We focus on pineal gland function and how the production of melatonin, the pineal hormone, is regulated. The work has broad implications in biology and is of special interest to clinical scientists studying human diseases relating to circadian rhythms, including endocrine pathologies, sleep disturbances, mood disorders, and deficiencies in alertness.
Arylalkylamine N-acetyltransferase, the melatonin rhythm enzyme
Ganguly, Coon, Namboodiri, Falcón, Ho, Pollard, Weller, Klein; in collaboration with Chemineau, Cole, Dawid, Dyda, Gothilf, Hickman, Iuvone, Jaffe, Koonin, Malpaux, Schomerus, Shi, Toyama, Zhen
We have been investigating arylalkylamine N-acetyltransferase (AANAT), the second enzyme in melatonin synthesis from serotonin (see Figure 26.1), and identified this enzyme as critical to the control of the rhythm in melatonin synthesis. In all species examined thus far, the large increase in melatonin synthesis at night causes an increase in the production of melatonin.
Our cloning of the enzyme allowed significant further advances in understanding the control of the enzyme through transcriptional and translational control. Transcriptional mechanisms include interactions of cyclic AMP response elements in the AANATpromoter; other response elements in the promoter also appear to be involved, making expression of the enzyme highly tissue-specific. Such transcriptional events control both tissue specificity and, in some but not all vertebrates, the rhythmic control of expression of the AANAT gene. For example, we determined that rodents undergo an approximately 100-fold increase in the expression of the AANATgene at night while ungulates and primates experience a much smaller increase, indicating that transcriptional mechanisms are not essential in controlling melatonin synthesis in all vertebrates. With regard to post-translational regulation of the enzyme, sequence analysis has revealed that all AANATs contain C- and N-terminal sites for phosphorylation by cyclic AMP–dependent protein kinase. We have found that the sites are critical in the control of the stability and biological half-life of the enzyme.
Phosphorylation of the sites controls binding of AANAT to a protective binding partner, 14-3-3 protein. Binding protects the enzyme from destruction while reversal of the binding can lead to destruction. Thus, phosphorylation-dependent binding of AANAT to 14-3-3 governs the balance between destruction and protection. In collaboration with NIDDK, we determined the molecular basis of the binding interaction by analyzing of the crystal structure formed by AANAT and 14-3-3- proteins. Recent research focused on one of the phosphorylation sites of AANAT, which is located in the C-terminal region. Joan Weller developed highly specific antisera, which revealed that the C-terminal site is phosphorylated at night and that the phosphorylation state of the enzyme decreases immediately after animals are exposed to light. This process is thought to lead to destruction of the enzyme.
Surajit Ganguly explored the functional role of phosphorylation of AANAT by establishing a highly sensitive method to measure binding of AANAT to 14-3-3. The method underlines the importance of phosphorylation and that the entire AANAT molecule appears to contribute substantially to the binding affinity toward 14-3-3 protein. Ganguly found that both the C- and N-terminal PKA sites are important for binding 14-3-3 proteins. An on/off mechanism centered around the C-terminal PKA site switches the enzyme from the active to inactive state. In a collaborative effort with Philip Cole, we used novel synthetic approaches to examine the role of phosphorylation in controlling AANAT, resulting in AANATs that contain non-hydrolyzable phosphate at the N-terminal PKA site. The biological half-life of such AANATs indicates that the non-hydrolyzable phosphate at the N-terminal PKA site prevents destruction of AANAT. Steven Coon studied the role of such post-translational regulatory mechanisms in the monkey pineal AANAT. He found large changes in the abundance of AANAT protein and activity, whereas AANAT mRNA, that is, gene expression, does not change. Accordingly, it appears that, in primates, the primary mechanism for regulating AANAT protein is post-translational.
Appelbaum L, Toyama R, Dawid IB, Klein DC, Baler R, Gothilf Y. Zebrafish serotonin-N-acetyltransferase-2 gene regulation: pineal-restrictive downstream module (PRDM) contains a functional E-box and three photoreceptor conserved elements. Mol Endocrinol2004;18:1210-1221.
