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

Charles W Morgan1, Juan E Diaz2, Samantha G Zeitlin2

  • 1Chemistry and Chemical Biology Graduate Program, University of California, San Francisco, CA 94143; Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143.

Proceedings of the National Academy of Sciences of the United States of America
|June 25, 2015
PubMed
Summary
This summary is machine-generated.

Selective activation of the caspase-activated DNase (CAD) alone does not cause cell death during apoptosis. DNA damage hallmarks appear, but CAD requires cooperation with DNA repair inhibition for DNA laddering.

Keywords:
DNA damageICADTEV proteaseapoptosissite-specific proteolysis

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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.