Biochimica et Biophysica Acta 1824 (2012) 237–245

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /bbapap

Review

Structure and function of tripeptidyl peptidase II, a giant cytosolic protease☆

Beate Rockel a,⁎, Klaus O. Kopec b, Andrei N. Lupas b, Wolfgang Baumeister a

a Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germanyb Department I — Protein Evolution, Max Planck Institute for Developmental Biology, Spemannstr. 35, D-72076 Tübingen, Germany

☆ This article is part of a Special Issue entitled: Pdiscovery of lysosome.⁎ Corresponding author at: Max Planck Institute of

Molecular Structural Biology, Am Klopferspitz 18, D-Tel.: +49 89 8578 2698; fax: +49 89 8578 2641.

E-mail addresses: rockel@biochem.mpg.de (B. Rockeklaus.kopec@tuebingen.mpg.de (K.O. Kopec), andrei.lup(A.N. Lupas), baumeist@biochem.mpg.de (W. Baumeiste

1570-9639/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.bbapap.2011.07.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 April 2011Received in revised form 29 June 2011Accepted 1 July 2011Available online 13 July 2011

Keywords:Tripeptidyl peptidase IICytosolic proteolysisHybrid structureProtein evolution

Tripeptidyl peptidase II is the largest known eukaryotic peptidase. It has been described as a multi-purposepeptidase, which, in addition to its house-keeping function in intracellular protein degradation, plays a role inseveral vital cellular processes such as antigen processing, apoptosis, or cell division, and is involved indiseases like muscle wasting, obesity, and in cancer. Biochemical studies and bioinformatics have identifiedTPPII as a subtilase, but its structure is very unusual: it forms a large homooligomeric complex (6 MDa) with aspindle-like shape. Recently, the high-resolution structure of TPPII homodimers (300 kDa) was solved and ahybrid structure of the holocomplex built of 20 dimers was obtained by docking it into the EM-density. Here,we summarize our current knowledge about TPPII with a focus on structural aspects. This article is part of aSpecial Issue entitled: Proteolysis 50 years after the discovery of lysosome.

roteolysis 50 years after the

Biochemistry, Department of82152 Martinsried, Germany.

l),as@tuebingen.mpg.der).

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

The ubiquitin–proteasome system constitutes the main pathway forprotein degradation in eukaryotic cells [1]. Itsmost downstreamelement,the 26S proteasome, has been studied in great detail even though a highresolution structureof theholocomplex is still not available. The structureof its proteolytic core complex, the 20S proteasome, has long been solvedand in conjunctionwithmutagenesis has clarified the role of 14α- and14β-subunits in protein breakdown [2]. In recent years, the group of giantpost-proteasomal proteases andespecially tripeptidyl peptidase II (TPPII)have also come into focus [3]. The proposed cellular role of the latter is incytosolic protein degradation downstream of the proteasome inconjunction with other exo- and endopeptidases [4]. Under conditionswhere the function of the proteasome is compromised, e. g. by inhibitors,but also in certain diseases, TPPII is upregulated and a number of studieshave been performed to reveal its role in health and disease states (see[5,6] for recent reviews). TPPII has been described as a ‘multi-purposepeptidase’ [7], and indeed, many functions have been ascribed to it, butin many cases its substrates or reaction partners have remainedobscure. Bioinformatic and biochemical studies had suggested early onthat the N-terminal part of TPPII is homologous to subtilisin [8] butthe function of the larger part of the polypeptide chain remainedenigmatic. Based on its similarity to subtilisin, homology models of the

active site region of TPPII have been created [9,10] and sequencecomparisons have been used to pinpoint potential functional regions[11]. New opportunities for the functional analysis of TPPII have beenopenedby the recent determinationof ahigh resolution structure of TPPIIusing a hybrid EM-X-ray crystallography approach, where the crystalstructure of TPPII dimers was docked into the structure of the TPPIIholocomplex obtained by cryo-electron microscopy [12]. In this reviewwe will summarize our current knowledge about this giant protease.

2. Cellular functions of TPPII

2.1. TPPII in cytosolic proteolysis

TPPII was discovered in 1983 in the extralysosomal fraction of ratliver during a search for peptidases with specificity to proteinsphosphorylated by cyclic AMP-dependent protein kinase [13]. Subse-quently it was found in many other tissues and also in red blood cells[14]. Its function – the removal of a tripeptide from the freeN-terminusof longer peptides – had up to then only been observed for TPPI, astructurally unrelated lysosomal peptidase. In addition to exopepti-dase activity, endopeptidase activity has also been ascribed to TPPII,but this activity is much lower than its exopeptidase activity [15,16].

So far only unfolded peptides have been reported to be cleaved byTPPII, the longest one, with a length of 41-residues, being Ova37–77[15]. Based on the type of substrates degraded and in analogy toTricorn protease [17], TPPII was assigned a role downstream of theproteasome in cellular protein degradation [4]; however, directexperimental evidence for this disassembly line is still lacking. Infact, the processing of proteasomal products is something TPPII has incommon with other peptidases like leucine aminopeptidase LAP [18],thimet oligopeptidase TOP [19], bleomycin hydrolase BH [20,21], or

238 B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

puromycin-sensitive aminopeptidase PSA [21,22]. Nonetheless, TPPIIappears to be the only peptidase capable of degrading peptides thatare longer than 15 residues [23,24].

Among the cellular functions attributed to TPPII, its function as aneuropeptidase inactivating the satiety hormone CCK8 is probablycharacterized best [25]. The cleavage of CCK8 to CCK5 in rat brain iscarried out by a membrane-associated version of TPPII, which wassuggested to be anchored in the lipid bilayer by a covalent glycosylphosphatidyl inositol link [25]. Its influence on satiety hasmadeTPPII aninteresting target for obesity-treatment and, indeed, TPPII-inhibition bytreatment with the specific inhibitor butabindide was shown to reducethe food intake in rats [25]. A role in fat metabolism was also proposedfor C. elegans TPPII, which surprisingly appeared to be independent ofthe presence of a functional proteolytic domain [26].

TPPII has been implicated in antigen processing, however the extentto which it is essential for the processing of antigenic peptides hasremained controversial (for reviews see [5,6,27]). TPPII was reported tobe involved in the generation of two viral epitopes [16,28], although aproteasomewith an altered specificity could also be responsible for thecreationof theseMHCclass-I peptides [29]. Altogether, the generation ofmost MHC class I-bound peptides appears to be independent of TPPII[27]. Nevertheless, the processing of peptides longer than 15 residuesrequires TPPII [23,24], but only a small fraction of the peptides releasedby the proteasome falls into that size range [24,30,31].

Fig. 1. 3D structure of the TPPII holocomplex. A) 3D-reconstruction of DmTPPII,segment numbers are indicated for one strand. B) DmTPPII rotated about 90° aroundthe longitudinal axis. Dimers in the strand on the left are highlighted in orange and redto visualize their stacking; the strand on the right was cut open in order to show theinternal cavity system of TPPII.

