Karen Toska

Tyrosine hydroxylase and Parkinson's disease.

A consistent neurochemical abnormality in Parkinsons disease (PD) is degeneration of dopaminergic neurons in substantia nigra, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation ofl-DOPA, the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Similarly, some patients with hereditaryl-DOPA-responsive dystonia, a neurological disorder with clinical similarities to PD, have mutations in the TH gene and decreased TH activity and/or stability. Thus, a logical and efficient treatment strategy for PD is based on correcting or bypassing the enzyme deficiency by treatment withl-DOPA, DA agonists, inhibitors of DA metabolism, or brain grafts with cells expressing TH. A direct pathogenetic role of TH has also been suggested, as the enzyme is a source of reactive oxygen species (ROS) in vitro and a target for radical-mediated oxidative injury. Recently, it has been demonstrated thatl-DOPA is effectively oxidized by mammalian TH in vitro, possibly contributing to the cytotoxic effects of DOPA. This enzyme may therefore be involved in the pathogenesis of PD at several different levels, in addition to being a promising candidate for developing new treatments of this disease.

Regulation of tyrosine hydroxylase by stress-activated protein kinases

Recombinant human tyrosine hydroxylase (hTH1) was found to be phosphorylated by mitogen and stress‐activated protein kinase 1 (MSK1) at Ser40 and by p38 regulated/activated kinase (PRAK) on Ser19. Phosphorylation by MSK1 induced an increase in Vmax and a decrease in Km for 6‐(R)‐5,6,7,8‐tetrahydrobiopterin (BH4), while these kinetic parameters were unaffected as a result of phosphorylation by PRAK. Phosphorylation of both Ser40 and Ser19 induced a high‐affinity binding of 14‐3‐3 proteins, but only the interaction of 14‐3‐3 with Ser19 increased the hTH1 activity. The 14‐3‐3 proteins also inhibited the rate of dephosphorylation of Ser19 and Ser40 by 82 and 36%, respectively. The phosphorylation of hTH1 on Ser19 caused a threefold increase in the rate of phosphorylation of Ser40. These studies provide new insights into the possible roles of stress‐activated protein kinases in the regulation of catecholamine biosynthesis.

Interaction of phosphorylated tyrosine hydroxylase with 14-3-3 proteins: evidence for a phosphoserine 40-dependent association.

Tyrosine hydroxylase (TH) has been reported to require binding of 14‐3‐3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14‐3‐3 proteins BMH1/BMH2, as well as 14‐3‐3 ζ and a mixture of sheep brain 14‐3‐3 proteins. Phosphorylation of Ser31 did not result in 14‐3‐3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14‐3‐3 isoforms BMH1/BMH2 and sheep brain 14‐3‐3, but not for 14‐3‐3 ζ. On phosphorylation of both Ser19 and Ser40, binding to the 14‐3‐3 ζ isoform also occurred, and the binding affinity to BMH1 and sheep brain 14‐3‐3 increased. Both phosphoserine‐specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14‐3‐3 proteins. This was also found when heparin was added, or after proteolytic removal of the N‐terminal 37 amino acids of Ser40‐phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14‐3‐3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14‐3‐3 proteins.

Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mössbauer and X-ray absorption spectroscopy.

Abstract Full-length human tyrosine hydroxylase 1 (hTH1) and a truncated enzyme lacking the 150 N-terminal amino acids were expressed in Escherichia coli and purified either with or without (6×histidine) N-terminal tags. After reconstitution with 57Fe(II), the Mössbauer and X-ray absorption spectra of the enzymes were compared before and after dehydration by lyophilization. Before dehydration, >90% of the iron in hTH1 had Mössbauer parameters typical for high-spin Fe(II) in a six-coordinate environment [isomer shift δ(1.8–77 K)=1.26–1.24 mm s–1 and quadrupole splitting ΔEQ=2.68 mm s–1]. After dehydration, the Mössbauer spectrum changed and 63% of the area could be attributed to five-coordinate high-spin Fe(II) (δ=1.07 mm s–1 and ΔEQ=2.89 mm s–1 at 77 K), whereas 28% of the iron remained as six-coordinate high-spin Fe(II) (δ=1.24 mm s–1 and ΔEQ=2.87 mm s–1 at 77 K). Similar changes upon dehydration were observed for truncated TH either with or without the histidine tag. After rehydration of hTH1 the spectroscopic changes were completely reversed. The X-ray absorption spectra of hTH1 in solution and in lyophilized form, and for the truncated protein in solution, corroborate the findings derived from the Mössbauer spectra. The pre-edge peak intensity of the protein in solution indicates six-coordination of the iron, while that of the dehydrated protein is typical for a five-coordinate iron center. Thus, the active-site iron can exist in different coordination states, which can be interconverted depending on the hydration state of the protein, indicating the presence or absence of a water molecule as a coordinating ligand to the iron. The present study explains the difference in iron coordination determined by X-ray crystallography, which has shown a five-coordinate iron center in rat TH, and by our recent spectroscopic study of human TH in solution, which showed a six-coordinated iron center.

Conformational stability and activity analysis of two hydroxymethylbilane synthase mutants, K132N and V215E, with different phenotypic association with acute intermittent porphyria.

