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Feb 20, 2014 - cleavage sites of a family of serine proteases: the granzymes. We and others previously assigned clear murine orthologues for all 5...
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Holistic View on the Extended Substrate Specificities of Orthologous Granzymes Kim Plasman,†,‡ Sebastian Maurer-Stroh,§,∥ Kris Gevaert,†,‡ and Petra Van Damme*,†,‡ †

Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium § Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore 138671 ∥ School of Biological Sciences (SBS), Nanyang Technological University (NTU), Singapore 637551 ‡

S Supporting Information *

ABSTRACT: As proteases sculpt the proteome in both homeostatic and pathogenic processes, unraveling their primary signaling pathways and key substrates is of utmost importance. Hence, with the development of procedures enriching for proteolysis-indicative peptides and the availability of more sensitive mass spectrometers, protease degradomics technologies are ideally suited to gain insight into a protease’s substrate repertoire and substrate-specificity profile. Especially, knowledge on discriminating sequence features among closely related homologues and orthologues may aid in identifying key targets and developing protease-specific inhibitors. Although clever labeling strategies allow one to compare the substrate repertoires and critical protease−substrate recognition motifs of several proteases in a single analysis, comprehensive views of (differences in) substrate subsite occupancies of entire protease families is lacking. Therefore, we here describe a hierarchical cluster analysis of the positional proteomics determined cleavage sites of a family of serine proteases: the granzymes. We and others previously assigned clear murine orthologues for all 5 human granzymes. As such, hierarchical clustering of the sequences surrounding granzyme cleavage sites reveals detailed insight into granzyme-specific differences in substrate selection and thereby deorphanizes the substrate specificity profiles and repertoires of the human and murine orthologous granzymes A, B, H/C, M, and K. KEYWORDS: granzyme, positional proteomics, degradomics, N-terminal COFRADIC, protease, substrate, substrate specificity

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INTRODUCTION TO PROTEASE DEGRADOMICS

GRANZYME BIOLOGY Cytotoxic lymphocytes (CLs) are key effector cells of the immune system. Natural killer cells (NK cells) and cytotoxic Tlymphocytes (CD4+ and CD8+) are CLs that can elicit apoptosis in sensitive target cells such as virally infected cells25 and transformed cells.26 Apoptosis can be elicited by receptor/ligand mediated signaling (i.e., Fas/FasL signaling) resulting in the intracellular activation of the cell death inducing caspase cascade.27 In addition, dense cytoplasmic cytotoxic granules, also referred to as secretory lysosomes, provide further cytotoxic potential to CLs.28 Regulated exocytosis of this granule content delivers the pore-forming protein perforin and granule-specific trypsin-like serine proteases, granzymes,29−32 to target cells. Although the underlying molecular mechanism of action is still debated, perforin permits granzyme access to target cells.33 Elucidation of true physiological functions of granzymes has been challenged since mismatching between the origins of the granzymes and the target cells used, in addition to the use of nonphysiological granzyme

Proteases either process proteins into stable fragments or shred them into amino acids (protein catabolism). These enzymes play key roles in homeostasis and pathogenesis; therefore, not surprisingly, elucidating their specific functions in such processes, among others, by identifying their physiological substrates is of utmost importance.1,2 Proteolytic cleavage results in the creation of new termini referred to as the protein neo-N- and neo-C-termini. Over the past years, a great deal of effort was put into the development of procedures that allow for the specific isolation of such neo-terminal peptides,3−5 which resulted in several proteomics-based strategies that enable studying protease specificities in great detail or to catalogue their substrates,5−14 with the most recent development being the quantitative profiling of substrate cleavage.15,16 Our laboratory developed the COFRADIC (COmbined FRActional DIagonal Chromatography) technology by which N- and C-terminomes can be isolated,5,9,17 and over the past few years, this technology was, among others, extensively applied to identify substrates and specificity profiles of human and mouse granzymes.5,15,18−24 © 2014 American Chemical Society