Falcón J, Gothilf Y, Coon SL, Boeuf G, Klein DC. Genetic, temporal and developmental differences between melatonin rhythm generating systems in the teleost fish pineal organ and retina. J Neuroendocrinol 2003;15:378-382.
Iuvone PM, Tosini G, Pozdeyev N, Haque R, Klein DC, Chaurasia SS. Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Prog Retinal Eye Res 2005;24:433-456.
Zheng W, Schwarzer D, LeBeau A, Weller JL, Klein DC, Cole PA. Cellular stability of serotonin N-acetyltransferase conferred by Ser205 phosphono-difluoromethylene alanine (Pfa) substitution. J Biol Chem 2005;280:10462-10467.
Characterization of 14-3-3 proteins in the pineal gland
Shi, Ganguly, Klein; in collaboration with Aitken
Qiong Shi used a comparative approach to analyze each 14-3-3 isoform. In collaboration with Alistair Aitken, he determined the developmental appearance of each isoform in the pineal gland based on both mRNA and protein analysis; the latter revealed marked differences in the relative abundance of each isoform. In addition, Shi identified changes in the cellular distribution of some but not all isoforms following adrenergic activation. We also identified the most efficient 14-3-3 isoform as regards binding and activation of AANAT; the most abundant form of 14-3-3 in the pineal gland is also the most potent in binding and activating the enzyme.
Besseau L, Benyassi A, Møller M, Coon SL, Weller JL, Boeuf G, Klein DC, Falcón J. Melatonin pathway: breaking the “high-at-night” rule in trout retina. Exp Eye Research 2005 [Epub ahead of print].
Ganguly S, Weller JL, Ho A, Chemineau P, Malpaux B, Klein DC. Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc Natl Acad Sci USA 2005;102:1222-1227.
Global analysis of gene expression
Coon, Gaildrat, Ganguly, Morin, Kim, Weller, Klein; in collaboration with Baler, Blackshaw, Carter, Hogenesch, Humphries, Møller, Munson
We initiated several projects aimed at (1) obtaining a global picture of differences in gene expression that occur on a night/day basis and (2) identifying genes that are highly enriched in the pineal gland. Initially, we analyzed a relatively small number of genes but are now using microarrays, which permit the examination of tens of thousands of genes and expressed sequence tags (ESTs). In addition to using commercially available microarrays, we participated in the NICHD initiative to develop a zebrafish microarray that will soon be commercially available.
In collaboration with Peter Munson, Steven Coon and other members of the section already identified a set of genes highly expressed in the pineal gland of several species. Those of several vertebrates include the genes known to be associated with melatonin production and visual signal transduction as well as several genes new to the pineal literature. The research is increasing our knowledge of the biochemical profile of the pineal gland conserved across species and is pointing to new transcriptional pathways controlled by previously unrecognized transcription factors. By analyzing the transcription factors and the promoters of genes that are either upregulated at night or highly expressed in the pineal gland, we will be able to construct a regulatory network that describes the cascade of transcription factors underlying the control of pineal gene expression. In addition to obtaining a more complete understanding of gene expression, we identified potentially new and important pathways involved in cell-cell communication and signal transduction in the pineal gland. Moreover, expression of some genes suggests new functions for the pineal gland. The available data indicate that many genes are regulated by a similar mechanism, as outlined in Figure 26.3.
Humphries A, Weller J, Klein DC, Baler R, Carter DA. NGFI-B (Nurr77/Nr4a1) orphan nuclear receptor in rat pinealocytes: circadian expression involves an adrenergic-cyclic AMP mechanism. J Neurochem2004;91:946-955.
MAP kinase signaling in the pineal gland
Klein, Weller; in collaboration with Carter, Ho
The norepinephrine-driven increase in mitogen-activated protein kinase (MAPK) activity is part of the mechanism that regulates arylalkylamine N-acetyltransferase (AA-NAT) activity in the rat pineal gland. In collaboration with Anthony Ho, the leading expert on MAPK signaling in the pineal gland, and his group, we identified a gene via microarray screening that encodes a phosphatase that acts on MAPK. We detected a very rapid nocturnal increase in the expression of a MAPK phosphatase, MAP kinase phosphatase-1 (MKP-1), that was blocked by maintaining animals in constant light or treating them with propranolol. Acting through both alpha- and beta-adrenergic receptors, norepinephrine regulated MKP-1 expression. The results demonstrate a nocturnal increase in pineal MKP-1 expression in the pineal gland that is under the control of a photoneural system. Given that substrates of MKP-1 can influence AA-NAT activity, MKP-1 is likely involved in the regulation of the nocturnal AA-NAT signal and melatonin production.
