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Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like protease

Jernej Murn1, Uros Urleb, Irena Mlinaric-Rascan

  • 1Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia.

Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|October 28, 2004
PubMed

Insights

Serine proteases play dual roles in apoptosis. Inhibiting them triggers caspase-dependent cell death, while chymotrypsin-like activity is essential for DNA fragmentation during apoptosis.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Serine proteases are implicated in apoptosis, but their precise mechanisms remain unclear.
  • Understanding these roles is crucial for developing targeted therapies for diseases involving programmed cell death.

Purpose of the Study:

  • To investigate the apoptosis-inducing potential of serine protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK).
  • To elucidate the specific roles of serine proteases in both the initiation and execution phases of apoptosis.

Main Methods:

  • Utilized TPCK and TLCK to inhibit chymotrypsin-like and trypsin-like proteases, respectively.
  • Assessed apoptosis induction through biochemical markers (PARP-1, ICAD cleavage) and morphological changes.
  • Investigated the involvement of caspases using the inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (z-VAD.fmk).
  • Examined the requirement of serine protease activity for internucleosomal DNA fragmentation in WEHI 231 cells.

Main Results:

  • TPCK and TLCK induced apoptosis, characterized by PARP-1 and ICAD proteolysis and mitochondrial dysfunction.
  • The apoptosis induced by these inhibitors was dependent on caspase activation, as it was blocked by z-VAD.fmk.
  • Chymotrypsin-like protease activity was found to be essential for internucleosomal DNA fragmentation in apoptotic cells.
  • TPCK treatment abrogated DNA fragmentation in anti-IgM or TLCK-induced apoptosis, highlighting the role of chymotrypsin-like proteases in this terminal event.

Conclusions:

  • Serine proteases have distinct roles in apoptosis: inhibiting anti-apoptotic proteases initiates the caspase cascade, while chymotrypsin-like activity mediates DNA fragmentation.
  • These findings provide a deeper understanding of the complex mechanisms governing programmed cell death and the involvement of serine proteases.

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