Portal de Periódicos da CAPES

Protease signalling: the cutting edge

2012; Springer Nature; Volume: 31; Issue: 7 Linguagem: Inglês

10.1038/emboj.2012.42

1460-2075

Boris Turk, Vito Türk, Vito Türk,

Ubiquitin and proteasome pathways

EMBO Member's Review24 February 2012free access Protease signalling: the cutting edge Boris Turk Corresponding Author Boris Turk Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia Center of Excellence CIPKEBIP, Ljubljana, Slovenia Center of Excellence NIN, Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia Search for more papers by this author Dušan Turk Dušan Turk Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia Center of Excellence CIPKEBIP, Ljubljana, Slovenia Search for more papers by this author Vito Turk Vito Turk Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia Center of Excellence CIPKEBIP, Ljubljana, Slovenia Search for more papers by this author Boris Turk Corresponding Author Boris Turk Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia Center of Excellence CIPKEBIP, Ljubljana, Slovenia Center of Excellence NIN, Ljubljana, Slovenia Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia Search for more papers by this author Dušan Turk Dušan Turk Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia Center of Excellence CIPKEBIP, Ljubljana, Slovenia Search for more papers by this author Vito Turk Vito Turk Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia Center of Excellence CIPKEBIP, Ljubljana, Slovenia Search for more papers by this author Author Information Boris Turk 1,2,3,4, Dušan Turk1,2 and Vito Turk1,2 1Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Ljubljana, Slovenia 2Center of Excellence CIPKEBIP, Ljubljana, Slovenia 3Center of Excellence NIN, Ljubljana, Slovenia 4Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia *Corresponding author. Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Jamova 39, Ljubljana 1000, Slovenia. Tel.: +386 1 477 3772; Fax: +386 1 477 3984; E-mail: [email protected] The EMBO Journal (2012)31:1630-1643https://doi.org/10.1038/emboj.2012.42 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research. Introduction to proteases Proteases control a great variety of physiological processes that are critical for life, including the immune response, cell cycle, cell death, wound healing, food digestion, and protein and organelle recycling. Their action is strictly controlled and imbalances in their activities have been found to be critical in a number of pathologies, such as cardiovascular diseases, inflammation, cancer, and neurodegenerative diseases, thereby suggesting proteases as suitable and valuable drug targets (Turk, 2006; Drag and Salvesen, 2010). In humans, there are almost 600 proteases and about 80 more can be found in the mouse, representing ∼2% of the genome. Proteases differ remarkably in their properties. Some are as small as 20–30 kDa and composed of essentially the catalytic domain(s) alone, such as the cysteine cathepsins, whereas some can be extremely large, multidomain protein complexes, such as the proteasome and tripeptidyl peptidase I. They vary significantly in their specificities from being highly selective, such as the proteases of the blood coagulation cascade which activate the subsequent protein in the cascade, or the caspases and granzyme B which cleave their substrates exclusively after Asp residues, to the highly non-specific, such as those involved in protein turnover and which include the cathepsins and the proteasome, as well as most exopeptidases. The different selectivitiy of the proteases has been widely exploited in different analytical applications from amino-acid sequencing and mass spectrometry analysis, where proteases like trypsin, which cleaves all unfolded protein substrates after every Arg and Lys residues only, to biotechnological applications such as removal of the tags from the recombinant proteins where proteases like thrombin or enterokinase have been frequently used. Based on their different catalytic mechanisms, there are five major classes of proteases known in mammals including serine, cysteine, metallo, aspartic, and threonine proteases. Whereas cysteine, serine, and metalloproteases are widespread with ∼150–200 members identified in humans, threonine and aspartic proteases are much scarcer with <30 members of each class being identified (Lopez-Otin and Overall, 2002; Overall and Blobel, 2007; Lopez-Otin and Bond, 2008). However, not all proteases catalyse the cleavage of α-peptide bonds between the naturally occurring amino acids. For example, deubiquitinating enzymes (DUBs), a large group comprising almost 100 members in humans mostly belonging to cysteine proteases, hydrolyse the isopeptide bond(s) between ubiquitin