Figure 1.
A genetic system for monitoring Caspase-1 activity in yeast cells.
(A) Engineered yeast were created that express a type-1 transmembrane protein (Fas-d-S1-TA) in which the Fas devoid of the death domain (Fas-d) is followed by a Caspase-1 target site (S1-WEHD), and a transcriptional activator (TA-consisting of LexA DNA binding domain and B42 activation domain). lexA operators are located upstream of lacZ (2 operators) and LEU2 (6 operators) reporter genes, respectively. The cells expressing 6op-LEU2/2op-lacZ/TEF-Fas-d-S1-TA stimulate Caspase-1 activity reporter, because expression of active Caspase-1 (over-expressing a full-length pro-Caspase-1 construct) results in Fas-d-S1-TA cleavage at the Caspase-1 target site (S1), releasing the transcriptional activator (TA), which enters the nucleus and activates lacZ and LEU2 reporter gene transcription. (B) A version of Fas-d-S1-TA in which the P1 aspartate is changed to glycine (Fas-d-G1-TA) cannot be cleaved by active Caspase-1. The cells expressing 6op-LEU2/2op-lacZ/TEF-Fas-d-G1-TA in which the glycine substitution is found serve as false-positive reporters for molecules that activate lacZ and LEU2 gene expressions independent of cleavage at the Caspase-1 target site. (C) Substrate specifities of the caspases expressed in yeast. Plasmids encoding various cleavable transcription factor substrates, harboring different tetrapeptide cleavage sequences optimized for specific Caspases (Fas-d-S1-TA, Fas-d-S2-TA, Fas-d-S3-TA, Fas-d-S6-TA, Fas-d-S8-TA, and Fas-d-S9-TA) were created by substituting the Caspase-1 cleavage site (S1-WEHD) with the Caspase-2 cleavage site (S2-DEHD), the Caspase-3 cleavage site (S3-DEVD), the Caspase-6 cleavage site (S6-TEVD), the Caspase-8/Caspase-10 cleavage site (S8-LETD), and the Caspase-9 cleavage site (S9-LEHD), respectively, and were expressed with the lacZ and LEU2 reporter genes. A construct encoding a Fas-LexA/B42 transcription factor containing a pseudo-site (G1) with the non-cleavable sequence WEHG was also generated. The resultant yeast strains EGY48, expressing 6op-LEU2/2op-lacZ/TEF-Fas-d-G1-TA, 6op-LEU2/2op-lacZ/TEF-Fas-d-S1-TA, 6op-LEU2/2op-lacZ/TEF-Fas-d-S2-TA, 6op-LEU2/2op-lacZ/TEF-Fas-d-S3-TA, 6op-LEU2/2op-lacZ/TEF-Fas-d-S6-TA, 6op-LEU2/2op-lacZ/TEF-Fas-d-S8-TA, or 6op-LEU2/2op-lacZ/TEF-Fas-d-S9-TA, were transformed with Caspase expression plasmids. Substrate specificities were determined for the ten Caspases (C1–C10). If cells express the active Caspases with the suitable cleavage sites, the cells can grow in the selection medium (without leucine) and they hydrolyze X-gal to become blue. Expression of the Caspases (p424-ADH-Caspase1-FLAG, p424-ADH-HA-Caspase2, p424-ADH-Caspase3, p424-TEF-Caspase4, p424-ADH-Caspase5, p424-TEF-HA-Caspase6, p424-ADH-Caspase7, p424-CYC1-Caspase8-HA/TEF-HA-FADD, p424-ADH-HA-Caspase9-FLAG, and p424-CYC1-Caspase10-FLAG/TEF-HA-FADD) had no effects on the cell growth when plated on regulate (leucine-containing) medium.
Figure 2.
Schematic representation of a 2-component system for caspase1 activators in yeast cells.