2.2. TPPII and its role in diseases

TPPII-activity is increased in skeletal muscle during sepsis-inducedmuscle wasting [32] as well as during cancer cachexia [33]. Responsiblefor the accelerated proteolysis under such catabolic conditions is theubiquitin proteasome-system [34,35], supporting the notion that TPPIIfunctions downstream of the proteasome and is co-induced with it.TPPII is also upregulated in tumor cells, a finding that might have animpact on cancer therapy: EL4 thymomaorEL4 lymphomacells adaptedto proteasome-inhibition aswell as Burkitt's lymphoma cells, where theproteasome appears to be functionally impaired, show increased TPPIIactivity [15,36,37]. From such observations it was concluded that TPPIImay allow survival of these cells by compensating for a loss ofproteasome function [15,36]. Indeed, it was shown that in Burkitt'slymphoma cells, protein turnover is unaffected and that ubiquitinatedproteins do not accumulate [37] unless the cells are treated with thecovalent serine protease inhibitor AAF-CMK, which inhibits TPPII[37,38]. However, AAF-CMK is not specific for TPPII, since it affectsalso other proteases like the proteasome [39]. In the presence of thespecific TPPII inhibitor butabindide or siRNA against TPPII no suchaccumulation occurs, implying that TPPII cannot substitute for theproteasome in the cleavage of ubiquitinated proteins [38].

Burkitt's lymphoma cells are apoptosis-resistant, but apoptosis canbe induced by TPPII-inhibition with AAF-CMK [37]. For EL-4 lymphomacells adapted to proteasome-inhibition apoptosis-resistance and in-creased growth-rate was ascribed to an impaired degradation ofinhibitors of apoptosis (IAP) and both features could be induced byTPPII-upregulation after TPPII-transfection [40]. HEK293 cells are yetanother cell line for which apoptosis-resistance and accelerated growthupon overexpression of TPPII were shown. Such TPPII-overexpressingHEK293-cells could survive the effect of the spindle poison nocodazoleand showed ahigher degree of aneuploidyaswell asmore structural andnumerical centrosome abnormalities than control cells [41,42]. Also thecell-division errors observed inBurkitt's lymphoma cells seem todependon TPPII, since the observed c-MYC induced centriole overduplicationcan be avoided by TPPII inhibitors like butabindide or by siRNA-mediated protein knock-down [43]. A participation of TPPII in celldivision, as suggested by these experiments, might be the reason for itsobserved localization in the vicinity of daughter centrioles in latemitosisand between daughter and mother centrioles during G2 phase [43].

In several malignant cell lines TPPII translocated into the nucleusupon γ-irradiation and the production of reactive oxygen species(ROS), which suggested a role for TPPII in DNA-repair [44,45].However, this translocation as well as the accumulation of p53remains controversial, since they were not observed in EL4 cells, COScells, and transformed fibroblasts [46,47], a discrepancy that wasattributed to different levels of ROS and sub-optimal cell densities [44].

2.3. TPPII-deficient species

In order to investigate the importance of TPPII for cell survival, anumber of TPPII-deficient species have been created. A T-DNAmutantof Arabidopsis defective in TPPII expression showed no phenotypicabnormalities [48] and likewise, a TPPII-knockout strain of S. pombewas viable and did not have any obvious growth defects [11,49].Suppressing TPPII expression by siRNA in C. elegans resulted indecreased fat stores in adult worms; however, reduced CCK8-degradation was not detectable and therefore no connection to satietycontrol could be established [26]. Divergent observations werereported for TPPII-deficient mice: McKay et al. [26] failed to obtainhomozygotic TPPII-deficient mice due to early embryonic lethality.However, their tpp2 heterozygous mutants were lean compared withwild-type littermates, while their food intake was normal. Kawaharaet al. [50] produced gene-trapped mice with an expression level ofTPPII reduced by N90% compared to wild-type. These mice with agene-trap disrupting tpp2 were viable, fertile, and normal inappearance and behavior. In contrast, Huai et al. [51] describeknockout mice homozygotic for tpp2−/−, which were viable but inwhich the TPPII-deficiency activated cell-type specific death programs.As a consequence the mice had a decreased life-span. Also, how TPPII-deficiency affects Drosophila is not clear. In a screen of lethal mutantson the second chromosome of D. melanogaster for those that couldenhance aweak Ras1 eggshell phenotype, one insertion disrupted twogenes, Nrk, a neurospecific receptor tyrosine kinase and TPPII.Whether the lethality is attributable to either of the two disruptedgenes alone or to the additive effect of both remains unclear [52].

239B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

3. TPPII structure

3.1. Quaternary structure of TPPII

The first electron micrographs of negatively stained TPPII particlesisolated from human erythrocytes were already published in 1996and showed that the subunits assemble into a large oligomericstructure [53]. Also Drosophila TPPII (DmTPPII) is a 6 MDa complexwith a spindle-like shape. It is composed of two 60 nm long twistedstrands build of 10 stacked dimers, where each strand encloses acentral cavity system that is accessible through lateral openings[54,55] (Fig. 1). TPPII dimers possess only 10% of the specific activity ofthe spindle [56,57] but upon assembly their specific activity increaseswith each newly formed interface, suggesting a contact-inducedactivation mechanism [57]. Compared to long, single strands thespindles are thermodynamically stabilized. This is accomplished by a

Fig. 2. High resolution structure of DmTPPII. A) Domain composition ofDmTPPII and comparisonsites D44, H272, S462 are shown as red asterisks; orange: insertionwithin the active site (residue1099–1354); gray blocks: Loops that arenot present in the crystal structure ofDmTPPII (PDB ID: 3are shown as red asterisks. B)DmTPPIImonomer shown in ribbon representation. Domain colordescribed above, one of themonomers is shown in ribbon— the other in surface representation.(PDB ID: 1CSE). Active site residuesD44, H272, S462, aswell as thehelix connected to S462 and lto S221 and loop “L” of subtilisin are shown in blue. E, F) Conformation of thehelix connected to tloop L2 residues bound to the active site as well as the location of the double-Glu motif (E312, E

‘double-clamp’, a structural feature allowing reciprocal interactionsbetween dimers positioned at the ends of the two strands [55].

