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Updated: Jun 26, 2025
Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation
Published on: January 16, 2019
Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase
Martin Grønbæk-Thygesen1, Vasileios Voutsinos1, Kristoffer E Johansson1
1Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Abstract:
Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.
Insights
Unstable proteins, like those causing Canavan disease, can be toxic. This study reveals how protein stability, degradation pathways, and cell health are interconnected, offering insights into disease mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Unstable proteins can lead to toxic interactions and cellular damage.
- The protein quality control network targets destabilized proteins for degradation.
- Aspartoacylase (ASPA) variants are linked to Canavan disease, a fatal neurological disorder.
Purpose of the Study:
- To systematically investigate the impact of single amino acid substitutions and nonsense variants on ASPA abundance and stability in human cells.
- To understand the relationship between protein stability, degradation, and cellular toxicity.
- To identify degradation signals (degrons) within ASPA.
Main Methods:
- High-throughput assessment of ~98% of possible single amino acid substitutions and nonsense variants of cytosolic ASPA in human cells.
- Analysis of protein degradation via the ubiquitin-proteasome pathway.
- Correlation of variant abundance with predicted thermodynamic stability and evolutionary conservation.
Main Results:
- Determined the cellular abundance of 6152 ASPA variants.
- Identified that most low-abundance variants are degraded by the ubiquitin-proteasome system and can become toxic upon prolonged expression.
- Demonstrated that buried degradation signals, particularly in the C-terminal region, regulate protein stability and degradation.
- Correlated variant abundance with predicted stability, conservation, and disease linkage.
Conclusions:
- ASPA variant abundance is strongly influenced by protein stability and degradation pathways.
- The C-terminal region of ASPA acts as a degron, impacting protein turnover.
- This data aids in interpreting Canavan disease variants and understanding the link between protein stability, degradation, and cell fitness.
