Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners.

A porous Si (pSi) microparticle-based delivery system is investigated, and the intrinsic luminescence from the particles is employed as a probe to monitor the release of a model protein payload, bovine serum albumin (BSA). The microparticles consist of a core Si skeleton surrounded by a SiO shell. Two types of pSi are tested, one with smaller (10 nm) pores and the other with larger (20 nm) pores. The larger pore material yields a higher mass loading of BSA (3 vs 20%). Two different methods are used to load BSA into these nanostructures: the first involves loading by electrostatic physisorption, and the second involves trapping of BSA in the pSi matrix by local precipitation of magnesium silicate. Protein release from the former system is characterized by a burst release, whereas in the latter system, release is controlled by dissolution of the pSi/magnesium silicate matrix. The protein release characteristics are studied under accelerated (0.1 M aqueous KOH, 21 °C) and physiologically relevant (phosphate-buffered saline, pH 7.4, 37 °C) conditions, and the near-infrared photoluminescence signal from the pSi skeleton is monitored as a function of time and correlated with protein release and silicon dissolution. The thickness of the Si core and the SiO shell are systematically varied, and it is found that the luminescence signature can be tuned to provide a signal that either scales with protein elution or that changes rapidly near the end of useful life of the delivery system. Although payload release and particle dissolution are not driven by the same mechanism, the correlations between luminescence and payload elution for the various formulations can be used to define design rules for this self-reporting delivery system.

Enzyme replacement therapy (ERT) is a therapeutic approach envisioned decades ago for the correction of genetic disorders, but ERT has been less successful for the correction of disorders with neurological manifestations. In this work, we have tested the functionality of nanoparticles (NP) composed of maltodextrin with a lipid core to bind and stabilize tyrosine hydroxylase (TH). This is a complex and unstable brain enzyme that catalyzes the rate-limiting step in the synthesis of dopamine and other catecholamine neurotransmitters. We have characterized these TH-loaded NPs to evaluate their potential for ERT in diseases associated with TH dysfunction. Our results show that TH can be loaded into the lipid core maltodextrin NPs with high efficiency, and both stability and activity are maintained through loading and are preserved during storage. Binding to NPs also favored the uptake of TH to neuronal cells, both in cell culture and in the brain. The internalized NP-bound TH was active as we measured an increase in intracellular L-Dopa synthesis following NP uptake. Our approach seems promising for the use of catalytically active NPs in ERT to treat neurodegenerative and neuropsychiatric disorders characterized by dopamine deficiency, notably Parkinson's disease.

Tyrosine hydroxylase (TH) is the enzyme catalyzing the rate-limiting step in the synthesis of dopamine in the brain. Developing enzyme replacement therapies using TH could therefore be beneficial to patient groups with dopamine deficiency, and the use of nanocarriers that cross the blood–brain barrier seems advantageous for this purpose. Nanocarriers may also help to maintain the structure and function of TH, which is complex and unstable. Understanding how TH may interact with a nanocarrier is therefore crucial for the investigation of such therapeutic applications. This work describes the interaction of TH with porous silicon nanoparticles (pSiNPs), chosen since they have been shown to deliver other macromolecular therapeutics successfully to the brain. Size distributions obtained by dynamic light scattering show a size increase of pSiNPs upon addition of TH and the changes observed at the surface of pSiNPs by transmission electron microscopy also indicated TH binding at pH 7. As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7. However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH. TH activity was unaffected by the binding to pSiNPs most probably because the active site stays available for catalysis, in agreement with calculations of the surface electrostatic potential pointing to the most positively charged regulatory domains in the tetramer as the interacting regions. These results reveal pSiNPs as a promising delivery device of enzymatically active TH to increase local dopamine synthesis.