Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

14-3-3 proteins in pineal photoneuroendocrine transduction: how many roles?

Recent studies suggest that a common theme links the diverse elements of pineal photoneuroendocrine transduction--regulation via binding to 14-3-3 proteins. The elements include photoreception, neurotransmission, signal transduction and the synthesis of melatonin from tryptophan. We review general aspects of 14-3-3 proteins and their biological function as binding partners, and also focus on their roles in pineal photoneuroendocrine transduction.

Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina.

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N-acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.

14-3-3 Proteins and photoneuroendocrine transduction: role in controlling the daily rhythm in melatonin.

This paper describes the role 14-3-3 proteins play in vertebrate photoneuroendocrine transduction. 14-3-3 proteins form a complex with arylalkylamine N-acetyltransferase (AANAT), the enzyme which turns melatonin production on during the day and off at night. Complex formation is triggered at night by cAMP-dependent phosphorylation of the enzyme, and results in activation and protection against proteolysis. This enhances melatonin production >10-fold. Light exposure results in dephosphorylation of the enzyme and disassociation from 14-3-3, leading to destruction and a rapid drop in melatonin production and release and circulating levels.

Characterization of the Saccharomyces cerevisiae homolog of the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (EC 2.3.1.87).

Arylalkylamine N-acetyltransferase (AANAT, serotonin N-acetyltransferase, EC ) plays a unique transduction role in vertebrate physiology by converting information about day and night into a hormonal signal: melatonin. Only vertebrate members of the AANAT family have been functionally characterized. Here a putative AANAT from Saccharomyces cerevisiae (scAANAT) was studied to determine whether it possessed the catalytic activity of the vertebrate enzyme. scAANAT is 47% similar to ovine AANAT, but lacks the regulatory N- and C-terminal flanking regions conserved in all vertebrate AANATs. It was found to have enzyme activity generally typical for AANAT family members, although the substrate preference pattern was somewhat broader, the specific activity was lower, and the pH optimum was higher. Deletion of scAANAT reduced arylalkylamine acetylation by S. cerevisiae extracts, indicating that scAANAT contributes significantly to this process. The scAANAT sequence conformed to the three-dimensional structure of ovine AANAT catalytic core; however, an important structural element (loop 1) was found to be shorter and to lack a proline involved in substrate binding. These differences could explain the lower specific activity of scAANAT, because of the importance of loop 1 in catalysis. Data base analysis revealed the presence of putative AANATs in other fungi but not in the nearly complete genomes of Drosophila melanogaster or Caenorhabditis elegans. These studies indicate that the catalytic and kinetic characteristics of fungal and vertebrate enzymes can be considered to be generally similar, although some differences exist that appear to be linked to changes in one structural element. Perhaps the most striking difference is that fungal AANATs lack the regulatory domains of the vertebrate enzyme, which appear to be essential for the regulatory role the enzyme plays in photochemical transduction.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC ). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN --> RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the K(m) for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Crystal structure of the 14-3-3zeta:serotonin N-acetyltransferase complex. a role for scaffolding in enzyme regulation.

Serotonin N-acetyltransferase (AANAT) controls the daily rhythm in melatonin synthesis. When isolated from tissue, AANAT copurifies with isoforms epsilon and zeta of 14-3-3. We have determined the structure of AANAT bound to 14-3-3zeta, an association that is phosphorylation dependent. AANAT is bound in the central channel of the 14-3-3zeta dimer, and is held in place by extensive interactions both with the amphipathic phosphopeptide binding groove of 14-3-3zeta and with other parts of the central channel. Thermodynamic and activity measurements, together with crystallographic analysis, indicate that binding of AANAT by 14-3-3zeta modulates AANAT's activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding.

Regulation of arylalkylamine N-acetyltransferase-2 (AANAT2, EC 2.3.1.87) in the fish pineal organ: evidence for a role of proteasomal proteolysis.

