Phenylketonuria is an inherited disorder characterised by the inability to break down the amino acid L-phenylalanine (L-Phe). The disease is primarily caused by mutations in the gene that encodes phenylalanine hydroxylase and there are >1000 human described variants, but the R261Q mutation is one of the most common mutations. A team of researchers from Norway and Switzerland used a mouse model with the desired R261Q mutation to study the effect of this mutation and the molecular basis of Phenylketonuria.
In their study, Aubi et al. found that the male R261Q mice had higher body weight compared to the female cohort and all mice presented had an increase in basal blood L-Phe levels. All mice had lipid metabolism alterations and oxidative stress, but no neurological alterations. In the liver, total levels of mutated PAH were decreased compared to healthy mice, but showed increased ubiquitination, indicating protein aggregation or misfolding.
Bioinformatics analysis of PAH amyloidogenesis properties hinted that PAH has predicted a high (>50%) propensity to form intermolecular cross-β (amyloid-like) aggregates. To confirm their suspicions, the researchers studied amyloid-like aggregates formation by the mutant R261Q-PAH using Amytracker. A fluorescence assay with purified recombinant mutant protein compared to Wt protein mutant showed increased intensity over time indicating amyloid-like aggregation accumulation over time (5h). (Image: Amytracker 680 assay with recombinant purified proteins WT-PAH (purple) and p.R261Q-PAH (ochre), with 1 mg/ml of each protein in 20 mM Na-Hepes, 200 mM NaCl, pH 7 and incubation at 37 °C. Image from Supplementary Figure 6A by Aubi et al. (2021) Nature Communications, 12(1), 2073 (CC BY 4.0)).
This publication reports the complex nature of PKU, as loss-of-function mutation can be accompanied by toxic gain-of-function (aggregation & oxidative stress) for specific PKU-associated mutations. Amytracker was useful to characterize the formation of amyloid-like structures by the R261Q-PAH mutant protein.