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concentrations, all led to inconsistencies in reports.34 Recently, some of these inconsistencies were explained by differences in substrate specificities, substrate and inhibitor repertoires, and cytotoxicity potentials among granzyme orthologues,18,22,35−38 all implying that key granzyme targets and thus the primary signaling pathways affected by granzymes when executing their function may differ. Overall, and given the implication of granzymes in cytotoxic and inflammatory processes,39,40 exploration of the primary signaling pathways targeted and used by granzymes is of great interest to the scientific community.

proteins identified by N-terminal COFRADIC,5,22 this database holds 97 literature-curated human cleavage sites that were identified by alternative means such as consensus sequence driven substrate identification and alternative proteome surveys (e.g., 2D gel electrophoresis) coupled or not to mutation analyses or N-terminal sequencing. Overall, the P4-P4′ specificity profiles from these two data sets are quite similar (Figure 1, top and middle) being V/I-E-P/N-D↓S-G-x-x and V/I-E-A/P-D↓S-x-x-x for the literature-curated and neo-Nterminal data sets, respectively. To probe for more subtle differences in these data sets, a differential iceLogo (Figure 1, bottom) was created. This shows that cleavage at Glu residues was more prominent in the N-terminomics data set (14% versus 1%). Besides, in the non-N-terminomics data sets, Val is about 1.5-fold more present in the P4 position (i.e., 38% versus 26%), which is, next to the P1 position, the major substrate specificity determinant inferring granzyme B cleavage susceptibility. Other differences include the close to 2-fold reduction in Pro occurrence in the P2 position (i.e., 23% versus 13%) and the decreased occurrence of Asn in the P2 position (16% to 2%) in our N-terminal data sets. In the P4′ position, the occurrence of the acidic amino acids Glu and Asp increases from 12% to 32%. The decreased occurrence of Asn in the P2 position in our proteomics data set is explained by the fact that except for one, all other motifs (15 motifs) harboring a P2 Asn were identified using a biased bioinformatics screen for putative granzyme B cleavage sites holding a P2 Asn in internal Nglycosylation (glycan) sequons (i.e., NDS or NDT).52 It is reasonable to assume that all other observed substrate biases are caused by differences in substrate cleavage efficiency. While being very powerful for elucidating protease specificities and substrate repertoires, the ever increasing sensitivity of mass spectrometers yields a wealth of protease substrate cleavage data, which might also include rare cleavage events.8,22 When probing for hGrB cleavage efficiencies using N-terminal COFRADIC, we observed a 3-fold increase in the occurrence of Pro in the P2 position when comparing efficient to inefficient cleavages events. In addition, Val in the P4 position also correlated with increased cleavage efficiency, while cleavage events at Glu were categorized as inefficient events.15 Such observations thus hint to the fact that lists of granzyme B substrates identified by positional proteomics might contain inefficiently and rarely cleaved substrates next to efficiently cleaved substrates. Assuming that the latter might point to physiologically more relevant substrates, we recently developed a strategy to discern efficiently from inefficiently cleaved substrates.15