or ubiquitin-like proteins and pro-proteins or target proteins (Reyes-Turcu et al, 2009). In addition to different catalytic mechanisms, proteases can cleave their substrates either at the termini, such as carboxypeptidases and aminopeptidases, or within the polypeptide chain, such as endopeptidases. Although exopeptidases and endopeptidases usually differ significantly, lysosomal cysteine cathepsins are an example where nature has been very economical. With just a handful of small changes, such as insertions of small structural elements including the loops and the remaining parts of propeptide regions onto an endopeptidase scaffold, endopeptidases have been converted into exopeptidases. Moreover, cathepsin B can act as both an endopeptidase or an exopeptidase, based on the opening or closing of the ‘occluding loop’, which depends on the pH of the local millieu (Turk et al, 2000, 2001c). Another important factor that affects protease activity and substrate selection is their localization. Approximately half of all proteases are extracellular, whereas the remainder proteases are intracellular, with a minor proportion being intramembrane, localized in plasma and organelle membranes. Interestingly, cysteine proteases that usually require reducing conditions for their activity are predominantly found intracellularly, whereas majority of the metalloproteases are extracellular. Collectively, all these different properties and abilities of proteases explain why they provide an excellent way of regulating cellular processes throughout the body (Lopez-Otin and Overall, 2002; Overall and Blobel, 2007; Lopez-Otin and Bond, 2008). Protease signalling Although proteases were not traditionally regarded as signalling molecules, this view is dramatically changing. However, there are important differences between protease signalling and the other types of cellular signalling, for example, receptor signalling or kinase signalling. Protease signalling is irreversible and the signal is transmitted through the cleavage(s) of protein substrates resulting in their activation, inactivation, or modulation of function (Turk, 2006). Proteases can thus not only activate proteins such as cytokines, or inactivate them such as numerous repair proteins during apoptosis, but also expose cryptic sites, such as occurs with β-secretase during amyloid precursor protein processing, shed various transmembrane proteins such as occurs with metalloproteases and cysteine proteases, or convert receptor agonists into antagonists and vice versa such as chemokine conversions carried out by metalloproteases, dipeptidyl peptidase IV and some cathepsins. In addition to the catalytic domains, a great number of proteases contain numerous additional domains or modules that substantially increase the complexity of their functions (Lopez-Otin and Overall, 2002; Lopez-Otin and Bond, 2008). A number of proteases act in cascades that allow much better amplification of the signal and more stringent regulation. Typical examples are the blood coagulation cascade, apoptosis, as well as activation of a number of metalloproteases from ADAM and MMP families by furin-like proprotein convertases (Turk, 2006). As it is often very difficult to draw the borders between different protease signalling events, it was suggested to use the terms protease networks or protease webs (Doucet et al, 2008; Krüger, 2009). However, regardless of the mechanism or the pathway, the key signalling event in protease signalling is the change of function of a physiological substrate (Turk, 2006). A proper understanding of the protease signalling pathways therefore requires identification of the physiological substrates of proteases or protease degradomes (Lopez-Otin and Overall, 2002). To qualify as a physiological substrate of a protease, it is neither sufficient for a protein to contain the recognition sequence for a protease nor for it to be cleaved in an in-vitro assay, as practiced in the early days of biochemical studies of proteases. The substrate has to colocalize with the active protease, that is, be present in the same cellular compartment or at the same extracellular location, and then subsequently to be processed. Moreover, a number of studies have demonstrated that a large number of cellular proteins reside in multiprotein complexes, which could further limit their accessibility to proteases (Gavin et al, 2002; Janin and Seraphin, 2003). However, it is unclear at the moment, how many proteins undergoing proteolytic processing are indeed present in such complex forms. There are quite a few examples known where a protein substrate is in a complex during the cleavage reaction, such as ICAD (inhibitor of caspase-activated DNase) that is in a complex with CAD (caspase-activated