Price DM, Chik CL, Weller J, Humphries A, Carter DA, Klein DC, Ho AK. Mitogen activated protein kinase phosphatase-1 (MKP-1): >100-fold nocturnal and norepinephrine-induced changes in the rat pineal gland. FEBS Lett 2004;577:220-226.
Regulation of S-adenosyl methionine, phosphodiesterase 4B2, and acetyl coenzyme A synthesis
Kim, Morin, Klein; in collaboration with Charlton, Møller
S-adenosylmethionine is the co-factor of the last enzyme in the melatonin synthesis pathway. Work by Jong-so Kim showed that the expression of methionine adenosyl transferase 2a (MAT2a), the enzyme that produces this co-factor in the pineal gland, rises at night and is accompanied by an increase in enzyme activity and enzyme protein, which are both linked to the increased requirement for this methyl donor. The molecular basis of the increase involves neural stimulation of the pineal gland by norepinephrine, which results in the elevation of cyclic AMP. Regulation of the synthesis of S-adenosylmethionine by neural mechanisms has not been previously described, even though the co-factor plays a central role in the synthesis and metabolism of many transmitters (catecholamines, indoles, histamine, and so forth). Accordingly, evidence from the pineal gland suggesting that the activity of MAT2a can be regulated by a neural circuit via a cyclic AMP mechanism points to the possibility that the activity of MAT2a might be regulated by transmitters in other brain regions; furthermore, the levels of S-adenosylmethionine might be controlled through pharmacological manipulation of MAT2a expression. Thus, it is reasonable to view the serotonin-to-melatonin pathway as controlled by the same neural and second-messenger system at two points: AANAT and S-adenosymethionine production. The clinical impact of our work lies in the likelihood that strong and rapid control of melatonin production occurs downstream of the enzyme that was thought to be the only regulated element of the serotonin-to-melatonin pathway.
The results of microarray analysis of the pineal gland suggested that expression of the gene encoding a cyclic AMP–selective phosphodiesterase, termed PDE4B2, is elevated at night. Jong-So Kim found that mRNA encoding this isoform is over five times higher at night than during the day and that expression in the pineal gland is higher than in other tissues, as confirmed by Morten Møller using in situ hybridization (see Figure 26.5). Kim has also discovered that the increase in mRNA is associated with an increase in protein and activity, thus influencing the accumulation of cyclic AMP. Expression of the PDE4B2 gene is controlled by the same neural pathway that regulates AANAT and MAT; in addition, cyclic AMP controls expression of the PDE4B2gene, representing a negative feedback mechanism. The microarray analysis is the first demonstration of a negative feedback mechanism involving cyclic AMP destruction in the pineal gland, which represents an internal interval timer or means of telling time.
Fabrice Morin investigated ATP-dependent citrate lyase, the enzyme that regulates the formation of acetyl coenzyme A, and established that the pineal gland expresses much higher levels of the enzyme than other tissues and that, in some species, the abundance of mRNA encoding the enzyme changes daily. Morin obtained evidence of a physical interaction between ATP-dependent citrate lyase and AANAT, which would bring the source of acetyl coenzyme A directly into contact with AANAT and promote efficient acetylation of serotonin and melatonin. It appears possible that phosphorylation might regulate the association and could provide an important element in the general activation of the pineal gland in supporting the nocturnal increase in melatonin production. Morin also demonstrated that inhibition of ATP-dependent citrate lyase reduces melatonin synthesis, establishing that acetyl coenzyme A production is a potential regulatory site in melatonin synthesis.