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SUBSTRATE SPECIFICITIES OF GRANZYMES Until recently, the substrate and specificity profiles of granzymes, other than those of granzyme B (GrB), were poorly characterized. Initial attempts made use of peptide-based strategies by means of positional scanning−synthetic combinatorial libraries (PS-SCL)18,35,41−43 and phage display.19,37 Substrate specificities determined in these ways were mostly limited to P4−P2′ substrate positions (Pn-...-P2-P1-P1′-P2′-...Pn′; nomenclature according to Schechter and Berger70) and did not always correlate with specificities in known substrates, as was shown for the dimeric GrA43 and GrB.41,44,45 Interestingly, upon GrB incubation, no or little cleavage of di/tripeptidic substrates was observed as compared to the cleavage of tetrapeptide motifs, with strongly decreasing cleavage efficiencies when deviating from the optimal GrB tetrapeptide motif Ile-Glu-Pro-Asp. These findings hint to an absolute dependency on extended substrate recognition beyond the P1 position for efficient proteolysis of substrates by GrB. Recent positional proteomics studies found differences in substrate selection for human GrA (hGrA),21 hGrH,20 and hGrK (Plasman et al., unpublished work) and hGrM18,23 and their murine counterparts mGrA,24 mGrC,19,20 mGrK (Plasman et al., unpublished work), and mGrM.18 Further, by mutating specific sites in granzyme substrates, a conserved but hitherto underappreciated specificity-determining role for extended substrate regions in steering granzyme cleavage susceptibility was found.20 In addition and despite the sometimes only subtle differences in the specificity profiles of orthologous granzymes, their substrate repertoires only partially overlap, and differences in cleavage efficiencies were found. Most of the protease cleavage data stored in public repositories such as MEROPS,46 CutDB,47 TopFIND,48 DegraBase,49 and TOPPR50 are derived from mass spectrometry based proteome analyses. These proteome analyses unavoidably introduce biases in the physical property and amino acid composition of the peptides they identify. In Nterminal COFRADIC, His and Arg residues are typically underrepresented in the identified peptides, which is, respectively, caused by applying strong cation exchange (SCX) and the use of trypsin.17 However, these particular biases can be overcome by the use of proteases other than trypsin (e.g., chymotrypsin and endoproteinase Glu-C) and avoiding the SCX pre-enrichment step.51 Previously, we and others demonstrated that enrichment of C-terminal peptides not only enables substrate screening of carboxyproteases but moreover also allows one to overcome some of these biases, leading to the identification of substrates which are a priori not assessable by N-terminal approaches.3,5 To probe for the existence of any other yet unknown biases, we studied the hGrB protein cleavages stored in MEROPS.46 Next to the 725 unique P4-P4′ hGrB cleavage sites in human

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GRANZYME-SPECIFIC DIFFERENCES IN SUBSTRATE SELECTION Hierarchical clustering allows for detailed analysis of differences in the protease substrate subsite occupancy. We here clustered the extended granzyme cleavage motifs of the orthologous granzymes A, B, H/C, M, and K identified by N-terminal COFRADIC to screen for discriminating subsite features for each of these granzymes. Amino acid sequences covering P4 to P9′ were considered since based on individual granzyme specificities,53 these cleavage regions harbor the critical protease−substrate recognition motif (Figure 2 and Table S1, Supporting Information). Although extended specificity profiles were observed for all granzymes, interestingly, orthologous granzymes cluster together at positions beyond P1−P4, which 1786

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KEY DETERMINANTS STEERING GRANZYME CLEAVAGE SUSCEPTIBILITY ARE P1, P1′, AND P2′ As apparent from the cluster analyses, positions P1, P1′, and P2′ are the most specific and distinctive, given the highest restriction of amino acid occurrences at these subsites (Figure 2). Obviously, P1 displays the most stringent subsite requirements confirming Asp-ase activity of GrB (Figure 2), while granzymes A and K clearly act as tryptases targeting substrates at Arg and Lys, though all prefer Arg residues. Whereas GrM was originally considered to be a Met-ase, we showed that GrM preferentially cleaves after Leu.18,23 Besides GrM’s P1 specificities of Leu and Met, the chymases hGrH and mGrC also recognize and cleave substrates carrying Phe and Tyr at this position. The accommodation of such bulky aromatic amino acids in the proteases’ S1 pocket is explained by the presence of a P1-specificity-determining, small Gly residue in the S1 pocket of hGrH.56 The possibility of hydrogen bond formation with a hGrH S1 Asn residue suggested a higher cleavage susceptibility of P1 Tyr over Phe substrates, whereas substrate profiling showed a two times more frequent occurrence of P1 Phe cleavages for hGrH (30% versus 14%). mGrC displays similar P1 preferences (i.e., Phe (44%), Leu (31%), Met (13%), and Tyr (12%)) as compared to hGrH. The partial redundant P1 specificity profiles of granzymes M and H/ C are also reflected by the shared P1 Leu-specific cleavage events.18,20,23 The repertoire of amino acids tolerated in the P1′ position is mainly limited to the small amino acids Ala and Ser in addition to Lys, with hGrH and mGrC having a pronounced preference for Ser, being 30% on average while, for example, in the P2′ position the percentage of Ser occurrence in all granzyme cleavage events is only 12% (Figure 2). Lys in the P1′ position is mainly tolerated by the tryptases and GrM. In the P2′ position, the overall profile partially matches the P1′ specificity profile observed, except that Leu in the P2′ position is highly preferred by the tryptases (up to 38%), while this amino acid is almost completely absent in hGrH and mGrC substrates (i.e., less than 1%) (Figure 2). The latter granzymes preferentially cleave substrates harboring Gly in the P2′ position (33% as opposed to the average of 12% observed for other granzyme cleavages). These observations are well in line with structural analyses as to avoid steric clashes between substrate amino acid residues with limited conformational flexibility and GrA43 and furthermore validate the size-restricted S2′ binding pocket of hGrH.56

