Structural mechanism for tyrosine hydroxylase inhibition by dopamine and reactivation by Ser40 phosphorylation.
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ABSTRACT:
SUBMITTER: Bueno-Carrasco MT
PROVIDER: S-EPMC8748767 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis; it uses tetrahydrobiopterin and molecular oxygen to convert tyrosine to DOPA. Its amino terminal 150 amino acids comprise a domain whose structure is involved in regulating the enzyme's activity. Modes of regulation include phosphorylation by multiple kinases at four different serine residues, and dephosphorylation by two phosphatases. The enzyme is inhibited in feedback fashion by the catecholamine neurotransmitters. Dopamine binds to TyrH competitively with tetrahydrobiopterin, and interacts with the R domain. TyrH activity is modulated by protein-protein interactions with enzymes in the same pathway or the tetrahydrobiopterin pathway, structural proteins considered to be chaperones that mediate the neuron's oxidative state, and the protein that transfers dopamine into secretory vesicles. TyrH is modified in the presence of NO, resulting in nitration of tyrosine residues and the glutathionylation of cysteine residues.
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Project description:Parkinson's disease (PD) is characterized by a progressive loss of dopamine-producing neurons in the midbrain, which results in decreased dopamine levels accompanied by movement symptoms. Oral administration of l-3,4-dihydroxyphenylalanine (L-dopa), the precursor of dopamine, provides initial symptomatic relief, but abnormal involuntary movements develop later. A deeper understanding of the regulatory mechanisms underlying dopamine homeostasis is thus critically needed for the development of a successful treatment. Here, we show that p21-activated kinase 4 (PAK4) controls dopamine levels. Constitutively active PAK4 (caPAK4) stimulated transcription of tyrosine hydroxylase (TH) via the cAMP response element-binding protein (CREB) transcription factor. Moreover, caPAK4 increased the catalytic activity of TH through its phosphorylation of S40, which is essential for TH activation. Consistent with this result, in human midbrain tissues, we observed a strong correlation between phosphorylated PAK4S474, which represents PAK4 activity, and phosphorylated THS40, which reflects their enzymatic activity. Our findings suggest that targeting the PAK4 signaling pathways to restore dopamine levels may provide a new therapeutic approach in PD.
| S-EPMC8334344 | biostudies-literature
Project description:DNA from four cattle breeds was used to re-sequence all of the exons and 56% of the introns of the bovine tyrosine hydroxylase (TH) gene and 97% and 13% of the bovine dopamine β-hydroxylase (DBH) coding and non-coding sequences, respectively. Two novel single nucleotide polymorphisms (SNPs) and a microsatellite motif were found in the TH sequences. The DBH sequences contained 62 nucleotide changes, including eight non-synonymous SNPs (nsSNPs) that are of particular interest because they may alter protein function and therefore affect the phenotype. These DBH nsSNPs resulted in amino acid substitutions that were predicted to destabilize the protein structure. Six SNPs (one from TH and five from DBH non-synonymous SNPs) were genotyped in 140 animals; all of them were polymorphic and had a minor allele frequency of > 9%. There were significant differences in the intra- and inter-population haplotype distributions. The haplotype differences between Brahman cattle and the three B. t. taurus breeds (Charolais, Holstein and Lidia) were interesting from a behavioural point of view because of the differences in temperament between these breeds.
