ARTICLE pubs.acs.org/jpr
Proteomic Identification of Protease Cleavage Sites Characterizes Prime and Non-prime Specificity of Cysteine Cathepsins B, L, and S Martin L. Biniossek,† Dorit K. N€agler,‡ Christoph Becker-Pauly,§ and Oliver Schilling*,† †
Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany Division of Clinical Chemistry and Clinical Biochemistry, Department of Surgery, Ludwig-Maximilians-University, Munich, Germany § Institute of Zoology, Cell and Matrix Biology, Johannes Gutenberg-University, Mainz, Germany ‡
bS Supporting Information ABSTRACT: Cysteine cathepsins mediate proteome homeostasis and have pivotal functions in diseases such as cancer. To better understand substrate recognition by cathepsins B, L, and S, we applied proteomic identification of protease cleavage sites (PICS) for simultaneous profiling of prime and non-prime specificity. PICS profiling of cathepsin B endopeptidase specificity highlights strong selectivity for glycine in P30 due to an occluding loop blocking access to the primed subsites. In P10 , cathepsin B has a partial preference for phenylalanine, which is not found for cathepsins L and S. Occurrence of P10 phenylalanine often coincides with aromatic residues in P2. For cathepsin L, PICS identifies 845 cleavage sites, representing the most comprehensive PICS profile to date. Cathepsin L specificity is dominated by the canonical preference for aromatic residues in P2 with limited contribution of prime-site selectivity determinants. Profiling of cathepsins B and L with a shorter incubation time (4 h instead of 16 h) did not reveal time-dependency of individual specificity determinants. Cathepsin S specificity was profiled at pH 6.0 and 7.5. The PICS profiles at both pH values display a high degree of similarity. Cathepsin S specificity is primarily guided by aliphatic residues in P2 with limited importance of prime-site residues. KEYWORDS: proteome-derived peptide library, protease specificity, subsite cooperativity
1. INTRODUCTION Cysteine cathepsins are a family of papain-like proteases with 11 members in man. Traditionally, cysteine cathepsins are considered to function in lysosomal protein degradation. However, a large body of data increasingly supports extralysosomal activity such as at the plasma membrane1 and in the pericellular milieu.2 As lysosomal proteases with a catalytically active sulfhydryl group, cysteine cathepsins typically require acidic and reducing conditions for optimal activity. Cathepsins are involved in numerous physiological and pathological processes. Cathepsin B has a causative role in carcinogenesis and metastasis.35 Cathepsin L is involved in epidermal68 and cardiac homeostasis,9,10 prohormone processing,11,12 and autophagy.13 It is a putative tumor suppressor in the K14-HPV16 mouse model of epidermal carcinogenesis,6 whereas it contributes to cell proliferation and tumor growth in a mouse model of pancreatic islet cell carcinogenesis.5 Cathepsin S plays a major role in antigen presentation1416 and is involved tumor formation and angiogenesis in the aforementioned pancreatic island cell cancer model. Protease specificity relies largely on the recognition of amino acid sequences that encompass the scissile peptide bond. For numerous proteases, specificity determinants are observed up to three amino acids amino- or carboxyterminally to the scissile peptide bond. The Schechter and Berger nomenclature numbers substrate amino acids (referred to as P) and enzyme substrate binding pockets (referred to as S) according to their position in r 2011 American Chemical Society
relation to the scissile peptide bond with C-terminally located residues being denoted as “primed” sites (P0 or S0 ).17 We have recently introduced proteomic identification of protease cleavage sites (PICS)18 as an efficient method for protease specificity profiling (Figure 1) based on the identification of hundreds of protease cleavage products from proteome-derived peptide libraries. PICS simultaneously determines prime and non-prime specificity thereby yielding a comprehensive portrayal of protease active site specificity. In contrast, many peptide library approaches focus on specificities either amino- and carboxyterminal to the scissile peptide bond.19 However, inhibitor studies for cathepsin B revealed mutual interactions between prime and non-prime subsites, which prevent a simplistic “addition” of prime and non-prime specificities if these are determined independently.20 This underlines the necessity for concurrent prime and non-prime subsite mapping. By determining individual cleavage sequences, PICS is also suited to probe subsite cooperativity as has been shown for HIV protease I.18 PICS employs database-searchable, proteome-derived peptide libraries and harnesses biological sequence diversity together with highthroughput peptide sequence identification by liquid chromatographytandem mass spectrometry (LCMS/MS). For PICS, cellular proteomes are digested into oligopeptides with specific endoproteases such as trypsin or Glu-C. After chemical protection Received: July 1, 2011 Published: October 03, 2011 5363
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cathepsins with enzyme-specific differences. We show for the first time that endopeptidase specificity of cathepsin B is guided by a strong preference for glycine in P30 . Cathepsins L and S display only minor prime-site specificities.
2. EXPERIMENTAL PROCEDURES 2.1. PICS Peptide Library Preparation
PICS peptide libraries were prepared as described elsewhere.21 Briefly, libraries were prepared from HEK293 cell lysates by tryptic or GluC digestion, followed by inactivation of the digestion protease. Free thiols were protected with iodoacetamide and primary amines (N-terminal α-amines and lysine ε-amines) were protected by dimethylation. Peptide libraries were purified and stored at 80 °C. 2.2. Enzyme Preparation
Figure 1. PICS workflow. To prepare PICS peptide libraries, cellular proteomes are digested with a specific endoprotease such as trypsin. Following inactivation of the digestion protease, free thiols are carboxyamidomethylated, and primary amines are dimethylated, a sterically small modification that perseveres the basic character. After cleanup, the PICS library is incubated with a cathepsin test protease. C-terminal cleavage products possess a free neo N-terminus, which is biotinylated to enable selective recovery with immobilized streptavidin. Sequence determination of C-terminal cleavage products occurs via LCMS/ MS analysis, whereas the corresponding N-terminal sequences are inferred bioinformatically. Figure adapted from ref 18.
