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In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening
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Caspase cleaves Drosophila BubR1 to modulate spindle assembly checkpoint function and lifespan of the organism

Natsuki Shinoda1, Misuzu Horikoshi1, Yusuke Taira1

  • 1Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan.

The FEBS Journal
|May 8, 2023
PubMed

Insights

Caspase cleavage of BubR1 protein limits the spindle assembly checkpoint and organism lifespan. Modifying this cleavage in flies extended their lifespan, highlighting caspase substrate analysis importance.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Caspases are crucial enzymes involved in both cell death and non-lethal cellular processes, cleaving over 1500 human protein substrates.
  • The physiological roles of caspase substrate cleavage, particularly in non-lethal contexts, are not well understood.
  • BubR1, a component of the spindle assembly checkpoint, is a known caspase substrate, but its function upon cleavage remains unclear.

Purpose of the Study:

  • To investigate the physiological consequences of caspase-mediated BubR1 cleavage in vivo.
  • To elucidate the mechanism of substrate specificity in caspase-mediated non-lethal cleavage.
  • To determine the role of BubR1 cleavage in regulating the spindle assembly checkpoint and organismal lifespan.

Main Methods:

  • Utilized Drosophila melanogaster as a model organism to study caspase cleavage of BubR1.
  • Employed proximity labeling to identify interacting partners of caspases and BubR1.
  • Generated cleavage-resistant BubR1 mutants in flies to assess functional consequences.

Main Results:

  • Identified Drice, a caspase, as a primary enzyme cleaving Drosophila BubR1 between its KEN box motif and kinase domain.
  • Proximity labeling revealed Drice, but not Dcp-1, is in close proximity to BubR1, suggesting proximity influences substrate preference.
  • Cleavage of BubR1 resulted in altered subcellular localization and protein-protein interactions of its fragments.
  • Flies expressing cleavage-resistant BubR1 exhibited prolonged kinetochore localization and extended lifespan.

Conclusions:

  • Caspase-mediated cleavage of BubR1 acts as a negative regulator of the spindle assembly checkpoint and organismal lifespan.
  • Protein proximity plays a role in determining caspase substrate specificity in non-lethal cellular events.
  • Individual in vivo analysis of caspase substrates is essential for understanding the biological significance of non-lethal caspase activity.
Keywords:
DrosophilaBubR1TurboIDageingcaspase

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