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Project description:BackgroundParkinson's disease (PD) is characterized by selective and progressive dopamine (DA) neuron loss in the substantia nigra and other brain regions, with the presence of Lewy body formation. Most PD cases are sporadic, whereas monogenic forms of PD have been linked to multiple genes, including Leucine kinase repeat 2 (LRRK2) and PTEN-induced kinase 1 (PINK1), two protein kinase genes involved in multiple signaling pathways. There is increasing evidence to suggest that endogenous DA and DA-dependent neurodegeneration have a pathophysiologic role in sporadic and familial PD.MethodsWe generated patient-derived dopaminergic neurons and human midbrain-like organoids (hMLOs), transgenic (TG) mouse and Drosophila models, expressing both mutant and wild-type (WT) LRRK2 and PINK1. Using these models, we examined the effect of LRRK2 and PINK1 on tyrosine hydroxylase (TH)-DA pathway.ResultsWe demonstrated that PD-linked LRRK2 mutations were able to modulate TH-DA pathway, resulting in up-regulation of DA early in the disease which subsequently led to neurodegeneration. The LRRK2-induced DA toxicity and degeneration were abrogated by wild-type (WT) PINK1 (but not PINK1 mutations), and early treatment with a clinical-grade drug, α-methyl-L-tyrosine (α-MT), a TH inhibitor, was able to reverse the pathologies in human neurons and TG Drosophila models. We also identified opposing effects between LRRK2 and PINK1 on TH expression, suggesting that functional balance between these two genes may regulate the TH-DA pathway.ConclusionsOur findings highlight the vital role of the TH-DA pathway in PD pathogenesis. LRRK2 and PINK1 have opposing effects on the TH-DA pathway, and its balance affects DA neuron survival. LRRK2 or PINK1 mutations can disrupt this balance, promoting DA neuron demise. Our findings provide support for potential clinical trials using TH-DA pathway inhibitors in early or prodromic PD.
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Project description:Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.
| S-EPMC6433060 | biostudies-literature
Project description:Billions of neurons are interconnected in the central nervous system (CNS). Identification of specific neuronal circuit is indispensable for understanding the relationship between structure and function in the CNS. The midbrain dopamine (DA) neuron system consists of the retrorubral area (A8), the substantia nigra (SN; A9) and the ventral tegmental area (VTA; A10). We hypothesized that genetic methods using cell-type-specific promoters may offer the possibility to express tracer molecules in DA neurons to facilitate neuronal tracing. To address this, we used the 2.5 kb rat tyrosine hydroxylase (TH) promoter in adenovirus or adeno-associated virus (AAV) to express tracers specifically in DA neurons. We found that stereotaxic injection of TH promoter containing adenoviral construct resulted in cell-type-specific transgene expression in the noradrenaline (NA) neurons of the locus coeruleus (LC). However, it caused a significant toxicity to DA neurons in the SN. In contrast, stereotaxic injection of TH promoter containing AAV to the SN resulted in cell-type-specific transgene expression in DA neurons with no detectable toxicity. Taken together, our results demonstrate that it is possible to selectively trace DA neuronal circuits in rodent brains using the TH promoter in the context of AAV.
| S-EPMC2747767 | biostudies-literature
Project description:The mesotelencephalic dopamine system shows substantial genetic variation which fundamentally affects normal and pathological behaviors related to motor function, motivation, and learning. Our earlier radioenzyme assay studies demonstrated significantly higher activity of tyrosine hydroxylase (TH), the first and rate limiting enzyme in the biosynthesis of catecholamine neurotransmitters, in the substantia nigra-ventral tegmental area of BALB/cJ mice in comparison with that of C57BL/6ByJ mice. Here, using quantitative immunoblotting and immunocytochemistry, we tested the hypothesis that mesencephalic TH protein content and number of nigral TH-positive neurons show strain-dependent differences in C57BL/6ByJ and BALB/cJ parallel to those observed in the TH activity studies. Immunoblotting experiments detected significantly higher mesencephalic TH protein content in BALB/cJ in comparison to C57BL/6ByJ (P<0.05). Immunocytochemical studies demonstrated that the number of TH-positive cells in substantia nigra was 31.3% higher in BALB/cJ than that in C57BL/6ByJ (P<0.01), while the average dopamine neuron volume was not significantly different. In a search for candidate genes that modulate TH content and the size of mesencephalic dopamine neuron populations we also studied near-isogenic mouse sublines derived from the C57BL/6ByJ and BALB/cJ progenitor strains. A whole-genome scan with 768 single nucleotide polymorphism markers indicated that two sublines, C4A6/N and C4A6/B, were genetically very similar (98.3%). We found significantly higher mesencephalic TH protein content in C4A6/B in comparison to C4A6/N (P=0.01), and a tendency for higher number of dopamine neurons in the substantia nigra in C4A6/B in comparison to C4A6/N, which, however, did not reach statistical significance. To identify the genetic source of the TH content difference we analyzed the single nucleotide polymorphism (SNP) genotype data of the whole-genome scan, and detected two small differential chromosome segments on chr. 13 and chr. 14. Microarray gene expression studies and bioinformatic analysis of the two differential regions implicated two cis-regulated genes (Spock1 and Cxcl14, chr. 13), and two growth factor genes [bone morphogenetic protein 6 (Bmp6) (chr. 13), and fibroblast growth factor 14 (Fgf14) (chr. 14)]. Taken together, the results suggest that (1) nigral dopamine neuron number and TH protein content may be genetically associated but further studies are needed to establish unequivocally this linkage, and (2) Spock1, Cxcl14, Bmp6, and Fgf14 are novel candidates for modulating the expression and maintenance of TH content in mesencephalic dopamine neurons in vivo.