3.2. Crystal structure of TPPII dimers

The concentration-dependent size of TPPII oligomers induces apolymorphism, which is not conducive to crystallization [57]. However,TPPII spindles can be dissociated by cold-treatment and reassembly ofthe dimers can be prevented by the presence of detergents such as octylglucoside, which made it possible to obtain crystals [12]. TPPII mono-mers are 128–150 kDa in size, dependent on the species. The 150-kDamonomer of DmTPPII can be divided into three basic domains (Fig. 2A):The N-terminal domain represents the subtilisin domain, which in TPPIIis interrupted by a long insertion between the catalytic D44 and H272residues. The central domain ismainly composed of β-strands. Together,these two domains form a ring-structure with a central hole, which

with subtilisin. Upper bar: Yellow: subtilisin-like domain of TPPII (residues 1–522), actives 75–266); green: central domain (residues 523–1098); blue: C-terminal domain (residuesLXU). Lower bar: yellow: subtilisin Carlsberg aligned to dmtppII, active sites D32, H64, S221s are as described in A). C)DmTPPII dimer shown in two orientations. Domain colors are asD) Overlay of the high resolution structure of TPPII (yellow)with subtilisin Carlsberg (gray)oop L2 of TPPII are shown in red, active site residues D32,H64, S221 and thehelix connectedhe active site serine in subtilisin (E) andTPPII (F). G) Active-site region of TPPII showing the343). The N-terminal continuation of L2 is indicated by a dotted red line next to L457 (P3).

240 B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

contains the active site on one side (Fig. 2B). The insertion connects thering-structurewith the C-terminal domain, which ismainlyα-helical. Ina TPPII-dimer, two monomers are arranged in a C2-symmetry relatedposition (Fig. 2C) and through this arrangement the holes in the centersof the rings are covered reciprocally and the C-terminal domainsassemble into a handle-like structure.

The active site residues D44 and H272 of DmTPPII are in aconformation very similar to that of the corresponding residues D32andH64 of subtilisin Carlsberg, but in contrast to S221 in subtilisin, S462of TPPII is pointing away from the other two residues of the catalytictriad (Fig. 2D). This difference in orientation is correlated with adifference in the position of the two serine residues: S221 in subtilisin islocated at the N-terminal end of anα-helix. S462 in TPPII is also close totheN-terminal endof ahelix but–unlikeS221 in subtilisin– it is notpartof the helix (Fig. 2E and F). At the N-terminal side of S221 in subtilisinthere is a stretch comprising ~20 residues that form a rather rigidstructure composed of two antiparallel β-sheets (loop “L” in Fig. 2D). Incontrast, most of the 20 residues at the N-terminal end of S462 in TPPIIbelong to the flexible loop L2 with no defined density in the crystalstructure (Fig. 2D); only residues L457-N459arevisible; theyoccupy thesubstrate-binding cleft of TPPII (Fig. 2D and G). The combination of adisplaced active-site serine residue and internal loop-residues bound tothe substrate-binding cleft is believed to be the reason for the observedlow activity of TPPII dimers [56,57].

The main activity of TPPII is that of a tripeptidyl peptidase. Thestructural realization of the molecular ruler ensuring the separation ofexactly a tripeptide is a ‘double-Glu’motif, which had been ascribed toTPPII already earlier [10] and which is also found in prolyl tripeptidylpeptidase [58], dipeptidyl peptidase IV [59], tricorn-interactingaminopeptidase F1 factor [60], and aminopeptidase N [61]. In TPPII,the two glutamate residues E312 and E343 of the double-Glu motifform a ridge that prevents the binding of a fourth residue to thesubstrate-binding site thus limiting access to three residues only. Atthe same time they interact with the positively charged N-terminus ofthe substrate. Since TPPII can also act as an endopeptidase, longerpeptides must be able to bind to the peptide binding cleft as well. Theconformation of the three residues of the internal loop L2 that arebound to the substrate-binding cleft in the crystal structure mayindicate how an endopeptidase substrate can be accommodated: theP3-residue L457 does not possess a free N-terminus and cannot built a

Fig. 3. Hybrid structure of the TPPII spindle. A) Crystal structure of theDmTPPII-dimer docked iribbon representation. Thecolor codecorresponds toFig. 2A.B)Schematicdrawingof the cavitytheactive sites.H:Handles, CC: Catalytic chamber,AC:Antechamber, F: Foyer. Arrows label the eof TPPII: The stacking of the dimers leads to the formation of the cavity system, the reorientat

salt-bridge with the double-Glu motif, the P4 residue cannot bind tothe blocked S4 position and as a consequence P3 would have to bendout of the cleft (Fig. 2G; [12]).

3.3. Hybrid structure of the TPPII spindle

The hybrid structure of TPPII revealed the location of the domainsof TPPII within the spindle architecture of the complex (Fig. 3A) andshowed that the N-terminal and central domains are located at theinner, concave side of the spindle, whereas the C-terminal domainsconstitute the outer, convex side [12]. Through the stacking ofthe dimers into strands, the active sites, which are located onthe surface in dimers (see Fig. 2), are sequestered inside a largecavity system traversing the strands. Substrates must enterthrough an opening at the handles into the foyer and proceedthrough the antechamber to reach either of the two catalyticchambers sandwiched between each two dimers (Fig. 3B and [12]).Constrictions at entry and exit sites of the antechambers are approx.30 Å by 15 Å restricting access to unfolded polypeptides. In additionto compartmentalizing the active sites, the stacking of dimers intostrands also induces activation, during which the S462 is moved toits catalytically active position and the L2 residues bound to thesubstrate-binding cleft are displaced. Most likely, the interactionof the flexible loop L2 with the neighboring dimer triggers thisrearrangement (Fig. 3C and [12]).

4. Functional regions in TPPII

Whereas the N-terminal third of the TPPII sequence had beenidentified as its proteolytic domain long time ago [62], the role of its C-terminal two-thirds remained enigmatic. From the hybrid structure ofTPPII it is obvious that a large portion of the polypeptide chain isinvolved in the formation of the unique cavity system of the complex(see Fig. 2C). Whether parts of the sequence are also involved in otherfunctions like substrate-binding or interaction with co-factors, iscurrently unknown. For some parts of the sequences there isstructural and functional information based on sequence analysis,mutations and biochemical evidence [11] (Fig. 4). The residues of theactive site [62,63] and the double-Glu motif [10,12] have beenconfirmed by biochemical studies and site-directed mutagenesis. The

nto the EM-map of the DmTPPII spindle (mesh representation). TPPII dimers are shown insystemthat is created through the stackingof thedimers into strands.Reddots: Locationsofntrances into thecavity systemat either site of thehandles of adimer. C)Activation-schemeion of loop L2 and also to the correct placement of the active-site serine residue S462.

241B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

insertion within the catalytic site, which has been reported to beinvolved in complex formation [64], is indeed located at the dimer–dimer interface in spindles. A special role in complex formation wasattributed to G252, a residue within the insertion, since the mutationG252R prevented the assembly of dimers into spindles [64], eventhough at high protein concentrations assembly does proceed [57]. Arole in the assembly-dependent activation of DmTPPII has beenproposed for residues L457-N459 and L603-R610. Residues L457-N459, which are connected to a flexible loop, are bound to thesubstrate-binding cleft in dimers [12]. It has been hypothesized thatduring assembly they are removed from the cleft due to theinteraction of the flexible loop with residues L603-R610 of theadjacent dimer [12]. The region around K1219 appears to beconserved in arthropods [11] and the corresponding region in HsTPPIIcontains the first 18 residues of a 20 kDa fragment, which can beproduced by chymotryptic cleavage of HsTPPII [65]. When thecleavage is carried out in the presence of CaEGTA, the 20 kDafragment is not observed; instead, a 30 kDa fragment is produced,

Fig. 4. Functional regions in TPPII. Functional regions in the TPPII sequence (amino acid sequFlybase ID FBpp0086888) are highlighted (and numbered) in both the sequence (A) and in thmissing in the crystal structure: loops L1, L2 and L3, aswell as 17 residues at the beginning of tresolution structure of DmTPPII-monomers in ribbon-representation, the locations of the funmonomers in surface representation in two orientations. Red (1): active site residues (D44,active site residues D44 andH272, residues Y69-L263, corresponding to residues 68–255 inHsdescribed to be critical for complex formation [64]. Orange (4): Residues L457-N459 are bounsupposedly involved in the activation of DmTPPII [12]. Bright green (7): R1012-V1013, in a(HsTPPII 983–984) in chordate and are affecting complex formation [66,67]. Dark blue (8): K1fragment, which can be produced if TPPII is cleaved by chymotrypsin in the absence of Caproduced instead of K1219-N1238 after chymotrypsin cleavage of TPPII in the presence of Cabeen described as hypervariable in mammals [11,66]. For all functional regions – except the

which corresponds to residues 939–956 in HsTPPII. This suggests thatthe corresponding regions can undergo conformational changes[8,11], which is quite interesting, since they reside in differentdomains of the complex. The region corresponding to residues 1137–1157 in HsTPPII was described as hypervariable in mammals [11,66]and in the crystal structure of DmTPPII dimers it represents a loopregion connecting two helices of the C-terminal domain. In HEK293cells the cDNA of a potential splicing variant of TPPII has beenidentified and expressed. The additional 13 residues located betweenresidues 983 and 984 in HsTPPII seem to lead to the formation of alarger TPPII complex or to aggregation [66,67]. In DmTPPII thecorresponding residues are located at the dimer–dimer contactregion, where it is quite likely that they influence complex formation.

5. Evolution of TPPII

Although TPPII is thought to be specific to eukaryotes, it is clearly amember of the subtilisin family and thus homologous to many

ence of the expressed Drosophila TPPII, http://uniprot.org/uniprot/Q9V6K1 Isoform 2, ore crystal structure (B–C) of DmTPPII. The underlined residues belong to regions that arehe sequence and a stretch of 14 residues in the insertion betweenD44 andH272. B)High-ctional regions highlighted in A) are indicated. C) High-resolution structure of DmTPPII-H272, and S462) and double-Glu motif (E312, E343) [12], Crème (2): insertion withinTPPII (DH-insert) [64].Magenta: G260 (3), corresponding to G252 inHsTPPII, whichwasd to the active site in the crystal structure ofDmTPPII [12]. Olive (5): Residues L603-R610splicing variant of TPPII, 13 residues are inserted between the corresponding residues219-N1238, corresponding region inHsTPPII represents the first 18 residues of a 20 kDaEGTA [11,65]. Dark green (6): S967-T973, corresponding region in HsTPPII 939–946 isEGTA [8,11], Cyan (9): L1257-K1262, corresponding region in HsTPPII (1137–1157) hasDH-insert – residues are shown in stick representation for better visibility.

242 B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

prokaryotic proteins. To explore this relationship, we performed abioinformatic analysis using structural (DALI [68]) and sequencecomparisons (HHpred [69]), aswell as clustering (CLANS [70])methods(Fig. 5).

As a first step, we compared the sequences of all subtilisins in thenon-redundant database and clustered them by the significance oftheir pairwise matches. The resulting map (Fig. 5A and B) shows acompact cluster for eukaryotic TPPII proteins (green in Fig. 5A and B),with two embedded bacterial proteases that presumably originated bylateral transfer (pink): a protein from Blastopirellulamarina, annotatedincorrectly as pyrolysin but previously reported by Eriksson et al. as aTPPII homolog [11], and a protein from Planctomyces brasiliensis.Closely connected to eukaryotic TPPII are a cluster of actinobacterialproteases (yellow) and the pyrolysins of euryarchaea (red) – whichcan thus be considered the bacterial and archaeal orthologs of TPPII,respectively – as well as a few peripheral sequences from environ-mental microorganisms (Symbiobacterium, Thermincola, Planctomyces,Chloroherpeton). The STABLEprotease of crenarchaea (stalk-associatedarchaebacterial endoprotease [71]) is a divergent satellite cluster toTPPII, branching off from the pyrolysins. The TPPII group is connectedto the main subtilisin groups via a large cluster of sequences foundmainly in actinobacteria (blue in Fig. 5A and C), which we will refer toin the following as the anchor group (the second group of eukaryoticproteases originating from this anchor group – light blue in Fig. 5A andC – is membrane-bound transcription factor site-1 protease). Thebroad range of organisms containing TPPII-like proteins suggests that aprimitive form of TPPII was already present in the last universalcommon ancestor (LUCA).

In order to gain an understanding of the domain structure of thisearly form of TPPII and its evolution to the giant, multi-domainprotease found in eukaryotes, we performed a domain analysis of thegroups of TPPII relatives identified by clustering (colored clusters inFig. 5A). TPPII was previously described by sequence analysis ascomposed of three parts: (i) the subtilisin domain, including theinsertion; (ii) a group of domains consisting of β-sheets; and (iii) ahelical C-terminal part [12]. For the present analysis, we used a moreprecise, structure-derived division of TPPII into eight domains, whichwe named A–H. The domains are arranged along the sequence fromN- to C-terminus as A′–B′–C–B″–A″–D–E–F′–G–F″–H (Fig. 5D and E).In case of domain insertions, letters with single and double primesindicate the N- and C-terminal parts of one domain, respectively. Thisnew partition offers a finer-grained view than the previously usedone, but the two correspond directly: part (i) contains A, B, and C, part(ii) comprises the four β-sheet domains D, E, F, and G, and part (iii) issynonymous for H.

The N-terminal domain A is the subtilisin-like domain anduniversally present. Within the catalytic triad of A, domain B isinserted and domain C is again inserted inside domain B, which yieldsthe rather unusual telescopic arrangement A′–B′–C–B″–A″. Insertdomain B adopts an unusual fold so far not captured in SCOP or CATH.Its closest structural relative appears to be another insert domain,which protrudes from the catalytic domain of oligosaccharyltransfer-ase (OST), a multidomain enzyme that catalyzes the co-translationaltransfer of an oligosaccharide from a lipid donor to an asparagineresidue in nascent polypeptide chains [72]. We were unable to detecthomologues of domain B by sequence comparisons outside the TPPIIgroup and no other proteins of the same fold were found using DALI.All proteins of the TPPII group have domain B (Fig. 5C), including thebacterial and archaeal orthologs, and the satellite clusters (STABLE,Thermaerobacter). Domain C is not so much a domain as an elongated,structured protrusion within domain B; it is difficult to detect bysequence comparisons due to its small size and poor conservation, sonot much can be concluded from its apparent absence in manyproteins containing domain B.

The four domains D, E, F, and G in the central part (ii) of TPPII areall β-domains, with G inserted into domain F. Domains D and F are

typical IG folds of the PapD superfamily, E is a jelly-roll β-sandwich ofunclear origin, and G is homologous to the collagen-binding domain ofclass 1 collagenase by structure and sequence. Domain F may beinvolved in dimerization, since in TPPII dimers it localized at themonomer–monomer interface. Clearly, domain D is present in allTPPII relatives analyzed here, including the actinobacterial anchorcluster (blue in Fig. 5A and C), and distinguishes these proteins fromother subtilisins. Domain E is present in the TPPII group, including itssatellites, and domains F and G are only reliably detected in theeukaryotic TPPII core cluster. Most proteins analyzed here containother, seemingly unrelated domains following domains D and E.

The C-terminal part (iii) of TPPII, i.e. domain H, is a solenoid of fivehairpinswith strong similarity to tetratricopeptide repeats (TPR),whichare often involved in protein–protein-interactions [73,74]. This issuggestive, since domain H is the region flanking the entrance to thecavity systemof TPPII. Indeed, the last twohairpins are clearly identifiedas TPR and the domain as a whole is recognized by HMM–HMMsequence comparisons as a TPR-like solenoid. Domain H seems to bespecific for the eukaryotic TPPII cluster, howeverwenote thatpyrolysinsalso seem to contain a much shorter version of domain H consisting ofonly two TPR hairpins, which is not necessarily of homologous origin, asit could have arisen by an independent fusion event.

Except for TPPII, which is intracellular, all other TPPII-relatedproteases have signal peptides (as predicted with SignalP, [75]; Fig. 5C).In addition, most of them have a propeptide located N-terminally to thesubtilisindomain,whichappears tobea subtilisin-specific intramolecularchaperone [76]. Its cleavage leads to activation of the subtilases [76],which may explain its absence in TPPII, where activation is coupled toassembly [57]. Indeed, this is one of several indications that higheroligomeric assemblymight be specific to eukaryotic TPPII. Thus, domainsF (which may mediate dimerization) and H (which interacts with theinsert domain C to give TPPII monomers their peculiar shape for spindleassembly) are also specific to these proteins.

These analyses suggest that the earliest form of a TPPII-likeprotease was an extracellular protease containing domains A and D.The TPPII branch proper subsequently acquired domains B and E, andthe eukaryotic form of the protein finally lost the signal sequence andpropeptide, while adding domains F, G, and H. It is attractive toconsider that the re-localization of the protease to the cytosolfollowing loss of the signal sequence provided the evolutionarypressure for segregating the active sites by self-compartmentalizationand thus for recruiting domains F and H.

6. Conclusions

In recent years, substantial progress has been made in elucidatingthe structure and function of TPPII. Structural studies have revealed anintriguing architecture but why TPPII forms an assembly of suchextraordinary size remains amystery. The high-resolution structure ofTPPII obtained by using a hybrid EM-X-ray crystallography approachhas revealed an extensive network of internal cavities and channels,which sequester the active sites from the cytosolic environment andcontrol access to them. The structure suggests a mechanism by whichthe essentially inactive dimers are converted into active ones uponassembly; activation follows sequestration of the active sites [12] — acommon theme among self-compartmentalizing proteases. A double-Glu motif in the catalytic domain provides a molecular ruler for thecleavage of tripeptides, as verified by mutational studies [10,12]. Aputative endoproteolytic activity and its functional significance awaitfurther clarification.

The list of physiological functions ascribed to TPPII in health anddisease is long but in almost all cases the exact role of TPPII remainedenigmatic. The grand challenge for the years to come is to firmlyestablish the role of TPPII in cellular processes. In the case of theproteasome the dissection of its physiological functions was greatlyfacilitated by the availability of specific proteasome inhibitors [77,78].

Fig. 5. Analysis of TPPII-like proteins: Domain composition and cluster map. A, B) Cluster maps. We used the buildali.pl script of HHpred with the sequence of TPPII as query to searchthe non-redundant database of NCBI for TPPII homologs. The script performs iterative PSI-BLAST runs and contains heuristics to reduce false positive matches caused bynonhomologous sequence segments at the end of PSI-BLAST matches. The obtained sequences were clustered to equilibrium in CLANS [70] at a P-value cutoff of e-25 using defaultsettings. In the cluster maps, dots represent proteins, while the gray lines represent BLAST p-values— the darker a line, the more significant the p-value. Both panels show the samemap but with different color schemes. In A), proteins are colored according to their group membership, while in B they are colored by superkingdom (blue = Archaea, yellow =Bacteria, green=Eukaryota). Subtilisin Carlsberg is marked in black as reference outgroup. Proteins shown inwhite are distantly related to TPPII andwere not analyzed in this study.The blow-up shows amagnified view of the TPPII cluster. C) Table of domain compositions of TPPII-like proteins. Rows correspond to the different groups (color-coded as in A), whilecolumns correspond to the constituent domains, detected by SignalP [75] and HHpred [69]. D) Eight-domain composition of TPPII. Domain borders for the basic domains areindicated below the colored bar, domain borders for the inserted domains are indicated above the colored bar. A′ denotes the N-terminal, A″ denotes the C-terminal part of domain A,the same code is used for the divided domains B and F. The dashed line between domains F″ and H represents themissing loop L3. E) Ribbon representation of a TPPII monomer (left)and octamer (right) color coded as described in D. The dashed line between domains F and H corresponds to loop L3.

243B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

244 B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

The TPPII inhibitor butabindide [25] is of limited usefulness for in vivostudies because it is inhibited by serum factors necessitating the use ofcells adapted to serum-free media [23].

Traditionally, functional studies with enzymes are performed afterisolation and purification. However, enzymes do not function inisolation. They are embedded in interaction networks and key tounderstanding this cellular role is an understanding of the spatio-temporal organization of these networks. To some extend this can beachieved by perturbing the relevant networks, for example by theapplication of inhibitory molecules. Or it can be approached byidentifying interaction partners, either one by one, or by using largescale (‘omics’) approaches. This may or may not allow to put togetherthe puzzle. Another strategy which could be pursued is visualproteomics using light optical [79,80] or electron microscopy[81,82]. Given the size of TPPII it should be possible to identify andlocalize TPPII in cryotomograms and relate it to other known cellularand molecular structure. A potential problem is the low abundance ofTPPII in many cell types. At this stage it would be very useful to knowmore about the relative abundance of TPPII in different cellularterritories — a challenge for quantitative proteomics.

