Journal of Neural Transmission

, Volume 116, Issue 11, pp 1355–1362

Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catecholamines

Authors

  • A. Nakashima
    • Department of Physiology, School of MedicineFujita Health University
  • N. Hayashi
    • Institute for Comprehensive Medical ScienceFujita Health University
  • Y. S. Kaneko
    • Department of Physiology, School of MedicineFujita Health University
  • K. Mori
    • Department of Physiology, School of MedicineFujita Health University
  • E. L. Sabban
    • Department of Biochemistry and Molecular BiologyNew York Medical College
    • Department of Pharmacology, School of MedicineFujita Health University
  • A. Ota
    • Department of Physiology, School of MedicineFujita Health University
Basic Neurosciences, Genetics and Immunology - Review Article

DOI: 10.1007/s00702-009-0227-8

Cite this article as:
Nakashima, A., Hayashi, N., Kaneko, Y.S. et al. J Neural Transm (2009) 116: 1355. doi:10.1007/s00702-009-0227-8

Abstract

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine to l-dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal actions of CA. Thus, TH is related to various neuro-psychiatric diseases such as TH deficiency, Parkinson’s disease (PD), and schizophrenia. Four isoforms of human TH (hTH1–hTH4) are produced from a single gene by alternative mRNA splicing in the N-terminal region, whereas two isoforms exist in monkeys and only a single protein exist in all non-primate mammals. A catalytic domain is located within the C-terminal two-thirds of molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain. The catalytic activity of TH is end product inhibited by CA, and the phosphorylation of Ser residues (Ser19, Ser31, and especially Ser40 of hTH1) in the N-terminus relieves the CA-mediated inhibition. Ota and Nakashima et al. have investigated the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability by producing various mutants of the N-terminus of hTH1. The expression of the following three enzymes, TH, GTP cyclohydrolase I, which synthesizes the tetrahydrobiopterin cofactor of TH, and aromatic-l-amino acid decarboxylase, which produces DA from l-dopa, were induced in the monkey striatum using harmless adeno-associated virus vectors, resulting in a remarkable improvement in the symptoms affecting PD model monkeys Muramatsu (Hum Gene Ther 13:345–354, 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.

Keywords

Gene therapyMutantsN-terminusParkinson’s diseaseSchizophreniaTyrosine hydroxylase

Abbreviations

AADC

Aromatic l-amino acid decarbxylase

BH4

(6R)-l-erythro-5,6,7,8-tetrahydrobiopterin

DA

Dopamine

hTH1–hTH4

Human tyrosine hydroxylase types 1–4

PD

Parkinson’s disease

TH

Tyrosine hydroxylase

Introduction

Tyrosine hydroxylase (TH; EC 1.14.16.2 tyrosine 3-monooxygenase) (Nagatsu et al. 1964; Reviews, Nagatsu 1991, 1995, 2006) catalyzes the conversion of l-tyrosine to l-dopa, which is the initial and rate-limiting step in the biosynthesis of catecholamines [CA; dopamine (DA), noradrenaline, and adrenaline], and plays a central role in the neurotransmission and hormonal action of CA. Thus TH is related to various neuropsychiatric diseases such as l-dopa-responsive and non-responsive dystonia due to GTP cyclohydrolase I deficiency (Segawa’s disease; Ichinose et al. 1994a), TH deficiency (Lüdecke et al. 1996), Parkinson’s disease (PD; Nagatsu 1993), manic depressive illness (Meloni et al. 1995), and schizophrenia (Thibaut et al. 1997; Reviews, Kobayashi and Nagatsu 2005).