Kim J-S, Coon SL, Blackshaw S, Cepko CL, Møller M, Mukda S, Zhao W-Q, Charlton CG, Klein DC. Methionine adenosyltransferase (MAT): adrenergic-cyclic AMP mechanism regulates a daily rhythm in pineal expression. J Biol Chem 2005;280:677-684.
Induction of membrane protein
Gaildrat, Klein; in collaboration with Ganapathy, Inui
One outcome of the microarray studies was the finding that the expression of the oligopeptide transporter (PEPT1) gene is markedly increased at night (about 100-fold). In collaboration with Vadivel Ganapathy, Pascaline Gaildrat found that expression of PEPT1 in the pineal gland at night produces a truncated version of the gene (see Figure 26.6). Regulation reflects neural cyclic AMP activation of the pineal gland and reflects particularly large changes in both the mRNA encoding the protein and the protein itself, which represents a unique mechanism of regulation of a membrane protein; it is also unusual that the product is relatively unstable and disappears rapidly. Gaildrat also discovered that expression of the gene product is mostly restricted to the pineal gland. He identified a section of the gene that appears to be responsible for this pattern of tissue distribution and, for the night/day pattern of expression, a section that shares features with the AANAT gene with respect to cyclic AMP response elements and putative sites for the binding of CRX/OTX transcription factors; the sites are located in an internal promoter. Thus, a mechanism exists to provide the pinealocyte selectively with a membrane-linked function, which may involve a regulatory role of the PEPT1 product that is linked to melatonin production.
Gaildrat P, Møller M, Mukda S, Humphries A, Carter DA, Ganapathy V, Klein DC. A novel pineal-specific product of the oligopeptide transporter Pept1 gene: circadian expression mediated by cAMP activation of an intronic promoter. J Biol Chem 2005;280:16851-16860.
Metal biology
Shi, Klein
Enzymes involved in indole synthesis and metabolism require metals. The concentration of divalent ions, including copper and zinc, are governed by metallothionines, small proteins dedicated to binding and buffering these ions. Qiong Shi discovered that an adrenergic cyclic AMP mechanism in the pineal gland governs expression of one form of these proteins. He determined that both the protein and mRNA encoding the enzyme are under the control of this second messenger. He has extended his work with the use of a zinc chelator, which blocked the synthesis of N-acetylserotonin, serotonin, and melatonin, suggesting that zinc has a important role in a common pathway, perhaps one involving the production of serotonin. The findings have implications for several clinical areas because of serotonin’s importance as a transmitter and its role in the immune system and because of potential variations in zinc availability that can cause zinc deficiency.
NeuroD-1, CRX, and OTX expression in the pineal gland
Muñoz, Shi, Klein; in collaboration with Møller
In collaboration with Morten Møller and colleagues, Qiong Shi and Estela Muñoz studied the three transcription factors NeuroD1, CRX, and OTX to determine whether they are involved in developmental and adult expression of genes in the pineal gland. In situ hybridization and Northern blot analysis established that they are expressed during development and remain at elevated levels in the adult. The Northern blot shown in Figure 26.7 reveals that the factors are highly expressed in the adult and appear simultaneously in early pineal development of the rat.
Pineal retinal evolution, AANAT, and the formation of conjugates of arylalkylamine and retinaldehye
Klein, Coon; in collaboration with Kirk
According to our theory of the evolution of the pineal gland, both the pineal gland and retina evolved from the same primitive photoreceptor cell after that cell acquired AANAT and HIOMT, the enzymes required to make melatonin. It was thought that these enzymes were originally important only in detoxification of arylalkylamines, which can be dangerous in all tissues because of the reactivity of the amine and that of the aldehyde, which arises from oxidation of the amine. Detoxification led to the production of melatonin and eventually to the development of the rhythm in melatonin as a day/night signal. Our theory proposes, however, that the requirement for high levels of melatonin was destructive to the primitive photoreceptor because it required high levels of serotonin, the melatonin precursor, which was especially toxic to photoreceptor function. Moreover, serotonin can react with and remove retinaldehyde, the key photodetection molecule. The theoretical product formed by such a reaction would contain two molecules of retinaldehyde and one molecule of serotonin [N -bis-retinyl-serotonin (A2S); see Figure 26.8]; homologous compounds would be formed from other arylalkylamines. These products belong to a larger family of N- bis-retinyl compounds, including bis-retinal-ethanolamine. The latter is thought to be toxic to the retina through the effects of the retinal side chains.