Figure 1. IceLogo representations of human granzyme B substrate specificity profiles. IceLogo representations were created using 725 (top) and 97 (middle) aspartate/glutamate P4-P4′ human granzyme B cleavage motifs of the cleavage sites identified by means of N-terminal COFRADIC (of the 732 unique hGrB specific neo-N-terminal peptides previously reported, 729 could be mapped against the Swiss-Prot database release 2013_08, and of these, 725 unique (and thus non-redundant) P4-P4′ cleavage motifs could be deduced) and the literature-curated, non-COFRADIC cleavage sites reported in release 9.9 (August 2013) of MEROPS,5,22 respectively. Multiple sequence alignments of peptide substrate motifs are given as 1−8 corresponding to cleavage site residues P4 to P4′ with the P1 cleavage site at position 4. Statistically significant residues with a p-value ≤ 0.05 are plotted, and amino acids heights are indicative for the degree of conservation at the indicated position. The frequency of the amino acid occurrence at each position in the sequence set was compared with that of the human protein sequences stored in the Swiss-Prot (version 2013_08) database. Residues with a statistically significant lower frequency of occurrence as compared with Swiss-Prot are indicated at the negative side of the x-axis, and missing residues are shown in pink (bottom). A differential iceLogo representation was created upon comparing both data sets. Statistically significant residues (p ≤ 0.05) are plotted with the size of the amino acids proportional to the difference in occurrence between both data sets.

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DISCRIMINATIVE GRANZYME SUBSTRATE DETERMINANTS BEYOND P1, P1′, AND P2′ In the P4 position, clear preferences for the orthologous granzyme B and M pairs can be observed; GrM recognition sites preferentially hold Lys (29% for hGrM and 25% for mGrM), and GrB recognition sites prefer Val (24% for hGrB and 20% for mGrB) (Figure 2), while the other granzyme cleavage motifs on average only hold 6% of Lys and 8% of Val in the P4 position. Lys is also found enriched, though to a lesser extent, in hGrH and mGrC (11% in both cases) cleavage motifs, while (highly) unfavored by GrA and GrK orthologues and almost not tolerated by GrB (2% on average). In addition, hGrH shows an enrichment of Pro (16%) at this position as compared to the P4 occupancy at other granzyme cleavage sites

until recently was the motif considered to steer granzyme substrate specificity.42,54,55 1787

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Figure 2. continued

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Figure 2. Cluster analysis of the extended P4 to P9′ substrate specificity profiles of the orthologous human and mouse granzymes analyzed by means of positional proteomics. Each dendrogram in the cluster represents the hierarchical Euclidian distance structure between the position-specific substrate specificity profiles that was computed using hierarchical clustering with the complete linkage algorithm and indicates the degree of similarity in the positional substrate specificity profiles of the 10 granzymes analyzed. In nearly all cases, clustering of subsite specificities follows the clustering of granzyme orthologues.

Figure 3. Structural alignment of the 5 human granzymes A, B, H, K, and M. While the overall fold and binding pocket core in all granzyme structures are well conserved, there is a notable flexibility in top surface loops comprising the extended substrate binding pocket. Color legend: gray, structure conserved; magenta, bound substrate peptides from different structures; red, unique structure in hGrA; blue, unique structure in hGrK; green, unique structure in hGrB; cyan, unique structure in hGrM; orange, unique structure in hGrH. Structural comparison and visualization were done in YASARA.67 The following PDB structures were used: hGrA (PDB: 1op857), hGrK (PDB: 1mza60), hGrB (PDB: 1iau58), hGrM (PDB: 2zgj59), and hGrH (PDB: 3tjv56). Structural alignments were done with MUSTANG.68

(9% on average), codetermining the slight differences observed in the delineated hGrH/mGrC substrate specificity profiles.20 The most striking observation in the P3 position is the shared preference for cleavage motifs holding Glu for both orthologous granzyme B (18% on average) and M (18% on average) pairs (Figure 2). A similar preference in hGrH substrates could be observed, and analogous to the slightly deviating specificity in the P4 position with its murine counterpart, P3 Glu occurrence was 2-fold increased in hGrH versus mGrC substrates (i.e., 17% versus 8%).