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Project description:Many hypotheses have been proposed to explain how a glutamate to valine substitution in sickle haemoglobin (HbS) can cause sickle cell disease (SCD). We propose and document a new mechanism in which elevated tyrosine phosphorylation of Band 3 initiates sequelae that cause vaso-occlusion and the symptoms of SCD. In this mechanism, denaturation of HbS and release of heme generate intracellular oxidants which cause inhibition of erythrocyte tyrosine phosphatases, thus permitting constitutive tyrosine phosphorylation of Band 3. This phosphorylation in turn induces dissociation of the spectrin-actin cytoskeleton from the membrane, leading to membrane weakening, discharge of membrane-derived microparticles (which initiate the coagulation cascade) and release of cell-free HbS (which consumes nitric oxide) and activates the endothelium to express adhesion receptors). These processes promote vaso-occlusive events which cause SCD. We further show that inhibitors of Syk tyrosine kinase block Band 3 tyrosine phosphorylation, prevent release of cell-free Hb, inhibit discharge of membrane-derived microparticles, increase sickle cell deformability, reduce sickle cell adhesion to human endothelial cells, and enhance sickle cell flow through microcapillaries. In view of reports that imatinib (a Syk inhibitor) successfully treats symptoms of sickle cell disease, we suggest that Syk tyrosine kinase inhibitors warrant repurposing as potential treatments for SCD.
| S-EPMC7606656 | biostudies-literature
Project description:Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsin-like enzymes and on the increase in catalytic efficiency.
| S-EPMC2286772 | biostudies-literature
Project description:BackgroundDopamine (DA) is a neurotransmitter that plays roles in movement, cognition, attention, and reward responses, and deficient DA signaling is associated with the progression of a number of neurological diseases, such as Parkinson's disease. Due to its critical functions, DA expression levels in the brain are tightly controlled, with one important and rate-limiting step in its biosynthetic pathway being catalyzed by tyrosine hydroxylase (TH), an enzyme that uses iron ion (Fe2+) as a cofactor. A role for metal ions has additionally been associated with the etiology of Parkinson's disease. However, the way dopamine synthesis is regulated in vivo or whether regulation of metal ion levels is a component of DA synthesis is not fully understood. Here, we analyze the role of Catsup, the Drosophila ortholog of the mammalian zinc transporter SLC39A7 (ZIP7), in regulating dopamine levels.ResultsWe found that Catsup is a functional zinc transporter that regulates intracellular zinc distribution between the ER/Golgi and the cytosol. Loss-of-function of Catsup leads to increased DA levels, and we showed that the increased dopamine production is due to a reduction in zinc levels in the cytosol. Zinc ion (Zn2+) negatively regulates dopamine synthesis through direct inhibition of TH activity, by antagonizing Fe2+ binding to TH, thus rendering the enzyme ineffective or non-functional.ConclusionsOur findings uncovered a previously unknown mechanism underlying the control of cellular dopamine expression, with normal levels of dopamine synthesis being maintained through a balance between Fe2+ and Zn2+ ions. The findings also provide support for metal modulation as a possible therapeutic strategy in the treatment of Parkinson's disease and other dopamine-related diseases.
| S-EPMC8564973 | biostudies-literature