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Processing of the Nedd2 precursor by ICE-like proteases and granzyme B
N L Harvey1, J A Trapani, T Fernandes-Alnemri
1Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, Australia.
Summary
The Nedd2 protein precursor (pro-Nedd2) is activated by cleavage into smaller subunits, suggesting its role in apoptosis. This activation process involves ICE-like proteases and granzyme B, indicating Nedd2
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nedd2/Ich-1 is a mammalian cysteine protease within the ICE-like protease family.
- ICE-like proteins are homologous to C. elegans CED-3 and are implicated in apoptosis execution.
- Active Interleukin-1 beta converting enzyme (ICE) is a tetramer formed from cleaved pro-enzyme subunits.
Purpose of the Study:
- To investigate the activation mechanism of the Nedd2 protein precursor (pro-Nedd2).
- To identify proteases responsible for pro-Nedd2 processing and its role in apoptosis.
Main Methods:
- Analysis of pro-Nedd2 cleavage using cell extracts from normal and apoptotic NIH-3T3 cells.
- Inhibition assays using specific inhibitors of ICE-like proteases.
- In vitro processing of pro-Nedd2 by various proteases including CPP32, ICE, Mch2, Nedd2, and Granzyme B.
Main Results:
- Pro-Nedd2 is cleaved into p19 and p12 subunits by extracts from apoptotic cells, indicating processing activity.
- This cleavage activity is inhibited by ICE-like protease inhibitors.
- Pro-Nedd2 can be processed in vitro by CPP32, ICE, Mch2, Nedd2, and notably, Granzyme B.
Conclusions:
- Nedd2 activation depends on cleavage by upstream ICE-like proteases within a proteolytic cascade.
- Cleavage of pro-Nedd2 by Granzyme B suggests Nedd2 acts as a downstream effector in cytotoxic T lymphocyte (CTL)-mediated cell killing.