Engineered cellular gene-replacement platform for selective and inducible proteolytic profiling

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Apoptosis involves caspases cleaving over 1,500 proteins, making individual roles difficult to study.
• Caspase-activated DNase (CAD) is a key player in apoptosis, activated by caspase-mediated cleavage of its inhibitor, ICAD.
• CAD activation leads to DNA laddering, a hallmark of programmed cell death.

Purpose of the Study:

• To investigate the specific role of CAD in apoptosis using a novel inducible system.
• To develop and validate a posttranscriptional gene replacement (PTGR) method for precise protein engineering in cells.
• To determine if CAD activation alone is sufficient for inducing apoptotic DNA fragmentation.

Main Methods:

• Developed a PTGR system to knock down endogenous ICAD and replace it with a protease-inducible engineered allele.
• Utilized tobacco etch virus (TEV) protease for site-specific and inducible activation of CAD.
• Applied this system to human cancer cell lines to study CAD function.

Main Results:

• Selective, inducible activation of CAD alone did not trigger cell death.
• Hallmarks of DNA damage were observed in cancer cells upon CAD activation.
• Data suggest that cooperative action between CAD and inhibition of DNA repair mechanisms is essential for DNA laddering.

Conclusions:

• CAD activation alone is insufficient to induce apoptosis; it requires concurrent inhibition of DNA repair pathways.
• The PTGR approach offers a versatile tool for studying protein function and engineering cellular processes.
• This method enables precise replacement of wild-type proteins with engineered variants for advanced cell engineering research.