DNase). Following ICAD cleavage by caspases during apoptosis, CAD is released from the complex, thereby initiating DNA fragmentation in the nucleus (Enari et al, 1998). However, no detailed studies have been performed to specifically address this question. This also raises a question as to the number of proteases active when in complexes, and how many can act alone. Clearly, proteases like the proteasome, γ-secretase as well as several serine proteases involved in blood coagulation such as the prothrombinase complex (a complex between Factor Xa and Factor Va required for thrombin activation) require complex formation to be able to process their physiological substrates. In a similar manner to the substrates, no real systematic studies have been performed to address these questions. Every single protein synthesized is degraded by the proteasome and/or lysosomal proteases during its recycling or degradation and is therefore by default a physiological substrate of these proteases; a general degradation mechanism that is not generally considered as part of protease signalling. Consequently, to prevent undesired proteolysis, proteases involved in protein recycling and degradation are physically separated from the majority of other proteins by being contained within lysosomes or in a self-compartment (proteasome). Identification of physiological protease substrates The identification of physiological protease substrates is currently one of the major challenges in protease research. Initial studies essentially used a bottom-up approach, that is, identification of the protease responsible for the processing of an orphan substrate, thereby simultaneously validating the results. The first such studies, performed over half a century ago, led to the discoveries of the renin-angiotensinogen system (Page and Helmer, 1940) and angiotensin-converting enzyme (ACE; Skeggs et al, 1956). This approach was also successfully applied to identification of the proteases in the blood coagulation cascade (Davie and Ratnoff, 1964), furin as the processing enzyme of many prohormones in mammals, caspase-1 as the interleukin-1β processing enzyme (Thornberry et al, 1992), dipeptidyl peptidase IV as the processing enzyme of insulin-related hormones (Demuth et al, 2005) and intramembrane-cleaving proteases (Weihofen and Martoglio, 2003; Wolfe, 2009). This approach is still in use, and has recently led to the identification of cathepsin L/V as the histone H3-processing enzyme (Duncan et al, 2008). The usefulness of this approach is further demonstrated by the fact that a number of proteases identified in this way have also been validated as drug targets. Moreover, ACE inhibitors are still the most commonly used protease-targeting drugs (Turk, 2006; Drag and Salvesen, 2010). The applicability of this approach is, however, limited as it is very labour intensive. The majority of proteases process more than one substrate, resulting in a functional redundancy that may mask the validation process. Therefore, additional approaches have been developed over the years, such as combinatorial fluorescent substrate libraries, positional scanning libraries based on covalent inhibitors, and phage display peptidic libraries (Matthews and Wells, 1993; Thornberry et al, 1997; Turk et al, 2001a). These approaches generated vast amount of data from which information could only be extracted with the simultaneous development of bioinformatic tools. Using these approaches, substantial success has been achieved in determining substrate specificities of several proteases, such as caspases (Thornberry et al, 1997). This latter seminal work identified the DXXD↓X amino-acid sequence as the consensus cleavage sequence for caspases-3 and -7 with an Asp residue in the P1 position being absolutely required. This information was then extensively used in a number of subsequent studies on identification of natural caspase-3 substrates. This approach proved highly successful since mutation of a selected Asp residue in the target protein was usually found sufficient not only to validate the protein as a caspase target, but also the selected cleavage site. Such studies were therefore of great help in the identification of substrate specificities of proteases and important for the development of in-vitro assays to follow protease activities in vitro or in complex samples and as a result they have been heavily employed in academic and industrial laboratories (Figure 1; Nicholson, 2000). Figure 1.Binding of a substrate to a protease. (A) Schematic representation of substrate binding. The basic differences between binding of a small peptidic or peptidomimetic substrate and a larger peptide or protein substrate are shown. Small substrate binds tightly to the