(A) The zymogen (inactive) pro-caspase-1 protein is expressed in yeast, with the cleavable reporter containing the S1 site (WEHD), by regulating the expression level (p413-TEF-Fas-d-S1-TA/ΔTEF1-Caspase1-FLAG) in the yeast cell (EGY191 expressing 2op-LEU2/2op-lacZ). The reporter genes are silent in the absence of Asc. Co-expression of Asc at high levels from p424-TEF-HA-Asc activates Caspase-1, resulting in S1 site cleavage, releasing the transcription factor from the membrane to enter the nucleus and activate reporter genes. Co-expression of Asc at high levels from p424-TEF-HA-Asc activates Caspase1, resulting in S1 site cleavage, and the transcription factor is released from the membrane to enter the nucleus and activate reporter genes. (B) Yeast transformants were plated on leucine-deficient medium containing X-gal. For controls, the “empty” version of the plasmid was always introduced so that cells were subjected to identical selection conditions. The recipient yeast strain EGY191 containing 2op-LEU2/2op-lacZ with substrate plasmid TEF-Fas-d-S1-TA, with or without a gene encoding pro-Caspase-1 (ΔTEF1-Caspase1-FLAG) were transformed with p424-TEF-HA-Asc or empty vector (-). Only the combination of Caspase-1 plus Asc resulted in reporter gene activation, indicating that Asc itself did not cut the S1 site. (C) Experiments were performed as above, except the recipient yeast strain EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid ΔTEF2-Fas-d-S2-TA, with or without a gene encoding pro-Caspase-2 (ΔGPD1-HA-Caspase2-FLAG), was transformed with p424-TEF-HA-RAIDD or empty vector (-). (D) The recipient yeast strain EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid GPD-Fas-d-S8-TA, with or without a gene encoding pro-Caspase-8 (CYC1-Caspase8-HA), was transformed with p424-TEF-HA-FADD or empty vector (-). (E) The recipient yeast EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid TEF-Fas-d-S9-TA, with or without a gene encoding pro-Caspase-9 (TEF-HA-Caspase9), was transformed with p424-TEF-HA-Apaf* expressing an active mutant of Apaf-1 or empty vector (-). (F) The recipient yeast strain EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid GPD-Fas-d-S8-TA, with or without a gene encoding pro-Caspase-10 (ADH-Caspase10-FLAG), was transformed with p424-TEF-HA-FADD or empty vector (-). (G, H) The recipient yeast EGY191 containing 2op-LEU2/2op-lacZ with substrate plasmid ΔTEF3-Fas-d-S3-7-TA, with or without genes encoding pro-Caspase-3 (ΔCYC2-Caspase3) or the recipient yeast EGY48 containing 6op-LEU2/2op-lacZ with substrate plasmid TEF-Fas-d-S3-7-TA, with or without genes encoding pro-Caspase-7 (CYC1-Caspase7), was transformed with p424-ADH-HA-Caspase-9-FLAG or empty vector (-).
Figure 3.
3-component systems for Caspase-8 activation in yeast.
The zymogen pro-Caspase-8 is expressed (from CYC1-promoters) with substrate containing LETD cleavage element S8 (from plasmid p413-TEF-Fas-d-S8-TA) in the yeast EGY48 expressing 6op-LEU2. (A) A small amount of FADD is also expressed from ΔADH1 promoter on a low-copy plasmid without activating the Caspase-8. Fas is expressed at high levels from ADH promoter on a high copy plasmid. (B) FADD and Fas are both expressed at high levels from TEF and ADH promoters, respectively, from high copy plasmids. (C,D) To validate the system from (A) for cloning cDNAs that activate Caspase-8 or -10 in a FADD-dependent manner (the death receptor cloning system), we established yeast transformants expressing Caspase-8 or -10, with low levels of FADD or corresponding empty vectors (-). Then, the cells (FADD/Caspase-8 or -10) were transformed with a Fas-expressing vector (p424-ADH-Fas) or the vector only (-). The yeast transformants included EGY48-6op-LEU2/2op-lacZ/TEF-Fas-d-S8(LETD)-TA/CYC1-Caspase8-HA (C) and EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG (D), without (-) or with a small amount of FADD, and were transformed with the a Fas-expressing vector or the empty vector (-). (E,F) To test the system from (B), The yeast transformants included EGY48-6op-LEU/2op-lacZ/TEF-Fas-d-S8 (LETD)-TA/CYC1-Caspase8-HA (E), and EGY48-6op-LEU2/2op-lacZ/GPD-Fas-d-S8(LETD)-TA/CYC1-Caspase10-FLAG (F) without (-) or with large amounts of Fas or DR5, and were transformed with the a FADD-expressing vector in a large amount under TEF prompter or the empty vector (-). (G–I) Examples of cDNA cloning results. Yeast strain EGY48 containing 6op-LEU2 and 2op-lacZ reporter genes and expressing the LETD-containing substrate (expressed from pTEF-Fas-d-S8-TA) with or without pro-Caspase-8 (CYC1-Caspase8-HA), and/or FADD (ΔADH1-FADD). Cells were subsequently transformed with various cDNA libraries (Figs. S9–pone.0007655.s012S11) and clones that activated the reporter genes were characterized by recovery of cDNA library plasmids and retransformation into yeast expressing FADD and pro-Caspase-8 (G), pro-Caspase-8 without FADD (H), or neither (I). Among the positive clones were #XA514 (n = 13) encoding a fragment of DR4 (G210-E468), #XA512 (n = 15) encoding a fragment of DR5 (A43-S411), #XD108 (n = 8) encoding a fragment of DR5 (Y99-S411), and #XD422 (n = 3) encoding a fragment of DR5 (A192-S411), and #XD402 (n = 7) encoding a fragment of DR5 (V124-S440), all of which activated the lacZ reporter gene in cells containing FADD and Caspase-8 (G) but not in cells lacking FADD (H) or both (I). Also obtained were cDNAs #XD103 encoding a fragment of Caspase-8 (M1-R233), #X8716 encoding a full-length FLIPL, and #XA411 encoding a full-length Caspase-2. Assays were performed in duplicate, growing cells for 4 days on plates.
