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Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes which are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on the Opn4 (melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have five Opn4 genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history of Opn4 genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon and gar) fishes. Our synteny analysis suggests that the intron-less Opn4 gene emerged by retroduplication after branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-less Opn4 gene became abundantly used without substantial changes of the molecular properties of the Opn4 protein. This stepwise evolutionary model of Opn4 genes is quite similar to that of rhodopsin genes in the Actinopterygii. The unique acquisition of rhodopsin and Opn4 retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments.

Apigenin is a flavonoid with antioxidant and anticancer effects. It has been reported that apigenin inhibits proliferation, migration, and invasion and induces apoptosis in cultured lung cancer cells. However, there is little information on the involvement of microRNAs (miRNAs) in its effects. miRNA microarray analysis and polymerase-chain-reaction analysis of miRNAs revealed that treatment of human lung cancer A549 cells with apigenin up-regulated the level of miR-34a-5p. Furthermore, mRNA microarray analysis and the results of three microRNA target prediction tools showed that Snail Family Transcriptional Repressor 1 (SNAI1), which inhibits the induction of apoptosis, had its mRNA expression down-regulated in A549 cells treated with apigenin. Our findings suggest that apigenin might induce apoptosis by down-regulation of SNAI1 through up-regulation of miR-34a-5p in A549 cells.

Misao Mizuno, Keita Sato, Takahiro Yamashita, Kazumi Sakai, Yasushi Imamoto, Yumiko Yamano, Akimori Wada, Hideyo Ohuchi, Yoshinori Shichida, and Yasuhisa Mizutani, Chromophore Structure in an Inactive State of a Novel Photosensor Protein Opn5L1: Resonance Raman Evidence for the Formation of a Deprotonated Adduct at the 11th Carbon Atom, The Journal of Physical Chemistry B, 127(10), 2169-2176, March 16, 2023, © 2023 American Chemical Society. DOI: 10.1021/acs.jpcb.2c08780, Opsins are photosensitive G protein-coupled receptor proteins and are classified into visual and nonvisual receptors. Opn5L1 is a nonvisual opsin that binds all-trans retinal as a chromophore. A unique feature of Opn5L1 is that the protein exhibits a photocyclic reaction upon photoexcitation. Determining the chromophore structures of intermediates in the photocycle is essential for understanding the functional mechanism of Opn5L1. A previous study revealed that a long-lived intermediate in the photocycle cannot activate the G protein and forms a covalent bond between the retinal chromophore and a nearby cysteine residue. However, the position of this covalent bond in the chromophore remains undetermined. Here, we report a resonance Raman study on isotopically labeled samples in combination with density functional theory calculations and reveal that the 11th carbon atom of the chromophore of the intermediate forms a covalent linkage to the cysteine residue. Furthermore, vibrational assignments based on the isotopic substitutions and density functional theory calculations suggested that the Schiff base of the intermediate is deprotonated. The chromophore structure determined in the present study well explains the mechanism of the photocyclic reaction, which is crucial to the photobiological function of Opn5L1.

Opsins are universal photoreceptive proteins in animals and can be classified into three types based on their photoreaction properties. Upon light irradiation, vertebrate rhodopsin forms a metastable active state, which cannot revert back to the original dark state via either photoreaction or thermal reaction. By contrast, after photoreception, most opsins form a stable active state which can photoconvert back to the dark state. Moreover, we recently found a novel type of opsins whose activity is regulated by photocycling. However, the molecular mechanism underlying this diversification of opsins remains unknown. In this study, we showed that vertebrate rhodopsin acquired the photocyclic and photoreversible properties upon introduction of a single mutation at position 188. This revealed that the residue at position 188 contributes to the diversification of photoreaction properties of opsins by its regulation of the recovery from the active state to the original dark state.

Opsins are G protein-coupled receptors specialized for photoreception in animals. Opn5 is categorized in an independent opsin group and functions for various non-visual photoreceptions. Among vertebrate Opn5 subgroups (Opn5m, Opn5L1 and Opn5L2), Opn5m and Opn5L2 bind 11-cis retinal to form a UV-sensitive resting state, which is inter-convertible with the all-trans retinal bound active state by photoreception. Thus, these opsins are characterized as bistable opsins. To assess the molecular basis of the UV-sensitive bistable property, we introduced comprehensive mutations at Thr188, which is well conserved among these opsins. The mutations in Opn5m drastically hampered 11-cis retinal incorporation and the bistable photoreaction. Moreover, T188C mutant Opn5m exclusively bound all-trans retinal and thermally self-regenerated to the original form after photoreception, which is similar to the photocyclic property of Opn5L1 bearing Cys188. Therefore, the residue at position 188 underlies the UV-sensitive bistable property of Opn5m and contributes to the diversification of vertebrate Opn5 subgroups.