Tyrosine Transporter Research Articles

Transcriptional convergence after repeated duplication of an amino acid transporter gene leads to the independent emergence of the black husk/pericarp trait in barley and rice.

The repeated emergence of the same trait (convergent evolution) in distinct species is an interesting phenomenon and manifests visibly the power of natural selection. The underlying genetic mechanisms have important implications to understand how the genome evolves under environmental challenges. In cereal crops, both rice and barley can develop black-coloured husk/pericarp due to melanin accumulation. However, it is unclear if this trait shares a common origin. Here, we fine-mapped the barley HvBlp gene controlling the black husk/pericarp trait and confirmed its function by gene silencing. The result was further supported by a yellow husk/pericarp mutant with deletion of the HvBlp gene, derived from gamma ray radiation of the wild-type W1. HvBlp encodes a putative tyrosine transporter homologous to the black husk gene OsBh4 in rice. Surprisingly, synteny and phylogenetic analyses showed that HvBlp and OsBh4 belonged to different lineages resulted from dispersed and tandem duplications, respectively, suggesting that the black husk/pericarp trait has emerged independently. The dispersed duplication (dated at 21.23 MYA) yielding HvBlp occurred exclusively in the common ancestor of Triticeae. HvBlp and OsBh4 displayed converged transcription in husk/pericarp tissues, contributing to the black husk/pericarp trait. Further transcriptome and metabolome data identified critical candidate genes and metabolites related to melanin production in barley. Taken together, our study described a compelling case of convergent evolution resulted from transcriptional convergence after repeated gene duplication, providing valuable genetic insights into phenotypic evolution. The identification of the black husk/pericarp genes in barley also has great potential in breeding for stress-resilient varieties with higher nutritional values.

High value ferulic acid biosynthesis using modular design and spent coffee ground in engineered Escherichia coli chassis

Sophisticated genetic engineering enables microbial hosts to derive high-value aromatics in a green manner. Ferulic acid (FA) is one of the noteworthy aromatics due to its potent pharmacokinetic properties. However, the current approaches to FA biosynthesis still decamp from time- and cost-effectiveness. Herein, FA pathway was artificially reconstructed in Escherichia coli using modular designs. Comprehensive screening of E. coli lineages was reckoned for efficient synthesis of p-coumaric acid (pCA) as a precursor and FA eventually. The modular design was further advanced by harboring tyrosine transporter, adapting the heterologous codon, utilizing pCA symporter, and enriching FADH2 cofactor pools via in vivo regeneration. Taken together with simultaneous optimization of culture condition, a remarkable FA yield of 972.6 mg/L with 89.4 % conversion was achieved in 48 h, circumventing the time-consuming issue. Moreover, this study successfully exported inexpensive precursor from spent coffee ground for the first time, paving the economical way of FA biosynthesis.

The tyrosine transporter of Toxoplasma gondii is a member of the newly defined apicomplexan amino acid transporter (ApiAT) family.

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites.

Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3.

Summary Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium‐dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3‐dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5‐3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5‐3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non‐phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5‐3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

Proinflammatory Cytokines and Oxidative Stress Decrease the Transport of Dopamine Precursor Tyrosine in Human Fibroblasts

Background: Proinflammatory cytokines and oxidative stress responses have been extensively implicated in the pathophysiology of neuropsychiatric disorders over the past 2 decades. Moreover, disturbed transport of the dopamine precursor (i.e., the amino acid tyrosine) has been demonstrated, in different studies, across fibroblast cell membranes obtained from neuropsychiatric patients. However, the role and influences of proinflammatory cytokines and oxidative stress, and the reasons for disturbed tyrosine transport in neuropsychiatric disorders, are still not evaluated. Aims: The present study aimed to assess the role of proinflammatory cytokines and oxidative stress, indicated in many neuropsychiatric disorders, in tyrosine transportation, by using human skin-derived fibroblasts. Methods: Fibroblasts obtained from a healthy control were used in this study. Fibroblasts were treated with proinflammatory cytokines (IL-1β, IFN-γ, IL-6, TNF-α), their combinations, and oxidative stress, optimized for concentrations and incubation time, to analyze the uptake of ¹⁴C-tyrosine compared to untreated controls. Results and Conclusion: This study demonstrates that proinflammatory cytokines and oxidative stress decrease the transport of tyrosine (47% and 33%, respectively), which can alter dopamine synthesis. The functionality of the tyrosine transporter could be a new potential biomarker to target for discovering new drugs to counteract the effects of proinflammatory cytokines and oxidative stress in the pathophysiology of neuropsychiatric disorders.