Figure 4.
(A) Yeast strain EGY48 containing 6op-LEU2/2op-lacZ expressing pro-Caspase-1 (driven by ΔTEF3 promoter, which expresses at low levels) with membrane tethered transcription factor substrate containing WEHD peptide linker (S1 substrate) were transformed with plasmids encoding adapter protein Asc (expressed at low levels from a CYC promoter) and upstream activators either NLRP1ΔLRR or NLRP3ΔLRR (expressed at high levels from TEF or GAL1 promoters). When Caspase-1 is activated via the combination of Asc and NLRP1 or NLRP3, the reporter is cleaved, releasing the chimeric LexA/B42 transcription factor to leave the membrane and enter the nucleus, where it induces expression of LEU2 and lacZ genes. (B) EGY48 cells (6op-LEU2, 2op-lacZ) were transformed with plasmids encoding the S1 substrate, pro-Caspase-1 (C1) expressed from ΔTEF3 promoter, Asc (low) from CYC promoter, and NLRP3ΔLRR from TEF promoter. Control transformants received the corresponding empty plasmids (-). Cells were grown for 2 days on leucine deficient plates containing X-gal and galactose (to induce NLRP3ΔLRR expression). (C) The EGY48 recipient (6op-LEU2, 2op-lacZ) yeast cell strain contained plasmids encoding substrates with either the WEHD tetrapeptide cleavable linker (S1 substrate expressed from TEF-Fas-d-S1(WEHD)-TA transcriptional unit) or WEHG non-cleavable substrate (G1 substrate expressed from TEF-Fas-d-G1(WEHG)-TA) in plasmids encoding either wild-type (“C1/WT”) or Cys285 mutated Caspase-1 (“C1/C285→G285”) (expressed from ΔTEF3-Caspase-1-FLAG or ΔTEF3-Caspase-1(C285→G285)-FLAG transcriptional units). Cells were transformed with the plasmids expressing a small amount of Asc (CYC-Asc), a large amount of Asc (TEF-Asc), a small amount of NLRP1ΔLRR (CYC-NLRP1), a large amount of NLRP1ΔLRR (TEF-NLRP1), various combinations, or empty vectors (-). Yeast transformants were plated on leucine-deficient medium containing X-gal. The large amount of Asc (expressed from TEF promoter) activated Caspase-1 by itself. NLRP1ΔLRR was unable to activate Caspase-1 by itself, albeit expressed at high levels (confirmed by immunoblotting [not shown]). NLRP1ΔLRR activated pro-Caspase-1 in the presence of a small amount of Asc, under conditions where the amount of Asc expressed (from CYC promoter) was insufficient to independently activate Caspase-1.
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 (•) 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 (ΔTEF1-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 (▴) or z-VAD-fmk (•) 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 ΔADH1 promoter) and a large amount of Fas (from ADH promoter), were incubated in selection media (SD/X-gal) with the indicated concentrations of calpeptin (▴) or z-VAD-fmk (•), or equivalent volume of DMSO (○), in 384-well plates for 3–4 days. For (A–C), relative absorbance at 620 nm was measured (mean ± std dev; n = 3−5 independent experiments). (* indicates p<0.001).
Figure 6.
HTS Implementation of yeast-based, 3-component Caspase assays.
