title: Cis-suppression to arrest protein aggregation in mammalian cells.
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.
authors.affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia, 22904-4741.
authors.forename: Simpson
authors.initials: S
authors.lastname: Gregoire
authors.forename: Shaojie
authors.initials: S
authors.lastname: Zhang
authors.forename: Joseph
authors.initials: J
authors.lastname: Costanzo
authors.forename: Kelly
authors.initials: K
authors.lastname: Wilson
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Fernandez
authors.forename: Inchan
authors.initials: I
authors.lastname: Kwon
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011489:Protein Denaturation; D017510:Protein Folding; D055503:Protein Multimerization; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1
chemical_list: D050505:Mutant Proteins; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1
keywords: RosettaDesign; mammalian cells; mutation; protein aggregation; protein engineering; superoxide dismutase