TH is a pteridine-dependent monooxygenase, and requires tyrosine and molecular oxygen as substrates and (6R)-l-erythro-tetrahydrobiopterin (BH4) and ferrous iron as cofactors. TH is a homotetramer composed of four identical subunits of Mr 60 kDa, each containing one BH4 and Fe2+ (Fig. 1: A modified model from Goodwill et al. 1997).
https://static-content.springer.com/image/art%3A10.1007%2Fs00702-009-0227-8/MediaObjects/702_2009_227_Fig1_HTML.jpg
Fig. 1

The crystal structures of rat tyrosine hydroxylase corresponding to human tyrosine hydroxylase type 1 (hTH1). (A modified model from Goodwill et al. 1997)

The structures of the human TH (hTH) gene, four mRNAs, and corresponding four isoform proteins of human TH (hTH1–hTH4) are shown in Fig. 2a (Kobayashi et al. 1988). The four isoforms are produced from a single gene by alternative mRNA splicing in the N-terminus, whereas two isoforms in monkeys and only a single protein exist in all non-primate mammals (Ichinose et al. 1993; Haycock 2002). A catalytic domain is located within the C-terminal two-thirds of the molecule, whereas the part of the enzyme controlling enzyme activity is assigned to the N-terminal end as the regulatory domain (Abate and Joh 1991). The homo-tetramer structure is formed by the C-terminal domain. As shown in Fig. 2b, the levels of hTH1–4 mRNAs in postmortem human brains were found to be markedly decreased in the substantia nigra containing nigro-striatal DA (A9) neurons in PD, whereas there were no significant changes in schizophrenia (Ichinose et al. 1994b). These results suggest that TH protein levels in the nigro-striatal DA neurons of postmortem PD brain are markedly decreased in PD, but not in schizophrenia. Furthermore, Mogi et al. (1988) found that the decrease in total TH protein level in the striatum measured by enzyme-immunoassay was greater than that in the total enzyme activity. The results suggest a compensatory activation of TH in nigro-striatal DA neurons in PD brain following the reduction in the TH protein content, probably by increased phosphorylation and activation, as described in the following.
https://static-content.springer.com/image/art%3A10.1007%2Fs00702-009-0227-8/MediaObjects/702_2009_227_Fig2_HTML.gif
Fig. 2

a The structures of the human tyrosine hydroxylase (hTH) gene, four isoform mRNAs, and proteins (hTH1–hTH4) produced by alternative splicing in the N-teminal region of the hTH gene. b mRNA contents of the four types of human tyrosine hydroxylase in the substantia nigra of the brain from controls, patients with Parkinson’s disease, and patients with schizophrenia. The numbers of samples were 12 for control, 7 for Parkinson’s disease and 8 for schizophrenia. The results are displayed as mean ± SEM *p < 0.05 compared with corresponding control value

TH, as the first enzyme for the biosynthesis of CA, is regulated in highly complex ways. In chronic regulation such as under long-term stress, TH is regulated by enzyme induction at the transcriptional level (Nankova et al. 1994; Kumer and Vrana 1996; Sabban and Kvetnansky 2001; Nakashima et al. 2003; Sabban et al. 2006). Moreover, TH is post-transcriptionally regulated during chronic stress and drug treatment (Czyzyk-Krzeska et al. 1997; Alterio et al. 2001; Wong and Tank 2007; Tank et al. 2008). TH activity is regulated acutely by the feedback inhibition by the end product CA: without preincubation of TH with DA, the direct inhibition of TH requires high millimolar concentrations of DA and is competitive with the pterin cofactor (Nagatsu et al. 1964), but with preincubation of TH with DA, the inhibition requires low micromolar concentrations of DA, suggesting strong binding of DA to TH causing inactivation (Okuno and Fujisawa 1991; Fujisawa and Okuno 2005).

The phosphorylation of Ser residues (Ser19, Ser31, and especially Ser40 of TH) in the N-terminus relieves the CA-mediated inhibition (Fujisawa and Okuno 2005). The information concerning the tertiary structure of the TH molecule should be critical to elucidate the function of the N-terminus in CA inhibition. However, there has been no information concerning the tertiary structure of its N-terminus because the crystallization of the TH molecule was performed with the truncated form of rat TH (corresponding to hTH1) lacking the first 155N-terminal amino acid residues (Goodwill et al. 1997). The full-length TH protein has not yet been successfully crystallized. The N-terminus of TH is supposedly located on the surface of the molecule, because TH subunits are gathered together by the interactions of their C-termini (Fig. 1). Recently, intrinsically disordered proteins are attracting a great deal of attention (Dyson and Wright 2005), and TH might be one of them whose N-terminal protein is locally unfolded, although there is no evidence yet.

