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A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction
Published on: August 27, 2019
Amelioration of protein misfolding disease by rapamycin: translation or autophagy?
Andreas Wyttenbach1, Sarah Hands, Matthew A King
1Southampton Neuroscience Group, School of Biological Sciences, Southampton, UK. aw3@soton.ac.uk
Abstract:
Rapamycin is an inhibitor of mTOR, a key component of the mTORC1 complex that controls the growth and survival of cells in response to growth factors, nutrients, energy balance and stresses. The downstream targets of mTORC1 include ribosome biogenesis, transcription, translation and macroautophagy. Recently it was proposed that rapamycin and its derivatives enhance the clearance (and/or reduce the accumulation) of mutant intracellular proteins causing proteinopathies such as tau, alpha-synuclein, ataxin-3, and full-length or fragments of huntingtin containing a polyglutamine (polyQ) expansion, by upregulating macroautophagy. We tested this proposal directly using macroautophagy-deficient fibroblasts. We found that rapamycin inhibits the aggregation of a fragment of huntingtin (exon 1) containing 97 polyQs similarly in macroautophagy-proficient (Atg5(+/+)) and macroautophagy-deficient (Atg5(-/-)) cells. These data demonstrate that autophagy is not the only mechanism by which rapamycin can alleviate the accumulation of misfolded proteins. Our data suggest that rapamycin inhibits mutant huntingtin fragment accumulation due to inhibition of protein synthesis. A model illustrates how a modest reduction in polyQ synthesis can lead to a long-lasting reduction in polyQ aggregation. We propose that several mechanisms exist by which rapamycin reduces the accumulation and potential toxicity of misfolded proteins in diseases caused by protein misfolding and aggregation.
Insights
Rapamycin reduces toxic protein aggregation, like that seen in Huntington's disease, by inhibiting protein synthesis, not solely through autophagy. This finding suggests multiple pathways for rapamycin's therapeutic potential.
Area of Science:
- Molecular Biology
- Cellular Biology
- Neuroscience
Background:
- Rapamycin inhibits mTORC1, a complex regulating cell growth and survival.
- mTORC1 targets include ribosome biogenesis, transcription, translation, and macroautophagy.
- Rapamycin was proposed to enhance clearance of mutant proteins in proteinopathies via macroautophagy.
Purpose of the Study:
- To directly test if rapamycin enhances clearance of mutant intracellular proteins by upregulating macroautophagy.
- To investigate the role of macroautophagy in rapamycin's effect on polyglutamine (polyQ) protein aggregation.
Main Methods:
- Utilized macroautophagy-deficient (Atg5(-/-)) and proficient (Atg5(+/+)) fibroblasts.
- Assessed rapamycin's effect on the aggregation of a huntingtin fragment with a 97 polyQ expansion.
Main Results:
- Rapamycin inhibited huntingtin fragment aggregation similarly in both macroautophagy-proficient and deficient cells.
- Autophagy is not the sole mechanism for rapamycin to reduce misfolded protein accumulation.
- Rapamycin appears to inhibit mutant huntingtin fragment accumulation by suppressing protein synthesis.
Conclusions:
- Rapamycin alleviates misfolded protein accumulation through mechanisms beyond macroautophagy.
- Inhibition of protein synthesis is a key mechanism for rapamycin's effect on polyQ aggregation.
- Multiple pathways likely contribute to rapamycin's reduction of toxic protein aggregation in neurodegenerative diseases.
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