Astakine 2—the Dark Knight Linking Melatonin to Circadian Regulation in Crustaceans

Abstract

Daily, circadian rhythms influence essentially all living organisms and affect many physiological processes from sleep and nutrition to immunity. This ability to respond to environmental daily rhythms has been conserved along evolution, and it is found among species from bacteria to mammals. The hematopoietic process of the crayfish Pacifastacus leniusculus is under circadian control and is tightly regulated by astakines, a new family of cytokines sharing a prokineticin (PROK) domain. The expression of AST1 and AST2 are light-dependent, and this suggests an evolutionarily conserved function for PROK domain proteins in mediating circadian rhythms. Vertebrate PROKs are transmitters of circadian rhythms of the suprachiasmatic nucleus (SCN) in the brain of mammals, but the mechanism by which they function is unknown. Here we demonstrate that high AST2 expression is induced by melatonin in the brain. We identify RACK1 as a binding protein of AST2 and further provide evidence that a complex between AST2 and RACK1 functions as a negative-feedback regulator of the circadian clock. By DNA mobility shift assay, we showed that the AST2-RACK1 complex will interfere with the binding between BMAL1 and CLK and inhibit the E-box binding activity of the complex BMAL1-CLK. Finally, we demonstrate by gene knockdown that AST2 is necessary for melatonin-induced inhibition of the complex formation between BMAL1 and CLK during the dark period. In summary, we provide evidence that melatonin regulates AST2 expression and thereby affects the core clock of the crustacean brain. This process may be very important in all animals that have AST2 molecules, i.e. spiders, ticks, crustaceans, scorpions, several insect groups such as Hymenoptera, Hemiptera, and Blattodea, but not Diptera and Coleoptera. Our findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.

Seasonality influences cuticle melanization and immune defense in a cricket: support for a temperature-dependent immune investment hypothesis in insects

“…For instance, manipulation of photoperiod in vertebrate systems invariably alters immune function under laboratory conditions (Nelson and Demas, 1996). Photoperioddriven circadian cycles have also been shown to influence invertebrate immune function (Stone et al, 2012;Watthanasurorot et al, 2013), with PO activity in the crayfish brain peaking in the early morning hours (Noonin et al, 2013). On a side note, it may be that these daily cycles in PO activity underlie the deposition of daily cuticle growth rings found in some arthropods.…”

The New Face of the Old Molecules: Crustin Pm4 and Transglutaminase Type I Serving as RNPs Down-Regulate Astakine-Mediated Hematopoiesis

“…Expression of shrimp astakine is shown to be down-regulated by binding of hemocytic proteins, crustin Pm4 and STG I to astakine 3′-UTR at post-transcriptional level. Interestingly, the expression of crayfish astakine 2 is up-regulated by melatonin, which affects the core clock of crayfish brain, during the dark period of circadian rhythm [9]. Whether expression of the intracellular crustin Pm4 and STGI proteins will be affected during the dark period of circadian rhythm is worthy to further study.…”

“…Serving as hematopoietic growth factors, astakine 1 may be involved in the early developmental stage and astakine 2 in the late stage in crayfish. In a recent study, the expression of crayfish astakine 2 is up-regulated by melatonin in brain during the dark period of the circadian rhythm [9]. However, the intracellular regulatory mechanism of astakine is still unknown.…”

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“…Melatonin and circadian rhythm: High AST2 expression is induced by melatonin in the brain 34 . Findings further reveal an ancient evolutionary role for the prokineticin superfamily protein that links melatonin to direct regulation of the core clock gene feedback loops.…”

“…Melatonin regulates the circadian rhythm and that this regulation is mediated by AST2 during the dark phase. AST2 acts as novel negative feedback regulator of CLK-BMAL1 activity 34 .…”