Regulatory role of N-terminus of hTH1 in enzyme activity

As described earlier, phosphorylation of Ser residues in the N-terminus appears to be one of the most important mechanisms to regulate the catalytic activity of the enzyme in vivo (Hufton et al. 1995; Dunkley et al. 2004; Fujisawa and Okuno 2005).

Among the three phosphorylation sites in the N-terminus of hTH1; i.e., Ser19, Ser31, and Ser40, only Ser31 and Ser40 are readily phosphorylated to activate hTH1 in vitro (Haycock and Wakade 1992; Sutherland et al. 1993; Haycock et al. 1998).

Ota et al. (1995; 1996) produced a series of N-terminal deletion mutants of hTH1 and reported that the N-terminal amino acid residues of TH contain the key sequence in mediating the inhibitory action of CA. Ota et al. (1997) further produced various N-terminus-deleted mutants of hTH1 to elucidate the regulatory role of the N-terminus in enzyme activity. A deletion of up to 39 amino acid residues was enough to abolish the inhibitory effects of DA. The results indicated that the amino acid sequence corresponding to the region Gly36-Arg37-Arg38-Gln39-Ser40 may have the critical role in determining the special configuration of the N-terminal regulatory domain. Nakashima et al. (1999a, 2000) identified the positive charge of Arg37–Arg38 as critical in determining the efficiency of the DA inhibition from the perspective of the secondary structure of the N-terminal 1–60 amino acid region of hTH1. The replacement of Arg by electrically neutral Gly and/or negatively charged Glu was enough to abolish the inhibitory effect of DA on the catalytic activity, although these mutations did not display changes in the circular dichroism spectrum. The prediction of the secondary structure of the N-terminal residues 1–60 by computer software specified the location of the Arg37–Arg38 sequence in the turn intervening between the two α-helices (residues 16–29 and residues 41–59; Fig. 3). The amino acid residues 30–40 are presumed to possess flexible mobility to allow the proper conformation of the regulatory site, as surmised from the susceptibility to proteolysis (McCulloch and Fitzpatrick 1999; Nakashima et al. 1999b).
https://static-content.springer.com/image/art%3A10.1007%2Fs00702-009-0227-8/MediaObjects/702_2009_227_Fig3_HTML.gif
Fig. 3

Prediction of the secondary structure of the N-terminal 1–60 amino acids of hTH1 (Nakashima et al. 2000)

Nakashima et al. (2002) further examined the in vivo role of the sequence Arg37–Arg38 of hTH1 in the feedback inhibition by the end product DA in AtT-20 neuroendocrine cells. These cells lack the TH enzyme and possess a large amount of aromatic l-amino acid decarboxylase (AADC) which can convert l-dopa into DA. Thus, hTH1 that is transfected into these cells would be inhibited by the end product DA accumulating in these cells. Nakashima et al. generated the following mutants of hTH1: (A) RR-GG, Arg37–Arg38 replaced by Gly37–Gly38; (B) RR-EE, Arg37–Arg38 replaced by Glu37–Glu38; (C) S40D, Ser40 replaced by Asp40; and (D) S40A, Ser40 replaced by Ala40. AtT-20 cells were transfected with wild-type or these mutated TH constructs. 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. Nakashima et al. also found that del-38 mutant of hTH1 had significantly higher affinity to BH4 to remove DA bound by preincubation than del-35. These results also suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintain the characteristics of the turn and is responsible for the enzyme inhibition by DA. It is also likely responsible for the efficiency in the l-dopa production of the N-terminus 1–60 amino acid substitution mutants that were expressed in the mammalian AtT-20 cells. The reports that phosphorylation of Ser40 increases the affinity for BH4 and reduces the affinity for CA (Haavik et al. 1990; Le Bourdellès et al. 1991) also support the hypothesis that the decreased DA inhibition in the TH mutants causes increased catalytic activity by replacement of inhibitory DA with activating BH4 cofactor, as schematically shown in Fig. 4.
https://static-content.springer.com/image/art%3A10.1007%2Fs00702-009-0227-8/MediaObjects/702_2009_227_Fig4_HTML.gif
Fig. 4

