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 (SOD1A4V) 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 SOD1A4V as a key residue responsible for SOD1A4V 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 SOD1A4V variants with a significantly reduced aggregation propensity inside cells were selected. The enhanced stability and reduced aggregation propensity of the three novel SOD1A4V variants were verified using cell fractionation and in vitro stability assays.

A Chemical Chaperone‐Based Drug Candidate is Effective in a Mouse Model of Amyotrophic Lateral Sclerosis (ALS)

“…Therefore, the apparent reduction of intracellular aggregates of hA4V SOD1 in the presence of 14 is likely due to the promoted degradation of the entire human SOD1 species. [29] To further probe this point, we tested the ability of compound 14 to activate the proteasome. Given the large number of misfolded/aggregated proteins implicated in ALS, the activation of one of the most important disposal mechanisms of the cell (even in a nonspecific manner) is a viable approach to combating the disease.…”

“…Flow cytometric analysis of cellular fluorescence was performed as previously reported. [29] Two days posttransfection, the transfected HEK293T cells were trypsinized, washed twice with 1 PBS and re-suspended in 500 mL 1 PBS. The fluorescence intensities of the HEK293T cells expressing SOD1 variant-EGFP fusion protein were measured using a C6 flow cytometer (BD Biosciences, San Jose, CA, USA).…”

“…[29] Two days post-transfection, the transfected HEK293T cells were examined by fluorescence microscopy using a VistaVision inverted fluorescence microscope (VWR, Radnor, PA, USA). Images were captured using a DC-2C digital camera.…”

“…[29] Cells were seeded on six-well plates one day prior to transfection. Once the cells grew to 80-90 % confluence, they were transfected with 3.5 mg of the appropriate plasmid via the calcium phosphate precipitation method.…”

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

“…Complementary to high-throughput screening, we used the semi-rational design approach to arrest the aggregation of a human copper, zinc superoxide dismutase mutant containing alanine to valine mutation at position 4 (SOD1 A4V ) inside mammalian cells by enhancing the thermodynamic conformational stability [23]. Although wild-type SOD1 (SOD1 WT ) is very stable and does not form intracellular aggregates, the A4V mutation greatly increases the aggregation-propensity.…”