Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation

João Leandro (1, 2) , Nina Simonsen (1) , Jaakko Saraste (1) , Paula Leandro (1) , Torgeir Flatmark (1)

1 Department of Biomedicine
2 Metabolism and Genetics Group

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 ~145-300Å). 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 -helix 1 which perturbs the wild-type - sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and nonamyloid 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.

Mots clés: hPAH; rPAH; PKU; -Phe; BH; IPTG; MBP; SEC; WT; TM; DM; ANS; DMSO; EM; Phenylketonuria; phenylalanine hydroxylase; pG46S; polymerization; fibril formation; chaperones

João Leandro, Nina Simonsen, Jaakko Saraste, Paula Leandro, Torgeir Flatmark. Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation. Biochimica et Biophysica Acta - Molecular Basis of Disease, 2010, 1812 (1), pp.106. ⟨10.1016/j.bbadis.2010.09.015⟩. ⟨hal-00642449⟩