In the primitive photoreceptor, the formation of A2S, due to its toxicity, would reduce photosensitivity and because A2S would remove retinal. Segregation of the processes into the pinealocyte and retinal photoreceptor made it possible for melatonin production and photodetection to evolve and improve. In collaboration with Ken Kirk, Steven Coon and David Klein synthesized A2S and related compounds by using LC/MS/MS to monitor their formation. They are testing the hypothesis that the compounds’ formation in the retina is a function of AANAT activity. They are also examining whether the compounds might play a role in human retinal disease such as macular degeneration.
Iyer LM, Aravind L, Coon SL, Klein DC, Koonin EV. Evolution of cell-cell signaling in animals: did late horizontal gene transfer from bacteria have a role? Trends Genet 2004;20:292-299.
Klein DC. The 2004 Aschoff/Pittendrigh Lecture: theory of the origin of the pineal gland—a tale of conflict and resolution. J Biol Rhythms2004;19(4):264-279.
1CNRS, Université Curie, Banyuls-sur-Mer, France
2University of Alberta, Edmonton, Canada
3Uniformed Services University of the Health Sciences, Bethesda, MD
4Uniformed Services University of the Health Sciences, Bethesda, MD
Collaborators
Alastair Aitken, PhD, University of Edinburgh, Edinburgh, UK
Ruben Baler, PhD, Laboratory of Cellular and Molecular Regulation, NIMH, Bethesda, MD
William B. Benjamin, PhD, SUNY, Stony Brook, NY
Seth Blackshaw, PhD, Harvard Medical School, Boston, MA
David Carter, PhD, University of Wales, Cardiff, UK
Clivel G. Charlton, PhD, Florida A&M University, Tallahassee, FL
Philippe Chemineau, PhD, INRS, Nouzilly, France
Constance L. Chik, MD, University of Alberta, Edmonton, Canada
Nelson Chong, PhD, University of Leicester, Leicester, UK
Philip Cole, MD, PhD, The Johns Hopkins University, Baltimore, MD
Igor Dawid, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda, MD
Fred Dyda, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Vadivel Ganapathy, PhD, Medical College of Georgia, Augusta, GA
Yoav Gothilf, PhD, Tel Aviv University, Tel Aviv, Israel
Allison Hickman, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Anthony Ho, PhD, University of Alberta, Edmonton, Canada
John Hogenesch, PhD, Genome Institute of Novartis Foundation, San Diego, CA
Ann Humphries, PhD, University of Wales, Cardiff, UK
Ken-Ichi Inui, PhD, Kyoto University Hospital, Kyoto, Japan
P. Michael Iuvone, PhD, Emory University School of Medicine, Atlanta, GA
Howard Jaffe, PhD, Laboratory of Neurochemistry, NINDS, Bethesda, MD
Ken Kirk, PhD, Laboratory of Chemistry, NIDDK, Bethesda, MD
Eugene V. Koonin, PhD, National Center for Biotechnology Information, NLM, Bethesda, MD
Benoit Malpaux, PhD, INRS, Nouzilly, France
Sandford Markey, PhD, Laboratory of Neurotoxicology, NIMH, Bethesda, MD
Morten Møller, PhD, Panum Institutet, Copenhagen, Denmark
Randall T. Moon, PhD, Howard Hughes Medical Institute, University of Washington, Seattle, WA
Peter Munson, PhD, National Center for Biotechnology Information, NLM, Bethesda, MD
Tomas Obsil, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Benjamin Ron, PhD, Israel Oceanographic and Limnological Research, Eilat, Israel
Christof Schomerus, PhD, J.W. Goethe Universität, Frankfurt, Germany
Reiko Toyama, PhD, Laboratory of Molecular Genetics, NICHD, Bethesda, MD
Weiping Zhen, PhD, The Johns Hopkins University, Baltimore, MD
For further information, contact klein@helix.nih.gov.