Cis-suppression to arrest protein aggregation in mammalian cells
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Published on: February 27, 2018
Simpson Gregoire1, Shaojie Zhang, Joseph Costanzo
1Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, 22904-4741.
Abstract:
Protein misfolding and aggregation are implicated in numerous human diseases and significantly lower production yield of proteins expressed in mammalian cells. Despite the importance of understanding and suppressing protein aggregation in mammalian cells, a protein design and selection strategy to modulate protein misfolding/aggregation in mammalian cells has not yet been reported. In this work, we address the particular challenge presented by mutation-induced protein aggregation in mammalian cells. We hypothesize that an additional mutation(s) can be introduced in an aggregation-prone protein variant, spatially near the original mutation, to suppress misfolding and aggregation (cis-suppression). As a model protein, we chose human copper, zinc superoxide dismutase mutant (SOD1(A4V) ) containing an alanine to valine mutation at residue 4, associated with the familial form of amyotrophic lateral sclerosis. We used the program RosettaDesign to identify Phe20 in SOD1(A4V) as a key residue responsible for SOD1(A4V) conformational destabilization. This information was used to rationally develop a pool of candidate mutations at the Phe20 site. After two rounds of mammalian-cell based screening of the variants, three novel SOD1(A4V) variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1(A4V) variants were verified using cell fractionation and in vitro stability assays.
Insights
Researchers developed a novel strategy to prevent protein aggregation in mammalian cells. By introducing specific mutations, they successfully reduced aggregation in a model protein linked to amyotrophic lateral sclerosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Protein misfolding and aggregation are linked to human diseases and reduced protein production yields in mammalian cells.
- Strategies to control protein aggregation in vivo are crucial but underexplored.
- Mutation-induced protein aggregation poses a significant challenge in protein expression.
Purpose of the Study:
- To develop and validate a protein design strategy for suppressing mutation-induced protein aggregation in mammalian cells.
- To identify specific mutations that can mitigate misfolding and aggregation of disease-associated proteins.
- To investigate the concept of cis-suppression by introducing mutations near the original destabilizing mutation.
Main Methods:
- Utilized RosettaDesign software to identify key residues involved in protein destabilization.
- Introduced targeted mutations at identified sites in a model protein, human copper, zinc superoxide dismutase mutant (SOD1(A4V)).
- Employed a two-round mammalian cell-based screening process to select variants with reduced aggregation.
Main Results:
- Identified Phenylalanine 20 (Phe20) as a critical residue in SOD1(A4V) aggregation.
- Successfully engineered three novel SOD1(A4V) variants exhibiting significantly reduced intracellular aggregation.
- Confirmed enhanced stability and decreased aggregation propensity of selected variants through cell fractionation and in vitro assays.
Conclusions:
- Demonstrated the efficacy of a rational design and selection approach for suppressing protein aggregation in mammalian cells.
- Validated the concept of cis-suppression for mitigating mutation-induced protein aggregation.
- The developed strategy offers a promising tool for improving protein production and addressing protein misfolding diseases.
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