The cleavage susceptibility of human and mouse GrM substrates appears further determined by the presence of Pro in the P2 position (24% on average), while tolerance of Pro by other granzymes does not exceed the overall preference for other tolerated amino acids (Figure 2). The most deviating specificity between orthologues at the P2 position is the preference for Phe (21%) by mGrC, which can also be observed for hGrH though to a lesser extent (17%). From P3′ to P7′ a strong preference for acidic residues and more specifically Glu by the granzymes B, H, and C appears (Figure 2). Although acidic residues are also tolerated by 1789

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Figure 4. Surface distribution of different types of amino acid residues (A) and electrostatic potential (B) of the 5 human granzymes. Surface residues are colored according to their physical properties (A): KRH, blue; DE, red; QN, green; STY, cyan; other, gray and bound substrate peptides from different structures, magenta. Panel B shows the same structure orientation colored according to the electrostatic potential: blue, positively; red, negatively charged. Structural comparison and visualization were done in YASARA.67 The following PDB structures were used: hGrA (PDB: 1op857), hGrK (PDB: 1mza60), hGrB (PDB: 1iau58), hGrM (PDB: 2zgj59), and hGrH (PDB: 3tjv56). Structural alignments were done with MUSTANG,68 and electrostatic surface potentials were calculated with the particle mesh Ewald method69 implemented in YASARA.

structures allowing for a detailed inspection of conformational differences are now available for the 5 human granzymes A, B, H, K, and M.56−60 Interestingly, in their structural alignment, we see that the overall fold and binding pocket core structure are well conserved, but nonetheless there is noticeable flexibility in top surface loops including those in contact with extended substrate motif residues (Figure 3). Since the cluster analyses identified distinct substrate charge preferences shared among some of the granzymes, we also investigated the distribution of charged amino acids at the surface of the different granzymes (Figure 4A) as well as the surface electrostatic potential (Figure 4B). Although the surface loop conformations differ among the 5 granzymes, characteristic surface charge patterns are shared among different granzymes, thereby likely influencing their preferences for specific charges at extended substrate motif positions. More specifically, the substrate pocket around P3′ and also the substrate entry region (P3) show clear differences with neutral or slightly negatively charged potential in granzymes A and K on the one hand and granzymes B, H, and M being strongly positively charged on the other. Indeed, the occurrence of negatively charged Glu at the P3 and P3′ substrate positions is higher in granzymes B, H, and M as compared to granzymes A and K. At the same time, all granzymes seem to be mainly positively charged at the surface of the substrate exit region beyond P5′, which may contribute to the more general preferences for negative charges observed from the P6′ to P9′ positions. In summary, the different granzymes differ in their detailed surface loop structures but partially share electrostatic potential patterns governing groupspecific preferences especially for negative charges at several extended substrate motif positions.

granzymes A and K, unlike for granzymes B, H, and C, no clear presence of extended acidic substrate patches can be observed. Such an acidic motif was shown to serve as a critical substrate determinant in steering granzyme B, H, and C cleavage susceptibility.20 From P8′ onward, deducing subsite specificities becomes more difficult and likely more error prone since the N-terminal COFRADIC technology enriches peptides ending on Arg, a biased preference as shown by the primed site unbiased assessment of granzyme B cleavage specificity using neo-Ctermini.5 Opposed to the general extended specificity profiles observed for granzymes B, H/C, and M, for granzymes A and K no striking subsite requirements, except for residues in the P1 and P2′ positions, were found. This observation is well in line with the suggested implication of macromolecular specificitydetermining substrate regions or exosites given the dimeric structure of GrA.57 Despite the fact that crystal structures of pro-hGrK revealed a monomeric state, its less restricted specificity profile (other than positions P1 and P2′) suggest that exosite interactions may also influence GrK macromolecular substrate selection.