non-prime binding sites whereas the usually bulky fluorophore/chromophore (leaving group) binds to the S1′ site while the other prime sites remain empty (1). Larger peptidic substrates bind to both prime site and non-prime site binding sites, while the interaction with some individual binding sites can be looser (2). Exosites on protease surface serve as additional binding elements for large substrates to strengthen the interaction with the protease and allow recognition (3). They can be also used for discrimination between the different substrates. (B) Small substrate-mimicking inhibitors that helped tremendously in elucidating the substrate binding mechanism(s) often bind the same way as the small substrates (some more differences with metalloproteases), except that in covalent inhibitors the leaving group is replaced by a warhead (see A for a schematic representation). Crystal structure of caspase-1 in complex with the inhibitor YVAD-CHO (Wilson et al, 1994; 1ICE) revealed the insight into the mechanism of substrate binding to the active site of caspases as an example of such small molecule (substrate or inhibitor) binding. The surface of caspase 1 is shown in light grey. In the active site cleft, the non-primed and primed parts are shown as cyan and blue surface. The structure of the inhibitor (shown as a ball-and-stick model) has revealed the S1, S2, and S3 substrate binding sites. The non-hydrogen atoms C, N, and O are shown in blue, dark blue, and red, respectively. Figure was prepared with MAIN (Turk, 1992) and rendered with POV-Ray. Download figure Download PowerPoint However, these studies were found to have a limited potential in identification of physiological protein substrates largely due to differences in the binding of small substrate molecules when compared with large protein substrates. Small peptidic substrates with a fluorophore attached only explore a small part of the non-prime substrate residues (i.e., substrate residues towards the N-terminus of the substrate from the scissile bond), whereas the prime residues (i.e., residues towards the C-terminus of the substrate from the scissile bond) remained largely unexplored (Figure 1). Another reason is that proteases may interact with their protein substrates through a considerably larger surface area as compared with the small peptidyl substrates (Figure 1). In a case study, this was demonstrated for the caspases. A recent proteomic study thus enabled discrimination between the substrate specificities of caspases-3 and -7 based on an undecapetide motif (P6-P5′; Demon et al, 2009). A further difficulty is imposed by those proteases that use exosites (i.e., those parts of the protease that are distal to the catalytic site that participate in substrate binding; Figure 1) for an efficient recognition of various physiological substrates, such as thrombin (Bar-Shavit et al, 1983) or various metalloproteases including ADAMTS-4 (ADAM with thrombospondin motif-4) and most MMPs (Overall and Blobel, 2007). The problem was at least partially addressed for MMPs with the approach called exosite scanning, which used MMP exosite domains as baits in the yeast-two hybrid screens to identify potential substrates (Overall et al, 2002). More recently, advanced proteomics studies were found superior in this aspect. Here, research went in two directions: one devoted to identification of physiological substrates of proteases and the other devoted to determination of extended substrate specificities of proteases. Initial studies using DIGE and other forms of two-dimensional electrophoresis for sample separation resulted in identification of a few physiological substrates, such as demonstrated for caspases (Brockstedt et al, 1998; Gerner et al, 2000), granzymes A and B (Bredemeyer et al, 2004), MMP-14 (Hwang et al, 2004), and ADAMTS1 (Canals et al, 2006). On the other hand, non-gel-based methods such as combined fractional diagonal chromatography (COFRADIC; Gevaert et al, 2003), proteomic identification of protease cleavage sites (PICS; Schilling and Overall, 2008), terminal amine isotopic labelling of substrates (TAILS; Kleifeld et al, 2010), ICAT and iTRAQ variations (Tam et al, 2004; Dean and Overall, 2007; Enoksson et al, 2007), or genetically engineered enzyme use (Mahrus et al, 2008) were found to be superior, leading to identification of a great number of protease substrates, including those of caspases, granzyme B, and various MMPs (Van Damme et al, 2005, 2009; Mahrus et al, 2008; Morrison et al, 2009). These methods, in addition to identifying cleavage sites within protein substrates, enable determination of protease specificities, especially when used on the larger peptidic fragments that are generated from proteins by trypsin, endo GluC