Regulatory Domain Of Tyrosine Hydroxylase Research Articles

α-synuclein enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis.

Parkinson's disease (PD) results from degeneration of dopamine and norepinephrine neurons due to α-synuclein aggregates that likely have their origin in the gut. Tyrosine hydroxylase (TH) catalyses the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A second enzyme, DOPA decarboxylase (DDC), catalyzes the conversion of L-DOPA to dopamine. A third enzyme, dopamine ß-hydroxylase (DBH), catalyzes the conversion of dopamine to norepinephrine. To analyze possible interactions of α-synuclein with TH, DDC and DBH, we performed in silico protein-protein docking. Protein data bank (pdb) entries were searched on the RCSB Protein Data Bank. We identified four structures that allowed us to examine the relationship of α-synuclein with TH, DDC, and DBH: (1) Human micelle-bound alpha-synuclein, (2) solution structure of the regulatory domain of tyrosine hydroxylase (Rattus norvegicus), (3) crystal structure of human aromatic L-amino acid decarboxylase (DOPA decarboxylase) in the apo form and (4) crystal structure of human dopamine ß-hydroxylase at 2.9 angstrom resolution. We used the ClusPro server (https://cluspro.org) for protein-protein docking. The protein structures were visualized with PyMOL v 2.3.4. α-synuclein partially enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis. α-synuclein may dock too far away from DOPA decarboxylase to affect its function directly. Our in silico finding of α-synuclein partly enfolding tyrosine hydroxylase and dopamine ß-hydroxylase suggests that α-synuclein docking inhibition could increase dopamine and norepinephrine biosynthesis, ameliorating PD symptoms. Small molecules that bind to α-synuclein have already been identified. Further studies may lead to new small molecule drugs that block α-synuclein enfolding of tyrosine hydroxylase and dopamine ß-hydroxylase.

Quantitation of Human 14-3-3[formula omitted] Dimerization and the Effect of Phosphorylation on Dimer-monomer Equilibria

14-3-3 proteins are universal regulatory proteins and their function depends on their oligomeric form which may alter between the monomeric, homodimeric and heterodimeric states. The populations of individual oligomeric forms are controlled by Kd values of the dimer–monomer equilibria between the involved isoforms. This complex picture is extended by post-translational modifications, e.g. phosphorylation. In this work, we describe the equilibria between monomers, homo- and heterodimers of the 14-3-3ζ isoform in the unmodified and phosphorylated form. To cover a wide range of dimerization affinities, we combined solution NMR, microscale thermophoresis, native PAGE, and a set of novel fluorescence assays. Using a FRET based assay, we also determined the kinetic parameters of dimerization. We found that phosphorylation of 14-3-3ζ at Ser58 increases its homodimeric Kd value by 6 orders of magnitude. The presented assays allow to efficiently monitor 14-3-3ζ dimerization as a function of external factors, such as temperature, salt concentration, and client protein binding. For instance, we obtained values of both transient and equilibrium thermodynamic constants for the dimerization, and observed a substantial decrease of 14-3-3ζ dimer dissociation rate upon binding to the doubly phosphorylated regulatory domain of tyrosine hydroxylase. In summary, our work provides a conceptual framework to characterise the isoform exchanges of homo- and heterodimers which can significantly deepen our knowledge about the regulatory function of 14-3-3 proteins.

The Monomeric Species of the Regulatory Domain of Tyrosine Hydroxylase Has a Low Conformational Stability.

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1-71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol(-1)). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements.

A novel prion protein-tyrosine hydroxylase interaction.

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences. It is the main cause of a group of neurological diseases collectively called transmissible spongiform encephalopathies that severely affect both humans and a variety of mammals. Much effort has been directed to understanding the molecular basis of PrP activity, both in physiological and pathological terms. In this context, identification of neuronally-relevant interactors of PrP may play a crucial role. We recently discovered a specific, high-affinity (nanomolar KD) interaction with tyrosine hydroxylase (TH), a enzyme catalyzing the rate-limiting step in the synthesis of the neurotransmitter dopamine. Using molecular biological, biochemical and biophysical techniques we identified the C-terminal structured domain of PrP and the Nterminal regulatory domain of TH as interacting domains between these two proteins. This interaction does not affect TH activity in vitro, although co-expression experiments in HeLa and Chinese hamster ovary cells revealed that PrP is able to internalize TH. Moreover, TH modulated the level of expression of PrP and its localization at the plasma membrane. This novel interaction between two proteins of central importance in nervous system function may shed new light on our understanding of PrP in neurological diseases.

