Rapid Profiling of Disease Alleles Using a Tunable Reporter of Protein Misfolding

Abstract

Many human diseases are caused by genetic mutations that decrease protein stability. Such mutations may not specifically affect an active site, but can alter protein folding, abundance, or localization. Here we describe a high-throughput cell-based stability assay, IDESA (intra-DHFR enzyme stability assay), where stability is coupled to cell proliferation in the model yeast, Saccharomyces cerevisiae. The assay requires no prior knowledge of a protein's structure or activity, allowing the assessment of stability of proteins that have unknown or difficult to characterize activities, and we demonstrate use with a range of disease-relevant targets, including human alanine:glyoxylate aminotransferase (AGT), superoxide dismutase (SOD-1), DJ-1, p53, and SMN1. The assay can be carried out on hundreds of disease alleles in parallel or used to identify stabilizing small molecules (pharmacological chaperones) for unstable alleles. As demonstration of the general utility of this assay, we analyze stability of disease alleles of AGT, deficiency of which results in the kidney stone disease, primary hyperoxaluria type I, identifying mutations that specifically affect the protein-active site chemistry.

Liver peroxisomal alanine:glyoxylate aminotransferase and the effects of mutations associated with Primary Hyperoxaluria Type I: An overview

“…Besides an about 30-50% reduction in the catalytic efficiency of the transamination reaction [12,35], the protein encoded by the minor haplotype (AGT-Mi) mainly differs from that encoded by the major one (AGT-Ma) for a reduced thermodynamic and kinetic stability [12,16,18]. The P11L substitution has proven to be responsible for AGT-Mi destabilization because it raises the aggregation propensity of AGT expressed in bacteria [35,36], increases the susceptibility to proteolytic degradation [36], reduces yeast growth in complementation experiments [16,37], decreases the thermal denaturation midpoint of AGT in both the holo-and apo-forms, and destabilizes the dimeric structure of apoAGT in equilibrium denaturation studies [12,17,18]. Accordingly, differential scanning calorimetry analyses have shown that the kinetic stability of AGT drops by about 150-fold in the presence of the P11L polymorphism, while it increases by about 30-fold in the presence of the I340M polymorphism [17].…”

“…On the other hand, the purification and characterization of the variants expressed in E. coli are suitable to dissect and quantify the effect of a mutation on the functional properties of AGT as well as on its secondary, tertiary and quaternary structures [11][12][13]17,18,35,36,[51][52][53][54][55][56][57][58]. Yeast and cell-free systems allow a rapid but semi-quantitative determination of the impact of each mutation on the intracellular activity, stability and assembly of the protein [37,53,54,59]. Thus, only the combination of more than one approach can provide a detailed picture of the molecular defect of a pathogenic variant.…”

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine

“…For the remaining mutations the allele in which are present has not yet been identified. Expression analyses in E. coli, yeast and/or mammalian cellular systems of~60 PH1-related variants have allowed to establish differences in their kinetic behavior and/or coenzyme binding mode and affinity, degradation and/or aggregation propensity, expression level, thermodynamic and/or kinetic stability and subcellular localization [2,5,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Among the variants on the background of the minor allele, only G170R, F152I, G161S, G161C, G41R and I244T have been in depth characterized [13][14][15][16]18,22].…”

Effects of alanine:glyoxylate aminotransferase variants and pyridoxine sensitivity on oxalate metabolism in a cell-based cytotoxicity assay

“…Adapting the present method for drug screening would allow candidate drugs to be tested for their potency. Such an assay could find a place alongside current models for drug screening in PH, such as yeast assays and mouse models [52,62, 63]. In addition to a qualitative assessment, this system could also allow a more quantitative description of the cellular mechanism of action (protein expression, catalytic activity, subcellular targeting) as has been done recently [30,48], as well as a direct assessment of oxalate and glyoxylate produced by the cells.…”