Human Tyrosine Hydroxylase Type Research Articles

Dopamine Inhibition of Human Tyrosine Hydroxylase Type 1 Is Controlled by the Specific Portion in the N-Terminus of the Enzyme

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-DOPA, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by feedback inhibition by catecholamine products including dopamine. To investigate the specific portion of the N-terminus of TH that determines the efficiency of dopamine inhibition, wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants (del-35, del-38, and del-44, respectively) of human TH type 1 were expressed as a maltose binding protein fusion in Escherichia coli and purified as a tetrameric form by affinity and size-exclusion chromatography. The fused-form wild-type enzyme possessed almost the same specific enzymatic activity as the previously reported recombinant nonfused form. Although maximum velocities of all N-terminus-deleted forms were about one-fourth of the wild-type value, there was no difference in Michaelis constants for L-tyrosine or (6R)-(L-erythro-1',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7,8-tetrahy dropteridine (6RBPH4) among the four enzymes. The iron contents incorporated into the three N-terminus-deleted mutants were significantly lower than that of wild type. However, there was no substantial difference in incorporated iron contents among the three mutants. The deletion of up to no less than 38 amino acid residues in the N-terminus made the enzyme more resistant to dopamine inhibition than the wild-type or del-35 TH form. Dopamine bound to the del-38 more than to the del-35 TH form. However, when incubation with dopamine was followed by further inhibition with the cofactor 6RBPH4 dopamine was expelled more readily from the del-38 than from the del-35 TH form. These observations suggest that the amino acid sequence Gly36-Arg37-Arg38 plays a key role in determining the competition between dopamine and 6RBPH4 and affects the efficiency of dopamine inhibition of the catalytic activity.

RNAi of 14-3-3η protein increases intracellular stability of tyrosine hydroxylase

Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis, and its N-terminus plays a critical role in the intracellular stability of the enzyme. In the present study, we investigated the mechanism by which the N-terminus of human tyrosine hydroxylase type 1 (hTH1) affects the stability. The results obtained by using N-terminus-deleted hTH1 mutants identified the sequence up to Ala 23 as mediating the stability. The down-regulation of 14-3-3η proteins in PC12D cells exogenously expressing hTH1, enhanced the stability of the wild-type enzyme and that of the mutant lacking the N-terminus up to Ala 23. However, the stability of the mutant was reduced compared to the wild-type enzyme. The stability of the mutant with the N-terminus deleted up to Glu 43 was not affected by the down-regulation of 14-3-3η. These results suggest that the 14-3-3η protein regulates hTH1 stability by acting on the N-terminus.

The phosphorylation of Ser40 of tyrosine hydroxylase has no effect on the stability of the enzyme in PC12 cells

It is well established that the phosphorylation of tyrosine hydroxylase (TH) at Ser40 is critical in regulating the catalytic activity of the enzyme. However, the influence of the phosphorylation of Ser40 on the stability of TH protein has not been investigated. This study was performed to estimate such a possibility. Although the treatment of rat pheochromocytoma cell line PC12 cells with forskolin increased the amount of TH phosphorylated at Ser40 in the cells, it did not affect the total amount of TH in the cells. Next, human TH type 1 (hTH1) of wild-type and a mutant missing the first 52 amino acid residues were expressed as histidine-tagged forms in PC-12 cells, and then the cells were treated with forskolin. However, the phosphorylation of hTH1 at Ser40 did not affect the amount of the wild-type hTH1 protein present in PC12 cells. Finally, wild-type and a mutant in which Ser40 was replaced by Asp (S40D, a mimic of TH phosphorylated at Ser40) were expressed in PC-12 cells as histidine-tagged forms or untagged forms. Neither histidine-tagged nor untagged forms showed any difference in their amounts of wild-type hTH1 and S40D hTH1 present in the cells. Collectively, these results indicate the fact that the phosphorylation of Ser40 does not affect the stability of TH protein in PC12 cells.

The mutation of two amino acid residues in the N-terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT-20 cells.