In fish, individual photoreceptor cells in the pineal organ and retina contain complete melatonin rhythm generating systems. In the pike and seabream, this includes a photodetector, circadian clock, and melatonin synthesis machinery; the trout lacks a functional clock. The melatonin rhythm is due in part to a nocturnal increase in the activity of the arylalkylamine N-acetyltransferase (AANAT) which is inhibited by light. Two AANATs have been identified in fish: AANAT1, more closely related to AANATs found in higher vertebrates, is specifically expressed in the retina; AANAT2 is specifically expressed in the pineal organ. We show that there is a physiological day/night rhythm in pineal AANAT2 protein in the pike, and that light exposure at midnight decreases the abundance of AANAT2 protein and activity. In culture, this decrease is blocked by inhibitors of the proteasomal degradation pathway. If glands are maintained under light at night, treatment with these inhibitors increases AANAT2 activity and protein. Organ culture studies with the trout and seabream also indicate that the light-induced decrease of AANAT2 activity is prevented when proteasomal proteolysis is blocked. A cAMP-dependent pathway protects AANAT2 protein from degradation. These results provide a clue to understanding how light regulates the daily rhythm in melatonin secretion in fish photoreceptor cells and provides evidence that proteasomal proteolysis is a conserved element in the regulation of AANAT in vertebrates.

cAmp regulation of arylalkylamine N-acetyltransferase (AANAT, EC 2.3.1.87): a new cell line (1E7) provides evidence of intracellular AANAT activation.

Arylalkylamine N-acetyltransferase (serotonin N-acetyltransferase, AANAT, EC ) is the penultimate enzyme in melatonin synthesis. As described here, a cell line (1E7) expressing human AANAT (hAANAT) has been developed to study the human enzyme. 1E7 hAANAT is detectable in immunoblots as a 23-kDa band and is immunocytochemically visualized in the cytoplasm. The specific concentration of hAANAT in homogenates is comparable to that of the night rat pineal gland. Kinetics of AANAT extracted from 1E7 cells are the same as those of bacterially expressed hAANAT; both preparations of hAANAT are equally sensitive to the inhibitor CoA-S-N-acetyltryptamine. Studies of cAMP regulation indicate that treatment with forskolin, dibutyryl cAMP, isobutylmethylxanthine, or isoproterenol activate cellular hAANAT within intact 1E7 cells approximately 8-fold without markedly increasing the abundance of AANAT protein or the activity of AANAT in broken cell preparations; and, that forskolin, isobutylmethylxanthine and isoproterenol elevate cyclic AMP production. These observations extend our understanding of cAMP regulation of AANAT activity, because it is currently thought that this only involves changes in the steady-state levels of AANAT protein. This previously unrecognized switching mechanism could function physiologically to control melatonin production without changing AANAT protein levels.

Selective adrenergic/cyclic AMP-dependent switch-off of proteasomal proteolysis alone switches on neural signal transduction: an example from the pineal gland.

The molecular processes underlying neural transmission are central issues in neurobiology. Here we describe a novel mechanism through which noradrenaline (NA) activates its target cells, using the mammalian pineal organ as a model. In this neuroendocrine transducer, NA stimulates arylalkylamine N:-acetyltransferase (AANAT; EC 2.3.1. 87), the key enzyme regulating the nocturnal melatonin production. In rodents, AANAT protein accumulates as a result of enhanced transcription, but in primates and ungulates, the AANAT mRNA level fluctuates only marginally, indicating that other mechanisms regulate AANAT protein and activity. These were investigated in cultured bovine pinealocytes. AANAT mRNA was readily detectable in unstimulated pinealocytes, and levels did not change following NA treatment. In contrast, NA increased AANAT protein levels in parallel with AANAT activity, apparently through a cyclic AMP-mediated mechanism. Immunocytochemistry revealed that the changes in AANAT protein levels occurred in virtually all pinealocytes. Inhibition of AANAT degradation by proteasomal proteolysis alone was found to switch-on enzyme activity by increasing AANAT protein levels five- to 10-fold. Accordingly, under unstimulated conditions AANAT protein is continually synthesized and immediately destroyed by proteasomal proteolysis. NA appears to act via cyclic AMP to protect AANAT from proteolytic destruction, resulting in accumulation of the protein. These findings show that tightly regulated control of proteasomal proteolysis of a specific protein alone can play a pivotal role in neural regulation.

