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Updated: May 29, 2026
Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells
Published on: October 4, 2017
A screen to identify cellular modulators of soluble levels of an amyotrophic lateral sclerosis (ALS)-causing mutant
Balajee R Somalinga1, Gregory A Miller, Hiba T Malik
1Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Abstract:
The molecular pathology of many protein misfolding, toxic gain-of-function diseases, such as amyotrophic lateral sclerosis (ALS), is not well understood. Although protein misfolding and aggregation are common themes in these diseases, efforts to identify cellular factors that regulate this process in an unbiased fashion and on a global scale have been lacking. Using an adapted version of an extant β-gal-based protein solubility assay, an expression screen for cellular modulators of solubility of an ALS-causing mutant SOD1 was carried out in mammalian cells. Following fluorescence-activated cell sorting enrichment of a mouse spinal cord cDNA library for gene products that increased SOD1 solubility, high-throughput screening of the cDNA pools from this enriched fraction was employed to identify pools containing relevant modulators. Positive pools, containing approximately 10 cDNA clones each, were diluted and rescreened iteratively until individual clones that improved SOD1 folding/solubility were identified. Genes with profound effects in the solubility assay were selected for validation by independent biochemical assays. Six of 10 validated genes had a significant effect on SOD1 solubility and folding in a SOD1 promoter-driven β-gal assay, indicating that global screening of cellular targets using such protein solubility/folding assay is viable and can be adapted for other misfolding diseases.
Insights
This study developed a novel screening method to identify cellular factors influencing protein misfolding in diseases like amyotrophic lateral sclerosis (ALS). The approach successfully identified genes that improve the solubility and folding of mutant SOD1, offering new therapeutic targets.
Area of Science:
- Molecular Biology
- Neuroscience
- Genetics
Background:
- Protein misfolding and toxic gain-of-function are implicated in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS).
- Understanding the cellular factors regulating protein misfolding is crucial for developing effective treatments.
Purpose of the Study:
- To develop and validate a global screening method for identifying cellular modulators of protein solubility.
- To discover novel genes that can improve the folding and solubility of mutant SOD1, a key protein in ALS pathogenesis.
Main Methods:
- An adapted β-galactosidase-based protein solubility assay was employed in mammalian cells.
- A mouse spinal cord cDNA library was screened using fluorescence-activated cell sorting to enrich for modulators of SOD1 solubility.
- High-throughput screening and iterative rescreening were used to identify individual clones that enhance SOD1 folding/solubility.
Main Results:
- The screening successfully identified genes that significantly improve the solubility and folding of mutant SOD1.
- Six out of ten validated genes demonstrated a significant effect on SOD1 solubility and folding in a secondary assay.
- The study confirms the viability of global screening for cellular targets in protein misfolding diseases.
Conclusions:
- A robust and adaptable screening strategy for identifying modulators of protein misfolding diseases has been established.
- The identified genes represent potential therapeutic targets for ALS and other protein misfolding disorders.
- This approach can be applied to discover cellular factors in a wide range of misfolding-related diseases.
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