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Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant–microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.
- Top of page
- Experimental Procedures
- Supporting Information
Vacuolar processing enzymes (VPEs) are cysteine proteases that are responsible for processing and maturation of vacuolar proteins and are involved in both plant development and immunity. VPEs are also called legumains or asparaginyl endopeptidases, and are classified as members of family C13 in the MEROPS protease database (http://merops.sanger.ac.uk/) (Rawlings et al., 2012). The C13 family belongs to the CD clan, which also contains caspases (family C14A) and metacaspases (family C14B). Caspases are the main players in regulation of programmed cell death (PCD) in animals, whereas metacaspases are involved in PCD in plants and fungi (Tsiatsiani et al., 2011). Clan CD proteases contain a His–Cys catalytic dyad, and have strict substrate requirements for the amino acid preceding the cleavable bond (P1 position). For instance, VPEs cleave substrates preferably after Asn residues (hence the name asparaginyl endopeptidases), whereas caspases cleave substrates specifically after Asp residues (Crawford and Wells, 2011; Tsiatsiani et al., 2011).
VPEs are thought to be evolutionary related to caspases because they share structural homology despite their low sequence similarities (Chen et al., 1998; Hara-Nishimura et al., 2005; Hatsugai et al., 2006). For instance, motifs surrounding the catalytic amino acids are conserved between VPEs and caspases. Furthermore, autocatalytic conversion of the inactive precursor protein into functional VPE resembles the processing and activation of caspase–1 (Hara-Nishimura et al., 2005; Hatsugai et al., 2006). Maturation of Arabidopsis γVPE occurs in three steps (Kuroyanagi et al., 2002) (Figure 1a). The pre-protein precursor (preproVPE, ppVPE) carries a 22-amino-acid N-terminal signal peptide that is co-translationally removed in the ER to produce proVPE (pVPE). ppVPE and pVPE are both inactive. Transfer to the acidic vacuole causes self-catalytic conversion into the 43 kDa intermediate isoform (iVPE) by removal of the C-terminal inhibitory pro-peptide (CTPP). The subsequent removal of the N-terminal pro-peptide (NTPP) produces the mature 40 kDa isoform (mVPE). Both iVPE and mVPE are active proteases (Kuroyanagi et al., 2002).
Figure 1. Mechanism of VPE maturation and probes. (a) Maturation of γVPE. In the first step, the signal peptide (SP) is co-translationally removed from preproVPE (ppVPE), resulting in proVPE (pVPE). Next, the autoinhibitory C-terminal propeptide (CTPP) is removed, resulting in a 43 kDa active intermediate VPE (iVPE). Finally, the N-terminal propeptide (NTPP) is removed, resulting in an active 40 kDa mature VPE (mVPE). The catalytic residues (His and Cys) are indicated. Adapted from Kuroyanagi et al., 2002. (b) Structure of the aza-epoxide probes used in this study. AMS101 and bAMS101 differ only in the reporter tags: AMS101 carries a BOPIDY fluorescent reporter and bAMS101 carries a biotin affinity tag (full structure in Figure S1). Both probes have an asparagine at the P1 position and a proline at the P2 position. The nucleophilic trap in the epoxide is indicated with a dash circle. (c) Proposed reaction mechanism of aza-epoxide. The sulfur of the catalytic Cys of VPE acts as a nucleophile on the epoxide, resulting in a stable covalent and irreversible sulfoether bond.
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The well-known cysteine protease inhibitors E-64 and leupeptin do not inhibit VPE activity because they do not carry an Asn residue (Hatsugai et al., 2004; Rojo et al., 2004). VPEs are specifically inhibited by Ac-ESEN-CHO (Hatsugai et al., 2004), as well as by the general caspase inhibitor VAD-FMK and the caspase-1 inhibitors YVAD-CMK, YVKD-CMK and Ac-YVAD-CHO (Hatsugai et al., 2004; Rojo et al., 2004). Likewise, VPE cleaves the caspase-1 substrate YVAD but not the caspase-3 substrate DEVD, demonstrating that VPEs exhibit caspase-1-like activity (Hatsugai et al., 2004; Rojo et al., 2004). The fact that VPEs also interact with inhibitors and substrates carrying Asp residues at the P1 position has been explained by the acidic vacuolar pH, which causes partial protonation of the Asp side chain and eliminates its negative charge (Kato et al., 2005). Indeed, VPE can also process natural substrates after Asp residues, but with low rates (Hiraiwa et al., 1999).