The sequence Arg37-Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37-Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR-GG, Arg37-Arg38 replaced by Gly37-Gly38; (ii) RR-EE, Arg37-Arg38 replaced by Glu37-Glu38; (iii) S40D, Ser40 replaced by Asp40; and (iv) S40A, Ser40 replaced by Ala40. In a cell-free system, the level of the DA inhibition of the RR-EE mutant enzyme was to the same or smaller degree than that of the phosphorylation-mimicking S40D. Next, AtT-20 neuroendocrine cells were transfected with wild-type and mutated TH genes because these cells were earlier shown to be capable of fully converting L-3,4-dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT-20 cells expressing the TH gene was in the order: RR-EE > S40D > S40A = RR-GG > wild-type, which was in accordance with the observations for the cell-free system. These results suggest that the sequence Arg37-Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40-hTH1.

Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro.

Autologous bone marrow stromal cells engineered to produce 3,4,-dihydroxyphenylalanine (L-DOPA) can potentially be used as donor cells for neural transplantation in Parkinson's disease. Here, we examined the possibility of using several different promoters and either a self-inactivating retrovirus (pSIR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors were constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP reporter gene or a bicistronic sequence containing the genes for human tyrosine hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced with both standard retroviral vectors and pSIR containing the PGK promoter. Transduced rMSCs expressed GFP (90.4--94.4% of cells) or were able to synthesize and secrete L-DOPA (89.0--283 pmols/10(6) cells/h). After transduced rMSCs were plated at low density (3--6 cells/cm(2)), the cells expanded over 1000-fold in 3--4 weeks, and the rMSCs continued to either express GFP or produce L-DOPA. Furthermore, two high-expressing clones were isolated and expanded at low-density from rMSCs transduced with pSIR driven by the PGK promoter (97.0% GFP+ or 1096.0 pmols L-DOPA/10(6) cells/h).

Positive charge intrinsic to Arg 37-Arg 38 is critical for dopamine inhibition of the catalytic activity of human tyrosine hydroxylase type 1

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-3,4-dihydroxyphenylalanine, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N-terminal sequence Arg 37-Arg 38 of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg 37-Arg 38 site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum. The prediction of the secondary structure of N-terminal residues 1–60 by computer software specified the location of the Arg 37-Arg 38 sequence in the turn intervening between the two α-helices (residues 16–29 and residues 41–59). These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine.

Expression of human tyrosine hydroxylase type I in Escherichia coli as a protease-cleavable fusion protein. Short communication.

Wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants of human tyrosine hydroxylase type 1 (hTH1) fused to maltose-binding protein via the target sequence for a restriction protease were expressed in Escherichia coli and purified. The fused protein was treated with the restriction protease factor Xa or enterokinase to isolate hTH1 from the fused form. The treatment of fused wild-type and 35-amino acid-deleted mutant with factor Xa and enterokinase caused non-specific cleavages in the vicinity of the phosphorylation sites, Ser19 and Ser40, due to the flexible conformation of the N-terminus of hTH1.

Effects of dopamine on N-terminus-deleted human tyrosine hydroxylase type 1 expressed in Escherichia coli

N-Terminus-deleted mutants and wild-type human tyrosine hydroxylase type 1 were expressed in Escherichia coli ( E. coli) and utilized to investigate the dopamine-induced decrease in the enzyme catalytic activity and also to identify the specific portion in the N-terminus that affects the efficiency of the inhibitory action of dopamine. Supernatants of bacterial lysates were used as enzyme samples. The pH profiles of the enzyme catalytic activity were affected according to the degree of the deletion. The deletion up to 39 amino acid residues was enough to abolish the inhibitory effect of dopamine in the basic pH range. These results suggest that the inhibition by dopamine of tyrosine hydroxylase activity is closely related to the amino acid sequence in the N-terminus of the enzyme.

Regulation of N-terminus-deleted human tyrosine hydroxylase type 1 by end products of catecholamine biosynthetic pathway.