Characterization of the chicken serotonin N-acetyltransferase gene. Activation via clock gene heterodimer/E box interaction.

The abundance of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) mRNA in the chicken pineal gland exhibits a circadian rhythm, which is translated into a circadian rhythm in melatonin production. Here we have started to elucidate the molecular basis of the circadian rhythm in chicken AANAT (cAANAT). The 5'-flanking region of the cAANAT gene was isolated and found to contain an E box DNA element that confers strong luciferase reporter activity. In transfection experiments using chicken pineal cells, an E box mutation dramatically decreased reporter activity. Northern blot analysis indicated that several putative clock genes (bmal1, Clock, and MOP4) are co-expressed in the chicken pineal gland. bmal1 mRNA is expressed in a rhythmic manner in the chicken pineal gland, with peak levels at early subjective night, coincident with the increase in cAANAT expression. Co-transfection experiments in COS cells demonstrated that chicken BMAL1/CLOCK and human BMAL1/MOP4 heterodimers bound the AANAT E box element and enhanced transcription. These observations suggest that binding of clock gene heterodimers to the cAANAT E box is a critical element in the expression of the cAANAT gene in vitro.

GCN5-related N-acetyltransferases: a structural overview.

Hundreds of acetyltransferases exist. All use a common acetyl donor--acetyl coenzyme A--and each exhibits remarkable specificity for acetyl acceptors, which include small molecules and proteins. Analysis of the primary sequences of these enzymes indicates that they can be sorted into several superfamilies. This review covers the three-dimensional structures of members of one of these superfamilies, now referred to in the literature as the GCN5-related N-acetyltransferases (GNAT), reflecting the importance of one functional category, the histone acetyltransferases. Despite the diversity of substrate specificities, members of the GNAT superfamily demonstrate remarkable similarity in protein topology and mode of acetyl coenzyme A binding, likely reflecting a conserved catalytic mechanism.

Differential regulation of fos family genes in the ventrolateral and dorsomedial subdivisions of the rat suprachiasmatic nucleus.

Extensive studies have established that light regulates c-fos gene expression in the suprachiasmatic nucleus, the site of an endogenous circadian clock, but relatively little is known about the expression of genes structurally related to c-fos, including fra-1, fra-2 and fosB. We analysed the photic and temporal regulation of these genes at the messenger RNA and immunoreactive protein levels in rat suprachiasmatic nucleus, and we found different expression patterns after photic stimulation and depending on location in the ventrolateral or dorsomedial subdivisions. In the ventrolateral suprachiasmatic nucleus, c-fos, fra-2 and fosB expression was stimulated after a subjective-night (but not subjective-day) light pulse. Expression of the fra-2 gene was prolonged following photic stimulation, with elevated messenger RNA and protein levels that appeared unchanged for at least a few hours beyond the c-fos peak. Unlike c-fos and fra-2, the fosB gene appeared to be expressed constitutively in the ventrolateral suprachiasmatic nucleus throughout the circadian cycle; immunohistochemical analysis suggested that delta FosB was the protein product accounting for this constitutive expression, while FosB was induced by the subjective-night light pulse. In the dorsomedial suprachiasmatic nucleus, c-fos and fra-2 expression exhibited an endogenous circadian rhythm, with higher levels during the early subjective day, although the relative abundance was much lower than that measured after light pulses in the ventrolateral suprachiasmatic nucleus. Double-label immunohistochemistry suggested that some of the dorsomedial cells responsible for the circadian expression of c-Fos also synthesized arginine vasopressin. No evidence of suprachiasmatic nucleus fra-1 expression was found. In summary, fos family genes exhibit differences in their specific expression patterns in the suprachiasmatic nucleus, including their photic and circadian regulation in separate cell populations in the ventrolateral and dorsomedial subdivisions. The data, in combination with our previous results [Takeuchi J. et al. (1993) Neuron 11, 825-836], suggest that activator protein-1 binding sites on ventrolateral suprachiasmatic nucleus target genes are constitutively occupied by DeltaFosB/JunD complexes, and that c-Fos, Fra-2, FosB and JunB compete for binding after photic stimulation. The differential regulation of fos family genes in the ventrolateral and dorsomedial suprachiasmatic nucleus suggests that their circadian function(s) and downstream target(s) are likely to be cell specific.