of primary amines (N-terminal α-amines and lysine ε-amines) and purification, these proteome-derived peptide libraries are used as a substrate screen for a test protease. Prime-site cleavage products possess newly generated, unprotected N-terminal αamines, which are biotinylated to enable affinity isolation and LCMS/MS based sequence identification. The sequence of the corresponding non-prime cleavage product is determined bioinformatically. The large array of cleaved peptide sequences is then parsed to calculate positional occurrences, which are typically shown as heatmaps and sequence logos. Positional occurrences are optionally corrected for natural amino acid abundance to avoid bias stemming from unequal amino acid distribution in cellular proteomes. The PICS workflow is summarized in Figure 1. PICS specificity profiles are in very good agreement with other protease specificity data.18 Proteases with broad specificity often prefer groups of structurally related amino acids in a particular subsite. Such groups include small residues (glycine, alanine, and in some cases serine), medium to large aliphatic residues (leucine, methionine, and in some cases isoleucine), aromatic residues (tryptophan, phenylalanine, and tyrosine), basic residues (lysine and arginine), and acidic residues (aspartate and glutamate). In this study, we apply PICS to profile prime and non-prime specificity of cathepsins B, L, and S. PICS profiling of cathepsin L alone yields >840 cleavage sequences. The characteristic S2 specificity for hydrophobic residues is found for all three cysteine
Human cathepsin B was expressed in Pichia pastoris (strain GS115) and purified as described previously. 22 The cDNA (cDNA) for human procathepsin L was amplified by PCR from a human placenta cDNA library (BD Biosciences Clontech, Palo Alto, CA) using gene-specific primers 50 -CCGCTCGAGAAAAGAGAGGCTGAAGCTACTCTAACATTTGATCACAGTTT-30 and 50 -ATTTGCGGCCGCTCACACAGTGGGGTAGCTGG-30 and Expand DNA polymerase (Roche Applied Science, Mannheim, Germany). Cathepsin L was expressed in Pichia pastoris (strain GS115) and purified according to the procedure described for cathepsin B.22 The purified enzymes were stored at 4 °C in the presence of 100 μM of the reversible inhibitor Smethyl methane thiosulfonate (Merck, Darmstadt, Germany). The concentration of active enzyme was determined by titration with E-64.23 Recombinant, active, human cathepsin S was from R & D Systems (Abingdon). 2.3. PICS Assay
Cathepsins B and L were activated using 2 mM dithiothreitol (DTT) in 50 mM sodium phosphate, pH 6.0, 200 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA). This buffer was also used for the PICS assays. Cathepsin S was additionally assayed in 50 mM HEPES pH 7.5, 200 mM NaCl, 5 mM EDTA, 2 mM DTT. PICS libraries were used at a concentration of 0.51.0 μg/μL at a total amount of 300 μg. Enzyme/library ratios were 1:100 for cathepsins B and S and 1:300 for cathepsin L. Incubation was at 37 °C for 16 or 4 h respectively. After the assay, cleavage products were isolated and prepared for LC MS/MS as described elsewhere.21 2.4. Nanoflow HPLCMS/MS
Mass spectrometric measurements were performed on an Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to an Ultimate3000 micro pump (Dionex, Idstein, Germany). High pressure liquid chromatography (HPLC) column tips (fused silica) with 75 μm inner diameter (New Objective, USA) were self-packed24 with Reprosil-Pur 120 ODS-3 (Dr. Maisch, Ammerbuch, Germany). The system was equipped with a PepMap100 C18 precolumn with 300 μm i.d. 5 mm (Dionex, Idstein, Germany). A gradient of A [0.5% acetic acid (ACS Reagent, Sigma, Germany) in water] and B [0.5 acetic acid in 80% acetonitrile (ACN, HPLC gradient grade, SDS, Peypin, France)] with increasing organic proportion was used for peptide separation (main separation ramp: 330% B within 70 min, flow rate 300 nL/min). The mass spectrometer switched data-dependent automatically between MS and MS/MS. 5364
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Journal of Proteome Research Each MS scan was followed by a maximum of 5 MS/MS scans. The resolution for the FTMS mode was set to 60.000. 2.5. Data Analysis
Orbitrap data were converted to mzXML format25 with msconvert (ProteoWizard version 2.0.190526). MS and MS/MS data were centroided. Data were analyzed by X!Tandem (version 2010.01.01)27 in conjunction with PeptideProphet (version 4.3.1)28 at a 95% confidence level for each peptide and a decoy search strategy.29 The human International Protein Index (IPI, version 3.42)30 was used. Including decoy sequences, the database comprised 144680 entries. Mass tolerance was 10 ppm for parent ions and 0.5 Da for fragment ions. Non-enzyme constraint searches were applied. Static modifications are cysteine carboxyamidomethylation (+57.02 Da), lysine dimethylation (+28.03 Da), and thioacylation of peptide amino termini (+88.00 Da). Generation of PICS specificity profiles and subsite cooperativity analysis were supported by a web-based PICS service as described elsewhere.21 Briefly, prime-site cleavage sequences are rendered non-redundant and extended N-terminally until the first potential cleavage site of the digestive protease. Positional frequencies are normalized to natural amino acid abundances based on the composition of the human proteome as defined by the IPI database. 2.6. Structural Analysis
Modeling was performed with DeepView SwissPDB Viewer 3.731 based on the crystal structures of cathepsin B (1HUC).32 Energy minimization, modeling, and molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081).33
3. RESULTS AND DISCUSSION 3.1. Overview of PICS Profiles
PICS specificity profiles with tryptic peptide libraries were acquired for cysteine cathepsins B, L, and S. In order to discern subsite specificity from random fluctuations of positional frequencies, we focus on residues exceeding their natural abundance more than two-fold.21 A corresponding cut-off has been applied for the heatmap-style representation of positional frequencies but not for the sequence logo type depiction (Figure 2 and 3). The present study focuses on tryptic peptide libraries as these function more robustly than GluC or chymotryptic peptide libraries,18,34 hence allowing for good interprotease comparison.35 Tryptic PICS libraries lack internal arginine or lysine and do not profile specificity for basic residues. Therefore, tryptic PICS profiles do not allow for any statement with regard to cathepsin specificity for basic residues. Several synthetic substrates for cysteine cathepsins comprise a basic residue in P1. However, the high number of cleavage products, ranging from 376 for cathepsin B to 845 for cathepsin L, illustrates efficient proteolysis of peptides lacking internal basic residues. This observation is reminiscent of PICS profiling of matrix metalloprotease (MMP)-2.18 Although MMPs have a partial preference for basic residues in P20 , a single PICS profiling experiment of MMP-2 with a tryptic peptide library yielded over 400 cleavage sequences and correctly identified all nonbasic specificity determinants.18 In addition to standard PICS specificity profiles with 16 h incubation time, we also probed specificity of cathepsins B and L after only 4 h incubation.
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3.2. Cathepsin B Endopeptidase Specificity
Cathepsin B functions as both an endopeptidase and dipeptidyl-carboxypeptidase.3638 The PICS approach focuses on endoprotease specificity: PICS employs LCMS/MS for the identification of prime-site cleavage fragments. High-confidence spectrum-to-sequence assignment by LCMS/MS typically requires peptides of at least seven residues, which represents the minimum length of a prime-side cleavage products identified in this study. PICS profiling of cathepsin B with a tryptic peptide library identified 376 cleavage sequences (16 h incubation time, PICS specificity profile in Figure 2 and 3, sequences listed in Supplementary Table 1). The major cathepsin B endoprotease specificity determinant is observed in P30 : here, glycine alone accounts for 45% of all residues and exceeds its natural abundance 6.7-fold. This is the highest positional occurrence of glycine observed by PICS so far. It represents an important endoproteolytic cathepsin B specificity determinant that has so far not been identified. Cathepsin B possesses a unique “occluding loop” (Figure 4), which restricts the prime-site extent of its active site and limits endopeptidase activity while favoring dipeptidyl carboxypeptidase activity.22 The preference for P30 glycine potentially reflects a sterically small S30 subsite together with the requirement of conformational flexibility for peptidic substrates in order to “bend away” from the occluding loop. In addition, binding of elongated peptidic substrates to cathepsin B possibly involves displacement of the occluding loop.39 A number of physiological, endoproteolytic cathepsin B substrates40 comprise a P30 glycine residue, including aggrecan,37 anti-leukoproteinase,41 BID,42 procathepsin B, 43 MARCKS, 44 osteocalcin,45 PARP-1, 46 sphingosine kinase-1,47 thyroglobulin,48 trypsinogen,49 and uPAR.50 In P3, individual amino acids do not exceed their natural abundance by more than 2-fold, characterizing S3 as a minor specificity determinant. Nevertheless, within P6P60 , both proline (10% of all cleavage sites) and leucine (17% of all cleavage sites) display the highest occurrence in this position. A partial P3 preference for aromatic and aliphatic residues has been previously reported.5154 In P2, cathepsin B displays the canonical cysteine cathepsin specificity for aromatic and aliphatic residues. However, in comparison to cathepsins L and S, P2 has a smaller contribution to cathepsin B active site specificity. Moreover, alanine is among the preferred P2 residues for cathepsin B but not for cathepsins L and S. Both distinguishing features are corroborated by combinatorial peptide library screens.52,55 In P1, the present PICS profile detects strong cathepsin B selectivity for glycine. Fluorescent peptidic substrates with a P1 glycine are efficiently cleaved by cathepsin B,56 and a series of irreversible cathepsin inhibitors comprise a P1 glycine.57 In the zymogen structure, a glycine residue of the propeptide is present in the S1 subsite.58 Endoproteolytic P1 preference for glycine is further corroborated by the observation that P1 side chains point away from the active site cleft and have very limited interactions with the protease.32 Cathepsin B endoproteolytic cleavage at a P1 glycine is reported for several proteins, including aggrecan,37 osteocalcin,45 and thyroglobulin.48 In P10 , cathepsin B displays dual specificity for either phenylalanine or glycine. Previous studies support a P10 preference for aromatic and aliphatic residues, albeit to a varying extent.59,60 Structural analysis of cathepsin B inhibitor complexes revealed a hydrophobic S10 pocket.6163 In the zymogen structure, the S10 pocket is occupied by a leucine residue.58 Cathepsins L and S do 5365
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Figure 2. PICS specificity profiles for cathepsins B, L, and S with 16 h incubation. PICS specificity profiles for P6P60 were determined as described in Experimental Procedures. Positional occurrences are shown as enrichment over natural abundance with human amino acid abundances being derived from the International Protein Index.30 For heatmap-style representation, only enrichments >2-fold are displayed. For the numerical representation, enrichments g2-fold are highlighted in yellow.
not share the P10 preference for phenylalanine. The P10 specificity study by Menard et al.59 further supports that this feature distinguishes cathepsin B from cathepsins L and S.
P10 phenylalanine occurs preferentially together with P2 phenylalanine (Figure 5): 25% of cleavage sequences with a P10 phenylalanine also possess a P2 phenylalanine compared to 5366
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Figure 3. Sequence logo depiction of cathepsin B, L, and S specificity. Sequence logos are generated with iceLogo.102
Figure 4. Cathepsin B occluding loop and substrate accommodation in active site. Structures for cathepsin B (A, C; PDB code 1HUC)32 and cathepsin L (B, PDB code 1ICF)83 are shown with gray-colored surfaces with the catalytically active cysteine in yellow. (A) Surface model of cathepsin B structure32 highlighting the occluding loop (residues Cys108Cys119) in red. (B) Comparison to cathepsin L structure,83 which lacks an occluding loop. (C) On the basis of PICS specificity data, the peptide GFGFVG (cyan) was docked into the active site cleft of cathepsin B. Higher magnification reveals hydrophobic interactions between aromatic residues in P2 and a hydrophobic patch at the upper rim of the active site cleft, potentially including Tyr 75 (orange). P1 aromatic residues are accommodated in a hydrophobic pocket involving Trp 221 (orange).
9% of all cleavage sequences. Similarly, the presence of a P2 phenylalanine increases the rate of P10 phenylalanine from 14% (all cleavage sequences) to 41%. Together, these data indicate positive cooperativity between aromatic residues in P2 and P10 .
Figure 5. Subsite cooperativity for cathepsin B cleavage sequences with aromatic residues in P2 and P10 . Cathepsin B cleavage sequences with a P2 Phe or P2 Tyr have 41% and 46% P10 Phe, respectively, whereas all cathepsin B cleavage sequences display 14% P10 Phe. Similarly, cleavage sequences with a P10 Phe possess 25% P2 Phe and 17% P2 Tyr compared to 7% P2 Phe and 6% P2 Tyr for all cathepsin B cleavage sequences. All three types of cleavage sequences (P2 Phe, P2 Tyr, and P10 Phe) also display an increase of P3 Gly of 1322%.
Structure based analysis of enzymesubstrate interactions for cathepsin B indicates that aromatic residues in P2 and P10 jointly anchor a peptide in the active site cleft (Figure 4 and section on structure modeling below). In P20 , strong preferences for individual residues are absent with the exception of a slight specificity for valine and isoleucine. 5367
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Journal of Proteome Research P20 has been suggested to lack major specificity determinants since P20 side chains point away from the protease.64 However, several specificity studies indicate that cathepsin B readily accommodates and prefers aliphatic and aromatic residues in P20 .54,57,60,65 At the same time, structural analysis of cathepsin B inhibitor complexes illustrates accommodation of P20 proline residues.62,63,66 In the cathepsin B zymogen, a leucine is the P20 equivalent.58 Cathepsins L and S do not share the minor P20 preference for valine and isoleucine, characterizing this trait as a distinguishing feature. In summary, we show for the first time that cathepsin B endopeptidase specificity is primarily guided by a strong selectivity for P30 glycine. Cathepsin B also differs from cathepsins L and S in partially preferring aromatic residues in P10 , a feature that involves positive cooperativity between P2 and S10 . 3.3. Cathepsin L Specificity
PICS profiling of cathepsin L with a tryptic library identified 845 peptidic cleavage sequences (16 h incubation time). To date, this represents the largest PICS data set determined in a single experiment. The resulting PICS specificity profile is shown in Figure 2 and 3, sequences are listed in Supplementary Table 2. Cathepsin L specificity is predominantly guided by P2. Here, cathepsin L has a strong preference for aromatic and, to a slightly lesser extent, aliphatic residues. P2 constitutes the major specificity determinant for cathepsin L. This observation is in line with results of previous studies that further demonstrate that cathepsin L specificity is largely based on P2 selectivity.52,53,6771 In the zymogen and in the structure of a cathepsin L inhibitor complex, the S2 subsite is occupied by a phenylalanine residue.72,73 In P3, major specificity determinants are absent, and the occurrence of individual amino acids does not exceed their natural abundance by more than 2-fold. Within P6 P60 , the highest occurrence of methionine is found in P3, while the occurrences of phenylalanine, isoleucine, and leucine are only surpassed by their predominant presence in P2. PICS profiling of cathepsin L suggests a minor P3 preference for hydrophobic amino acids in accordance with several previous studies.52,54,68,71,73,74 In P1, cathepsin L displays mixed selectivity for glutamine and glycine. The partial P1 preference for glutamine is corroborated by two synthetic peptide library screens.52,70 For cathepsin L, P1 glutamine exceeds its natural abundance 2.3-fold; similar values are found for cathepsin B (1.8-fold) and cathepsin S (1.9-fold at pH 6.0 and 2.3-fold at pH 7.5). Hence, a minor preference for glutamine is shared by all three cathepsins and does not represent a distinct feature of cathepsin L. Similar to cathepsin B, a propeptide glycine occupies the S1 subsite in the cathepsin L zymogen.72 A number of physiological cathepsin L cleavages sites with a P1 glutamine or glycine have been reported, including procathepsin L,75 collagen α-1(I),76 cystatin C,77 heparanase,78 thyroglobulin,48 and pro-uPA.79 In P10 , cathepsin L has a preference for glycine as well as serine or histidine. This is corroborated by previous studies that identified a partial P10 preference for small, neutral hydrophilic residues or basic amino acids.59,71,80 Cathepsin L does not share the cathepsin B preference for P10 phenylalanine. This observation is supported by a previous P10 specificity study.59 The slight preference for histidine is not confined to S10 but also is observed in P20 and P30 . Noteworthy, the positional frequency of histidine remains below 10%. However, histidine is comparably rare in proteome-derived peptide libraries (occurrence 2-fold in
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these positions. Two physiological cleavage sites with a P10 histidine have been reported,79,81 while P10 specificity for a cathepsin L inhibitor stems partially from a heterocyclic, albeit non-imidazole pyridine group.82 In P20 and P30 , only histidine exceeds its natural abundance more than 2-fold. A previously reported positional preference for hydrophobic residues in P20 is not corroborated by the present PICS study.54,57 Inhibition of cathepsin L by a p41 fragment involves positioning of a ligand histidine in the primed area of the active site cleft.83 Structural analysis illustrates that the p41 histidine points toward a cluster of acidic residues that flank the primed area of the active site cleft.83 Minor positional preferences are also found in P40 and P50 . These positions interact with subsites that are not part of the active site cleft but are located in more distant regions on the protease surface. For example, cathepsin K displays a preference for aspartic acid in P50 that has been attributed to a cationic patch at the protease “backside”.18 For cathepsin L, PICS indicates a preference for proline in P40 and for aspartate in P40 and P50 . The latter preference resembles the situation for cathepsin K and can be attributed to an accumulation of basic residues at the protease “backside”.83,84 Cathepsin L specificity was also probed with a GluC library, yielding 93 cleavage sequences (Supplementary Figure 1 and Supplementary Table 3). Here, cathepsin L displays a preference for dimethylated lysine in P1. Dimethylation preserves the basic character of lysine. Within P6P60 , arginine has its highest occurrence in P1. The P1 preference for basic residues is in agreement with further studies.52,69,70 The P2 preference for aromatic residues is less pronounced with the GluC library. In summary, cathepsin L specificity is primarily guided by a strong preference for aromatic as well as aliphatic residues in P2 with only limited prime-site specificity contributions. 3.4. Cathepsin S Specificity
Cathepsin S specificity was profiled for 16 h at pH 6.0 and pH 7.5, yielding 470 and 671 cleavage sequences, respectively. Cathepsin S differs from cathepsins B and L in retaining enzymatic activity at basic pH.68,8587 PICS profiling of cathepsins B and L at basic pH was not successful, most likely due to their rapid inactivation upon incubation at basic pH.68,85,86,8891 Overall, the different pH values had only marginal effects on cathepsin S specificity and yielded comparable PICS profiles (Figures 2 and 3 and Supplementary Tables 4 and 5). In agreement with a number of previous studies, PICS corroborates that P2 represents the major specificity determinant for cathepsin S.52,67,68,92,93 For P2, cathepsin S prefers predominantly aliphatic residues such as valine, isoleucine, leucine, and methionine. A preference for aromatic residues is less pronounced. In contrast, cathepsin L selects aromatic over aliphatic residues in P2. The different P2 preferences in the PICS profiles of cathepsins L and S are in line with earlier reports.52,53,55,67,68,87,9294 In the cathepsin S zymogen, the S2 subsite is occupied by a propeptide leucine95 rather than a phenylalanine as is the case for cathepsin L.72 The P2 pocket of cathepsin S is considered to be relatively large96 with binding of aliphatic or aromatic residues involving a conformational switch of a phenylalanine side chain.97 P3 contributes only marginally to cathepsin S specificity. Further studies underline the absence of pronounced specificity determinants in P3.52,53,55,68,92,93 In P1, cathepsin S prefers glycine as well as glutamine (see also discussion on partial P1 glutamine preference for cathepsin L). Moreover, within P3P30 , glutamate has its highest occurrence 5368
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Journal of Proteome Research in P1 (2.2-fold exceeding natural abundance at pH 6.0 and 1.6fold exceeding natural abundance at pH 7.5). In contrast, aspartate is not preferred in P,1 nor do cathepsins B or L display a mild preference for glutamate in P1. As for cathepsin B and L, the cathepsin S1 subsite has been described as shallow.95,97 Enzymesubstrate interactions are thought to involve only backbone atoms of the peptidic substrate.96 Additional studies indicate broad P1 specificity.52,68,92,93,98 In P10 , cathepsin S displays a preference for glycine that is more pronounced at pH 6.0 than at pH 7.5. At the latter pH, slight P1 0 preferences for glutamine (2.2 times its natural abundance at pH 7.5 and 1.9 times at pH 6.0) and serine (2.3 times its natural abundance at pH 1.2 times at pH 6.0) are also apparent. Cathepsin S cleavage assays with single proteins did not reveal explicit P1 preferences.68,98 P10 profiling experiments with peptide libraries indicate a mixed preference for either small or hydrophilic residues or aliphatic amino acids.59,92,93 Similar to cathepsin L and in line with a previous P10 specificity study,59 cathepsin S does not share the cathepsin B preference for P10 phenylalanine, although a structural study illustrates that an aromatic group can be accommodated in the S10 subsite.96 P20 appears to not contribute to cathepsin S selectivity. At both pH values, P20 specificity determinants are absent. A cathepsin S inhibitor study suggested a P20 preference for aromatic residues,57 while a second study, based on peptidic substrates, substantiates the lack of P20 specificity.92 In P30 , PICS detects minor preferences for proline and histidine at both pH values and a slight preference for aspartate. An apparent preference for P30 proline is also found for cathepsin L with a positional occurrence corresponding to 2.0 times its natural abundance. In all cases, P30 proline is often preceded by P20 lysine or arginine. Since trypsin does not cleave N-terminal to proline, these represent missed tryptic cleavages upon library generation that are now selected as basic residues in P20 by cathepsin L or S. As described earlier, structural studies of cathepsin L point toward a cluster of acidic residues at the upper rim of the prime-site area of the active site cleft.83 However, a similar cluster is not evident for cathepsin S.99 A previously suggested, subtle P30 preference for aliphatic and aromatic residues92 is not corroborated by the present PICS study. In summary, cathepsin S specificity is primarily guided by a preference for aliphatic residues in P2 with only limited primesite specificity contributions. Its specificity profile appears to be pH-independent. 3.5. Structural Model of Substrate Binding to Cathepsin B
To better understand substrate recognition by cathepsin B, we modeled binding of the peptide GlyP3-PheP2-GlyP1-PheP10 -ValP20 GlyP30 to its active site cleft (Figure 4). The peptide sequence represents the positional preferences of cathepsin B, taking into account positive cooperativity between aromatic residues in P2 and P10 . Based on this model, P10 phenylalanine binds to a hydrophobic pocket that is partially formed by Trp 221 at the lower rim of the active site cleft. P2 phenylalanine interacts with a hydrophobic patch at the upper rim of the active site cleft, possibly involving Tyr 75. A similar hydrophobic interaction was observed for cathepsin B accommodating its endogenous inhibitor stefin A.39 While conformational details of substrate recognition by cathepsin B are likely to be further guided by an induced fit, both the structure model and the PICS profile for cathepsin B indicate that aromatic residues in P2 and P10 are able to jointly anchor a peptidic substrate in the active site cleft.
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The present model of substrate binding to cathepsin B was intentionally built without a priori knowledge of individual subsites. It is validated by previous structural studies which substantiate Trp 221 as a component of the S10 subsite63,64 and Tyr 75 as a component of the S2 subsite.32,63 It has also been suggested that Tyr 75 is part of the cathepsin B S3 subsite.51 We note that the occurrence of P2 phenylalanine or P2 tyrosine coincides with an increased rate of P3 glycine and a decreased rate of aromatic and aliphatic residues in P3 (Figure 5). Hence, accommodation of hydrophobic residues in the S2 subsite appears to reduce acceptance of aromatic residues in the S3 subsite. This “negative cooperativity” potentially involves structural flexibility and minor repositioning of Tyr 75, which thereby stabilizes hydrophobic interactions in either S3 or S2. In fact, Tyr 75 is located at the outer edge of S2 in proximity of S3 (Figure 4). 3.6. PICS Profiling of Cathepsins B and L with Shorter Incubation of 4 h
PICS profiling experiments typically employ an incubation time of 16 h.21,35 For cathepsins B and L, we performed an additional PICS profiling experiment with a shorter 4 h incubation time (Figure 6) in order to discern potential overdigestion effects resulting from prolonged incubation. Overdigestion might result in fewer defined specificity profiles. Fewer cleavage products were identified for both proteases: 214 cleaved peptides for cathepsin B and 302 cleaved peptides for cathepsin L. The resulting PICS profiles bear close resemblance to the PICS profiles obtained with 16 h incubation time. Cathepsin B displays strong selectivity for glycine in P1 and P30 at both incubation times. The preference for aromatic residues in P2 in P10 is less pronounced at 4 h: phenylalanine exceeds its natural abundance 1.7-fold in P2 and 1.3-fold in P10 . Positive cooperativity between P2 phenylalanine and P10 phenylalanine is observed, albeit to a lesser extent in comparison to the 16 h incubation (data not shown). For cathepsin L, comparison of the 4 and 16 h incubation PICS profiles shows a more pronounced preference for P1 glycine and a slightly less pronounced preference for P2 aromatics at 4 h. Overall, there are no major differences between the PICS profiles at 4 and 16 h, which illustrates that the present PICS profiles are not skewed by overdigestion stemming from prolonged incubation. For both cathepsin B and L, the prototypical preference for P2 aromatics was observed slightly less prominently at 4 h in comparison to 16 h incubation. PICS distinguishes between preferences for aromatic or aliphatic residues. This is highlighted by PICS profiling of cathepsin G (prefers large aliphatic and aromatic residues in P1) in comparison to neutrophil elastase (prefers small aliphatic residues in P1).18 The present comparison of P2 specificities for cathepsins L and S further stresses this point. However, we consider the slight under-representation of P2 aromatics at 4 h to be too subtle to prove a time-dependency of this specificity determinant. The primary reason for our assessment is that aromatic residues feature as major P2 specificity determinants at both time points. Nevertheless, this example highlights the usefulness of time course studies for PICS analyses. 3.7. Inter-cathepsin Comparison and Perspective
Our study presents the simultaneous profiling of prime and non-prime preferences for cysteine-type cathepsins B, L, and S. For all three cathepsins combined, PICS identified >2900 cleavage 5369
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Figure 6. PICS specificity profiles for cathepsins B and L with 4 h incubation. PICS specificity profiles for P6P60 were determined as described in Experimental Procedures. Positional occurrences are shown as enrichment over natural abundance with human amino acid abundances being derived from the International Protein Index.30 For heatmap-style representation, only enrichments >2-fold are displayed. Sequence logos are generated with iceLogo.102
sequences. Selectivity determinants were found N- and C-terminal to the scissile peptide bond. Cathepsins B, L, and S share a number of common specificity features: P3 plays a minor role in substrate selectivity, while P2 is a major specificity determinant. All three cathepsins prefer glycine in P1 and P10 . Several unique and distinguishing features were also identified: In P2, cathepsin L and to a lesser extent cathepsin B, prefer aromatic residues, while cathepsin S primarily selects for aliphatic residues. In P10 , cathepsin B has partial preference for phenylalanine that is not present for cathepsins S and L. Similarly, in P20 , cathepsin B has a minor preference for aliphatic residues that is not present for cathepsins S and L. Most importantly, cathepsin B endopeptidase specificity is dominated by a P30 selectivity for glycine that has been identified for the first time. The present study further validates PICS as an important tool for protease specificity profiling and highlights its suitability to probe proteases with rather low specificity. The straightforward PICS workflow enables comparative specificity profiling under differing experimental conditions such as pH or incubation time. PICS has particular strengths in simultaneously determining prime and non-prime specificity as well as in the detection of potential subsite cooperativity. Limitations of the PICS strategy presently comprise the usage of dimethylated lysine, which is not recognized by some proteases. PICS strategies employing unprotected lysine have been proposed.18 The application of specific digestion proteases results in PICS peptides lacking specific amino acids at internal positions. PICS libraries generated with nonspecific proteases have not yet been tested.
In-depth knowledge of proteases specificity determinants is a fundamental prerequisite for the prediction of potential cleavage sites in silico.100,101 While these procedures potentially suffer from high false-discovery rates,18 they provide a “reasoning environment for protease activity” and generate lead hypotheses.100 Moreover, cell-contextual cleavage sites typically adhere to the active site specificity of the executing protease.21 Hence, detailed characterization of protease selectivity determinants is useful in corroborating physiological, proteinaceous cleavage events. In summary, PICS profiling of cathepsin specificity is a first step toward characterizing cathepsin activity in vivo by proteomic and bioinformatic means.
’ ASSOCIATED CONTENT
bS
Supporting Information Supplementary figure and tables. This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes
The LC-MS/MS data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash:9vW9vw2eBchoa+zJvgpvBnFDRZuoYWUh+lMm/ +tuQuF8olpOFNP/RLSgqem0fUO2fFuk/1CZv4zwuVQVpr2et9N2XpQAAAAAAAACvg== and zBwzs1VugEbcFXq5Ir oi1ghudu7v2Hmb0jTfSpP/2sdudlhO8xHwHWEUv8XkvtoksNWMey9/4q62BlJxHLV079MvzuQAAAAAAAABqA==. The hash may be used to prove exactly what files were published as part of this manuscript0 s data set, and the hash may also be used to check that the data has not changed since publication. 5370
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’ AUTHOR INFORMATION Corresponding Author
*Tel: +49 761 203 9615. E-mail: oliver.schilling@mol-med. uni-freiburg.de. The authors declare no conflict of interest.
’ ACKNOWLEDGMENT O.S. is supported by an Emmy-Noether grant of the Deutsche Forschungsgemeinschaft (DFG) (SCHI 871/2). C.B.-P. is supported by DFG grant BE4086/1-2. The authors thank C. Peters for critical discussion and Heidi Br€auner, Franz Jehle, and Bettina Mayer for excellent technical assistance. The Excellence Cluster Centre for Biological Signaling Studies (BIOSS) is acknowledged for usage of the Orbitrap XL mass spectrometer. ’ ABBREVIATIONS C-terminal, carboxy-terminal; cDNA, cDNA; LCMS/MS, liquid chromatographytandem mass spectrometry; N-terminal, amino-terminal; P, non-prime side; P0 , prime side; PICS, proteomic identification of protease cleavage sites ’ REFERENCES (1) Jane, D. T.; Morvay, L.; Dasilva, L.; Cavallo-Medved, D.; Sloane, B. F.; Dufresne, M. J. Cathepsin B localizes to plasma membrane caveolae of differentiating myoblasts and is secreted in an active form at physiological pH. Biol. Chem. 2006, 387 (2), 223–34. (2) Roshy, S.; Sloane, B. F.; Moin, K. Pericellular cathepsin B and malignant progression. Cancer Metastasis Rev. 2003, 22 (23), 271–86. (3) Sevenich, L.; Schurigt, U.; Sachse, K.; Gajda, M.; Werner, F.; M€uller, S.; Vasiljeva, O.; Schwinde, A.; Klemm, N.; Deussing, J.; Peters, C.; Reinheckel, T. Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (6), 2497–502. (4) Vasiljeva, O.; Korovin, M.; Gajda, M.; Brodoefel, H.; Bojic, L.; Kr€uger, A.; Schurigt, U.; Sevenich, L.; Turk, B.; Peters, C.; Reinheckel, T. Reduced tumour cell proliferation and delayed development of highgrade mammary carcinomas in cathepsin B-deficient mice. Oncogene 2008, 27 (30), 4191–9. (5) Gocheva, V.; Zeng, W.; Ke, D.; Klimstra, D.; Reinheckel, T.; Peters, C.; Hanahan, D.; Joyce, J. A. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev. 2006, 20 (5), 543–56. (6) Dennem€arker, J.; Lohm€uller, T.; Mayerle, J.; Tacke, M.; Lerch, M. M.; Coussens, L. M.; Peters, C.; Reinheckel, T. Deficiency for the cysteine protease cathepsin L promotes tumor progression in mouse epidermis. Oncogene 2010, 29 (11), 1611–21. (7) Reinheckel, T.; Hagemann, S.; Dollwet-Mack, S.; Martinez, E.; Lohm€uller, T.; Zlatkovic, G.; Tobin, D. J.; Maas-Szabowski, N.; Peters, C. The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling. J. Cell Sci. 2005, 118 (Pt 15), 3387–95. (8) Tobin, D. J.; Foitzik, K.; Reinheckel, T.; Mecklenburg, L.; Botchkarev, V. A.; Peters, C.; Paus, R. The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling. Am. J. Pathol. 2002, 160 (5), 1807–21. (9) Spira, D.; Stypmann, J.; Tobin, D. J.; Petermann, I.; Mayer, C.; Hagemann, S.; Vasiljeva, O.; G€unther, T.; Sch€ule, R.; Peters, C.; Reinheckel, T. Cell type-specific functions of the lysosomal protease cathepsin L in the heart. J. Biol. Chem. 2007, 282 (51), 37045–52. (10) Stypmann, J.; Gl€aser, K.; Roth, W.; Tobin, D. J.; Petermann, I.; Matthias, R.; M€onnig, G.; Haverkamp, W.; Breithardt, G.; Schmahl, W.; Peters, C.; Reinheckel, T. Dilated cardiomyopathy in mice deficient for
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the lysosomal cysteine peptidase cathepsin L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99 (9), 6234–9. (11) Friedrichs, B.; Tepel, C.; Reinheckel, T.; Deussing, J.; von Figura, K.; Herzog, V.; Peters, C.; Saftig, P.; Brix, K. Thyroid functions of mouse cathepsins B, K, and L. J. Clin. Invest. 2003, 111 (11), 1733–45. (12) Yasothornsrikul, S.; Greenbaum, D.; Medzihradszky, K. F.; Toneff, T.; Bundey, R.; Miller, R.; Schilling, B.; Petermann, I.; Dehnert, J.; Logvinova, A.; Goldsmith, P.; Neveu, J. M.; Lane, W. S.; Gibson, B.; Reinheckel, T.; Peters, C.; Bogyo, M.; Hook, V. Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (16), 9590–5. (13) Dennem€arker, J.; Lohm€uller, T.; M€uller, S.; Aguilar, S. V.; Tobin, D. J.; Peters, C.; Reinheckel, T. Impaired turnover of autophagolysosomes in cathepsin L deficiency. Biol. Chem. 2010, 391 (8), 913–22. (14) Shen, L.; Sigal, L. J.; Boes, M.; Rock, K. L. Important role of cathepsin S in generating peptides for TAP-independent MHC class I crosspresentation in vivo. Immunity 2004, 21 (2), 155–65. (15) Pl€uger, E. B. E.; Boes, M.; Alfonso, C.; Schr€oter, C. J.; Kalbacher, H.; Ploegh, H. L.; Driessen, C. Specific role for cathepsin S in the generation of antigenic peptides in vivo. Eur. J. Immunol. 2002, 32 (2), 467–76. (16) Beck, H.; Schwarz, G.; Schr€oter, C. J.; Deeg, M.; Baier, D.; Stevanovic, S.; Weber, E.; Driessen, C.; Kalbacher, H. Cathepsin S and an asparagine-specific endoprotease dominate the proteolytic processing of human myelin basic protein in vitro. Eur. J. Immunol. 2001, 31 (12), 3726–36. (17) Schechter, I.; Berger, A. On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem. Biophys. Res. Commun. 1968, 32 (5), 898–902. (18) Schilling, O.; Overall, C. M. Proteome-derived, databasesearchable peptide libraries for identifying protease cleavage sites. Nat. Biotechnol. 2008, 26 (6), 685–94. (19) Auf dem Keller, U.; Schilling, O. Proteomic techniques and activity-based probes for the system-wide study of proteolysis. Biochimie 2010, 92 (11), 1705–14. (20) Schaschke, N.; Assfalg-Machleidt, I.; Machleidt, W.; Turk, D.; Moroder, L. E-64 analogues as inhibitors of cathepsin B. On the role of the absolute configuration of the epoxysuccinyl group. Bioorg. Med. Chem. 1997, 5 (9), 1789–97. (21) Schilling, O.; Huesgen, P. F.; Barre, O.; Auf dem Keller, U.; Overall, C. M. Characterization of the prime and non-prime active site specificities of proteases by proteome-derived peptide libraries and tandem mass spectrometry. Nat. Protoc. 2011, 6 (1), 111–20. (22) N€agler, D. K.; Storer, A. C.; Portaro, F. C.; Carmona, E.; Juliano, L.; Menard, R. Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts. Biochemistry 1997, 36 (41), 12608–15. (23) Barrett, A. J.; Kirschke, H.; Cathepsin, B; Cathepsin, H; cathepsin, L. Methods Enzymol. 1981, 80 (Pt C), 535–61. (24) Olsen, J. V.; Ong, S.-E.; Mann, M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol. Cell. Proteomics 2004, 3 (6), 608–14. (25) Pedrioli, P. G. A.; Eng, J. K.; Hubley, R.; Vogelzang, M.; Deutsch, E. W.; Raught, B.; Pratt, B.; Nilsson, E.; Angeletti, R. H.; Apweiler, R.; Cheung, K.; Costello, C. E.; Hermjakob, H.; Huang, S.; Julian, R. K.; Kapp, E.; McComb, M. E.; Oliver, S. G.; Omenn, G.; Paton, N. W.; Simpson, R.; Smith, R.; Taylor, C. F.; Zhu, W.; Aebersold, R. A common open representation of mass spectrometry data and its application to proteomics research. Nat. Biotechnol. 2004, 22 (11), 1459–66. (26) Kessner, D.; Chambers, M.; Burke, R.; Agus, D.; Mallick, P. ProteoWizard: open source software for rapid proteomics tools development. Bioinformatics 2008, 24 (21), 2534–6. (27) Craig, R.; Beavis, R. C. TANDEM: matching proteins with tandem mass spectra. Bioinformatics 2004, 20 (9), 1466–7. (28) Keller, A.; Nesvizhskii, A. I.; Kolker, E.; Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 2002, 74 (20), 5383–92. 5371
dx.doi.org/10.1021/pr200621z |J. Proteome Res. 2011, 10, 5363–5373
Journal of Proteome Research (29) Martens, L.; Vandekerckhove, J.; Gevaert, K. DBToolkit: processing protein databases for peptide-centric proteomics. Bioinformatics 2005, 21 (17), 3584–5. (30) Kersey, P. J.; Duarte, J.; Williams, A.; Karavidopoulou, Y.; Birney, E.; Apweiler, R. The International Protein Index: an integrated database for proteomics experiments. Proteomics 2004, 4 (7), 1985–8. (31) Kaplan, W.; Littlejohn, T. G. Swiss-PDB viewer (Deep View). Briefings Bioinformatics 2001, 2 (2), 195–7. (32) Musil, D.; Zucic, D.; Turk, D.; Engh, R. A.; Mayr, I.; Huber, R.; Popovic, T.; Turk, V.; Towatari, T.; Katunuma, N. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 1991, 10 (9), 2321–30. (33) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25 (13), 1605–12. (34) Schilling, O.; Auf dem Keller, U.; Overall, C. M. Protease specificity profiling by tandem mass spectrometry using proteomederived peptide libraries. Methods Mol. Biol. 2011, 753, 257–72. (35) Becker-Pauly, C.; Barre, O.; Schilling, O.; Auf dem Keller, U.; Ohler, A.; Broder, C.; Sch€utte, A.; Kappelhoff, R.; St€ocker, W.; Overall, C. M. Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates. Mol. Cell. Proteomics 2011, 10 (9), M111–009233. (36) Br€omme, D. Papain-like cysteine proteases. Curr. Protoc. Protein Sci 2001, 21.2.1–21.2.14. (37) Fosang, A. J.; Neame, P. J.; Last, K.; Hardingham, T. E.; Murphy, G.; Hamilton, J. A. The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B. J. Biol. Chem. 1992, 267 (27), 19470–4. (38) Illy, C.; Quraishi, O.; Wang, J.; Purisima, E.; Vernet, T.; Mort, J. S. Role of the occluding loop in cathepsin B activity. J. Biol. Chem. 1997, 272 (2), 1197–202. (39) Renko, M.; Pozgan, U.; Majera, D.; Turk, D., Stefin A displaces the occluding loop of cathepsin B only by as much as required to bind to the active site cleft. FEBS J. 2010. (40) Rawlings, N. D.; Barrett, A. J.; Bateman, A. MEROPS: the peptidase database. Nucleic Acids Res. 2010, 38 (Database issue), D227–33. (41) Taggart, C. C.; Lowe, G. J.; Greene, C. M.; Mulgrew, A. T.; O’Neill, S. J.; Levine, R. L.; McElvaney, N. G.; Cathepsin, B L, and S cleave and inactivate secretory leucoprotease inhibitor. J. Biol. Chem. 2001, 276 (36), 33345–52. (42) Cirman, T.; Oresic, K.; Mazovec, G. D.; Turk, V.; Reed, J. C.; Myers, R. M.; Salvesen, G. S.; Turk, B. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J. Biol. Chem. 2004, 279 (5), 3578–87. (43) Quraishi, O.; Storer, A. C. Identification of internal autoproteolytic cleavage sites within the prosegments of recombinant procathepsin B and procathepsin S. Contribution of a plausible unimolecular autoproteolytic event for the processing of zymogens belonging to the papain family. J. Biol. Chem. 2001, 276 (11), 8118–24. (44) Spizz, G.; Blackshear, P. J. Identification and characterization of cathepsin B as the cellular MARCKS cleaving enzyme. J. Biol. Chem. 1997, 272 (38), 23833–42. (45) Baumgrass, R.; Williamson, M. K.; Price, P. A. Identification of peptide fragments generated by digestion of bovine and human osteocalcin with the lysosomal proteinases cathepsin B, D, L, H, and S. J. Bone Miner. Res. 1997, 12 (3), 447–55. (46) Gobeil, S.; Boucher, C. C.; Nadeau, D.; Poirier, G. G. Characterization of the necrotic cleavage of poly(ADP-ribose) polymerase (PARP-1): implication of lysosomal proteases. Cell Death Differ. 2001, 8 (6), 588–94. (47) Taha, T. A.; El-Alwani, M.; Hannun, Y. A.; Obeid, L. M. Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease. FEBS Lett. 2006, 580 (26), 6047–54. (48) Dunn, A. D.; Crutchfield, H. E.; Dunn, J. T. Thyroglobulin processing by thyroidal proteases. Major sites of cleavage by cathepsins B, D, and L. J. Biol. Chem. 1991, 266 (30), 20198–204.
ARTICLE
(49) Teich, N.; B€ odeker, H.; Keim, V. Cathepsin B cleavage of the trypsinogen activation peptide. BMC Gastroenterol. 2002, 2, 16. (50) Kobayashi, H.; Schmitt, M.; Goretzki, L.; Chucholowski, N.; Calvete, J.; Kramer, M.; G€unzler, W. A.; J€anicke, F.; Graeff, H. Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA). J. Biol. Chem. 1991, 266 (8), 5147–52. (51) Taralp, A.; Kaplan, H.; Sytwu, I. I.; Vlattas, I.; Bohacek, R.; Knap, A. K.; Hirama, T.; Huber, C. P.; Hasnain, S. Characterization of the S3 subsite specificity of cathepsin B. J. Biol. Chem. 1995, 270 (30), 18036–43. (52) Choe, Y.; Leonetti, F.; Greenbaum, D. C.; Lecaille, F.; Bogyo, M.; Br€omme, D.; Ellman, J. A.; Craik, C. S. Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J. Biol. Chem. 2006, 281 (18), 12824–32. (53) Greenbaum, D. C.; Arnold, W. D.; Lu, F.; Hayrapetian, L.; Baruch, A.; Krumrine, J.; Toba, S.; Chehade, K.; Br€ omme, D.; Kuntz, I. D.; Bogyo, M. Small molecule affinity fingerprinting. A tool for enzyme family subclassification, target identification, and inhibitor design. Chem. Biol. 2002, 9 (10), 1085–94. (54) Portaro, F. C.; Santos, A. B.; Cezari, M. H.; Juliano, M. A.; Juliano, L.; Carmona, E. Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P20 and P30 variations in extended substrates. Biochem. J. 2000, 347 (Pt 1), 123–9. (55) Gosalia, D. N.; Salisbury, C. M.; Ellman, J. A.; Diamond, S. L. High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays. Mol. Cell. Proteomics 2005, 4 (5), 626–36. (56) Del Nery, E.; Alves, L. C.; Melo, R. L.; Cesari, M. H.; Juliano, L.; Juliano, M. A. Specificity of cathepsin B to fluorescent substrates containing benzyl side-chain-substituted amino acids at P1 subsite. J. Protein Chem. 2000, 19 (1), 33–8. (57) Br€omme, D.; Kirschke, H. N-peptidyl-O-carbamoyl amino acid hydroxamates: irreversible inhibitors for the study of the S20 specificity of cysteine proteinases. FEBS Lett. 1993, 322 (3), 211–4. (58) Podobnik, M.; Kuhelj, R.; Turk, V.; Turk, D. Crystal structure of the wild-type human procathepsin B at 2.5 A resolution reveals the native active site of a papain-like cysteine protease zymogen. J. Mol. Biol. 1997, 271 (5), 774–88. (59) Menard, R.; Carmona, E.; Plouffe, C.; Br€omme, D.; Konishi, Y.; Lefebvre, J.; Storer, A. C. The specificity of the S10 subsite of cysteine proteases. FEBS Lett. 1993, 328 (12), 107–10. (60) Cezari, M. H. S.; Puzer, L.; Juliano, M. A.; Carmona, A. K.; Juliano, L. Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides. Biochem. J. 2002, 368 (Pt 1), 365–9. (61) Jia, Z.; Hasnain, S.; Hirama, T.; Lee, X.; Mort, J. S.; To, R.; Huber, C. P. Crystal structures of recombinant rat cathepsin B and a cathepsin B-inhibitor complex. Implications for structure-based inhibitor design. J. Biol. Chem. 1995, 270 (10), 5527–33. (62) Yamamoto, A.; Hara, T.; Tomoo, K.; Ishida, T.; Fujii, T.; Hata, Y.; Murata, M.; Kitamura, K. Binding mode of CA074, a specific irreversible inhibitor, to bovine cathepsin B as determined by X-ray crystal analysis of the complex. J. Biochem. 1997, 121 (5), 974–7. (63) Turk, D.; Podobnik, M.; Popovic, T.; Katunuma, N.; Bode, W.; Huber, R.; Turk, V. Crystal structure of cathepsin B inhibited with CA030 at 2.0-A resolution: A basis for the design of specific epoxysuccinyl inhibitors. Biochemistry 1995, 34 (14), 4791–7. (64) Turk, D.; Guncar, G.; Podobnik, M.; Turk, B. Revised definition of substrate binding sites of papain-like cysteine proteases. Biol. Chem. 1998, 379 (2), 137–47. (65) Krupa, J. C.; Hasnain, S.; N€agler, D. K.; Menard, R.; Mort, J. S. S20 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B. Biochem. J. 2002, 361 (Pt 3), 613–9. (66) Stern, I.; Schaschke, N.; Moroder, L.; Turk, D. Crystal structure of NS-134 in complex with bovine cathepsin B: a two-headed epoxysuccinyl inhibitor extends along the entire active-site cleft. Biochem. J. 2004, 381 (Pt 2), 511–7. 5372
dx.doi.org/10.1021/pr200621z |J. Proteome Res. 2011, 10, 5363–5373
Journal of Proteome Research (67) Br€omme, D.; Bonneau, P. R.; Lachance, P.; Storer, A. C. Engineering the S2 subsite specificity of human cathepsin S to a cathepsin L- and cathepsin B-like specificity. J. Biol. Chem. 1994, 269 (48), 30238–42. (68) Br€omme, D.; Steinert, A.; Friebe, S.; Fittkau, S.; Wiederanders, B.; Kirschke, H. The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B. Biochem. J. 1989, 264 (2), 475–81. (69) Puzer, L.; Cotrin, S. S.; Cezari, M. H. S.; Hirata, I. Y.; Juliano, M. A.; Stefe, I.; Turk, D.; Turk, B.; Juliano, L.; Carmona, A. K. Recombinant human cathepsin X is a carboxymonopeptidase only: a comparison with cathepsins B and L. Biol. Chem. 2005, 386 (11), 1191–5. (70) Puzer, L.; Cotrin, S. S.; Alves, M. F. M.; Egborge, T.; Araujo, M. S.; Juliano, M. A.; Juliano, L.; Br€omme, D.; Carmona, A. K. Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L. Arch. Biochem. Biophys. 2004, 430 (2), 274–83. (71) K€argel, H. J.; Dettmer, R.; Etzold, G.; Kirschke, H.; Bohley, P.; Langner, J. Action of cathepsin L on the oxidized B-chain of bovine insulin. FEBS Lett. 1980, 114 (2), 257–60. (72) Coulombe, R.; Grochulski, P.; Sivaraman, J.; Menard, R.; Mort, J. S.; Cygler, M. Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. EMBO J. 1996, 15 (20), 5492–503. (73) Shenoy, R. T.; Sivaraman, J. Structural basis for reversible and irreversible inhibition of human cathepsin L by their respective dipeptidyl glyoxal and diazomethylketone inhibitors. J. Struct. Biol. 2011, 173 (1), 14–9. (74) Gal, S.; Gottesman, M. M. The major excreted protein (MEP) of transformed mouse cells and cathepsin L have similar protease specificity. Biochem. Biophys. Res. Commun. 1986, 139 (1), 156–62. (75) Menard, R.; Carmona, E.; Takebe, S.; Dufour, E.; Plouffe, C.; Mason, P.; Mort, J. S. Autocatalytic processing of recombinant human procathepsin L. Contribution of both intermolecular and unimolecular events in the processing of procathepsin L in vitro. J. Biol. Chem. 1998, 273 (8), 4478–84. (76) Nosaka, A. Y.; Kanaori, K.; Teno, N.; Togame, H.; Inaoka, T.; Takai, M.; Kokubo, T. Conformational studies on the specific cleavage site of Type I collagen (alpha-1) fragment (157192) by cathepsins K and L by proton NMR spectroscopy. Bioorg. Med. Chem. 1999, 7 (2), 375–9. (77) Popovic, T.; Cimerman, N.; Dolenc, I.; Ritonja, A.; Brzin, J. Cathepsin L is capable of truncating cystatin C of 11 N-terminal amino acids. FEBS Lett. 1999, 455 (12), 92–6. (78) Abboud-Jarrous, G.; Atzmon, R.; Peretz, T.; Palermo, C.; Gadea, B. B.; Joyce, J. A.; Vlodavsky, I. Cathepsin L is responsible for processing and activation of proheparanase through multiple cleavages of a linker segment. J. Biol. Chem. 2008, 283 (26), 18167–76. (79) Goretzki, L.; Schmitt, M.; Mann, K.; Calvete, J.; Chucholowski, N.; Kramer, M.; G€unzler, W. A.; J€anicke, F.; Graeff, H. Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L. FEBS Lett. 1992, 297 (12), 112–8. (80) Melo, R. L.; Alves, L. C.; Del Nery, E.; Juliano, L.; Juliano, M. A. Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides. Anal. Biochem. 2001, 293 (1), 71–7. (81) Felbor, U.; Dreier, L.; Bryant, R. A.; Ploegh, H. L.; Olsen, B. R.; Mothes, W. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J. 2000, 19 (6), 1187–94. (82) Katunuma, N.; Murata, E.; Kakegawa, H.; Matsui, A.; Tsuzuki, H.; Tsuge, H.; Turk, D.; Turk, V.; Fukushima, M.; Tada, Y.; Asao, T. Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo. FEBS Lett. 1999, 458 (1), 6–10. (83) Guncar, G.; Pungercic, G.; Klemencic, I.; Turk, V.; Turk, D. Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S. EMBO J. 1999, 18 (4), 793–803. (84) Ong, P. C.; McGowan, S.; Pearce, M. C.; Irving, J. A.; Kan, W.-T.; Grigoryev, S. A.; Turk, B.; Silverman, G. A.; Brix, K.; Bottomley, S. P.; Whisstock, J. C.; Pike, R. N. DNA accelerates the inhibition of human cathepsin V by serpins. J. Biol. Chem. 2007, 282 (51), 36980–6.
ARTICLE
(85) Xin, X. Q.; Gunesekera, B.; Mason, R. W. The specificity and elastinolytic activities of bovine cathepsins S and H. Arch. Biochem. Biophys. 1992, 299 (2), 334–9. (86) Kirschke, H.; Wiederanders, B.; Br€omme, D.; Rinne, A. Cathepsin S from bovine spleen. Purification, distribution, intracellular localization and action on proteins. Biochem. J. 1989, 264 (2), 467–73. (87) Br€omme, D.; Bonneau, P. R.; Purisima, E.; Lachance, P.; Hajnik, S.; Thomas, D. Y.; Storer, A. C. Contribution to activity of histidine-aromatic, amide-aromatic, and aromatic-aromatic interactions in the extended catalytic site of cysteine proteinases. Biochemistry 1996, 35 (13), 3970–9. (88) Song, J.; Xu, P.; Xiang, H.; Su, Z.; Storer, A. C.; Ni, F. The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B. FEBS Lett. 2000, 475 (3), 157–62. (89) Mort, J. S.; Recklies, A. D.; Poole, A. R. Characterization of a thiol proteinase secreted by malignant human breast tumours. Biochim. Biophys. Acta 1980, 614 (1), 134–43. (90) Turk, B.; Dolenc, I.; Zerovnik, E.; Turk, D.; Gubensek, F.; Turk, V. Human cathepsin B is a metastable enzyme stabilized by specific ionic interactions associated with the active site. Biochemistry 1994, 33 (49), 14800–6. (91) Aronson, N. N.; Barrett, A. J. The specificity of cathepsin B. Hydrolysis of glucagon at the C-terminus by a peptidyldipeptidase mechanism. Biochem. J. 1978, 171 (3), 759–65. (92) L€utzner, N.; Kalbacher, H. Quantifying cathepsin S activity in antigen presenting cells using a novel specific substrate. J. Biol. Chem. 2008, 283 (52), 36185–94. (93) R€uckrich, T.; Brandenburg, J.; Cansier, A.; M€uller, M.; Stevanovic, S.; Schilling, K.; Wiederanders, B.; Beck, A.; Melms, A.; Reich, M.; Driessen, C.; Kalbacher, H. Specificity of human cathepsin S determined by processing of peptide substrates and MHC class IIassociated invariant chain. Biol. Chem. 2006, 387 (1011), 1503–11. (94) Br€omme, D.; Klaus, J. L.; Okamoto, K.; Rasnick, D.; Palmer, J. T. Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L. Biochem. J. 1996, 315 (Pt 1), 85–9. (95) Kaulmann, G.; Palm, G. J.; Schilling, K.; Hilgenfeld, R.; Wiederanders, B. The crystal structure of a Cys25 f Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions. Protein Sci. 2006, 15 (11), 2619–29. (96) McGrath, M. E.; Palmer, J. T.; Br€omme, D.; Somoza, J. R. Crystal structure of human cathepsin S. Protein Sci. 1998, 7 (6), 1294–302. (97) Pauly, T. A.; Sulea, T.; Ammirati, M.; Sivaraman, J.; Danley, D. E.; Griffor, M. C.; Kamath, A. V.; Wang, I.-K.; Laird, E. R.; Seddon, A. P.; Menard, R.; Cygler, M.; Rath, V. L. Specificity determinants of human cathepsin s revealed by crystal structures of complexes. Biochemistry 2003, 42 (11), 3203–13. (98) Coppini, L. P.; Barros, N. M. T.; Oliveira, M.; Hirata, I. Y.; Alves, M. F. M.; Paschoalin, T.; Assis, D. M.; Juliano, M. A.; Puzer, L.; Br€omme, D.; Carmona, A. K. Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor. Biol. Chem. 2010, 391 (5), 561–70. (99) Turkenburg, J. P.; Lamers, M. B. A. C.; Brzozowski, A. M.; Wright, L. M.; Hubbard, R. E.; Sturt, S. L.; Williams, D. H. Structure of a Cys25wSer mutant of human cathepsin S. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58 (3), 451–455. (100) Boyd, S. E.; Pike, R. N.; Rudy, G. B.; Whisstock, J. C.; Garcia de la Banda, M. PoPS: a computational tool for modeling and predicting protease specificity. J. Bioinform. Comput. Biol. 2005, 3 (3), 551–85. (101) Lohm€uller, T.; Wenzler, D.; Hagemann, S.; Kiess, W.; Peters, C.; Dandekar, T.; Reinheckel, T. Toward computer-based cleavage site prediction of cysteine endopeptidases. Biol. Chem. 2003, 384 (6), 899–909. (102) Colaert, N.; Helsens, K.; Martens, L.; Vandekerckhove, J.; Gevaert, K. Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 2009, 6 (11), 786–7.
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