The Solution Structure of the Regulatory Domain of Tyrosine Hydroxylase

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine in the biosynthesis of the catecholamine neurotransmitters. The activity of the enzyme is regulated by phosphorylation of serine residues in a regulatory domain and by binding of catecholamines to the active site. Available structures of TyrH lack the regulatory domain, limiting the understanding of the effect of regulation on structure. We report the use of NMR spectroscopy to analyze the solution structure of the isolated regulatory domain of rat TyrH. The protein is composed of a largely unstructured N-terminal region (residues 1–71) and a well-folded C-terminal portion (residues 72–159). The structure of a truncated version of the regulatory domain containing residues 65–159 has been determined and establishes that it is an ACT domain. The isolated domain is a homodimer in solution, with the structure of each monomer very similar to that of the core of the regulatory domain of phenylalanine hydroxylase. Two TyrH regulatory domain monomers form an ACT domain dimer composed of a sheet of eight strands with four α-helices on one side of the sheet. Backbone dynamic analyses were carried out to characterize the conformational flexibility of TyrH65–159. The results provide molecular details critical for understanding the regulatory mechanism of TyrH.

Fluorescence Spectroscopy as a Probe of the Effect of Phosphorylation at Serine 40 of Tyrosine Hydroxylase on the Conformation of Its Regulatory Domain

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by 3 orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W(3)F TyrH). The steady-state fluorescence anisotropy of F74W W(3)F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W(3)F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34 but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14 and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.

The Low Affinity Dopamine Binding Site on Tyrosine Hydroxylase: The Role of the N-Terminus and In Situ Regulation of Enzyme Activity

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.

The 14-3-3 Protein Affects the Conformation of the Regulatory Domain of Human Tyrosine Hydroxylase

Tyrosine hydroxylase (TH) catalyzes the first step in the biosynthesis of catecholamines. Regulation of TH enzyme activity is controlled through the posttranslational modification of its regulatory domain. The regulatory domain of TH can be phosphorylated at four serines (8, 19, 31, and 40) by a variety of protein kinases. Phosphorylation of Ser19 does not by itself increase TH activity but induces its binding to the 14-3-3 protein. That leads to the enhancement of TH activity with a still not fully understood mechanism. The main goal of this work was to investigate whether the 14-3-3 protein binding affects the conformation of the regulatory domain of human TH isoform 1 (TH1R). Site-directed mutagenesis was used to generate five single-tryptophan mutants of TH1R with the Trp residue located at five different positions within the domain (positions 14, 34, 73, 103, and 131). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser19 and Ser40 does not itself induce any significant structural changes in regions surrounding inserted tryptophans. On the other hand, the interaction between the 14-3-3 protein and phosphorylated TH1R decreases the solvent exposure of tryptophan residues at positions 14 and 34 and induces distinct structural change in the vicinity of Trp73. The 14-3-3 protein binding also reduces the sensitivity of phosphorylated TH1R to proteolysis by protecting its N-terminal part (first 33 residues). Circular dichroism measurements showed that TH1R is an unstructured protein with a low content of secondary structure and that neither phosphorylation nor the 14-3-3 protein binding changes its secondary structure.

Specificity of the MAP kinase ERK2 for phosphorylation of tyrosine hydroxylase

Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2 mol of phosphate into each subunit of tyrosine hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.

A site-specific mutation of tyrosine hydroxylase reduces feedback inhibition by dopamine in genetically modified cells grafted in parkinsonian rats.

Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.

Effects of substitution at serine 40 of tyrosine hydroxylase on catecholamine binding.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate of dissociation of inhibitory catecholamines [Ramsey, A. J., and Fitzpatrick, P. F. (1998) Biochemistry 37, 8980-8986]. To probe the structural basis for this effect and to ascertain the ability of other amino acids to functionally replace serine and serine phosphate, the effects of replacement of Ser40 with other amino acids were determined. Only minor changes in the Vmax value and the Km values for tyrosine and tetrahydropterin were seen upon replacement of Ser40 with alanine, valine, threonine, aspartate, or glutamate, in line with the minor effects of phosphorylation on steady-state kinetic parameters. More significant effects were seen on the binding of dopamine and dihydroxyphenylalanine. The affinity of the S40T enzyme for either catecholamine was very similar to that of the wild-type enzyme, while the S40E enzyme was similar to the phosphorylated enzyme. The S40D enzyme had an affinity for DOPA comparable to the phosphorylated enzyme but a higher affinity for dopamine than the latter. With both catecholamines, the S40V and S40A enzymes showed intermediate levels of activation. The results suggest that the serine hydroxyl contributes to the stabilization of the catecholamine-inhibited enzyme. In addition, the S40E enzyme will be useful in further studies of the effects of multiple phosphorylation on tyrosine hydroxylase, while the alanine enzyme does not provide an accurate mimic of the unphosphorylated enzyme.

Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins.

Tyrosine hydroxylase (TH) is phosphorylated by CaM kinase II and is activated in situ in response to a variety of stimuli that increase intracellular Ca(2+). We report here, using baculovirus-expressed TH, that the 14-3-3 protein binds and activates the expressed TH when the enzyme is phosphorylated at Ser-19, a site of CaM kinase II-dependent phosphorylation located in the regulatory domain of TH. Site-directed mutagenesis showed that a TH mutant in which Ser-19 was substituted by Ala retained enzymatic activity at the same level as the non-mutated enzyme, but was a poor substrate for CaM kinase II and did not bind the 14-3-3 protein. Likewise, a synthetic phosphopeptide (FRRAVpSELDA) corresponding to the part of the TH sequence, including phosphoSer-19, inhibited the interaction between the expressed TH and 14-3-3, while the phosphopeptide (GRRQpSLIED) corresponding to the site of cAMP-dependent phosphorylation (Ser-40) had little effect on complex formation. The complex was very stable with a dissociation constant of 3 nM. Furthermore, analysis of PC12nnr5 cells transfected with myc-tagged 14-3-3 showed that 14-3-3 formed a complex with endogenous TH when the cultured cells were exposed to a high K(+) concentration that increases intracellular Ca(2+) and phosphorylation of Ser-19 in TH. These findings suggest that the 14-3-3 protein participates in the stimulus-coupled regulation of catecholamine synthesis that occurs in response to depolarization-evoked, Ca(2+)-dependent phosphorylation of TH.

A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity.

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37 degrees C, TPH was much less stable (t1/2 = 28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by a t1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.

Relationship between Enzymatic Activity and Oligomerization State of Tyrosine Hydroxylase.

The native form of tyrosine hydroxylase (TH) is a homotetramer which consists of four identical subunits each with an MW of approximately 60 kD. The relationships between the catalytic activity of TH and oligomerization of the enzyme have not yet been characterized. We have investigated, by deletion and/or substitution mutagenesis, the involvement of the leucine zipper (LZ) motifs in the oligomer formation of TH and its relation to catalytic activity. Our results demonstrate that deletion of the carboxyl-terminal LZ (LZ-C) abolishes tetramer formation. Interruption of the other two LZ motifs (LZ-A and LZ-B), located in a central region of the catalytic domain by substitution of Leu to Pro at residues 294 and 301 or 386 and 393 has no effect on the tetramer formation of TH. However, the interruption of LZ-A and LZ-B abolishes TH enzymatic activity. The substitution of Leu residues 188 and 190 with Pro at the regulatory domain of TH reduces enzymatic TH activity without affecting tetramer formation. Thus, LZ-C is required for tetramer formation, while LZ-A and LZ-B seem to be involved in the catalytic activity without affecting the tetramer formation of TH. Copyright 1996 S. Karger AG, Basel

Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains.

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified from E. coli. The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparent Km for tyrosine and phenylalanine substrates, and a substantial increase in the Ki for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.

Effect of spermine on tyrosine hydroxylase activity before and after phosphorylation by cyclic AMP-dependent protein kinase

The effect of spermine on tyrosine hydroxylase (TH) activity purified from bovine adrenal medulla was examined before and after phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Before phosphorylation, spermine (less than 1 mM) inhibited the enzymatic activity, and negative cooperative effect of spermine on TH (Hill coefficient = 0.7) was observed from the kinetic analysis concerning 6-methyl-5,6,7,8-tetrahydropterin (6MPH4). Spermine interacted noncompetitively toward tyrosine and the Ki for spermine was calculated to be 68 microM. Phosphorylation abolished the ability of spermine to inhibit TH activity in a negative cooperative manner against the pterin cofactor, and also increased four-fold the Ki value against the substrate. These results suggest that spermine may inhibit TH activity by interacting with the pterin binding site of the enzyme molecule in a manner of negative cooperativity, and that this inhibition is reversed by the conformational change of regulatory domain of TH after phosphorylation by A-kinase.