Glutathione peroxidase (GPx) is a widespread protein superfamily found in many organisms throughout all kingdoms of life. Although it was initially thought to use only glutathione as reductant, recent evidence suggests that the majority of GPxs have specificity for thioredoxin. We present a thorough in silico analysis performed on 724 sequences and 12 structures aimed to clarify the evolutionary, structural, and sequence determinants of GPx specificity. Structural variability was found to be limited to only two regions, termed oligomerization loop and functional helix, which modulate both reduced substrate specificity and oligomerization state. We show that mammalian GPx-1, the canonic selenocysteine-based tetrameric glutathione peroxidase, is a recent “invention” during evolution. Contrary to common belief, cysteine-based thioredoxin-specific GPx, which we propose the TGPx, are both more common and more ancient. This raises interesting evolutionary considerations regarding oligomerization and the use of active-site selenocysteine residue. In addition, phylogenetic analysis has revealed the presence of a novel member belonging to the GPx superfamily in Mammalia and Amphibia, for which we propose the name GPx-8, following the present numeric order of the mammalian GPxs.

Daily light and temperature cycles are considered the most important zeitgebers for circadian clocks in many organisms. The influence of each single zeitgeber on the clock has been well studied, but little is known about any synergistic effects of both zeitgebers on the clock. In nature, light and temperature show characteristic daily oscillations with the temperature rising during the light phase and reaching its maximum in the late afternoon. Here, we studied behavioral and molecular rhythms in Drosophila melanogaster under simulated natural low light-dark (LD) and temperature (T) cycles that typically occur during the September equinox. Wild-type flies were either subjected to simulated LD or T cycles alone or to a combination of both. Behavioral rhythms and molecular rhythms in the different clock neurons were assessed under the 3 different conditions. Although behavioral rhythms entrained to all conditions, the rhythms were most robust under the combination of LD and T cycles. The clock neurons responded differently to LD and T cycles. Some were not entrained by T cycles alone; others were only slightly entrained by LD cycles alone. The amplitude of the molecular cycling was not different between LD alone and T cycles alone; but LD alone could set the pacemaker neurons to similar phases, whereas T cycles alone could not. The combination of the 2 zeitgebers entrained all clock neurons not only with similar phase but also enhanced the amplitude of Timeless cycling in the majority of cells. Our results show that the 2 zeitgebers synergistically entrain behavioral and molecular rhythms of Drosophila melanogaster.