Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland

Abstract

Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

The Genetics of Autism Spectrum Disorders

“…More recently, we observed a low level of the 14-3-3 proteins both in the blood platelet and pineal gland of patients with ASD (Pagan et al 2014). These ubiquitous chaperone proteins are known to form a protein complex, the 'melatoninosome,' involving AANAT and ASMT in pinealocytes (Obsil et al 2001;Maronde et al 2011). This interaction between 14-3-3 and AANAT and/or ASMT might be essential for the production of melatonin and an adequate balance of the serotonin-NAS-melatonin pathway.…”

“…This complex includes at least four proteins: AANAT, ASMT, 14-3-3 and S-antigen. The immunofluorescence confocal image of AANAT (green) and 14-3-3δ/ε (red) in the pinealocytes is adapted from Maronde et al (2011). The structure of the 14-3-3ζ homodimer binding to AANAT is adapted from Obsil et al (2001).…”

Role of AA-NAT and TPH1 in the Ghrelin-Dependent Regulation of Melatonin Secretion in Sheep during Different Seasons: An <em>In Vitro</em> Study

“…The nocturnal elevation of AA-NAT activity is dependent on AA-NAT gene transcription [38] and the role of the RNA-binding protein heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AA-NAT in rats [39]. However, in sheep [14,19,20], cattle [21] and humans [14], it mainly depends on changes in AA-NAT phosphorylation and post-translational modifications of the AA-NAT protein, such as proteasomal degradation. In present study, GHRL decreases p31T-AA-NAT expression and AA-NAT protein concentration in the ovine PG, and the effect of its activity was the strongest during the SD season.…”

“…The mammalian PG disseminates information about circadian time to peripheral tissues through the synthesis and release of melatonin (MEL), which is restricted to nighttime. The initiation of MEL synthesis begins within the well-known sympathetic pathways [14]. MEL is formed by a four-stage reaction.…”

“…The first two stages are associated with the conversion of tryptophan into serotonin (5-HT), which undergoes acetylation by serotonin N-acetyltransferase (AA-NAT) into N-3 acetylserotonin (NAS). Then, NAS is methylated by hydroxyindole O-methyltransferase (HIOMT) to generate the final biologically functional N-acetyl-5-methoxytryptamine [14].…”