DNAJC12 Stabilizes Phenylalanine Hydroxylase and Facilitates Its Substrate-Dependent Activation

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Phenylalanine hydroxylase (PAH) deficiency causes hyperphenylalaninemia (HPA) and phenylketonuria (PKU) due to enzyme dysfunction and instability.
• DNAJC12 protein variants are linked to HPA, highlighting the role of protein homeostasis in PAH function.
• Previous reports suggest DNAJC12 and PAH interact, but the underlying molecular mechanisms are not well understood.

Purpose of the Study:

• To elucidate the molecular and structural mechanisms of the DNAJC12-PAH complex formation.
• To investigate how DNAJC12 binding affects PAH stability, activity, and cofactor interaction.
• To explore the therapeutic potential of the PAH:DNAJC12 complex for HPA.

Main Methods:

• Biochemical assays to characterize DNAJC12 binding affinity to PAH.
• Structural analysis of the PAH:DNAJC12 complex.
• Enzyme activity assays to assess the impact of DNAJC12 on PAH function and cofactor BH4 interaction.
• Analysis of DNAJC12's effect on a PKU-associated PAH variant (PAH-p.R261Q).

Main Results:

• DNAJC12 binds PAH with higher affinity to its L-phenylalanine activated form.
• Four DNAJC12 monomers stabilize the PAH tetramer, preventing aggregation and enhancing substrate-induced activation.
• DNAJC12 stabilizes the PKU-associated PAH-p.R261Q variant and delays its aggregation.
• The interaction modulates PAH activity without affecting BH4 binding and stimulates Hsc70 ATPase activity.

Conclusions:

• The study provides the first detailed molecular characterization of the PAH:DNAJC12 complex.
• DNAJC12 binding enhances PAH stability and activity, offering mechanistic insight into DNAJC12 deficiency.
• The PAH:DNAJC12 complex represents a promising therapeutic target for treating hyperphenylalaninemia.