Genetic and Functional Study of L-Type Amino Acid Transporter 1 in Schizophrenia

Schizophrenia involves neural catecholaminergic dysregulation. Tyrosine is the precursor of catecholamines, and its major transporter, according to studies on fibroblasts, in the brain is the <smlcap>L</smlcap>-type amino acid transporter 1 (LAT1). The present study assessed haplotype tag single-nucleotide polymorphisms (SNPs) of the SLC7A5/LAT1 gene in 315 patients with psychosis within the schizophrenia spectrum and 233 healthy controls to investigate genetic vulnerability to the disorder as well as genetic relationships to homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG), the major catecholamine metabolites in the cerebrospinal fluid (CSF). Moreover, the involvement of the different isoforms of the system L in tyrosine uptake and LAT1 tyrosine kinetics were studied in fibroblast cell lines of 10 patients with schizophrenia and 10 healthy controls. The results provide suggestive evidence of individual vulnerability to schizophrenia related to the LAT1 SNP rs9936204 genotype. A number of SNPs were nominally associated with CSF HVA and MHPG concentrations but did not survive correction for multiple testing. The LAT1 isoform was confirmed as the major tyrosine transporter in patients with schizophrenia. However, the kinetic parameters (maximal transport capacity, affinity of the binding sites, and diffusion constant of tyrosine transport through the LAT1 isoform) did not differ between patients with schizophrenia and controls. The present genetic findings call for independent replication in larger samples, while the functional study seems to exclude a role of LAT1 in the aberrant transport of tyrosine in fibroblasts of patients with schizophrenia.

Homology modelling and virtual screening of P-protein in a quest for novel antimelanogenic agent and in vitro assessments.

An adequate knowledge on molecular mechanism of melanogenesis provides an opportunity to find the novel molecular targets for the discovery and development of new cosmetics. Among various genes, the OCA2 is being essential for proper melanin synthesis, and mutation or deletion of this gene leads to oculocutaneous albinism type 2. Thus, for this study, the product of this gene, that is P-protein, was targeted in quest for novel inhibitors as antimelanogenic agents. Based on pattern search of amino acid sequence and homology analysis, the protein structure was modelled. The role of this protein has been predicted as a tyrosine transporter of melanosomes. Thus, the molecular library was generated on the basis of tyrosine transporter inhibitor. Based on the dock score, 20 molecules have been considered as putative inhibitors for P-protein. Among these compounds, five molecules (compound #1, #4, #8, #13 and #17) were found to be quite effective as antimelanogenic without having any toxicity. Further investigations to establish the mechanism of action, the indirect methods such as tyrosinase assay, analysis for eumelanin and pheomelanins and investigation of mRNA levels were being carried out. The results from the studies offered a new lead in antimelanogenic therapy and may be very useful for further optimization work in developing them as novel depigmenting agents.

Improved Acid Stress Survival of Lactococcus lactis Expressing the Histidine Decarboxylation Pathway of Streptococcus thermophilus CHCC1524

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.

Complementary Genes That Cause Black Ripening Hulls in F1 Plants of Crosses between Indica and Japonica Rice Cultivars

The F1 plants of crosses between indica and japonica rice cultivars often have black hulls during ripening, even though both of the parental cultivars have yellow hulls. Complementary genes are suggested to be necessary for the black hull phenotype, and one of them is predicted to be Phr1, which encodes polyphenol oxidase. On the other hand, Bh4, which encodes a tyrosine transporter, is known to cause the black hull phenotype in wild rice species, Oryza rufipogon. However, the relationship between Bh4 and Phr1 in the black hull phenotype has not been elucidated. In this study, a genotype analysis of the segregating populations from the cross between an indica cultivar, Habataki, and a japonica cultivar, Arroz da Terra, indicated that only those plants that had both functional genes, Bh4 and Phr1, showed the black hull phenotype, suggesting that a complementarity of Bh4 and Phr1 was necessary for the black hull phenotype.

Dual Role for the Tyrosine Decarboxylation Pathway inEnterococcus faeciumE17: Response to an Acid Challenge and Generation of a Proton Motive Force

In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products.

The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis.

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.

Putative interhelical interactions within the PheP protein revealed by second-site suppressor analysis.