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STRUCTURAL RATIONALE BEHIND GRANZYME-SHARED SUBSTRATE SPECIFICITIES The structural differences describing granzyme-specific P1 preferences have been well documented previously56−59 and include different locked states and unlocking residues for some granzymes.19,60 In specific granzymes and orthologous granzyme pairs, we have found specific positively charged residues that, to a certain extent, can be responsible for the preferences of negatively charged residues in certain extended substrate motif positions.21,22,61 However, often, sequence conservation of these residues is not easily noticeable from structural granzyme alignments. Therefore, we wanted to investigate whether the substrate specificity profiles in extended substrate motif positions here found to be shared between some granzymes but not others can be rationalized in view of their 3D structures (Figures 3 and 4). High resolution crystal

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CONCLUSIONS AND PERSPECTIVES Understanding the substrate specificity of proteases is of crucial importance to unravel their mechanism of action. Although many tools are available to study protease substrate specificity at the peptide level, only rather recently proteomic approaches have been applied to protease degradomics research. Next to permitting the identification of key substrates in primary 1790

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signaling pathways,23 these positional proteomics endeavors have proven to more comprehensively capture the extended specificity profiles of proteases,62 including those of granzymes.18−24 Overall, results from peptide-based approaches applied to granzymes other than GrA and GrK42,43,63 did correlate well with the specificity profiles determined by positional proteomics. However, the former do typically not consider primed site specificities that are here shown to be of key importance in steering granzyme cleavage susceptibility. Overall, the specificity profiles revealed only subtle subsite specificity differences for orthologous granzymes; however, these have been found to result in different functional outcomes.22,35−37,64 Along this line, synthetic peptides holding one or several residues known to influence cleavage efficiencies by orthologous granzymes B were found exclusively cleaved by mGrB.22 This might thus pave the way for specific targeting of closely related proteases such as the different granzyme tryptases and executioner caspases by peptide-based inhibitors.65 In order to further elucidate the primary signaling pathways of granzymes and to designate critical determinants, kinetic degradome analyses and in vivo validations have proven indispensable.15 Along this line, recently, in addition to discovery proteomics, targeted proteomics66 has been used to allow for absolute quantification of a predefined set of proteolytically generated substrate fragments.16

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ASSOCIATED CONTENT

* Supporting Information IceLogo visualization of the non-redundant P4−P9′ cleavage site motifs of the human granzymes A, B, H, K, and M and their murine orthologues identified by N-terminal COFRADIC and positional amino acid occurrence (percentage) as deduced from extended P15 to P15′ granzyme cleavages identified. This material is available free of charge via the Internet at http:// pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B9000 Ghent, Belgium. Tel: +32-92649279. Fax: +32-92649496. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Sven Degroeve for technical assistance. K.P. is supported by a Ph.D. grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). P.V.D. is a Postdoctoral Fellow of the Research Foundation-Flanders (FWO-Vlaanderen). K.G. acknowledges support from the Research Foundation-Flanders (FWO-Vlaanderen), project number G.0440.10.

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REFERENCES

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ABBREVIATIONS

CD, cluster of differentiation; CL, cytotoxic lymphocyte; COFRADIC, combined fractional diagonal chromatography; Gr, granzyme; LC, liquid chromatography; SCX, strong cation exchange 1791

