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Ribosome-mediated folding of partially unfolded ricin A-chain
R H Argent1, A M Parrott, P J Day
1School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
The Journal of Biological Chemistry
|March 29, 2000
Summary
Ricin A-chain unfolds at body temperature, forming a molten globule. Ribosomes are essential for refolding ricin A-chain to regain its catalytic activity, leading to cell death.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Ricin is a cytotoxic protein internalized by mammalian cells.
- Ricin A-chain must cross the endoplasmic reticulum membrane to reach ribosomal targets.
- Unfolding and refolding are hypothesized steps for ricin A-chain translocation and activity.
Purpose of the Study:
- To biochemically and biophysically analyze the unfolding and refolding of ricin A-chain in vitro.
- To investigate the role of cellular components in ricin A-chain refolding and activity.
Main Methods:
- Biochemical assays
- Biophysical analyses
- In vitro refolding experiments
- Use of salt-washed ribosomes and ribosomal RNA
Main Results:
- Native ricin A-chain is unstable at pH 7.0 and 37°C, unfolding into a molten globule-like state.
- Partially unfolded ricin A-chain could not be refolded by buffer manipulation or substrate analogs alone.
- Salt-washed ribosomes were found to be essential for the refolding and restoration of catalytic activity of partially unfolded ricin A-chain.
Conclusions:
- The conformational stability of ricin A-chain is optimized for translocation from the endoplasmic reticulum.
- Ribosomes play a critical role in the cytosol by facilitating the refolding of ricin A-chain, enabling its cytotoxic function.
- This refolding mechanism contributes to ribosome depurination and subsequent cell death.