Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation |
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Authors: | João Leandro Nina SimonsenJaakko Saraste Paula LeandroTorgeir Flatmark |
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Institution: | a Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norwayb Metabolism and Genetics Group, iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal |
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Abstract: | The missense mutation pG46S in the regulatory (R) domain of human phenylalanine hydroxylase (hPAH), associated with a severe form of phenylketonuria, generates a misfolded protein which is rapidly degraded on expression in HEK293 cells. When overexpressed as a MBP-G46S fusion protein, soluble and fully active tetrameric/dimeric forms are assembled and recovered in a metastable conformational state. When MBP is cleaved off, G46S undergoes a conformational change and self-associates with a lag phase and an autocatalytic growth phase (tetramers ? dimers), as determined by light scattering. The self-association is controlled by pH, ionic strength, temperature, protein concentration and the phosphorylation state of Ser16; the net charge of the protein being a main modulator of the process. A superstoichiometric amount of WT dimers revealed a 2-fold enhancement of the rate of G46S dimer self-association. Electron microscopy demonstrates the formation of higher-order oligomers and linear polymers of variable length, partly as a branching network, and partly as individual long and twisted fibrils (diameter ~ 145-300 Å). The heat-shock proteins Hsp70/Hsp40, Hsp90 and a proposed pharmacological PAH chaperone (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of α-helix 1 which perturbs the wild-type α-β sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and non-amyloid fibril formation. The metastable conformational state of G46S as a MBP fusion protein, and its self-association propensity when released from MBP, may represent a model system for the study of other hPAH missense mutations characterized by misfolded proteins. |
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Keywords: | hPAH human phenylalanine hydroxylase rPAH rat phenylalanine hydroxylase PKU phenylketonuria l-Phe" target="_blank">l-Phe l-phenylalanine" target="_blank">l-phenylalanine BH4 l-erythro-5" target="_blank">(6R)-l-erythro-5 6 7 8-tetrahydrobiopterin IPTG d-galactoside" target="_blank">isopropyl-thio-β-d-galactoside MBP maltose binding protein SEC size-exclusion chromatography WT wild-type TM tetramer DM dimer ANS 8-anilino-1-naphthalenesulfonic acid DMSO dimethyl sulfoxide EM electron microscopy |
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