David C. Klein

Indole Metabolism in the Pineal Gland: A Circadian Rhythm in N-Acetyltransferase

The activity of N-acetyltransferase in the rat pineal gland is more than 15 times higher at night than during the day. This circadian rhythm persists in complete darkness, or in blinded animals, and is suppressed in constant lighting. The N-acetyltransferase rhythm is 180� out of phase with the serotonin rhythm and is similar to the norepinephrine and melatonin rhythms. Experiments in vitro indicate that norepinephrine, not serotonin, regulates the activity of N-acetyl-transferase through a highly specific receptor.

Pineal N-acetyltransferase and hydroxyindole-O-methyl-transferase: control by the retinohypothalamic tract and the suprachiasmatic nucleus

The visual pathway and central neural structures involved in the photic and endogenous regulation of the activity of pineal N-acetyltransferase and hydroxyindole-O-methyltransferase were investigated. The results indicate that the visual pathway regulating both enzymes is the retinohypothalamic tract, and that the inferior accessory optic tract is clearly not involved in the regulation of hydroxyindole-O-methyltransferase activity, as has been previously thought. In addition, the suprachiasmatic nucleus was found to be necessary for the generation of a rhythm in N-acetyltransferase activity in blinded animals, and to be responsible for the tonic elevation of hydroxyindole-O-methyltransferase activity in blinded animals. Finally, it was concluded that the rapid and large daily changes in N-acetyltransferase activity seen in a normal lighting cycle and the much slower and smaller changes in hydroxyindole-O-methyltransferase activity seen only after weeks in constant lighting conditions are mediated by the same neural tract; the different time courses of the effects of environmental lighting may be explained on the basis of different intracellular regulatory mechanisms.

Rapid Light-Induced Decrease in Pineal Serotonin N-Acetyltransferase Activity

Light acting by way of the eye causes the dark-induced activity of serotonin N-acetyltransferase in the pineal gland of the rat to decrease with a halving time of about 3 minutes. This effect, which is one of the more rapid physiological changes known to occur in the activity of any enzyme that metabolizes biogenic amines, appears to explain the rapid increase in the concentration of pineal serotonin that is caused by light exposure at night.

Crystal Structure of the 14-3-3ζ: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 AANATs activity and affinity for its substrates by stabilizing a region of AANAT involved in substrate binding.

Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis.

Pineal serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, or AA-NAT) generates the large circadian rhythm in melatonin, the hormone that coordinates daily and seasonal physiology in some mammals. Complementary DNA encoding ovine AA-NAT was cloned. The abundance of AA-NAT messenger RNA (mRNA) during the day was high in the ovine pineal gland and somewhat lower in retina. AA-NAT mRNA was found unexpectedly in the pituitary gland and in some brain regions. The night-to-day ratio of ovine pineal AA-NAT mRNA is less than 2. In contrast, the ratio exceeds 150 in rats. AA-NAT represents a family within a large superfamily of acetyltransferases.

Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina.

Circadian clocks are self-sustaining genetically based molecular machines that impose approximately 24h rhythmicity on physiology and behavior that synchronize these functions with the solar day-night cycle. Circadian clocks in the vertebrate retina optimize retinal function by driving rhythms in gene expression, photoreceptor outer segment membrane turnover, and visual sensitivity. This review focuses on recent progress in understanding how clocks and light control arylalkylamine N-acetyltransferase (AANAT), which is thought to drive the daily rhythm in melatonin production in those retinas that synthesize the neurohormone; AANAT is also thought to detoxify arylalkylamines through N-acetylation. The review will cover evidence that cAMP is a major output of the circadian clock in photoreceptor cells; and recent advances indicating that clocks and clock networks occur in multiple cell types of the retina.

Arylalkylamine N-Acetyltransferase: “the Timezyme”

Arylalkylamine N-acetyltransferase controls daily changes in melatonin production by the pineal gland and thereby plays a unique role in biological timing in vertebrates. Arylalkylamine N-acetyltransferase is also expressed in the retina, where it may play other roles in addition to signaling, including neurotransmission and detoxification. Large changes in activity reflect cyclic 3′,5′-adenosine monophosphate-dependent phosphorylation of arylalkylamine N-acetyltransferase, leading to formation of a regulatory complex with 14-3-3 proteins. This activates the enzyme and prevents proteosomal proteolysis. The conserved features of regulatory systems that control arylalkylamine N-acetyltransferase are a circadian clock and environmental lighting.

Natural melatonin `knockdown' in C57BL/6J mice: rare mechanism truncates serotonin N-acetyltransferase

Pineal melatonin synthesis (serotonin --> N-acetylserotonin --> melatonin) is severely compromised in most inbred strains of mice, in many cases because serotonin is not acetylated by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT). We have found that in the C57BL/6J strain, AANAT mRNA encodes a severely truncated AANAT protein, because a pseudo-exon containing a stop codon is spliced in. This is the first identification of a natural mutation which knocks down melatonin synthesis. The decrease in melatonin signaling may have been a selective factor in the development of laboratory strains of mice because melatonin can inhibit reproduction and modify circadian rhythmicity.

Avian melatonin synthesis: Photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N‐acetyltransferase (arylalkylamine N‐acetyltransferase; AA‐NAT; EC 2.3.1.87). Here we determined that the chicken AA‐NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA‐NAT mRNA was not detected in other tissues. The AA‐NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA‐NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6‐h light pulse late in subjective night in DD. Nocturnal AA‐NAT mRNA levels do not change during a 20‐min exposure to light, whereas this treatment dramatically decreases AA‐NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA‐NAT activity reflect, at least in part, clock‐generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light‐induced decrease in AA‐NAT activity.

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 2.3.1.87). 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 Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.