Highly conserved glycine residues within span I and span II of the phenylalanine and tyrosine transporter PheP were shown to be important for the function of the wild-type protein. Replacement by amino acids with increasing side chain volume led to progressive loss of transport activity. Second-site suppression studies performed with a number of the primary mutants revealed a tight packing arrangement between spans I and II that is important for function and an additional interaction between spans I and III. We also postulate that a third motif, GXXIG, present in span I and highly conserved within different members of the amino acid-polyamine-organocation family, may function as a dimerization motif. Surprisingly, other highly conserved residues, such as Y60 and L41, could be replaced by various residues with no apparent loss of activity.

Aberrant pH of Melanosomes in Pink-Eyed Dilution (p) Mutant Melanocytes

In past studies, we cloned the mouse p gene and its human homolog P, which is associated with oculocutaneous albinism type 2. Both mouse and human genes are expressed in melanocytes and encode proteins predicted to have 12 membrane-spanning domains with structural homology to known ion transporters. We have also demonstrated that the p protein is localized to the melanosomal membrane and does not function as a tyrosine transporter. In this study, immunohistochemistry and confocal microscopy were used to show that the p protein plays an important role in the generation or maintenance of melanosomal pH. Melanosomes (and their precursor compartments) were defined by antiserum directed against the melanosomal marker tyrosinase related protein 1. Acidic vesicles were identified by 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine incorporation, visualized with anti-dinitrophenol. In C57BL/6+/+ (wild-type) melanocytes, 94.2% of vesicles demonstrated colocalization of tyrosinase related protein 1 and 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine, indicating that almost all melanosomes or their precursors were acidic. By contrast, only 7%-8% of the staining vesicles in p mutant cell lines (pJ/pJ and pcp/p6H) showed colocalization of tyrosinase related protein 1 and 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine. Thus, without a functional p protein, most melanosomes and their precursors are not acidic. As mammalian tyrosinase activity in situ is apparently dependent on low pH, we postulate that in the absence of a low pH environment brought about by ionic transport mediated by the p protein, tyrosinase activity is severely impaired, leading to the minimal production of melanin that is characteristic of p mutants. Additionally (or alternatively), an abnormal pH may also impair the assembly of the normal melanogenic complex.

Normal Tyrosine Transport and Abnormal Tyrosinase Routing inPink-Eyed DilutionMelanocytes

The pink-eyed dilution phenotype in mice arises from mutations in thepgene; in humans, analogous mutations in thePgene result in oculocutaneous albinism type 2. Although the molecular mechanisms which underlie this phenotype remain obscure, it has been postulated that mutations inpresult in defective tyrosine transport into murine melanosomes, resulting in hypopigmentation and diminished coat color. However, we previously reported no difference in melanosomal tyrosine transport in unpigmented, melanoblast-like pink-eyed dilution (pcp/pcp), and in pigmented (melan-a) murine melanocytes. In this study, we utilized melan-p1 cells, more differentiated pink-eyed dilution (pcp/p25H) melanocytes which can be induced to produce melanin, to characterize the melanogenic lesion(s) more definitively. Uptake of [3H]tyrosine into melan-a melanosomes did not differ significantly from uptake into melanosomes derived from melan-p1 melanocytes, further arguing against its critical role as a tyrosine transporter. Pink-eyed dilution melanocytes incubated in high tyrosine concentrations became extremely pigmented as they became confluent and secreted large amounts of black material into the medium. Total cellular tyrosinase activity in melan-p1 melanocytes was significantly higher than that in melan-a melanocytes (which are wild-type at theplocus), but the localization of tyrosinase to melanosomes was impaired in melan-p1 melanocytes compared to melan-a melanocytes. These results indicate that mechanisms other than deficient tyrosine transport are involved in the pink-eyed dilution phenotype and that this protein may serve a chaperone-like or stabilizing function in melanocytes.

Tyrosine transporter in larval Leptinotarsa decemlineata midgut brush border membrane

An ion-dependent tyrosine transporter from Leptinotarsa decemlineata midgut was further characterized using brush border membrane vesicles. Tyrosine transport is not stimulated by Li +, Rb +, Cs +, Ca 2+, or Mg 2+. K + paired with SCN − stimulates tyrosine uptake, but K + paired with less permeant PO 4 2− or gluconate anions does not stimulate tyrosine uptake. KSCN stimulates initial tyrosine uptake in a concentration-dependent manner, reaching a maximum at 20 mM. Tyrosine transport is slower when moving against a positive electrical gradient than against a negative or neutral electrical gradient. In the presence of an inward K + gradient, tyrosine is not concentrated in BBMV above the equilibrium concentration. Tyrosine transport is highest at pH 6.4 and 7.4, and significantly lower at pH 9.4. This ion-dependent tyrosine transporter is insensitive to the amino acid analogs methylamino-isobutyric acid and 2-aminobicyclo-[2,2,11-heptane-2-carboxylic acid. None of eight plant lectins tested inhibited initial tyrosine uptake. The Bacillus thuringiensis Cry3A ∂-endotoxin, active against L. decemlineata in vivo, did not affect initial tyrosine uptake rate.

