The regulation of tyrosine hydroxylase by dopamine and selected binding partners
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Tyrosine hydroxylase (TH) catalyses the rate limiting step in the metabolic pathway of dopamine (DA) synthesis and is therefore important in physiological functions such as motor control, cognitive function, and the brain reward system. DA is the main regulator of TH activity through feedback inhibition. The mechanism of this inhibition has recently been further elucidated, adding to the knowledge about the structure, regulation, and stabilization of TH through the inhibition by DA, which its reversed by Ser40 phosphorylation. An α-helix in the N-terminal of TH is especially important for the DA inhibition. As DA levels are highly controlled, TH is regulated by several processes in addition to DA itself, notably by phosphorylation at different sites (Ser19, Ser31 and Ser40) and by several proteins that have been shown to regulate TH activity and stability. In this work, we have investigated the binding of three TH binding partners that have previously been shown to regulate TH, i.e. α-synuclein (α-syn), an abundant neuronal protein involved in vesicle trafficking, DNAJC12, the specific HSP40 co-chaperone of TH, and 14-3-3ζ, a regulatory phosphoprotein-binding protein that interacts with Ser19-phosphorylated (pSer19) TH. As the effect of these proteins on the binding of DA has not been previously investigated in detail, the aim of this work was therefore to study the interplay between the binding of these proteins on the inhibition of TH by DA. Furthermore, an additional aim in this thesis work was to characterize the complex formation between TH and α-syn. Our initial binding studies, using the methods SEC-MALS, native-PAGE coupled with immunoblotting, crosslinking and Bio-layer interferometry (BLI), did not provide any insight regarding the interaction between TH and α-syn. Furthermore, our studies on the effect of TH regulatory proteins on DA binding and feedback inhibition, using BLI and enzymatic assays, suggested that DA and DNAJC12 can interact and regulate TH simultaneously, whereas DA and 14-3-3ζ mutually affects the interaction between pSer19 and its counterpart, likely due to the role of the N-terminal in both interactions.
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