Acknowledgments

This work was supported by funding from the Deutsche For-schungsgemeinschaft (Ro-2036/5-1 and SFB 594) and by institutionalfunds from the Max Planck Society.

References

[1] A. Hershko, A. Ciechanover, The ubiquitin system, Annu. Rev. Biochem. 67 (1998)425–479.

[2] D. Voges, P. Zwickl, W. Baumeister, The 26S proteasome: a molecular machinedesigned for controlled proteolysis, Annu. Rev. Biochem. 68 (1999) 1015–1068.

[3] T.T. Yao, R.E. Cohen, Giant proteases: beyond the proteasome, Curr. Biol. 9 (1999)R551–R553.

[4] B. Tomkinson, Tripeptidyl peptidases: enzymes that count, Trends Biochem. Sci.24 (1999) 355–359.

[5] G. Preta, R. de Klark, R. Gavioli, R. Glas, The enigma of tripeptidyl-peptidase II: dualroles in housekeeping and stress, J. Oncol. Manag. 2010 (2010) 1–10.

[6] P. van Endert, Post-proteasomal and proteasome-independent generation of MHCclass I ligands, Cell. Mol. Life Sci. 68 (2011) 1553–1567.

[7] B. Tomkinson, A.C. Lindas, Tripeptidyl-peptidase II: amulti-purpose peptidase, Int.J. Biochem. Cell Biol. 37 (2005) 1933–1937.

[8] B. Tomkinson, A.K. Jonsson, Characterization of cDNA for human tripeptidylpeptidase-II — the N-terminal part of the enzyme is similar to subtilisin,Biochemistry 30 (1991) 168–174.

[9] H. De Winter, H. Breslin, T. Miskowski, R. Kavash, M. Somers, Inhibitor-basedvalidation of a homology model of the active-site of tripeptidyl peptidase II, J. Mol.Graph. Model. 23 (2005) 409–418.

[10] A.C. Lindas, S. Eriksson, E. Jozsa, B. Tomkinson, Investigation of a role for Glu-331and Glu-305 in substrate binding of tripeptidyl-peptidase II, Biochim. Biophys.Acta, Proteins Proteomics 1784 (2008) 1899–1907.

[11] S. Eriksson, O.A. Gutierrez, P. Bjerling, B. Tomkinson, Development, evaluation andapplication of tripeptidyl-peptidase II sequence signatures, Arch. Biochem.Biophys. 484 (2009) 39–45.

[12] C.K. Chuang, B. Rockel, G. Seyit, P.J. Walian, A.M. Schonegge, J. Peters, P.H. Zwart,W. Baumeister, B.K. Jap, Hybrid molecular structure of the giant proteasetripeptidyl peptidase II, Nat. Struct. Mol. Biol. 17 (2010) 990–996.

[13] R.M. Balow, U. Ragnarsson, O. Zetterqvist, Tripeptidyl aminopeptidase in theextralysosomal fraction of rat liver, J. Biol. Chem. 258 (1983) 11622–11628.

[14] R.M. Balow, B. Tomkinson, U. Ragnarsson, O. Zetterqvist, Purification, substrate-specificity, and classification of tripeptidyl peptidase-II, J. Biol. Chem. 261 (1986)2409–2417.

[15] E. Geier, G. Pfeifer, M. Wilm, M. Lucchiari-Hartz, W. Baumeister, K. Eichmann, G.Niedermann, A giant protease with potential to substitute for some functions ofthe proteasome, Science 283 (1999) 978–981.

[16] U. Seifert, C. Maranon, A. Shmueli, J.F. Desoutter, L. Wesoloski, K. Janek, P.Henklein, S. Diescher, M. Andrieu, H. de la Salle, T. Weinschenk, H. Schild, D.Laderach, A. Galy, G. Haas, P.M. Kloetzel, Y. Reiss, A. Hosmalin, An essential role fortripeptidyl peptidase in the generation of an MHC class I epitope, Nat. Immunol. 4(2003) 375–379.

[17] N. Tamura, F. Lottspeich, W. Baumeister, T. Tamura, The role of tricorn proteaseand its aminopeptidase-interacting factors in cellular protein degradation, Cell 95(1998) 637–648.

[18] J. Beninga, K.L. Rock, A.L. Goldberg, Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducingleucine aminopeptidase, J. Biol. Chem. 273 (1998) 18734–18742.

[19] X.Y. Mo, P. Cascio, K. Lemerise, A.L. Goldberg, K. Rock, Distinct proteolyticprocesses generate the C and N termini of MHC class I-binding peptides,J. Immunol. 163 (1999) 5851–5859.

[20] D. Bromme, A.B. Rossi, S.P. Smeekens, D.C. Anderson, D.G. Payan, Humanbleomycin hydrolase: molecular cloning, sequencing, functional expression, andenzymatic characterization, Biochemistry 35 (1996) 6706–6714.

[21] L. Stoltze, M. Schirle, G. Schwarz, C. Schroter, M.W. Thompson, L.B. Hersh, H.Kalbacher, S. Stevanovic, H.G. Rammensee, H. Schild, Two new proteases in theMHC class I processing pathway, Nat. Immunol. 1 (2000) 413–418.

[22] G.D. Johnson, L.B. Hersh, Studies on the subsite specificity of the rat-brainpuromycin-sensitive aminopeptidase, Arch. Biochem. Biophys. 276 (1990) 305–309.

[23] E. Reits, J. Neijssen, C. Herberts, W. Benckhuijsen, L. Janssen, J.W. Drijfhout, J.Neefjes, A major role for TPPII in trimming proteasomal degradation products forMHC class I antigen presentation, Immunity 20 (2004) 495–506.

[24] I.A. York, N. Bhutani, S. Zendzian, A.L. Goldberg, K.L. Rock, Tripeptidyl peptidase IIis the major peptidase needed to trim long antigenic precursors, but is notrequired for most MHC class I antigen presentation, J. Immunol. 177 (2006)1434–1443.

[25] C. Rose, F. Vargas, P. Facchinetti, P. Bourgeat, R.B. Bambal, P.B. Bishop, S.M.T. Chan,A.N.J. Moore, C.R. Ganellin, J.C. Schwartz, Characterization and inhibition of acholecystokinin-inactivating serine peptidase, Nature 380 (1996) 403–409.

[26] R.M. McKay, J.P. McKay, J.M. Suh, L. Avery, J.M. Graff, Tripeptidyl peptidase IIpromotes fat formation in a conserved fashion, EMBO Rep. 8 (2007) 1183–1189.