Melanosomal tyrosine transport in normal and pink-eyed dilution murine melanocytes.

Tyrosine is the endogenous substrate for melanin production within melanosomes, but the method of tyrosine transport into the melanosome has not been investigated. In the mouse, melanogenesis is disrupted by mutations in the p gene resulting in the pink-eyed dilution phenotype; it has been suggested that the p gene codes for a tyrosine transport protein. We determined that normal (melan-a) melanosome-rich granular fractions take up 10 microns [3H]tyrosine at 21.1 +/- 6.1 (SEM, standard error of the mean) pmol/min/mg protein (N = 7) compared with 21.3 +/- 5.8 SEM pmol/min/mg protein (N = 5) for pink-eyed dilution, whose plasma membrane tyrosine transport was also normal (Km 89 microM; Vmax 302 pmol/min/mg cell protein). We also demonstrated that pink-eyed dilution melanosomes are immature by virtue of their low density, high hexosaminidase activity, and lack of pigment. These data indicate that a tyrosine transport system exists in the melanosomal membrane and that the p gene does not encode a tyrosine transporter of critical importance.

A sodium- and potassium-stimulated tyrosine transporter from Leptinotarsa decemlineata midguts

Tyrosine transport across the midgut brush border membrane of Leptinotarsa decemlineata was characterized using brush border membrane vesicles (BBMV). The concentrations of tyrosine in potato foliage that are potentially available to the insect and the hemolymph tyrosine concentration were estimated. l-Tyrosine transport into BBMV is stimulated by SCN − and Cl − salts of K + and Na + under isosmotic conditions. Compared to ion-independent l-tyrosine transport, KSCN and NaSCN increased both the J max and K t for l-tyrosine uptake. Ion independent l-tyrosine uptake was strongly inhibited by leucine, phenylalanine, proline and serine and moderately inhibited by lysine, threonine, tryptophan and valine. The NaSCN stimulated tyrosine transporter was not affected by d-tyrosine and only cysteine inhibited uptake in the presence of NaSCN. This is the first report of ion-dependent amino acid transport in the Coleoptera and the first characterization of a tyrosine transporter in insects.

Molecular genetics of oculocutaneous albinism.

Albinism is a group of genetic disorders characterized by deficient synthesis of melanin pigment. In oculocutaneous albinism (OCA) the pigment deficiency involves the skin, hair, and eyes, whereas in ocular albinism (OA) the defect involves principally the visual system. Type I (tyrosinase-deficient) OCA results from deficient catalytic activity of tyrosinase, which catalyzes at least three steps in the melanin biosynthetic pathway. Type II (tyrosinase-positive) OCA results from abnormalities of the 'P' polypeptide, which may be a melanosomal tyrosine transporter. At least some forms of OA appear to represent mild presentations of types I and II OCA. The causes of several other forms of albinism have not yet been identified. Recent application of molecular genetic techniques to the study of these disorders has led to greatly improved knowledge of their molecular pathogenesis and relationships, and paves the way to improved diagnosis, carrier detection and prenatal diagnosis, and even to eventual treatment.

Diethylpyrocarbonate inhibition of sodium-glucose cotransport in kidney brush-border membrane vesicles

Exposure of kidney brush-border membrane vesicles to the acylating reagent diethylpyrocarbonate resulted in inactivation of the glucose transporter, as demonstrated by inhibition of sodium-coupled d-glucose transport and phlorizin binding. The transport site(s) was protected against inactivation by the simultaneous presence of sodium ions and d-glucose, and were partially protected by phlorizin. Transport activity was not restored by hydroxylamine; this rules out the possibility of diethylpyrocarbonate interaction with histidine, serine or tyrosine transporter residues. Dithiothreitol, a thiol protector, slightly prevented diethylpyrocarbonate inactivation. It is therefore suggested that (an) amino group(s) in the translocation complex is involved, at the level of the sugar transport site and the preferential protection of d-glucose against diethylpyrocarbonate inactivation related to a conformation change caused by the simultaneous binding of sodium and d-glucose to the cotransporter.