Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild-type PAH of 20 selected PKU-linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild-type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH4 ), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single-site missense variants using the full-length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease-linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.
Submitter: Shu-Ching Ou
Submission Date: March 22, 2019, 11:40 a.m.
|Structure ID||Release Date||Resolution||Structure Title|
|1PHZ||1998-11-11T00:00:00+0000||2.2||STRUCTURE OF PHOSPHORYLATED PHENYLALANINE HYDROXYLASE|
|5DEN||2015-08-25T00:00:00+0000||2.9||The First Structure of a Full-Length Mammalian Phenylalanine Hydroxylase Reveals the Architecture of an Auto-inhibited Tetramer|
|2PHM||1998-11-11T00:00:00+0000||2.6||STRUCTURE OF PHENYLALANINE HYDROXYLASE DEPHOSPHORYLATED|
|5FGJ||2015-12-20T00:00:00+0000||3.6||Structure of tetrameric rat phenylalanine hydroxylase, residues 1-453|
|5EGQ||2015-10-27T00:00:00+0000||2.5||Structure of tetrameric rat phenylalanine hydroxylase mutant R270K, residues 25-453|
|2PAH||1998-05-26T00:00:00+0000||3.1||TETRAMERIC HUMAN PHENYLALANINE HYDROXYLASE|
|1DMW||1999-12-15T00:00:00+0000||2.0||CRYSTAL STRUCTURE OF DOUBLE TRUNCATED HUMAN PHENYLALANINE HYDROXYLASE WITH BOUND 7,8-DIHYDRO-L-BIOPTERIN|
|6N1K||2018-11-08T00:00:00+0000||3.06||Full-length human phenylalanine hydroxylase (PAH) in the resting state|
|6HPO||2018-09-21T00:00:00+0000||1.67||Crystallographic structure of the catalytic domain of Human Phenylalanine Hydroxylase (hPAH CD) in complex with iron at 1.6 Angstrom|
|4PAH||1998-08-20T00:00:00+0000||2.0||HUMAN PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN DIMER WITH BOUND NOR-ADRENALINE INHIBITOR|