Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28gamma as an
R Araya1, R Takahashi, Y Nomura
1Laboratory for Motor System Neurodegeneration, RIKEN Brain Science Institute, Saitama 351-0198, Japan.
Abstract:
Caspase-3 and -7 represent executioner/effector caspases that directly cause apoptotic morphological changes by cleaving various death substrates. The substrates for caspases generally interact with active caspases, but not with inactive zymogens of caspase or procaspases. Here, to isolate proteins that interact with caspase-7, we established a yeast two-hybrid screening system using reversed-caspase-7, a constitutive active mutant of caspase-7 as a bait plasmid. Screening of an adult brain cDNA library led to isolation of proteasome activator 28 subunit, PA28gamma. In vitro translates of PA28gamma were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28gamma was cleaved during not only Fas-induced apoptosis of HeLa cells, but also cisplatin-induced cell death of MCF7 cells, which are devoid of caspase-3. These findings indicate that PA28gamma is an endogenous substrate for caspase-3 and -7 and that yeast two-hybrid screening using reversed-caspase is a novel and useful approach to clone substrates for effector caspases.
Insights
Researchers identified proteasome activator 28 subunit, PA28gamma, as a direct substrate for executioner caspases-3 and -7. This discovery utilized a novel yeast two-hybrid system for identifying caspase substrates during apoptosis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Caspase-3 and -7 are critical executioner caspases driving apoptosis by cleaving specific substrates.
- Identifying these substrates is crucial for understanding apoptotic pathways.
Purpose of the Study:
- To identify novel substrates interacting with active caspase-7.
- To establish a functional screening method for effector caspase substrates.
Main Methods:
- A yeast two-hybrid screening system was developed using a constitutively active caspase-7 mutant (reversed-caspase-7) as bait.
- An adult brain cDNA library was screened.
- In vitro cleavage assays and mutagenesis were performed on the identified protein.
Main Results:
- Proteasome activator 28 subunit, PA28gamma, was identified as a binding partner of caspase-7.
- PA28gamma was confirmed as a direct substrate cleaved by both caspase-3 and -7 in vitro.
- Mutating the DGLD80 cleavage site abolished caspase-mediated cleavage.
- Endogenous PA28gamma cleavage was observed during Fas-induced apoptosis and cisplatin-induced cell death.
Conclusions:
- PA28gamma is a novel endogenous substrate for executioner caspases-3 and -7.
- Yeast two-hybrid screening with active caspase mutants is an effective method for discovering effector caspase substrates.
