Signal transduction in the pineal gland is studied, with primary focus on the biochemistry and molecular biology of first enzyme in the serotonin -> melatonin pathway, serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87). The production of melatonin is increased at night in all vertebrates, due to a marked increase in AANAT activity in the pineal gland. As such, AANAT plays an essential role in vertebrate physiology in converting night into a circulating signal. Melatonin is also synthesized in the vertebrate retina, where it appears to enhance dark adaption. The genetic code of AANAT is several vertebrate classes has been determined and is being used to study the regulation of mRNA encoding the enzyme and to generate proteins and peptides to use as antigens to raise antisera against the protein. Using this information, it has been found that transcriptional mechanisms play a regulatory role in controlling melatonin production in some but not all species. However, in all species, it is becoming clear that a common mechanism regulating AANAT activity is second messenger control of proteasomal proteolysis. This mechanism appears to play a central role in producing the rapid light induced decrease in AANAT activity and melatonin production. It is of interest that in different vertebrate classes light acts through different mechanisms, yet all regulate proteasomal proteolysis of AANAT. The Section is interested in the biochemical and physicochemical analysis of AANAT and has determined the mechanism of catalytic action using expressed protein. Site directed mutagenesis indicates that one or more histidines located between the putative arylalkylamine binding domain and putative CoA binding site represent the catalytic domain. X-ray crystallographic analysis has established the 3-D structure of an active form of AANAT. Studies on the rat promoter has made it possible to identify elements involved in the tissue-specific expression of AANAT. With this information, it has been possible to construct a rat transgenic that expresses a reporter under the control of the AANAT promoter in the pineal gland and retina - on a day/night basis. The analysis of AANAT genes has resulted in the identification of a new AANAT gene subfamily - AANAT-2, recently discovered in fish. AANAT-1 - the well studied form - and AANAT-2 differ in tissue distribution, kinetics and circadian rhythm patterns. Each may have evolved to meet different demands for melatonin synthesis in the pineal gland and retina. The biology of AANAT-2 will be studied in the future. A major step forward has been the development of a new cell line expressing human AANAT. This has had a major impact on research, with the most important advance being evidence indicating a new level of regulation of AANAT activity - through an activation/inactivation mechanism. Recent work has resulted in the development of AANAT cell lines, which have provided a new tool to study regulation of the enzyme. Using this, it has been discovered that cAMP can modulate the activity of AANAT, without changing AANAT protein levels. Perhaps the most important recent advance in understanding the role AANAT plays in the regulation of melatonin synthesis is the discovery that AANAT exists in the cell in a complex composed of at least one phosphorylated AANAT molecule and two molecules of 14-3-3 protein. The complex has been characterized and crystallized. Analysis reveals the complex is held together by many interactions, but the key one - the switch - is a set of interactions involving a phosphothreonine of AANAT and residues on 14-3-3. The complex does not form if this threonine is not phosphorylated. Complex formation protects AANAT from degradation and activates it by maintaining the enzyme in an optimal serotonin binding configuration.
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