The N-terminal 52-, 70-, and 157-amino acids-deleted mutants and wild-type tyrosine hydroxylases were expressed in Escherichia coli and utilized to investigate the roles of the N-terminus in the catecholamine inhibition on enzyme activity. Their lysate's supernatants were used as enzyme samples. Three catecholamines, namely dopamine, norepinephrine, and epinephrine, affected both wild-type and mutant enzymes after preincubation in the mode of mixed inhibition, and the most marked alteration among the kinetic parameters produced by the deletion was the increase in the inhibition constants. The deletions also abolished the catecholamine-induced shift of the pH profile of the enzyme activity toward a more acidic pH optimum. All three mutants responded to catecholamines almost in the same way. These results suggest that the three catecholamine end products exert their inhibition on tyrosine hydroxylase to the same extent and that the N-terminal 52 amino acid residues contain the key sequence in mediating the inhibitory action.

GTP cyclohydrolase I gene, tetrahydrobiopterin, and tyrosine hydroxylase gene: their relations to dystonia and parkinsonism.

Catecholamine biosynthesis is regulated by tyrosine hydroxylase (TH) requiring tetrahydrobiopterin (BH4) as the cofactor. We found four (human TH type 1-4) and two isoforms (TH type 1 and 2) in humans and monkeys, while non-primate animals have a single TH corresponding to human TH type 1. BH4 is synthesized from GTP, and GTP cyclohydrolase I (GCH) is the first and regulatory enzyme. Mutations in GCH gene were found to cause both GCH deficiency with autosomal recessive trait and hereditary progressive dystonia with marked diurnal fluctuation (HPD) (Segawa's disease)/or DOPA-responsive dystonia (DRD) with autosomal dominant trait. When GCH activity is decreased to less than 20% of the normal value, the activity of TH in the nigrostriatal dopaminergic neurons may be first decreased resulting in decreases in TH activity and dopamine level, and in the symptoms of HPD/DRD. In contrast to HPD/DRD, juvenile parkinsonism (JP) have normal GCH activity. In Parkinson's disease (PD), GCH, TH, and dopamine in the striatum may decrease in parallel, as the secondary effects caused by cell death.

Deletion Mutagenesis of Human Tyrosine Hydroxylase Type 1 Regulatory Domain

A series of N-terminal deletion mutants of human tyrosine hydroxylase type 1 has been expressed in Escherichia coli to characterize the N-terminal regulatory domain. The mutants lacking the first 74 to 117 amino acids led to the precipitation into aggregates probably due to improper folding. All of the deletion mutants are active in the lysate supernatant and/or the pellet. The Michaelis constants for pterins are similar among all the mutants examined and the wild-type.

Four isoforms of tyrosine hydroxylase are expressed in human brain

In contrast to nonprimate species, the RNA for human tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, can undergo alternative splicing to produce four different types of mRNA. Although types 1 and 2 of these human tyrosine hydroxylase mRNAs have been identified in human brain, whether types 3 and 4 human tyrosine hydroxylase mRNAs are present in the central nervous system remains controversial. Furthermore, little is known about the expression of the protein products of these mRNAs in human brain. In this study we used antibodies raised against different octapeptide sequences from each of the predicted human tyrosine hydroxylase protein forms to determine the presence and distribution of each human tyrosine hydroxylase isoforms in several regions of human brain. Control immunocytochemical and blot immunolabeling experiments demonstrated that each antibody selectively recognized the human tyrosine hydroxylase isoform against which it was directed. In immunocytochemical studies, all four human tyrosine hydroxylase isoforms were clearly detectable in neurons of both the substantia nigra and locus coeruleus. The presence of all four isoforms in these nuclei was confirmed with blot immunolabeling studies. Single-label immunocytochemical studies of adjacent sections as well as dual-label comparisons of immunoreactivity for human tyrosine hydroxylase type 1 with type 2, type 3, or type 4 suggested that at least some neurons in these brain regions contain all four human tyrosine hydroxylase isoforms. In contrast, some neurons of the mesencephalon appeared to be selectively immunoreactive with the antibodies against type 1. In the caudate nucleus and putamen, the terminal zones of the dopaminergic projection from the substantia nigra, all four isoforms were detected, although in immunocytochemical studies type 1 appeared to be the predominant isoform present in axons and terminals. These findings demonstrate that human brain contains four distinct isoforms of human tyrosine hydroxylase and that the presence or relative amount of each isoform may differ among catecholaminergic cell populations and between catecholaminergic neurons and terminal fields. These patterns of expression may have important implications for understanding the regulation of catecholamine biosynthesis in human brain both in normal and pathological states.

