidases will emerge form the analysis of genome and withit an array of endogenous inhibitors to control theiractivities. At this time, it appears evident that one mustconsider that various modes of action and ways to controlpeptidase activity will follow suit. In turn, years to come byunraveling these mechanisms should lead to a muchimproved understanding of the interactions betweenpeptidases and inhibitors but may be more importantly so interms of protein-protein interactions. In that sense, it isrevealing that most of the emerging families of peptidasesbelong to the membrane peptidases and are controlled and/orpart of intriguing complex formation. So to the questionwhether "do we still have to study proregions andpeptidases?" the answer can only be definitely yes as there ismuch still to be understand.

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The author would first like to sincerely thank allmembers, past and present, of his laboratory. We would liketo thank Dr. I. Lindberg for providing PC2 activity. Wewould like to acknowledge Drs. James Cromlish and JohnS. Munzer for critical reading of the manuscript. Studiesreported here were made possible through funding by theCanadian Institutes of Health Research and the ProteinEngineering Network Center of Excellence.

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