(A) Validation of HTS assay for NLRP1-Asc-Caspase-1 system. Yeast cells (18 µl, typically at ≈2×105 cells/well) containing NLRP1ΔLRR/Asc/Caspase-1 were plated into wells of 384 well plates in media containing 1% galactose, 0.2% raffinose. Wells also contained 18 µL selection media with (blue squares) or without (purple diamonds) X-gal substrate (400 µg/mL final Concentration) plus 10% DMSO (4 µl) (to simulate compound addition). The plates were incubated for 72 hrs at 30°C and the OD620 was recorded. Values (mean±std dev) were calculated for the assay maximum and minimum. The Z' factor was calculated as described [45]. (B) Example of library screening plate for NLRP1-Asc-Caspase-1. Yeast cells (typically at ≈2×105 cells/well) containing NLRP1/Asc/Caspase-1 were plated in 384 well plates in Selection Media, without (purple triangles) or with (all other wells) 400 µg/ml X-gal (final concentration). Compounds in DMSO (∼10 µM final) had been pre-added to wells (diamonds). The plates were incubated for 72 hrs at 30°C and the OD620 was recorded, “Hits” were defined by >50% reduction (as shown in green diamonds). (C) Validation of HTS assay for Fas-FADD-Caspase-8 system. Yeast cells containing Fas/FADD/Caspase-8 were cultured in 384 well plates as above with (blue squares) or without (purple diamonds) X-gal substrate, the plates were incubated for 72 hrs at 30°C and analyzed as above. (D) Example of library screening plate for Fas-FADD-Caspase-8. Yeast cells containing Fas/FADD/Caspase-8 were plated at ≈2×105 cells/wells in 384 well plates in Selection Media, without (purple triangles) or with (all others) 400 µg/ml X-gal (final concentration), and without (triangles, squares) or with (diamonds) compounds in 10% DMSO (∼10 µM final). Plates were incubated for 48 hrs at 30°C and OD620 values recorded. “Hits” were defined by >50% reduction (shown as green diamonds). (E,F) Examples of compounds showing evidence of selective inhibition of Fas-FADD-Caspase-8. EGY48 yeast cell lines employed for deconvoluting hits included yeast expressing lacZ gene (▾) expresses LexA/B42 transcription factor from GAL1 promoter; cells grown in galactose media to induce promoter) to eliminate false-positives due to β-galactosidase inhibition and Asc/Caspase-1-expressing yeast (▪) to eliminate hits that interfere with a different Caspase. Yeast containing empty vector were also included (⧫). Yeast cells (at 2×105 cells/well) containing Fas/FADD/Caspase-8 (▴) or other strains were cultured in 384 well plates in a total volume of 40 µL Selection Media, with 400 µg/ml X-gal final concentration, without (“C”) or with various concentrations of compound CID-5154 or CID-3101. The plates were incubated for 2–4 days at 30°C and OD620 values recorded. (G) Example of compound showing evidence of inhibition of Caspase-8. An in vitro biochemical assay was used containing purified recombinant Caspase-8 and fluorigenic substrate Ac-IETD-AFC. Data represent relative fluorescence units (RFU) assessed in the absence or presence of various concentrations of compound (CID-2531).
Figure 7.
Yeast-based HTS assay for ATG4B.
(A) The basis for the yeast-based cleavable reporter assay for monitoring ATG4B activity is depicted. The membrane tethered transcription factor (TF) consists of the DNA-binding domain of the LexA protein and the transactivation domain of the B42 protein, fused to LC3 and the extracellular and transmembrane domains of Fas. ATG4B cleaves LC3 to release the chimeric LexA/B42 transcription factor, leaving the membrane and entering the nucleus, where it induces expression of LEU2 and LacZ genes. (B) EGY48 yeast transformed with plasmids encoding various proteases (ATG4B, ATG4B(C/A), Caspase-1) and various Fas-LexA/B42 substrates containing either LC3 protein or the WEHD tetrapeptide were streaked on standard media plates without (top) or with (bottom) Xgal substrate. Note that wild-type ATG4B but not mutant ATG4B(C/A) activated the lacZ reporter gene. (C) For HTS implementation, multiple replicate wells were prepared of yeast cells (100 µL/well at 104 cells/mL) expressing LC3-containing substrate and either ATG4B (red) or ATG4B(C/A) mutant (blue) in 384 well plates using media containing 1% galactose/2%raffinose, with X-gal substrate. Cells were cultured for 48 hrs at 30°C before recording OD 620 nm values.