Schematic presentation of possible regulation of hTH1 activity and stability by the N-terminus as indicated in the wild type and the mutants of the enzyme. Positive charge intrinsic to Arg37–Arg38 and phosphorylation/dephosphorylation of Ser40 are thought to be critical either in inhibition or in activation of TH. The deletion mutation of N-terminal 38-amino acids or point mutation of Arg37–Arg38 to Glu37–Glu38 increases the binding capacity of BH4 to TH molecule to increase the activity and possibly to increase the stability. The N-terminus of TH protein inhibits activity and may increase the stability by binding of the end product CA, and is activated and may be stabilized by binding of the cofactor BH4

Regulatory role of N-terminus of hTH1 in enzyme stability

Phosphorylation of Ser in the N-terminus of TH was reported to change the affinity of the cofactor BH4 and also the stability against denaturation of the enzyme due to the changes in the conformation of the molecule. Dunkley et al. (2004) reported that less than 5% of TH protein is phosphorylated at Ser40 under basal conditions and that depolarizing stimuli enhanced the rate of phosphorylation maximally to approximately 10%. Phosphorylation at Ser40 was reported to decrease the stability against denaturation of the molecule (Okuno and Fujisawa 1991; Gahn and Roskoski 1995; Muga et al. 1998). In contrast, phosphorylation at Ser31 increased the stability (Moy and Tsai 2004). On the other hand, Nakashima et al. (2005b) could not show evidence that phosphorylation of intracellular rat TH or hTH1 expressed in rat pheochromocytoma PC12 cells affects the intracellular stability against degradation of TH protein.

There have been controversial reports whether the BH4 cofactor stabilizes or inactivates TH protein in vivo. Binding of DA to rat TH, which removes BH4, inhibited the activity and also increased thermo stability and resistance toward proteolysis, indicating that removal of BH4 by DA stabilizes TH (Okuno and Fujisawa 1991; Martínez et al. 1996). Urano et al. (2006) have recently reported that hTH1 is inactivated by BH4 by forming insoluble aggregates. Choi et al. (2000) also reported that extracellularly added BH4 causes preferential death of DA cells probably by oxidative stress caused by BH4 and DA oxidation. On the other hand, there have been several reports indicating that BH4 administration stimulates TH activity in vivo (Nagatsu et al. 1994; Ota et al. 2007) and that BH4 deficiency in the hph-1 and Pts-knockout mice leads to TH protein and activity loss (Brand et al. 1996; Sumi-Ichinose et al. 2001, 2005), suggesting the protective role of BH4 for TH in vivo. Another pteridine-requiring monooxygenase, liver phenylalanine hydroxylase (PAH), is stabilized by BH4 (Blau and Erlandsen 2004; Thöny et al. 2004; Scavelli et al. 2005).

Thöny et al. (2008) have recently reported that both the cofactor BH4 and feedback inhibitor DA increase the kinetic stability of hTH1 in vitro and that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH4, i.e., its chaperone activity, and a secondary effect by increased synthesis and binding of CA ligands.

Døskeland and Flatmark (2002) reported that TH is a substrate for ubiquitin conjugation in a reconstituted in vitro system. The N-terminus of hTH1, which is supposedly located on the surface of the molecule, can be a target region for the ubiquitin-proteasome system. Nakashima et al. (2005a) reported that AtT-20 cells transfected with mutant hTH1 with deletion of the N-terminus from Met1 to Ala52 of hTH1 are able to produce a large amount of DA because of the high stability of the N-terminus-deleted hTH1 molecule. These results indicate that the stability against degradation of the hTH1 molecule transfected in AtT-20 cells is determined by the N-terminal region of hTH1. Computer-assisted analysis of the spatial configuration of hTH1 identified five newly recognized PEST (Pro, Glu/Asp, Ser, Thr) motifs, which confer rapid turnover of many short-lived regulatory proteins, in the amino acid sequences. One of the PEST motifs (PEST-I) in hTH1 molecules was located in the N-terminus sequence of Met1-Lys12 and it was predicted that deletion of this N-terminus region would alter the secondary structure within the catalytic domain. However, the deletion of this PEST motif did not cause any alteration in the stability (Nakashima et al. 2007). Nevertheless, the high stability against degradation of the N-terminus-deleted hTH1 mutants can be generated by a structural change in the catalytic domain, which would enable an efficient production of DA in mammalian cells expressing N-terminus deleted hTH1 (Nakashima et al. 2005a). Collectively, these results suggest that the N-terminus of hTH1 also regulates the stability against degradation of the enzyme in vivo.