[27] P. van Endert, Role of tripeptidyl peptidase II in MHC class I antigen processing —

the end of controversies? Eur. J. Immunol. 38 (2008) 609–613.[28] S. Guil, M. Rodriguez-Castro, F. Aguilar, E.M. Villasevil, L.C. Anton, M. Del Val, Need

for tripeptidyl-peptidase II in major histocompatibility complex class I viralantigen processing when proteasomes are detrimental, J. Biol. Chem. 281 (2006)39925–39934.

[29] E.J. Wherry, T.N. Golovina, S.E. Morrison, G. Sinnathamby, M.J. McElhaugh, D.C.Shockey, L.C. Eisenlohr, Re-evaluating the generation of a “proteasome-indepen-dent” MHC class I-restricted CD8 T cell epitope, J. Immunol. 176 (2006)2249–2261.

[30] I. Dolenc, E. Seemüller, W. Baumeister, Decelerated degradation of short peptidesby the 20S proteasome, FEBS Lett. 434 (1998) 357–361.

[31] A.F. Kisselev, T.N. Akopian, K.M. Woo, A.L. Goldberg, The sizes of peptidesgenerated from protein by mammalian 26 and 20 S proteasomes — implicationsfor understanding the degradative mechanism and antigen presentation, J. Biol.Chem. 274 (1999) 3363–3371.

[32] C.J. Wray, B. Tomkinson, B.W. Robb, P.O. Hasselgren, Tripeptidyl-peptidase IIexpression and activity are increased in skeletal muscle during sepsis, Biochem.Biophys. Res. Commun. 296 (2002) 41–47.

[33] A. Chand, S.M. Wyke, M.J. Tisdale, Effect of cancer cachexia on the activity oftripeptidyl-peptidase II in skeletal muscle, Cancer Lett. 218 (2005) 215–222.

[34] W.E. Mitch, A.L. Goldberg, Mechanisms of disease – mechanisms of musclewasting – the role of the ubiquitin-proteasome pathway, N. Engl. J. Med. 335(1996) 1897–1905.

[35] D. Attaix, S. Ventadour, A. Codran, D. Bechet, D. Taillandier, L. Combaret, Theubiquitin-proteasome system and skeletal muscle wasting, Essays in Biochemis-try, Vol 41: the Ubiquitin-Proteasome System, 41, 2005, pp. 173–186.

[36] R. Glas, M. Bogyo, J.S. McMaster, M. Gaczynska, H.L. Ploegh, A proteolytic systemthat compensates for loss of proteasome function, Nature 392 (1998) 618–622.

[37] R. Gavioli, T. Frisan, S. Vertuani, G.W. Bornkamm, M.G. Masucci, c-mycoverexpression activates alternative pathways for intracellular proteolysis inlymphoma cells, Nat. Cell Biol. 3 (2001) 283–288.

[38] E.M. Villasevil, S. Guil, L. Lopez-Ferreras, C. Sanchez, M. Del Val, L.C. Anton,Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chlor-omethylketone is independent of the inhibition of tripeptidyl peptidase II,Biochim. Biophys. Acta, Mol. Cell Res. 1803 (2010) 1094–1105.

[39] M. Bury, I. Mlynarczuk, E. Pleban, G. Hoser, J. Kawiak, C. Wojcik, Effects of aninhibitor of tripeptidyl peptidase II (Ala-Ala-Phe-chloromethylketone) and itscombination with an inhibitor of the chymotrypsin-like activity of the protea-some (PSI) on apoptosis, cell cycle and proteasome activity in U937 cells, FoliaHistochem. Cytobiol. 39 (2001) 131–132.

[40] X. Hong, L. Lei, R. Glas, Tumors acquire inhibitor of apoptosis protein (IAP)-mediated apoptosis resistance through altered specificity of cytosolic proteolysis,J. Exp. Med. 197 (2003) 1731–1743.

[41] V. Stavropoulou, J.J. Xie, M. Henriksson, B. Tomkinson, S. Imreh, M.G. Masucci,Mitotic infidelity and centrosome duplication errors in cells overexpressingtripeptidyl-peptidase II, Cancer Res. 65 (2005) 1361–1368.

[42] V. Stavropoulou, V. Vasquez, B. Cereser, E. Freda, M.G. Masucci, TPPII promotesgenetic instability by allowing the escape from apoptosis of cells with activatedmitotic checkpoints, Biochem. Biophys. Res. Commun. 346 (2006) 415–425.

[43] S. Duensing, S. Darr, R. Cuevas, N. Melquiot, A.G. Brickner, A. Duensing, K. Münger,Tripeptidyl peptidase II is required for c-MYC-induced centriole overduplicationand a novel therapeutic target in c-MYC-associated neoplasms, Genes & Cancer 1(2010) 883–892.

[44] G. Preta, R. de Klark, R. Glas, A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses,Biochem. Biophys. Res. Commun. 389 (2009) 575–579.

[45] G. Preta, R. de Klark, S. Chakraborti, R. Glas, MAP kinase-signaling controls nucleartranslocation of tripeptidyl-peptidase II in response to DNA damage and oxidativestress, Biochem. Biophys. Res. Commun. 399 (2010).

[46] E. Firat, C. Tsurumi, S. Gaedicke, J. Huai, G. Niedermann, Tripeptidyl peptidase IIplays a role in the radiation response of selected primary cell types but not basedon nuclear translocation and p53 stabilization, Cancer Res. 69 (2009) 3325–3331.

245B. Rockel et al. / Biochimica et Biophysica Acta 1824 (2012) 237–245

[47] C. Tsurumi, E. Firat, S. Gaedicke, J. Huai, P.K. Mandal, G. Niedermann, Viability andDNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts,Biochem. Biophys. Res. Commun. 386 (2009) 563–568.

[48] A.J. Book, P.Z. Yang, M. Scalf, L.M. Smith, R.D. Vierstra, Tripeptidyl peptidase II. Anoligomeric protease complex fromarabidopsis, Plant Physiol. 138 (2005) 1046–1057.

[49] A. Decottignies, I. Sanchez-Perez, P. Nurse, Schizosaccharomyces pombe essentialgenes: a pilot study, Genome Res. 13 (2003) 399–406.

[50] M. Kawahara, I.A. York, A. Hearn, D. Farfan, K.L. Rock, Analysis of the role oftripeptidyl peptidase II in MHC class I antigen presentation in vivo, J. Immunol.183 (2009) 6069–6077.

[51] J. Huai, E. Firat, A. Nil, D. Million, S. Gaedicke, B. Kanzler, M. Freudenberg, P. vanEndert, G. Kohler, H.L. Pahl, P. Aichele, K. Eichmann, G. Niedermann, Activation ofcellular death programs associated with immunosenescence-like phenotype inTPPII knockout mice, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 5177–5182.