or some other protease cleavage, or on total cellular lysates. This offers a significant advantage compared with the combinatorial libraries as it can be used to define not only non-prime site specificity, but also extended prime site specificity. However, methods like PICS have to be performed with care. Generation of the larger initial peptides by trypsin or any other selective protease might be problematic as it can result in an apparently completely different specificity of proteases under investigation that readily accept the same P1 residues as the protease used in peptide generation. A typical example is cysteine cathepsins, where basic residues are well accepted in the P1 position, when combined with the use of trypsin for peptide generation. Recently, the approach has been expanded allowing the C-termini of the cleavage sites within the substrates to be identified, thereby enhancing the possibility of identifying the cleavage site (Schilling et al, 2010; Van Damme et al, 2010). Another approach that simultaneously allows identification of cleavage sites and identification of stable fragments is Protein Topography and Migration Analysis Platform (PROTOMAP). Due to the identification of stable fragments, this approach at the same time offers an advantage of initial validation of cleavage sites (Dix et al, 2008). In addition, there are various bioinformatic methods that have been found to be indispensable in extracting information from the database libraries and have helped in identifying additional protease substrates, such as demonstrated for granzymes (Barkan et al, 2010). Unfortunately, none of the methods mentioned above is ideal and each suffers from certain limitations. A recent analysis of several cleavage site prediction programs, based on such experimental methods, has revealed that our understanding of protease processing is still relatively confined. In particular, a clear insight into the processing roles of non-specific proteases is still lacking (Verspurten et al, 2009). In addition to the non-specific proteases such as the cathepsins, investigating proteases with a requirement for an exosite (e.g., thrombin) is still a serious technological challenge. Even more challenging is the validation of these findings. In this aspect, mouse knockout/knock-in and transgenic models and the use of RNAi were found to be absolutely indispensable (Turk, 2006; Overall and Blobel, 2007). Alternatively, protease inhibitors can also be used in validation. However, they have to be used with extreme care as their specificity is often limited (Rozman-Pungerčar et al, 2003; Turk, 2006; Drag and Salvesen, 2010). All these combined efforts have generated a vast amount of data, which has greatly advanced our knowledge. Challenges in protease signalling The primary cleavage of a protein substrate is largely responsible for the change of its function (Turk, 2006). However, defining this primary cleavage event could not be satisfactorily resolved by the above-mentioned approaches due to the predominant use of long-term incubation times. More specifically, only the surface-exposed regions of substrates are accessible to proteases, whereas internal regions can only be cleaved after substantial structure disassembly takes place as a consequence of protein degradation and/or unfolding. And these secondary cleavages resulting from the prolonged incubation were usually observed, masking the true picture. Until recently, proteases were believed to initially process their substrates either at the termini or within the exposed loops, whereas cleavages within the secondary structure regions were considered unlikely. However, a recent study has demonstrated that cleavage can occur within the α helices far more frequently than expected, occurring almost as frequently as cleavages within the unstructured regions (Timmer et al, 2009). Using a very elegant approach, this group has overcome the major problem in such an experiment that of the sequential processing of a substrate. In order to reduce the number of cleavages they have used two highly specific proteases, GluC and caspase-3, which cleave specifically after Glu and Asp residues, respectively. In addition, they have designed a procedure by which only the single cleavage events were considered. The proteomic findings were verified in an in-vitro system using individual recombinant proteins. Since E. coli does not express proteases with Asp or Glu specificity, its proteome has been considered as unbiased. Interestingly, a kinetic comparison between the activity of mammalian caspase-3 on E. coli substrates and its natural mammalian substrates has revealed that the former were largely inferior to the natural substrates with up to a few orders of magnitude