Chaperone 14-3-3 protein is also known to bind TH protein. Itagaki et al. (1999) found that, although phosphorylation of Ser19 does not directly activate TH, chaperone 14-3-3 protein binds and activates TH at Ser19 phosphorylated by Ca2+/calmodulin-dependent protein kinase II (Ca/CaMPK II). These results indicate depolarization-evoked activation of TH in vivo and agree with the fact that Ca/CaMPK II mediates phosphorylation of TH by hormonal and electrical stimuli, which leads to elevation of intracellular Ca2+ levels. Recently, the role of 14-3-3 protein was highlighted by Sato et al. (2006).14-3-3η, an ubiquitous cytoplasmic chaperone protein, but not other members of the 14-3-3 family, was found to be a regulator of parkin, which is an E3 ubiquitin ligase (Shimura et al. 2000; Zhang et al. 2000), and functionally links two hereditary gene products, parkin and α-synuclein implicated in familial PD, PARK2 and PARK1. Chaperone 14-3-3η protein shares physical and functional characteristics with those of α-synuclein, a main component of intracellular Lewy bodies in PD brain and the causative protein of familial PD PARK1.

Recently, Nakashima et al. (2007) found that the N-terminus of hTH1 could control its own intracellular stability against degradation under the influence of chaperone 14-3-3η protein. The results obtained by using N-terminus-deleted hTH1 mutants identified the sequence up to Ala23 as mediating the stability. The down-regulation of 14-3-3η proteins by RNAi 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 Ala23. 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 Glu43 was not affected by the down-regulation of 14-3-3η. These results suggest that the 14-3-3η protein regulated hTH1 stability against degradation by acting on the N-terminus in PC12D cells and the N-terminus of the enzyme, up to Ala23, is an important sequence for this effect. The 14-3-3η protein is a possible regulator of the quantity of hTH1 protein in CA-producing cells. In contrast to this effect of 14-3-3η protein to decrease TH protein stability in PC12D cells, Obsilova et al. (2008) reported that human 14-3-3ξ protein binding to the N-terminus of hTH1 regulatory domain protects its N-terminus from proteolysis. Winge et al. (2008) reported that purified human tryptophan hydroxylase 2 (TPH2), another pteridine-requiring monooxygenase, is activated by phosphorylation of Ser19 by protein kinase A and 14-3-3 binding. The precise mechanism by which the 14-3-3η protein exerts its effect on the hTH1 stability still remains to be elucidated.

Possible implication of hTH1 mutants in gene therapy of Parkinson’s disease

We have reviewed the role of the N-terminus of TH enzyme in the regulation of both the catalytic activity and the intracellular stability. Recently, gene therapy by transfecting DA-synthesizing enzymes, TH and/or AADC, into the striatum to supplement deficient neurotransmitter DA, has been investigated in various animal models of PD, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropteridine (MPTP)-induced PD monkeys. The expression of the following three human enzymes: TH, GTP cyclohydrolase I, which synthesize the BH4 cofactor of TH, and AADC, in the striatum of MPTP-induced PD monkeys, using adeno-associated virus vectors, brought remarkable improvement in the symptoms affecting PD model monkeys (Muramatsu et al. 2002). Increased knowledge concerning the amino acid sequences of the N-terminus of TH that control enzyme activity and stability will extend the spectrum of the gene-therapy approach for PD.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (14580752, 16500247, 18500301) to A. Nakashima and (10670050) to A. Ota, by a grant from Fujita Health University, Japan, to A. Nakashima and A. Ota, and also by NIH grant NS 44218 to E.L. Sabban.

Copyright information

© Springer-Verlag 2009