Effect of (6R)- and (6S)-tetrahydrobiopterin on L-3,4-dihydroxyphenylalanine (DOPA) formation in NRK fibroblasts transfected with human tyrosine hydroxylase type 2 cDNA

l-erythro-5,6,7,8-Tetrahydrobiopterin (BH(4)), which is the cofactor of aromatic amino acid hydroxylases, plays an important role in the biosyntheses of monoamine neurotransmitters. BH(4) exists as natural (6R)- and unnatural (6S)-isomers. In our previous reports, only (6R)-isomer significantly stimulated cofactor activity for tyrosine, tryptophan and phenylalanine hydroxylases (TH, TPH, PAH) in whole animals or in tissue slices. In this study we have compared the in situ cofactor activity on TH between natural (6R)- and unnatural (6S)-isomers in clonal cells. We have transfected human TH type 2 cDNA into the normal rat kidney (NRK) fibroblasts. These cells expressed TH protein, but had neither DOPA decarboxylase (DDC) nor BH(4). Thus, TH activity was observed only in the presence of exogenous BH(4). We compared the difference in in situ DOPA formation by TH activity in the presence of (6R)- or (6S)-BH(4) in the human TH-transfected cells. The effect of exogenous BH(4) was also compared between (6R)- and (6S)-isomers in rat pheochromocytoma PC12h cells, which contained approximately 100 ?M endogenous (6R)-BH(4). The rate of uptake of both BH(4) isomers into these cells increased in proportion to the pterin cofactor concentrations in the incubation medium up to 400 ?M but was nearly saturated at 1 mM BH(4). TH-transfected NRK fibroblasts formed DOPA only in the presence of exogenously added (6R)- or (6S)-BH(4) dose-dependently and released DOPA into the medium. At a saturating concentration of 1 mM, (6R)-BH(4) was approximately three times as active as (6S)-BH(4). In contrast, in PC12h cells which contained endogenous (6R)-BH(4) (approximately 100 ?M), exogenous (6R)-BH(4) activated DOPA formation maximally at 500 ?M about 10-fold, while (6S)-BH(4) activated it only slightly, about 2.5-fold. These results suggest that (6S)-isomer has lower cofactor activity with TH in the cells than (6R)-isomer. This TH transfected fibroblasts should be useful to assess cofactor activities of tetrahydropteridines in the cell.

Retroviral transfer of a human tyrosine hydroxylase cDNA in various cell lines: regulated release of dopamine in mouse anterior pituitary AtT-20 cells.

Little is known about the molecular events mediating neurotransmitter release, a crucial step in synaptic transmission. In this paper, the biosynthesis and release of L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine were analyzed in three heterologous cell lines after retroviral-mediated gene transfer of tyrosine hydroxylase (EC 1.14.16.2), the rate-limiting enzyme in catecholamine synthesis. A recombinant retrovirus encoding human tyrosine hydroxylase type I as well as neomycin-resistance gene was used to infect a fibroblast (NIH 3T3), a neuroblastoma (NS20 Y), and a neuroendocrine (AtT-20) cell line. After selection in the presence of neomycin and in tyrosine-free medium, high levels of exogenous tyrosine hydroxylase activity were detected in extracts of the three cell lines. High-performance liquid chromatography of cell extracts and culture supernatants confirmed that the three cell lines hydroxylated tyrosine to form L-DOPA and released this metabolite into the culture medium. Interestingly, the neuroendocrine cell line AtT-20 synthesized not only L-DOPA but also dopamine. Evoked secretion studies established that AtT-20 cells released the transmitter upon depolarization in a regulated, calcium-dependent way. We discuss the implication of this approach for the analyses of neurotransmitter release as well as in the context of degenerative disorders such as Parkinson disease.

Isolation of a full-length cDNA clone encoding human tyrosine hydroxylase type 3.

Isolation of a full-length cDNA clone encoding human tyrosine hydroxylase type 3.