[Show abstract][Hide abstract] ABSTRACT: Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).
[Show abstract][Hide abstract] 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 in a protease's substrate repertoire and substrate specificity profile. Especially knowledge on discriminating sequence features among closely related homologs and orthologs may aid identifying key targets and developing protease-specific inhibitors. Although clever labeling strategies allow comparing 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 by positional proteomics determined cleavage sites of a family of serine proteases; the granzymes. We and others previously assigned clear murine orthologs 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.
Journal of Proteome Research 02/2014; · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The granzyme family of serine proteases are key effector molecules expressed by cytotoxic lymphocytes. The physiological role of granzyme A (GzmA) is controversial, with significant debate over its ability to induce death in target cells. Here we investigate natural inhibitors of GzmA. We employed substrate phage display and positional proteomics to compare substrate specificities of mouse and human GzmA at the peptide and proteome-wide level respectively, and used the resulting substrate specificity profiles to search for potential inhibitors from the intracellular serpin family. We identified Serpinb6b as a potent inhibitor of mGzmA. Serpinb6b interacts with mGzmA, but not hGzmA, with an association constant of 1.9 ± 0.8 × 10(5) M(-1)s(-1) and a stoichiometry of inhibition of 1.8. Mouse GzmA is over 5 times more cytotoxic than hGzmA when delivered into P815 target cells with streptolysin O, while transfection of target cells with a Serpinb6b cDNA increases the EC50 of mGzmA 13-fold, without affecting hGzmA cytotoxicity. Unexpectedly, we also found that Serpinb6b employs an exosite to specifically inhibit dimeric, but not monomeric mGzmA. The identification of an intracellular inhibitor specific for mGzmA only indicates that a lineage-specific increase in GzmA cytotoxic potential has driven cognate inhibitor evolution.
Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu(1280) separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.Cell Death and Differentiation advance online publication, 1 November 2013; doi:10.1038/cdd.2013.155.
Cell death and differentiation 11/2013; · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granzymes are structurally related serine proteases involved in cell death and immunity. To date four out of five human granzymes have assigned orthologues in mice; however for granzyme H, no murine orthologue has been suggested and its role in cytotoxicity remains controversial. Here, we demonstrate that, as is the case for granzyme C, human granzyme H is an inefficient cytotoxin which together with their similar pattern of GrB divergence and functional similarity strongly hint to their orthologous relationship. Besides analyzing the substrate specificity profile of granzyme H by substrate phage display, substrate cleavage susceptibility of human granzyme H and mouse granzyme C was assessed on a proteome-wide level. The extended specificity profiles of granzymes C and H (i.e. beyond cleavage positions P4-P4 primed) match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes as granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can simply be conferred by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility.
[Show abstract][Hide abstract] ABSTRACT: Positional proteomics refers to mass spectrometry (MS)-driven proteomics technologies by which peptides carrying the amino or carboxyl terminus of proteins are enriched from whole proteomes, and identified by means of MS. Proteases irreversibly modify their substrates by hydrolyzing peptide bonds and have thus profound effects on the biological processes steered by their substrates. Since proteases create novel termini in their substrates, positional proteomics is ideally suited for protease degradomics studies. We here review recent developments in the field of positional proteomics applied to protease research.
Current opinion in chemical biology 01/2013; · 8.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We here present The Online Protein Processing Resource (TOPPR; http://iomics.ugent.be/toppr/), an online database that contains thousands of published proteolytically processed sites in human and mouse proteins. These cleavage events were identified with COmbinded FRActional DIagonal Chromatography proteomics technologies, and the resulting database is provided with full data provenance. Indeed, TOPPR provides an interactive visual display of the actual fragmentation mass spectrum that led to each identification of a reported processed site, complete with fragment ion annotations and search engine scores. Apart from warehousing and disseminating these data in an intuitive manner, TOPPR also provides an online analysis platform, including methods to analyze protease specificity and substrate-centric analyses. Concretely, TOPPR supports three ways to retrieve data: (i) the retrieval of all substrates for one or more cellular stimuli or assays; (ii) a substrate search by UniProtKB/Swiss-Prot accession number, entry name or description; and (iii) a motif search that retrieves substrates matching a user-defined protease specificity profile. The analysis of the substrates is supported through the presence of a variety of annotations, including predicted secondary structure, known domains and experimentally obtained 3D structure where available. Across substrates, substrate orthologs and conserved sequence stretches can also be shown, with iceLogo visualization provided for the latter.
Nucleic Acids Research 10/2012; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution. Positional scanning libraries of tetrapeptide substrates revealed that mGrM is preferred to cleave after a methionine residue, whereas hGrM clearly favours a leucine residue at the P1 position. The kinetic optimal non-prime subsites of both granzymes were also distinct. Gel-based and complementary positional proteomics showed that hGrM and mGrM have a partially overlapping set of natural substrates and a diverged prime and non-prime consensus cleavage motif with leucine and methionine residues being major P1 determinants. Consistent with positional scanning libraries of tetrapeptide substrates, P1 methionine was more frequently used by mGrM as compared with hGrM. Both hGrM and mGrM cleaved α-tubulin with similar kinetics. Strikingly, neither hGrM nor mGrM hydrolysed mouse NPM (nucleophosmin), whereas human NPM was hydrolysed efficiently by GrM from both species. Replacement of the putative P1'-P2' residues in mouse NPM with the corresponding residues of human NPM restored cleavage of mouse NPM by both granzymes. This further demonstrates the importance of prime sites as structural determinants for GrM substrate specificity. GrM from both species efficiently triggered apoptosis in human but not in mouse tumour cells. These results indicate that hGrM and mGrM not only exhibit divergent specificities but also trigger species-specific functions.
[Show abstract][Hide abstract] ABSTRACT: Several mass spectrometry-driven techniques allow to map the substrate repertoires and specificities of proteases. These techniques typically yield long lists of protease substrates and processed sites with (potential) physiological relevance, but in order to understand the primary function of a protease, it is important to discern bystander substrates from critical substrates. Because the former are generally processed with lower efficiency, data on the actual substrate cleavage efficiency could assist in categorizing protease substrates. In this study, quantitative mass spectrometry following metabolic proteome labeling (SILAC), combined with the isolation of N-terminal peptides by Combined Fractional Diagonal Chromatography, was used to monitor fluxes in the concentration of protease-generated neo-N-termini. In our experimental setup, a Jurkat cell lysate was treated with the human serine protease granzyme B (hGrB) for three different incubation periods. The extensive list of human granzyme B substrates previously catalogued by N-terminal Combined Fractional Diagonal Chromatography (1) was then used to assign 101 unique hGrB-specific neo-N-termini in 86 proteins. In this way, we were able to define several sites as getting efficiently cleaved in vitro and consequently recognize potential physiologically more relevant substrates. Among them the well-known hGrB substrate Bid was confirmed as being an efficient hGrB substrate next to several other potential regulators of hGrB induced apoptosis such as Bnip2 and Akap-8. Several of our proteomics results were further confirmed by substrate immunoblotting and by using peptide substrates incubated with human granzyme B.
[Show abstract][Hide abstract] ABSTRACT: Proteolytic processing has recently received increased attention in the field of signal propagation and cellular differentiation. Because of its irreversible nature, protein cleavage has been associated with committed steps in cell function. One aspect of protease biology that boomed the past few years is the detailed characterization of protease substrates by both shotgun as well as targeted MS-driven proteomics techniques. The most promising techniques are discussed in this review and we further elaborate on the bioinformatics challenges that accompany mainly qualitative, MS-driven protease substrate degradome studies.
[Show abstract][Hide abstract] ABSTRACT: Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.