(Figures 3H, I), as expected [28], while the Caspase-2 clone activated the reporter independently of both FADD and Caspase-8 (Figure 3H, I), suggesting it directly cleaves the reporter protein containing the LETD sequence. In this regard, while the preferred cleavage sequence for Caspase-2 has been reported as DEHD, our comparisons of Caspase-2 on various cleavable reporters showed that LETD is also a substrate (Figure 1, S8), consistent with results of peptide library screens in which L at the P4 position was shown to be cleaved by Caspase-2 with ,40% the efficiency as D [20]. In another example of how this 3-component system can be employed for functional cloning, we expressed low levels pro-Caspase-10 and high levels of Fas, then screened cDNA libraries to clone adaptor molecules that link Fas to Caspase-10 (Figures S12-S15). The yeast cell line EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8/10(LETD)- TA/CYC1-Caspase10-FLAG/ADH-Fas failed to activate Caspase- 10 in the absence of FADD, as monitored by activation of the LEU2 and lacZ reporter genes. In contrast, Caspase-10 was activated when FADD was expressed in these engineered yeast cells (Figure S13). Screening 2.26106 clones from a HEK293 cell library resulted in 55 positives, of which 24 encoded FADD (Figures S14, S15). These and other cDNA library screening experiments demonstrated the robust performance of the cleavable reporter-based screening system for studying proteases, illustrated here for the DISC (Fas/FADD/ Caspase-8/10). Adaptation of the yeast-based Caspase activation assay to HTS We have implemented the yeast-based protease reporter system in HTS compatible formats by revising conditions from agar plates to liquid medium in microtiter wells. To this end, we measured b-galactosidase produced by yeast carrying the Caspase-cleavable reporter proteins, assaying the colorimetric product (OD620 nm) Yeast-Based Protease Technlgys derived from X-gal substrate in 384 well plates, as a measure of the lacZ reporter gene activity. Comparisons were made of b-galactosidase activity produced in the presence or absence of the broad-spectrum Caspase inhibitory compound, zVAD-fmk (benzoyl-Valinyl-Alaninyl-Aspartyl-fluoromethylketone). Among the variables that were initially interrogated were initial seeding cell density, time of culture, concentration of X-gal substrate, and supporting carbohydrate (raffinose vs mannose) (Figures S16 and S17). Note that we cannot employ glucose in yeast media because it represses the GAL1 promoter used in some plasmids. All variables tested alter the signal to noise ratio of the assay. For the Fas/FADD/ Caspase-8 multi-component assay, the best results were achieve with ,26105 cells per well starting density cultured for ,3 days in raffinose-containing media with 400 ug/mL concentration of X-gal substrate. For the 2-component system of Asc/Caspase-1, the same conditions were optimal, among those tested (not shown). For the NLRP1DLRR/Asc/Caspase-1 multi-component assay, the best results were achieved with ,26105 cells per well starting density cultured for ,3 days in raffinose-containing media with 400 mg/mL concentration of X-gal substrate. We further validated the 384 well assay format, using zVAD- fmk, a peptidyl inhibitor with broad-spectrum activity against animal Caspases. For some experiments, a Calpain inhibitor was employed in side-by-side experiments as a control. The broad- spectrum Caspase inhibitor zVAD-fmk inhibited b-galactosidase activity in a dose-dependent fashion, with the concentration required for achieving 50% inhibition ranging from ,1–10 mM in these assays, which were performed in 384 well format (Figure 5) (Table S1). In contrast, the Calpain inhibitor did not suppress activation of the lacZ reporter gene. These experiments verify that the 384 well version of the yeast-based Caspase-dependent reporter gene assay faithfully indicates the activity of an inhibitory Figure 5. Validation of yeast-based assay using pharmacological inhibitor of Caspases. (A) Caspase-1, single component assay. EGY48 yeast cells expressing S1 substrate (TEF-Fas-d-S1-TA) and high levels of Caspase-1 (TEF-HA-Caspase1-FLAG) were incubated for 3 days in selection media (SD/X-gal) with the indicated concentrations of z-VAD-fmk in DMSO (N) or DMSO only (#), using 384-well plates. (B) Asc/Caspase-1 two- component system. EGY48 yeast cells expressing S1 substrate (TEF-Fas-d-S1-TA) a low level of pro-Caspase-1 (DTEF1-Caspase1-FLAG) and high levels of Asc (TEF-HA-Asc) were incubated for 3 days in the selection media (SD/X-gal) in 384 well plates with the indicated concentrations of calpeptin (m) or z-VAD-fmk (N) or an equivalent volume of DMSO (#). (C) Fas/FADD/Caspase8 three-component system. EGY48 yeast cells expressing S8 substrate (TEF-S8-TA), low levels of pro-Caspase-8 CYC1-Caspase-8-HA, with a small amount of FADD (from DADH1 promoter) and a large amount of Fas (from ADH promoter), were incubated in selection media (SD/X-gal) with the indicated concentrations of calpeptin (m) or z-VAD-fmk (N), or equivalent volume of DMSO (#), in 384-well plates for 3–4 days. For (A–C), relative absorbance at 620 nm was measured (mean 6 std dev; n = 325 independent experiments). (* indicates p,0.001). doi:10.1371/journal.pone.0007655.g005 PLoS ONE | www.plosone.org 8 October 2009 | Volume 4 | Issue 10 | e7655