Four VPE-encoding genes have been identified in Arabidopsis: αVPE, βVPE, γVPE and δVPE (Kinoshita et al., 1999; Gruis et al., 2002). βVPE plays a key role in processing of storage proteins during seed maturation (Gruis et al., 2002; Shimada et al., 2003). δVPE is highly expressed at early stages in developing seeds (Gruis et al., 2002) and is required for PCD of cell layers during seed coat formation (Nakaune et al., 2005). VPEs are also required for cell death triggered by the fungal toxin fumonisin B1 (Kuroyanagi et al., 2005), and for a successful symbiosis of the fungus Piriformospora indica with Arabidopsis that involves cell death (Qiang et al., 2012). Likewise, γVPE knockout lines showed increased susceptibility to turnip mosaic virus and the nectrotrophic pathogen Botrytis cinerea (Rojo et al., 2004). Together, these data show that Arabidopsis VPEs are involved in the regulation of PCD during plant immunity, symbiosis and development.
VPEs also play important roles in other plant species. Experiments with tobacco mosaic virus in Nicotiana tabacum demonstrated that virus-induced hypersensitive cell death is blocked by VPE silencing and VPE inhibitors (Hatsugai et al., 2004). In monocots, VPEs are required for processing of glutelin, the dominant seed storage protein in rice (Wang et al., 2009; Kumamaru et al., 2010). A similar role is expected for nucellain, a VPE ortholog in barley seeds (Linnestad et al., 1998). VPEs also process seed storage proteins (albumins, globulins and ricin) in storage vacuoles in seeds of pumpkin and castor bean (Hara-Nishimura et al., 1991, 1993; Shimada et al., 2003). VPE is also thought to mediate the maturation of concanavalin A, the lectin of jackbean seeds, which involves deglycosylation, processing after Asn residue and formation of a de novo peptide bond (Abe et al., 1993; Min and Jones, 1994; Sheldon et al., 1996). In addition, VPEs process Asn–Gly bonds in the PV100 protein of pumpkin seeds, producing multiple functional seed proteins (Yamada et al., 1999).
The importance of VPEs, combined with their post-translational control of activity through cystatins, processing and pH, for example, calls for new and simple methods to directly monitor VPE activities in tissues or extracts of various plant species. The activity of enzymes may be monitored by using activity-based probes. Activity-based probes are reporter-tagged inhibitors that react with active site residues of enzymes in a mechanism-dependent manner (Cravatt et al., 2008; Edgington et al., 2011). Labeling reflects enzyme activities because the availability and reactivity of active sites are hallmarks of protein activities (Kobe and Kemp, 1999). Activity-based protein profiling has been extensively used in the animal field to study diverse protease families (Serim et al., 2012). More recently, activity-based probes have been introduced in plant research for the proteasome (Gu et al., 2010; Kolodziejek et al., 2011), metalloproteases (Lenger et al., 2012), serine hydrolases (Kaschani et al., 2009a, 2012b; Nickel et al., 2012) and glyceraldehyde dehydrogenases (Kaschani et al., 2012b). Activities of papain-like cysteine proteases (family C1A of clan CA) may be displayed using DCG-04, a biotinylated derivative of the broad-range cysteine protease inhibitor E-64 (Greenbaum et al., 2000). Since its introduction into plant science (Van der Hoorn et al., 2004), DCG-04 has been widely used to monitor papain-like cysteine protease activities in Arabidopsis (Van Esse et al., 2008; Wang et al., 2008; Kaschani et al., 2009b; Lampl et al., 2010; Gu et al., 2012; Richau et al., 2012; Shindo et al., 2012), tomato (Rooney et al., 2005; Tian et al., 2007; Shabab et al., 2008; Van Esse et al., 2008; Song et al., 2009; Kaschani et al., 2010; Hörger et al., 2012; Lozano-Torres et al., 2012), tobacco (Gilroy et al., 2007), maize (Van der Linde et al., 2012) and wheat (Martinez et al., 2007). These studies illustrate the wide applicability of activity-based probes in plant science. Although DCG-04 targets cysteine proteases, this probe does not label VPEs because DCG-04 does not carry an Asn or Asp residue at the P1 position.
Here we report the use of an activity-based probe to monitor VPE activities in plants. This probe displays highly specific labeling of mVPE and iVPE that is pH-dependent and is competed for by the caspase-1 inhibitor YVAD-CMK. Additionally, this probe is suitable for subcellular in vivo imaging of VPEs. Using this probe, we observed a previously unnoticed up-regulation of γVPE activity during compatible but not incompatible interactions of Arabidopsis with the biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa). Further studies demonstrated a role for VPEs during compatible Hpa interactions.