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human and mouse granzymes are structurally and functionally divergent. J. Cell Biol. 2006, 175 (4), 619−630. (38) de Poot, S. A.; Lai, K. W.; Hovingh, E. S.; Bovenschen, N. Granzyme M cannot induce cell death via cleavage of mouse FADD. Apoptosis 2013, 18, 533−534. (39) Anthony, D. A.; Andrews, D. M.; Watt, S. V.; Trapani, J. A.; Smyth, M. J. Functional dissection of the granzyme family: cell death and inflammation. Immunol Rev 2010, 235 (1), 73−92. (40) Heutinck, K. M.; ten Berge, I. J.; Hack, C. E.; Hamann, J.; Rowshani, A. T. Serine proteases of the human immune system in health and disease. Mol. Immunol. 2010, 47 (11−12), 1943−1955. (41) Harris, J. L.; Peterson, E. P.; Hudig, D.; Thornberry, N. A.; Craik, C. S. Definition and redesign of the extended substrate specificity of granzyme B. J. Biol. Chem. 1998, 273 (42), 27364−27373. (42) Mahrus, S.; Craik, C. S. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells. Chem. Biol. 2005, 12 (5), 567− 577. (43) Bell, J. K.; Goetz, D. H.; Mahrus, S.; Harris, J. L.; Fletterick, R. J.; Craik, C. S. The oligomeric structure of human granzyme A is a determinant of its extended substrate specificity. Nat. Struct. Biol. 2003, 10 (7), 527−534. (44) Andrade, F.; Casciola-Rosen, L. A.; Rosen, A. Granzyme Binduced cell death. Acta Haematol. 2004, 111 (1−2), 28−41. (45) Sun, J.; Whisstock, J. C.; Harriott, P.; Walker, B.; Novak, A.; Thompson, P. E.; Smith, A. I.; Bird, P. I. Importance of the P4′ residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop. J. Biol. Chem. 2001, 276 (18), 15177−15184. (46) Rawlings, N. D.; Barrett, A. J.; Bateman, A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2012, 40 (databaseissue), D343−D350. (47) Igarashi, Y.; Eroshkin, A.; Gramatikova, S.; Gramatikoff, K.; Zhang, Y.; Smith, J. W.; Osterman, A. L.; Godzik, A. CutDB: a proteolytic event database. Nucleic Acids Res. 2007, 35 (database issue), D546−D549. (48) Lange, P. F.; Huesgen, P. F.; Overall, C. M. TopFIND 2.0– linking protein termini with proteolytic processing and modifications altering protein function. Nucleic Acids Res. 2012, 40 (database issue), D351−D361. (49) Crawford, E. D.; Seaman, J. E.; Agard, N.; Hsu, G. W.; Julien, O.; Mahrus, S.; Nguyen, H.; Shimbo, K.; Yoshihara, H. A.; Zhuang, M.; Chalkley, R. J.; Wells, J. A. The DegraBase: a database of proteolysis in healthy and apoptotic human cells. Mol. Cell. Proteomics 2013, 12 (3), 813−824. (50) Colaert, N.; Maddelein, D.; Impens, F.; Van Damme, P.; Plasman, K.; Helsens, K.; Hulstaert, N.; Vandekerckhove, J.; Gevaert, K.; Martens, L. The Online Protein Processing Resource (TOPPR): a database and analysis platform for protein processing events. Nucleic Acids Res. 2013, 41 (database issue), D333−D337. (51) Vogtle, F. N.; Wortelkamp, S.; Zahedi, R. P.; Becker, D.; Leidhold, C.; Gevaert, K.; Kellermann, J.; Voos, W.; Sickmann, A.; Pfanner, N.; Meisinger, C. Global analysis of the mitochondrial Nproteome identifies a processing peptidase critical for protein stability. Cell 2009, 139 (2), 428−439. (52) Gahring, L.; Carlson, N. G.; Meyer, E. L.; Rogers, S. W. Granzyme B proteolysis of a neuronal glutamate receptor generates an autoantigen and is modulated by glycosylation. J. Immunol. 2001, 166 (3), 1433−1438. (53) Colaert, N.; Helsens, K.; Martens, L.; Vandekerckhove, J.; Gevaert, K. Improved visualization of protein consensus sequences by iceLogo. Nat. Methods 2009, 6 (11), 786−787. (54) Mahrus, S.; Kisiel, W.; Craik, C. S. Granzyme M is a regulatory protease that inactivates proteinase inhibitor 9, an endogenous inhibitor of granzyme B. J. Biol. Chem. 2004, 279 (52), 54275−54282. (55) Ruggles, S. W.; Fletterick, R. J.; Craik, C. S. Characterization of structural determinants of granzyme B reveals potent mediators of extended substrate specificity. J. Biol. Chem. 2004, 279 (29), 30751− 30759. 1792

