gene was not activated when substituting non-cleavable reporter constructs in which the aspartic acid in position P1 of the cleavable linker was converted to glycine or replacing vectors encoding wild- type Caspases with mutants in which the active site cysteine was changed to glycine or alanine (Figure S1). Taken together, these data demonstrate the performance of a one-component cleavable reporter gene system for measuring activity of all 10 human Caspases in yeast. We have employed this system for screening human cDNA libraries expressed from yeast plasmids, where the cleavable Fas- LexA-B42 fusion protein was expressed without a Caspase and cDNA libraries were screened for proteases that activate the reporter gene (Figures S2-S4). The libraries employed here were constructed by directional cloning into a high copy-number yeast plasmid containing GAL1 or ADH promoters (strong and moderate strength, respectively) for driving cDNA expression. Using the WEHD-containing reporter protein, we obtained eleven cDNAs that confirmed positive on repeated testing, including five that encoded Caspase-1 and six encoding Caspase-4 (Figures S2 and S4). Using the DEVD-containing reporter, we obtained twelve clones that confirmed positive, including three that encoded Caspase-3 and nine encoding Caspase-7 (Figures S3 and S4). These cDNA library screening results further validate the cleavable reporter system. Development of 2-component systems demonstrating function of Caspase activators in yeast Next, we attempted to develop 2-component systems for assaying Caspase activity in yeast, in which the inactive proforms of the Caspases were co-expressed with various activator proteins and in which the Caspases were expressed from weak promoters to achieve levels of the zymogens that avoid ‘‘spontaneous’’ activation. Figure 2 depicts the concept for Caspase-1, showing its preferred substrate WEHD. Among the 2-component systems interrogated were combinations of upstream initiator Caspases co- expressed with known activators, including: (1) pro-Caspase-1 plus Asc; (2) pro-Caspase-2 plus RAIDD; (3) pro-Caspase-9 plus a gain- of-function mutant of Apaf1; (4) pro-Caspase-8 plus FADD; and (5) pro-Caspase-10 plus FADD. In each of these cases, the upstream initiator Caspase, contains a N-terminal pro-domain (either CARD or DED) that binds a compatible CARD or DED in the activator protein. We also tested 2-component systems involving an upstream and downstream protease. For example, Caspase-9 is a direct upstream activator of downstream proteases, Caspases-3 and -7 [21]. We therefore over-expressed active Caspase-9 (high level expression) in combination with the inactive proforms of Caspase-3 or -7 (expressed at low levels) (Figures 2G, 2H, S5). In each case, an appropriate cleavable reporter protein was co-expressed with the 2-component systems in yeast containing LEU2 and lacZ reporter genes. For all pair-wise combinations of pro-Caspase and upstream activator, we ex- pressed the pro-Caspase at relatively low levels using a low-copy plasmid containing CEN/ARS (1 to 2 copies per cell) and using weak to moderate strength promoters (such as CYC1, and DGPD1), and the upstream activator at high levels using 2m plasmid origin-containing plasmids (20 to 50 copies per cell) with relatively strong promoters (TEF or ADH). For each pair of pro- Caspase and upstream activator, we empirically adjusted the number of lexA operators driving the LEU2 or lacZ reporter genes to achieve an acceptable signal:noise ratio. For example, for the combination of pro-Caspase-1 and Asc, we determined that 2 lexA operators for LEU2 were superior to 4 or 6 operators when using DTEF1-Caspase1-FLAG to express pro-Caspase-1 (Figure 2), and that 6 lexA operators for LEU2 were superior to 2 or 4 operators Yeast-Based Protease Technlgys when using DTEF3-Caspase1-FLAG to express pro-Caspase-1 (Figures S6A and S7). Successful activation of the cleavable reporter protein was achieved for each 2-component system (Figure 2). Expression of either the pro-Caspase alone or the upstream activator alone failed to result in activation of the LEU2 and lacZ reporter genes, as determined by ability to grow on leucine-deficient plates and b-galactosidase-positivity, confirming that both components are necessary for activating the reporter proteins. Expressing Caspases in which the active site cysteine was mutated to glycine or alanine failed to activate the cleavable reporter (Figures S1, S5, and S6). Moreover, pairing pro-Caspases with the wrong activators (e.g. Apaf1*+pro-Caspase-8; Asc+pro-Caspase-9) also failed to activate the reporters (Figure S7). Taken together, these data demonstrate the utility of yeast-based protease screening using 2 component systems. Development of a 3-component system – mammalian protease activating pathways reconstituted in yeast Having developed prototypical 2-component systems, we attempted to increase the complexity of reconstituted Caspase- activating systems in yeast, moving to a 3-component network. Two different mammalian Caspase-activating networks were explored. In the first, we attempted to recapitulate the proximal portion of the extrinsic pathway (so-called ‘‘death inducing signaling complex’’ [DISC]), expressing (1) death domain (DD)- containing TNF-family death receptor, Fas [CD95]; (2) bipartite adapter protein FADD, which contains DD and DED; and (3) the proform of a DED-containing protease, either Caspase-8 or Caspase-10, along with (4) a cleavable reporter protein containing the Caspase-8/10 substrate tetrapeptide LETD. Two strategies for reconstituting the Fas/FADD/pro-Caspase-8 network in yeast were compared. First, we tried expressing the bridging adapter protein FADD at low levels using a constitutive but weak promoter such that the amount of FADD was inadequate to achieve pro- Caspase-8 activation in the absence of Fas. For that system, Fas was expressed at high levels so that it can oligomerize with itself without requiring Fas Ligand (Figure 3A). By empirically comparing different strength promoters for driving expression of the 3 components and adjusting the number of lexA operators in the promoters of the LEU2 and lacZ reporter genes, we arrived at conditions that produced the desired result for the approach whereby low levels of constitutive FADD were complemented by expression of Fas (Figure 3C, 3D, S8). Second, we expressed low levels of either pro-Caspase-8 or pro-Caspse-10 with high levels of adapter protein FADD (Figure 3B), adding expression plasmids encoding death receptors such as Fas (CD95) or DR5 (TRAIL-R2) to this system. However, high FADD was sufficient to activate Caspase-8 and -10, with addition of death receptors only modestly further increasing reporter gene activity (Figures 3E, F). Another 3 component Caspase-activating network was similarly created for NLR-family proteins, using variations on the same approach. NLRs constitute a family of signaling proteins involved in innate immunity, several of which activate pro-inflammatory Caspases (reviewed in [22]). NLRs are activated by pathogen- derived ligands via leucine-rich repeat (LRR) domains that auto- inhibit these proteins in the absence of ligand, controlling oligomerization of these proteins that form multiprotein complexes termed ‘‘inflammasomes’’ [23]. We attempted to reconstitute in yeast the NLRP1 (NALP1) and NLRP3 (NALP3) inflammasome comprising 3-component systems including: (1) the CARD- containing protease, pro-Caspase-1; (2) the bipartite adapter Asc, which contains CARD and PYD domains; and (3) the NLR-family protein NLRP1/NALP1 (which contains PYD and CARD PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7655