RESUMO
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.
Assuntos
Nanopartículas/metabolismo , Silício/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Humanos , Porosidade , Eletricidade EstáticaRESUMO
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.
Assuntos
Encéfalo/metabolismo , Portadores de Fármacos/química , Nanopartículas/química , Neurônios/metabolismo , Polissacarídeos/química , Tirosina 3-Mono-Oxigenase/administração & dosagem , Tirosina 3-Mono-Oxigenase/farmacocinética , Animais , Linhagem Celular , Estabilidade Enzimática , Terapia Enzimática , Feminino , Humanos , Masculino , Modelos Moleculares , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/enzimologiaRESUMO
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.