The autosomal dominantly inherited disease AIP (acute intermittent porphyria) is caused by mutations in HMBS [hydroxymethylbilane synthase; also known as PBG (porphobilinogen) deaminase], the third enzyme in the haem biosynthesis pathway. Enzyme-intermediates with increasing number of PBG molecules are formed during the catalysis of HMBS. In this work, we studied the two uncharacterized mutants K132N and V215E comparative with wt (wild-type) HMBS and to the previously reported AIP-associated mutants R116W, R167W and R173W. These mainly present defects in conformational stability (R116W), enzyme kinetics (R167W) or both (R173W). A combination of native PAGE, CD, DSF (differential scanning fluorimetry) and ion-exchange chromatography was used to study conformational stability and activity of the recombinant enzymes. We also investigated the distribution of intermediates corresponding to specific elongation stages. It is well known that the thermostability of HMBS increases when the DPM (dipyrromethane) cofactor binds to the apoenzyme and the holoenzyme is formed. Interestingly, a decrease in thermal stability was measured concomitant to elongation of the pyrrole chain, indicating a loosening of the structure prior to product release. No conformational or kinetic defect was observed for the K132N mutant, whereas V215E presented lower conformational stability and probably a perturbed elongation process. This is in accordance with the high association of V215E with AIP. Our results contribute to interpret the molecular mechanisms for dysfunction of HMBS mutants and to establish genotype–phenotype relations for AIP.

Endogenous tetrahydroisoquinolines associated with Parkinson’s disease mimic the feedback inhibition of tyrosine hydroxylase by catecholamines

N‐methyl‐norsalsolinol and related tetrahydroisoquinolines accumulate in the nigrostriatal system of the human brain and are increased in the cerebrospinal fluid of patients with Parkinson’s disease. We show here that 6,7‐dihydroxylated tetrahydroisoquinolines such as N‐methyl‐norsalsolinol inhibit tyrosine hydroxylase, the key enzyme in dopamine synthesis, by imitating the mechanisms of catecholamine feedback regulation. Docked into a model of the enzyme’s active site, 6,7‐dihydroxylated tetrahydroisoquinolines were ligated directly to the iron in the catalytic center, occupying the same position as the catecholamine inhibitor dopamine. In this position, the ligands competed with the essential tetrahydropterin cofactor for access to the active site. Electron paramagnetic resonance spectroscopy revealed that, like dopamine, 6,7‐dihydroxylated tetrahydroisoquinolines rapidly convert the catalytic iron to a ferric (inactive) state. Catecholamine binding increases the thermal stability of tyrosine hydroxylase and improves its resistance to proteolysis. We observed a similar effect after incubation with N‐methyl‐norsalsolinol or norsalsolinol. Following an initial rapid decline in tyrosine hydroxylation, the residual activity remained stable for 5 h at 37 °C. Phosphorylation by protein kinase A facilitates the release of bound catecholamines and is the most prominent mechanism of tyrosine hydroxylase reactivation. Protein kinase A also fully restored enzyme activity after incubation with N‐methyl‐norsalsolinol, demonstrating that tyrosine hydroxylase inhibition by 6,7‐dihydroxylated tetrahydroisoquinolines mimics all essential aspects of catecholamine end‐product regulation. Increased levels of N‐methyl‐norsalsolinol and related tetrahydroisoquinolines are therefore likely to accelerate dopamine depletion in Parkinson’s disease.

Mechanisms of Tyrosine Hydroxylase Activation by Stress Activated Protein Kinases

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamines. The short-term regulation of TH is mediated by phosphorylation (pTH) and possibly also by feedback inhibition by catecholamines. In human TH, three sites, located in the N-terminal regulatory domain, are phosphorylated by several different protein kinases i.e. Ser19, Ser31 and Ser40 (1). Protein kinase pathways activated by mitogens and stress (such as UV-radiation, oxidant stress or heat shock) have by several groups been reported to be involved in TH regulation in vitro and in vivo (reviewed in (2)). Recently, two novel protein kinases, one regulated and activated by the p38/SAPK2 protein kinase (PRAK) (3) and another activated by both the mitogen and the stress activated (SAPK2) protein kinase pathway (MSK1) were identified (4). In this study, the in vitro effects of PRAK and MSK1 on TH phosphorylation and regulation were compared and the sites of phosphorylation identified. In addition, differences in thermal stability after phosphorylation on Ser19 and/or Ser40 were studied.

Interaction of Phosphorylated Tyrosine Hydroxylase with 14-3-3 Proteins: Effects on Phosphorylation Kinetics

Tyrosine hydroxylase (TH, EC 1.14.16.2) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Apart from tyrosine, the reaction requires dioxygen and the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). Feedback inhibition by catecholamines, cofactor availability and serine phosphorylation are considered to be important post-translational regulatory mechanisms of TH activity (1). The human TH isoforms (hTHl-4) can be phosphorylated on Serl9, 31 and 40 by a range of different kinases. Phosphorylation at Ser40 by cAMP dependent protein kinase (PKA) has been shown to increase the inhibitory constant of catecholamines, lower the Km for BH4 and increase Vmax of the enzyme, whereas phosphorylation by Ca2+/calmodulin dependent protein kinase II (CaMKU) enables binding of 14-3-3 proteins and activation (Vmax) (2, 3).