[52] J.D. Schnorr, R. Holdcraft, B. Chevalier, C.A. Berg, Ras1 interacts with multiple newsignaling and cytoskeletal loci in Drosophila eggshell patterning and morpho-genesis, Genetics 159 (2001) 609–622.

[53] E. Macpherson, B. Tomkinson, R.M. Balow, S. Hoglund, O. Zetterqvist, Supramolecularstructure of tripeptidyl peptidase II from human erythrocytes as studied by electronmicroscopy, and its correlation to enzyme activity, Biochem. J. 248 (1987) 259–263.

[54] B. Rockel, J. Peters, B. Kühlmorgen, R.M. Glaeser, W. Baumeister, A giant proteasewith a twist: the TPP II complex from Drosophila studied by electron microscopy,EMBO J. 21 (2002) 5979–5984.

[55] B. Rockel, J. Peters, S.A. Müller, G. Seyit, P. Ringler, R. Hegerl, R.M. Glaeser, W.Baumeister, Molecular architecture and assembly mechanism of Drosophilatripeptidyl peptidase II, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 10135–10140.

[56] B. Tomkinson, Association and dissociation of the tripeptidyl-peptidase II complex as away of regulating the enzyme activity, Arch. Biochem. Biophys. 376 (2000) 275–280.

[57] G. Seyit, B. Rockel, W. Baumeister, J. Peters, Size matters for the tripeptidylpepti-dase II complex from Drosophila — The 6-MDa spindle form stabilizes theactivated state, J. Biol. Chem. 281 (2006) 25723–25733.

[58] Y. Xu, Y. Nakajima, K. Ito, H. Zheng, H. Oyama, U. Heiser, T. Hoffmann, U.T. Gartner,H.U. Demuth, T. Yoshimoto, Novel inhibitor for prolyl tripeptidyl aminopeptidasefrom Porphyromonas gingivalis and details of substrate-recognition mechanism,J. Mol. Biol. 375 (2008) 708–719.

[59] M. Engel, T. Hoffmann, L. Wagner, M. Wermann, U. Heiser, R. Kiefersauer, R.Huber, W. Bode, H.U. Demuth, H. Brandstetter, The crystal structure of dipeptidylpeptidase IV(CD26) reveals its functional regulation and enzymatic mechanism,Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 5063–5068.

[60] P. Goettig, M. Groll, J.S. Kim, R. Huber, H. Brandstetter, Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalyticmechanism, EMBO J. 21 (2002) 5343–5352.

[61] A. Addlagatta, L. Gay, B.W. Matthews, Structure of aminopepidase N fromEscherichia coli suggests a compartmentalized, gated active site, Proc. Natl. Acad.Sci. U.S.A. 103 (2006) 13339–13344.

[62] B. Tomkinson, C. Wernstedt, U. Hellman, O. Zetterqvist, Active-site of tripeptidylpeptidase-II from human-erythrocytes is of the subtilisin type, Proc. Natl. Acad.Sci. U.S.A. 84 (1987) 7508–7512.

[63] H. Hilbi, E. Jozsa, B. Tomkinson, Identification of the catalytic triad in tripeptidyl-peptidase II through site-directed mutagenesis, Biochim. Biophys. Acta, ProteinsProteomics 1601 (2002) 149–154.

[64] B. Tomkinson, B.N. Laoi, K. Wellington, The insert within the catalytic domain oftripeptidyl-peptidase II is important for the formation of the active complex, Eur.J. Biochem. 269 (2002) 1438–1443.

[65] B. Tomkinson, O. Zetterqvist, Immunological cross-reactivity between humantripeptidyl peptidase-II and fibronectin, Biochem. J. 267 (1990) 149–154.

[66] B. Tomkinson, Characterization of cDNA for murine tripeptidyl-peptidase-IIReveals alternative splicing, Biochem. J. 304 (1994) 517–523.

[67] B. Tomkinson, M. Hansen, W.F. Cheung, Structure–function studies of recombi-nant murine tripeptidyl-peptidase II: the extra domain which is subject toalternative splicing is involved in complex formation, FEBS Lett. 405 (1997)277–280.

[68] L. Holm, C. Sander, Dali/FSSP classification of three-dimensional protein folds,Nucleic Acids Res. 25 (1997) 231–234.

[69] J. Soeding, A. Biegert, A.N. Lupas, The HHpred interactive server for proteinhomology detection and structure prediction, Nucleic Acids Res. 33 (2005)W244–W248.

[70] T. Frickey, A. Lupas, CLANS: a Java application for visualizing protein familiesbased on pairwise similarity, Bioinformatics 20 (2004) 3702–3704.

[71] J. Mayr, A. Lupas, J. Kellermann, C. Eckerskorn, W. Baumeister, J. Peters, Ahyperthermostable protease of the subtilisin family bound to the surface layer ofthe Archaeon Staphylothermus marinus, Curr. Biol. 6 (1996) 739–749.

[72] M. Igura, N. Maita, J. Kamishikiryo, M. Yamada, T. Obita, K. Maenaka, D. Kohda,Structure-guided identification of a new catalytic motif of oligosaccharyltransfer-ase, EMBO J. 27 (2008) 234–243.

[73] L.D. D'Andrea, L. Regan, TPR proteins: the versatile helix, Trends Biochem. Sci. 28(2003) 655–662.

[74] R.K. Allan, T. Ratajczak, Versatile TPR domains accommodate different modes oftarget protein recognition and function, Cell Stress Chaperones (2010).

[75] O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen, Locating proteins in the cellusing TargetP, SignalP and related tools, Nat. Protoc. 2 (2007) 953–971.

[76] R.J. Siezen, J.A.M. Leunissen, Subtilases: the superfamily of subtilisin-like serineproteases, Protein Sci. 6 (1997) 501–523.

[77] A.F. Kisselev, A.L. Goldberg, Proteasome inhibitors: from research tools to drugcandidates, Chem. Biol. 8 (2001) 739–758.

[78] M. Bogyo, E.W. Wang, Proteasome inhibitors: complex tools for a complexenzyme, in: P. Zwickl, W. Baumeister (Eds.), Proteasome-Ubiquitin ProteinDegradation Pathway, vol. 268, Springer, Berlin Heidelberg, 2002, pp. 185–208.

[79] L. Dehmelt, P.I.H. Bastiaens, Spatial organization of intracellular communication:insights from imaging, Nat. Rev. Mol. Cell Biol. 11 (2010) 440–452.

[80] F.S. Wouters, P.J. Verveer, P.I.H. Bastiaens, Imaging biochemistry inside cells,Trends Cell Biol. 11 (2001) 203–211.

[81] S. Nickell, C. Kofler, A.P. Leis, W. Baumeister, A visual approach to proteomics, Nat.Rev. Mol. Cell Biol. 7 (2006) 225–230.

[82] C.V. Robinson, A. Sali, W. Baumeister, The molecular sociology of the cell, Nature450 (2007) 973–982.