lower kcat/Km values. This suggests that proteases and their substrates have co-evolved and that this is crucial for successful signalling. Nevertheless, despite the high frequency of the cleavage sites found within helices, extended loops and unstructured interdomain linker regions still seem to be the preferable processing sites for the activation of proteins (Timmer et al, 2009). This is, however, true only for folded substrates. Two excellent examples of this are the activation of Bid in apoptosis and the inhibition mechanism of serpins. Bid has been found to be processed at different sites by a number of proteases (caspase-8, calpains, cathepsins B, L, S, H, D, granzyme B; Turk and Turk, 2009). The sites are all located within the same large loop (Chou et al, 1999). Similarly, serpins are initially cleaved by their target proteases within the reactive site loop (RSL) but remain trapped by covalent bond to the catalytic serine residue of the protease through the P1 residue. In the next step, the cleaved serpin undergoes a major conformational change in which the cleaved RSL inserts into the centre of the β-sheet, resulting in a huge protease movement to the opposite end of the serpin (∼70 Å) (Schulze et al, 1990; Huntington et al, 2000). Moreover, in the case of crossreactive serpins, which inhibit serine and/or cysteine proteases, the cleavage site varies within the loop depending on the protease (Schick et al, 1998; Al-Khunaizi et al, 2002). Even though the problem of the initial cleavage can be at least partially resolved by measuring cleavage kinetics as recently demonstrated for granzyme B (Plasman et al, 2011), there is still another, even larger, problem remaining: that of differentiation between the signalling and the bystander events. There is no guarantee that a rapidly cleaved protein is also a physiologically relevant substrate required for a signalling pathway to proceed, again indicating the importance of data validation. For example, several hundred caspase substrates have been identified. However, only for a handful of them it can be said with confidence that they are directly involved in apoptosis progression. Among these are the executioner caspase zymogens and Bid, which are activated, and PARP and ICAD (DFF45), which are inactivated (Timmer and Salvesen, 2007). Another problem is posed by those substrates that are activated through the initial cleavage, but become degraded through subsequent processing events. Although these initial fragments play an active role in signalling, they are very difficult to identify due to their transient appearance, as they disappear from the sample. As already mentioned above, proteases that bind their substrates through exosites represent an investigative challenge, as do multidomain proteases or proteases that change their properties upon binding to other proteins or other molecules. One example of the latter is thrombin that normally cleaves fibrinogen thereby finalizing blood clot formation. However, upon thrombomodulin binding, thrombin–thrombomodulin complex exhibits different specificity and instead activates the serine protease protein C, thereby acting as a negative feedback regulator of blood coagulation (Davie et al, 1991). The other example is glycosaminoglycans, which are known to substantially modify protease activities. When complexed to cathepsin K, chondroitin sulphate was shown to convert it into a very potent collagenase (Li et al, 2002), whereas heparin binding was found to substantially modify the properties of a number of serine proteases. Although these issues can be at least partially addressed using current proteomic technologies, no systematic studies have been performed so far. Orphan enzymes represent another, though technologically less difficult challenge. Using proteomic methods and subsequently validating them in cellular or in-vivo models may help in elucidating their biological functions through the discovery of their true physiological substrates (Overall and Blobel, 2007). In addition to qualitative assessment and identification of cleavage sites in substrates, a further challenge will be to quantitatively determine the extent of processing at a given site in a protein substrate. Although it should be possible to determine the extent of cleavage, its physiological relevance would remain questionable since for a physiological process to proceed, a complete processing at a single site is normally not required. A typical example can be seen with the executioner caspases such as caspase-3 that are never completely activated during apoptosis in a cellular system. Determining the actual threshold of a given physiological process therefore remains a major challenge for the future. Protein degradation as a si