Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation

Abstract

Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.

Repurposing carbamazepine for the treatment of amyotrophic lateral sclerosis in SOD1‐G93A mouse model

“…Mutant SOD1 aggregates are involved in the pathogenesis of ALS, and reducing mutant SOD1 protein aggregation is regarded as an effective strategy to treat ALS . Carbamazepine have been reported to promote the degradation of mutant α1‐antitrypsin Z protein, so we hypothesized possible changes of SOD1 protein aggregation in spinal cord of those CBZ‐treated TG mice.…”

Manipulating the substrate specificity of murine dihydrofolate reductase enzyme using an expanded set of amino acids

“…Third, RosettaDesign was used to calculate the conformational stability of mDHFR variants upon 2Nal incorporation. One critical issue in engineering enzymes is a loss of enzyme conformational stability when a mutation(s) is introduced into enzymes [53,54]. The reduced conformational stability of enzymes often results in misfolding and/or aggregation likely leading to a significant loss of catalytic activity.…”

“…mDHFR:ligand complex structures with minimum score were extracted from 500 structures. The conformational stabilities of mDHFR variant:ligand complex structures were calculated using the RosettaDesign version 3.4 as described previously with minor changes [35,53,54]. A fixed backbone protein design protocol has been successfully used for computational design of diverse proteins [55][56][57][58][59][60][61].…”

Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function

“…The LJR score is a measure of the steric compatibility of a mutation and, for a fixed-backbone technique, also represents the available free energy to drive structural changes to a more stable protein conformation [ 68 , 69 , 70 , 71 , 72 ]. The RosettaDesign LJR score was previously used to evaluate the conformational stability of human copper, zinc superoxide dismutase and was used successfully to predict mutations for both favorable and unfavorable conformational interactions [ 73 , 74 ].…”