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A genetic system for monitoring Caspase-1 activity in yeast cells.

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posted on 2009-10-30, 02:08 authored by Hideki Hayashi, Michael Cuddy, Vincent Chih-Wen Shu, Kenneth W. Yip, Charitha Madiraju, Paul Diaz, Toshifumi Matsuyama, Muneshige Kaibara, Kohtaro Taniyama, Stefan Vasile, Eduard Sergienko, John C. Reed

(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.