Amino acid residue at position 188 determines the UV-sensitive bistable property of vertebrate non-visual opsin Opn5.

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.

Vertebrate ancient-long opsin has molecular properties intermediate between those of vertebrate and invertebrate visual pigments.

VA/VAL opsin is one of the four kinds of nonvisual opsins that are closely related to vertebrate visual pigments in the phylogenetic tree of opsins. Previous studies indicated that among these opsins, parapinopsin and pinopsin exhibit molecular properties similar to those of invertebrate bistable visual pigments and vertebrate visual pigments, respectively. Here we show that VA/VAL opsin exhibits molecular properties intermediate between those of parapinopsin and pinopsin. VAL opsin from Xenopus tropicalis was expressed in cultured cells, and the pigment with an absorption maximum at 501 nm was reconstituted by incubation with 11-cis-retinal. Light irradiation of this pigment caused cis-to-trans isomerization of the chromophore to form a state having an absorption maximum in the visible region. This state has the ability to activate Gi and Gt types of G proteins. Therefore, the active state of VAL opsin is a visible light-absorbing intermediate, which probably has a protonated retinylidene Schiff base as its chromophore, like the active state of parapinopsin. However, this state was apparently photoinsensitive and did not show reverse reaction to the original pigment, unlike the active state of parapinopsin, and instead similar to that of pinopsin. Furthermore, the Gi activation efficiency of VAL opsin was between those of pinopsin and parapinopsin. Thus, the molecular properties of VA/VAL opsin give insights into the mechanism of conversion of the molecular properties from invertebrate to vertebrate visual pigments.

Chk2/Cds1 protein kinase blocks apoptosis during early development of Xenopus laevis.

Early Xenopus laevis embryos possess cell cycles that do not arrest at checkpoints in response to damaged DNA. At the midblastula transition (MBT), embryos with damaged DNA undergo apoptosis. After the MBT, DNA damage triggers cell cycle arrest rather than apoptosis. The transition from checkpoint-unregulated to checkpoint-regulated cycles makes Xenopus embryos compelling for studying mechanisms regulating response to genomic damage. The DNA damage checkpoint is mediated by the Chk2/Cds1 kinase. Conflicting evidence implicates Chk2 as an inhibitor or promoter of apoptosis. To better understand the developmental function of Chk2, we expressed wild-type (wt) and dominant-negative (DN) Chk2 in Xenopus embryos. Wt-Chk2 created a pre-MBT checkpoint due to degradation of Cdc25A and phosphorylation of cyclin-dependent kinases. Embryos expressing DN-Chk2 developed normally until gastrulation and then underwent apoptosis. Conversely, low doses of wt-Chk2 blocked radiation-induced apoptosis. Therefore, Chk2 operates at a switch between cell cycle arrest or apoptosis in response to genomic assaults.