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Journal of Proteome Research

Reviews

(56) Wang, L.; Zhang, K.; Wu, L.; Liu, S.; Zhang, H.; Zhou, Q.; Tong, L.; Sun, F.; Fan, Z. Structural insights into the substrate specificity of human granzyme H: the functional roles of a novel RKR motif. J. Immunol. 2012, 188 (2), 765−773. (57) Hink-Schauer, C.; Estebanez-Perpina, E.; Kurschus, F. C.; Bode, W.; Jenne, D. E. Crystal structure of the apoptosis-inducing human granzyme A dimer. Nat. Struct. Biol. 2003, 10 (7), 535−540. (58) Rotonda, J.; Garcia-Calvo, M.; Bull, H. G.; Geissler, W. M.; McKeever, B. M.; Willoughby, C. A.; Thornberry, N. A.; Becker, J. W. The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1. Chem. Biol. 2001, 8 (4), 357−368. (59) Wu, L.; Wang, L.; Hua, G.; Liu, K.; Yang, X.; Zhai, Y.; Bartlam, M.; Sun, F.; Fan, Z. Structural basis for proteolytic specificity of the human apoptosis-inducing granzyme M. J. Immunol. 2009, 183 (1), 421−429. (60) Hink-Schauer, C.; Estebanez-Perpina, E.; Wilharm, E.; FuentesPrior, P.; Klinkert, W.; Bode, W.; Jenne, D. E. The 2.2-A crystal structure of human pro-granzyme K reveals a rigid zymogen with unusual features. J. Biol. Chem. 2002, 277 (52), 50923−50933. (61) Plasman, K.; Maurer-Stroh, S.; Ahmad, J.; Hao, H.; Kaiserman, D.; Sirota, F. L.; Jonckheere, V.; Bird, P. I.; Gevaert, K.; Van Damme, P. Conservation of the extended substrate specificity profiles among homologous granzymes across species. Mol. Cell. Proteomics 2013, 12 (10), 2921−2934. (62) Plasman, K.; Van Damme, P.; Gevaert, K. Contemporary positional proteomics strategies to study protein processing. Curr. Opin. Chem. Biol. 2013, 17 (1), 66−72. (63) Bovenschen, N.; Quadir, R.; van den Berg, A. L.; Brenkman, A. B.; Vandenberghe, I.; Devreese, B.; Joore, J.; Kummer, J. A. Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A. J. Biol. Chem. 2009, 284 (6), 3504−3512. (64) de Poot, S. A.; Lai, K. W.; Hovingh, E. S.; Bovenschen, N. Granzyme M cannot induce cell death via cleavage of mouse FADD. Apoptosis 2013, 18 (4), 533−534. (65) Demon, D.; Van Damme, P.; Vanden Berghe, T.; Deceuninck, A.; Van Durme, J.; Verspurten, J.; Helsens, K.; Impens, F.; Wejda, M.; Schymkowitz, J.; Rousseau, F.; Madder, A.; Vandekerckhove, J.; Declercq, W.; Gevaert, K.; Vandenabeele, P. Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity. Mol. Cell. Proteomics 2009, 8 (12), 2700−2714. (66) Lange, V.; Picotti, P.; Domon, B.; Aebersold, R. Selected reaction monitoring for quantitative proteomics: a tutorial. Mol. Syst. Biol. 2008, 4, 222. (67) Krieger, E.; Koraimann, G.; Vriend, G. Increasing the precision of comparative models with YASARA NOVA–a self-parameterizing force field. Proteins 2002, 47 (3), 393−402. (68) Konagurthu, A. S.; Whisstock, J. C.; Stuckey, P. J.; Lesk, A. M. MUSTANG: a multiple structural alignment algorithm. Proteins 2006, 64 (3), 559−574. (69) York, D. M.; Wlodawer, A.; Pedersen, L. G.; Darden, T. A. Atomic-level accuracy in simulations of large protein crystals. Proc. Natl. Acad. Sci. U.S.A. 1994, 91 (18), 8715−8718. (70) Schechter, I.; Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967, 27 (2), 157−162.

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