Nurgul Carkaci-Salli

Complex molecular regulation of tyrosine hydroxylase.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is strictly controlled by several interrelated regulatory mechanisms. Enzyme synthesis is controlled by epigenetic factors, transcription factors, and mRNA levels. Enzyme activity is regulated by end-product feedback inhibition. Phosphorylation of the enzyme is catalyzed by several protein kinases and dephosphorylation is mediated by two protein phosphatases that establish a sensitive process for regulating enzyme activity on a minute-to-minute basis. Interactions between tyrosine hydroxylase and other proteins introduce additional layers to the already tightly controlled production of catecholamines. Tyrosine hydroxylase degradation by the ubiquitin–proteasome coupled pathway represents yet another mechanism of regulation. Here, we revisit the myriad mechanisms that regulate tyrosine hydroxylase expression and activity and highlight their physiological importance in the control of catecholamine biosynthesis.

Functional Domains of Human Tryptophan Hydroxylase 2 (hTPH2)

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NΔ150 and NΔ150/CΔ24) using isopropyl β-d-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH2-terminal regulatory domain. The solubility of hTPH2, NΔ150, and NΔ150/CΔ24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t½ at 37 °C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NΔ150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH2-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH2-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase Vmax values. These data identify the NH2-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.

Plasma Biomarkers in Pediatric Patients Undergoing Cardiopulmonary Bypass

It is critical to identify at-risk patients and minimize the deleterious effects of cardiopulmonary bypass (CPB) procedures in pediatric populations. The present study screened the plasma proteome of pediatric patients undergoing CPB procedures to identify potential clinical biomarkers related to tissue damage, inflammation, or other pathologies. Blood samples were collected at five different time points from 10 children undergoing a CPB procedure. Plasma was isolated and analyzed using two-dimensional differential in-gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry. Levels of differentially regulated proteins identified by two-dimensional differential in-gel electrophoresis, and related proteins were then measured in all time points and patients. As well, associated small molecules and ions were measured. The present study identified 13 proteins and protein isoforms altered in expression, including hemopexin, ceruloplasmin, inter-alpha inhibitor H4, and alpha-2-macroglobulin. Immunoblot analysis revealed significant decreases in each of these proteins during the CPB procedure. Significant changes in the levels of copper, iron, Hb, epinephrine, norepinephrine, and serotonin were observed. The potential markers of pathology (inflammation, oxidative stress) identified during this preliminary study may illuminate opportunities for preventative measures and/or treatments during and following CPB procedures in pediatric patients.

A retrospective chart review to identify perinatal factors associated with food allergies

BackgroundGut flora are important immunomodulators that may be disrupted in individuals with atopic conditions. Probiotic bacteria have been suggested as therapeutic modalities to mitigate or prevent food allergic manifestations. We wished to investigate whether perinatal factors known to disrupt gut flora increase the risk of IgE-mediated food allergies.MethodsBirth records obtained from 192 healthy children and 99 children diagnosed with food allergies were reviewed retrospectively. Data pertaining to delivery method, perinatal antibiotic exposure, neonatal nursery environment, and maternal variables were recorded. Logistic regression analysis was used to assess the association between variables of interest and subsequent food allergy diagnosis.ResultsRetrospective investigation did not find perinatal antibiotics, NICU admission, or cesarean section to be associated with increased risk of food allergy diagnosis. However, associations between food allergy diagnosis and male gender (66 vs. 33; p=0.02) were apparent in this cohort. Additionally, increasing maternal age at delivery was significantly associated with food allergy diagnosis during childhood (OR, 1.05; 95% CI, 1.017 to 1.105; p=0.005).ConclusionsGut flora are potent immunomodulators, but their overall contribution to immune maturation remains to be elucidated. Additional understanding of the interplay between immunologic, genetic, and environmental factors underlying food allergy development need to be clarified before probiotic therapeutic interventions can routinely be recommended for prevention or mitigation of food allergies. Such interventions may be well-suited in male infants and in infants born to older mothers.

TPH2 in the ventral tegmental area of the male rat brain.

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain. TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

Human TPH2 (hTPH2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.

The V81M variant of tyrosine hydroxylase is associated with more severe freezing of gait in Parkinson's disease

INTRODUCTION Many of the symptoms and signs of Parkinsons disease (PD) arise from the death of midbrain dopamine neurons that utilize tyrosine hydroxylase (TH) as the rate-limiting enzyme in catecholamine biosynthesis. METHODS We investigated whether the presence of a common TH polymorphism affects the clinical outcomes in 101 PD subjects. We further examined the effect of this polymorphism on the purified recombinant enzyme. RESULTS PD subjects homozygous for the common V81M polymorphism, have higher overall freezing of gait scores after controlling for disease duration, although this polymorphism does not associate with the occurrence of PD or FOG. In vitro functional assays on pure recombinant wild type TH and V81M TH revealed that the Km of the mutant enzyme for tyrosine was twice that of the wild-type. This polymorphism, however, did not change the stability of the enzyme, nor did it affect the Vmax or Km for the co-substrate BH4. CONCLUSION The data suggest that presence of a homozygous V81M polymorphism is associated with more severe FOG, possibly due to lower catecholamine synthetic capacity. Further studies are warranted to investigate the role of subtle changes in catecholamine availability in the development of FOG.

Gender-specific regulation of tyrosine hydroxylase in thymocyte differentiation antigen-1 knockout mice

Thymocyte differentiation antigen‐1 (Thy‐1) is a cell surface glycoprotein found on T cells and neurons and is involved in cell‐to‐cell interactions. In addition, Thy‐1 knockouts (KO) are a potential mouse model of restless legs syndrome (RLS) based on clinical observations and the role of dopamine in the disease. In this study, we analyzed the activity and quantity of tyrosine hydroxylase (TH; the rate‐limiting enzyme in dopamine production) and determined phosphorylation levels for the enzyme phosphoserine‐40 (pSer‐40). There was no significant difference in the total TH activity and pSer‐40 TH levels between Thy‐1 KO and control groups; however, TH specific activity was significantly lower (by 26%) in Thy‐1 KO mice. This difference is due in part to increased TH protein levels in this group (increased by 29%). When analyzed by gender, Thy‐1 KO female mouse striata contained less TH specific activity compared with control females (decreased by 41%) and male control or Thy‐1 KO animals (decreased by 30%). TH specific activity and pSer‐40 TH levels in male Thy‐1 KO and control displayed no differences. However, pSer‐40 TH was significantly higher in control females (38%) compared with control or Thy‐1 KO males. The Thy‐1 KO females exhibited significantly lower (28%) pSer‐40 TH (normalized to GAPDH or TH) than control females. Indeed, the Thy‐1 KO females had 50% of the pSer‐40 TH found in controls. Our results suggest a gender effect on TH specific activity, TH protein levels, and serine‐40 phosphorylation of TH in Thy‐1 KO female mice.