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

卷:1812

Article

Leandro, Joao^1,2  Simonsen, Nina¹  Saraste, Jaakko¹  Leandro, Paula²  Flatmark, Torgeir¹ 

[1] Univ Bergen, Dept Biomed, N-5009 Bergen, Norway

[2] Univ Lisbon, Fac Pharm, iMed UL, Metab & Genet Grp, P-1649003 Lisbon, Portugal

关键词: Phenylketonuria;    Phenylalanine hydroxylase;    pG46S;    Polymerization;    Fibril formation;    Chaperone;   

DOI  :  10.1016/j.bbadis.2010.09.015

来源: Elsevier

【 摘 要 】

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 G465 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 similar to 145-300 angstrom). 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--one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of alpha-helix 1 which perturbs the wild-type alpha-beta 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.(c) 2010 Elsevier B.V. All rights reserved.

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