TY - JOUR
T1 - A screen to identify cellular modulators of soluble levels of an amyotrophic lateral sclerosis (ALS)-causing mutant SOD1
AU - Somalinga, Balajee R.
AU - Miller, Gregory A.
AU - Malik, Hiba T.
AU - Wigley, W. Christian
AU - Thomas, Philip J.
N1 - Funding Information:
We thank Dr. David Russell, Dr. Stefan Andersson, and Daphne Head for initial discussions regarding cDNA library construction and screening. The research work was funded through grants from the ALS Association and a Sponsored Research Agreement from Reata Pharmaceuticals.
PY - 2011/10
Y1 - 2011/10
N2 - 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.
AB - 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.
KW - amyotrophic lateral sclerosis
KW - cDNA expression cloning screen
KW - protein solubility assay
KW - superoxide dismutase 1
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U2 - 10.1177/1087057111418505
DO - 10.1177/1087057111418505
M3 - Article
C2 - 21875953
AN - SCOPUS:80053540457
SN - 1087-0571
VL - 16
SP - 974
EP - 985
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 9
ER -