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein.

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.

Melatonin synthesis: arylalkylamine N-acetyltransferases in trout retina and pineal organ are different.

Serotonin N-acetyltransferase (AANAT) is the first enzyme in the conversion of serotonin to melatonin. Changes in AANAT activity determine the daily rhythm in melatonin secretion. Two AANAT genes have been identified in the pike, pAANAT-1 and pAANAT-2, expressed in the retina and in the pineal, respectively. The genes preferentially expressed in these tissues encode proteins with distinctly different kinetic characteristics. Like the pike, trout retina primarily expresses the AANAT-1 gene and trout pineal primarily expresses the AANAT-2 gene. Here we show that the kinetic characteristics of AANAT in these tissues differ as in pike. These differences include optimal temperature for activity (pineal: 12 degrees C; retina: 25 degrees C) and relative affinity for indoleethylamines compared to phenylethylamines. In addition, retinal AANAT exhibited substrate inhibition, which was not seen with pineal AANAT. The kinetic differences between AANAT-1 and AANAT-2 appear to be defining characteristics of these gene subfamilies, and are not species specific.

Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities.

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 +/- 24.4 vs. H: 344.1 +/- 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 +/- 6.7 vs. H: 119.1 +/- 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

Serotonin N-acetyltransferase mRNA levels in photoreceptor-enriched chicken retinal cell cultures: elevation by cyclic AMP.

Serotonin N-acetyltransferase (AA-NAT; arylalkylamine N-acetyltransferase; EC 2.3.1.87) is a key regulatory enzyme in the biosynthesis of melatonin. Previous studies have shown that the activity of this enzyme in the chicken retina is regulated by a cyclic AMP-dependent mechanism. In the present report, we investigated whether cyclic AMP can regulate the levels of AA-NAT mRNA in photoreceptor-enriched chick retinal cell cultures. AA-NAT mRNA levels were elevated by acute treatment with cyclic AMP protagonists, including forskolin; this response was blocked by H-89, a selective inhibitor of cyclic AMP-dependent protein kinase. Forskolin did not alter the rate of disappearance of AA-NAT mRNA in actinomycin D-treated cells, suggesting that cyclic AMP enhances transcription of the AA-NAT gene. Forskolin-induced elevation of AA-NAT mRNA levels was enhanced by cycloheximide, which decreased the degradation of the transcript in cells treated with actinomycin D. These studies indicate that the abundance of AA-NAT mRNA is regulated in part through a cyclic AMP-dependent mechanism.

Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function.

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.

Melatonin synthesis pathway: circadian regulation of the genes encoding the key enzymes in the chicken pineal gland and retina.

The mRNAs encoding three enzymes of the melatonin synthesis pathway (tryptophan hydroxylase (TPH), arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyl-transferase (HIOMT)) are expressed with a day/night rhythm in the chicken pineal gland and retina. TPH and AANAT mRNA levels reach their peak at night. HIOMT mRNA levels peak at night in the retina, but during the day in the pineal gland. In this tissue, the rhythm of TPH, AANAT and HIOMT mRNA levels persisted in constant darkness (DD), both in vivo and in vitro, indicating that the three genes are controlled by the circadian oscillator of the chicken pineal. In the retina, the rhythms of TPH and AANAT mRNA levels also persisted in DD in vivo, suggesting that they are driven by a circadian oscillator. In contrast, the rhythm of HIOMT mRNA in the retina appeared to be controlled only by light. The clones of chicken AANAT and HIOMT genes that we have isolated should help us to understand the molecular mechanisms of: 1) their transcriptional regulation by circadian oscillators and by light; 2) their tissue-specific expression in the pineal gland and the retina.

The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 A resolution with a bisubstrate analog.

Serotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis.