Convergent mechanism underlying the acquisition of vertebrate scotopic vision.

High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (kth) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth, mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision.

Protonation of Asp116 and distortion of the all-trans retinal chromophore in Krokinobacter eikastus rhodopsin 2 causes a redshift in absorption maximum upon dehydration.

Water is usually indispensable for protein function. For ion-pumping rhodopsins, water molecules inside the proteins play an important role in ion transportation. In addition to amino acid residues, water molecules regulate the colors of retinal proteins. It was reported that a sodium-pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2), showed a color change from red to purple upon dehydration under crystalline conditions. Here, we applied comprehensive visible and IR absorption spectroscopy and resonance Raman spectroscopy to KR2 in liposomes under hydration-controlled conditions. A large increase in the hydrogen-out-of-plane (HOOP) vibration at 947 (H-C11=C12-H Au mode) and moderate increases at 893 (C7-H and C10-H) and 808 (C14-H) cm-1 were observed under dehydrated conditions, which were assigned by using systematically deuterated retinal. Moreover, the Asn variant at Asp116, which functions as a counter ion for the protonated retinal Schiff base (PRSB), caused a large redshift in the absorption maximum and constitutive increase in the HOOP modes under hydrated and dehydrated conditions. The protonation of a counter ion at Asp116 clearly causes a redshift in the absorption maximum as the all-trans retinal chromophore twists upon dehydration. Namely, the results strongly suggested that water molecules are important for maintaining the hydrogen-bonding network at the PRSB and deprotonation state of Asp116 in KR2.

Targeting transglutaminase 2 mediated exostosin glycosyltransferase 1 signaling in liver cancer stem cells with acyclic retinoid.

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.

Characterization of retinal chromophore and protonated Schiff base in Thermoplasmatales archaeon heliorhodopsin using solid-state NMR spectroscopy.

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.

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.

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.

Solid-state NMR for the characterization of retinal chromophore and Schiff base in TAT rhodopsin embedded in membranes under weakly acidic conditions.

TAT rhodopsin extracted from the marine bacterium SAR11 HIMB114 has a characteristic Thr-Ala-Thr motif and contains both protonated and deprotonated states of Schiff base at physiological pH conditions due to the low pKa. Here, using solid-state NMR spectroscopy, we investigated the 13C and 15N NMR signals of retinal in only the protonated state of TAT in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (1'-rac-glycerol) (POPE/POPG) membrane at weakly acidic conditions. In the 13C NMR spectrum of 13C retinal-labeled TAT rhodopsin, the isolated 14-13C signals of 13-trans/15-anti and 13-cis/15-syn isomers were observed at a ratio of 7:3. 15N retinal protonated Schiff base (RPSB) had a significantly higher magnetic field resonance at 160 ppm. In 15N RPSB/λmax analysis, the plot of TAT largely deviated from the trend based on the retinylidene-halide model compounds and microbial rhodopsins. Our findings indicate that the RPSB of TAT forms a very weak interaction with the counterion.

Total Synthesis of Loroxanthin.

The first total synthesis of loroxanthin (1) was accomplished by Horner-Wadsworth-Emmons reaction of C25-apocarotenal 8 having a silyl-protected 19-hydroxy moiety with C15-phosphonate 25 bearing a silyl-protected 3-hydroxy-ε-end group. Preparation of apocarotenal 8 was achieved via Stille coupling reaction of alkenyl iodide 10 with alkenyl stananne 9, whereas phosphonate 25 was prepared through treatment of ally alcohol 23 with triethyl phosphite and ZnI2. The ally alcohol 23 was derived from the known (3R,6R)-3-hydroxy C15-aldehyde 20, which was obtained by direct optical resolution of racemate 20 using a semi-preparative chiral HPLC column.

Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR.

Bacteriorhodopsin (BR) functions as a light-driven proton pump that transitions between different states during the photocycle, such as all-trans (AT; BR568) and 13-cis, 15-syn (CS; BR548) state and K, L, M1, M2, N, and O intermediates. In this study, we used in situ photoirradiation 13C solid-state NMR to observe a variety of photo-intermediates and photoreaction pathways in [20-13C]retinal-WT-BR and its mutant [20-13C, 14-13C]retinal-D96N-BR. In WT-BR, the CS state converted to the CS* intermediate under photoirradiation with green light at -20 °C and consequently converted to the AT state in the dark. The AT state converted to the N intermediate under irradiation with green light. In D96N-BR, the CS state was converted to the CS* intermediate at -30 °C and consequently converted to the AT state. Simultaneously, the AT state converted to the M and L intermediates under green light illumination at -30 °C and subsequently converted to the AT state in the dark. The M intermediate was directly excited to the AT state by UV light illumination. We demonstrated that short-lived photo-intermediates could be observed in a stationary state using in situ photoirradiation solid-state NMR spectroscopy for WT-BR and D96N-BR, enabling insight into the light-driven proton pump activity of BR.

Evolutionary adaptation of visual pigments in geckos for their photic environment.

Vertebrates generally have a single type of rod for scotopic vision and multiple types of cones for photopic vision. Noteworthily, nocturnal geckos transmuted ancestral photoreceptor cells into rods containing not rhodopsin but cone pigments, and, subsequently, diurnal geckos retransmuted these rods into cones containing cone pigments. High sensitivity of scotopic vision is underlain by the rod's low background noise, which originated from a much lower spontaneous activation rate of rhodopsin than of cone pigments. Here, we revealed that nocturnal gecko cone pigments decreased their spontaneous activation rates to mimic rhodopsin, whereas diurnal gecko cone pigments recovered high rates similar to those of typical cone pigments. We also identified amino acid residues responsible for the alterations of the spontaneous activation rates. Therefore, we concluded that the switch between diurnality and nocturnality in geckos required not only morphological transmutation of photoreceptors but also adjustment of the spontaneous activation rates of visual pigments.

Structure of a retinal chromophore of dark-adapted middle rhodopsin as studied by solid-state nuclear magnetic resonance spectroscopy.

Middle rhodopsin (MR) found from the archaeon Haloquadratum walsbyi is evolutionarily located between two different types of rhodopsins, bacteriorhodopsin (BR) and sensory rhodopsin II (SRII). Some isomers of the chromophore retinal and the photochemical reaction of MR are markedly different from those of BR and SRII. In this study, to obtain the structural information regarding its active center (i.e., retinal), we subjected MR embedded in lipid bilayers to solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectroscopy. The analysis of the isotropic 13C chemical shifts of the retinal chromophore revealed the presence of three types of retinal configurations of dark-adapted MR: (13-trans, 15-anti (all-trans)), (13-cis, 15-syn), and 11-cis isomers. The higher field resonance of the 20-C methyl carbon in the all-trans retinal suggested that Trp182 in MR has an orientation that is different from that in other microbial rhodopsins, owing to the changes in steric hindrance associated with the 20-C methyl group in retinal. 13Cζ signals of Tyr185 in MR for all-trans and 13-cis, 15-syn isomers were discretely observed, representing the difference in the hydrogen bond strength of Tyr185. Further, 15N NMR analysis of the protonated Schiff base corresponding to the all-trans and 13-cis, 15-syn isomers in MR showed a strong electrostatic interaction with the counter ion. Therefore, the resulting structural information exhibited the property of stable retinal conformations of dark-adapted MR.

[Chemical Biology Studies Using Vitamin A Analogs].

"Retinoid" is the general term for vitamin A derivatives and chemical compounds that act like vitamin A. Vitamin A are composed of four isoprene units and are named according to their terminal functional group, such as retinol (OH, 1), retinal (CHO, 2), and retinoic acid (CO2H, 3). Vitamin A usually refers to retinol. In the past few decades, major advances in research on vitamin A have improved our understanding of its fundamental roles and physiological significance in living cells. In this review, three types of chemical biology studies using vitamin A analogs are described: (1) conformational studies of the chromophore in retinal proteins (rhodopsin, phoborhodopsin, and retinochrome), especially the conformation around the cyclohexene ring; (2) structure-activity relationship studies of retinoic acid analogs to create new signaling molecules for activating nuclear receptors; and (3) development of a new channelrhodopsin with an absorption maximum at longer wavelength to overcome the various demerits of channelrhodopsins used in optogenetics, as well as the stereoselective synthesis of retinoid isomers and their analogs using a diene-tricarbonyliron complex or a palladium-catalyzed cross-coupling reaction between vinyl triflates and stannyl olefins.

Identification of Paracentrone in Fucoxanthin-Fed Mice and Anti-Inflammatory Effect against Lipopolysaccharide-Stimulated Macrophages and Adipocytes.

SCOPE: Fucoxanthin is converted to fucoxanthinol and amarouciaxanthin A in the mouse body. However, further metabolism such as cleavage products (i.e., apocarotenoids) remains unclear. The fucoxanthin-derived apocarotenoid in vivo is investigated and the anti-inflammatory effect of apocarotenoids with fucoxanthin partial structure such as allenic bond and epoxide residue against activated macrophages and adipocytes in vitro is evaluated. METHODS AND RESULTS: LC-MS analysis indicates the presence of paracentrone, a C31 -allenic-apocarotenoid, in white adipose tissue of diabetic/obese KK-Ay and normal C57BL/6J mice fed 0.2% fucoxanthin diet for 1 week. In lipopolysaccharide-activated RAW264.7 macrophages, paracentrone as well as C26 - and C28 -allenic-apocarotenoids suppresses the overexpression of inflammatory factors. Further, apo-10'-fucoxanthinal, a fucoxanthin-derived apocarotenoid which retained epoxide residue, exhibits a most potent anti-inflammatory activity through regulating mitogen-activated protein kinases and nuclear factor-κB inflammatory signal pathways. In contrast, ß-apo-8'-carotenal without allenic bond and epoxide residue lacks suppressed inflammation. In 3T3-L1 adipocytes, paracentrone, and apo-10'-fucoxanthinal downregulate the mRNA expression of proinflammatory mediators and chemokines induced by co-culture with RAW264.7 cells. CONCLUSION: Dietary fucoxanthin accumulates as paracentrone as well as fucoxanthinol and amarouciaxanthin A in the mouse body. Allenic bond and epoxide residue of fucoxanthin-derived apocarotenoids have pivotal roles for anti-inflammatory action against activated macrophages and adipocytes.

Visual adaptation of opsin genes to the aquatic environment in sea snakes.

BACKGROUND: Evolutionary transitions from terrestrial to aquatic life history cause drastic changes in sensory systems. Indeed, the drastic changes in vision have been reported in many aquatic amniotes, convergently. Recently, the opsin genes of the full-aquatic sea snakes have been reported. However, those of the amphibious sea snakes have not been examined in detail. RESULTS: Here, we investigated opsin genes and visual pigments of sea snakes. We determined the sequences of SWS1, LWS, and RH1 genes from one terrestrial, three amphibious and four fully-aquatic elapids. Amino acid replacements at four and one spectra-tuning positions were found in LWS and RH1, respectively. We measured or predicted absorption of LWS and RH1 pigments with A1-derived retinal. During their evolution, blue shifts of LWS pigments have occurred stepwise in amphibious sea snakes and convergently in both amphibious and fully-aquatic species. CONCLUSIONS: Blue shifted LWS pigments may have adapted to deep water or open water environments dominated by blue light. The evolution of opsins differs between marine mammals (cetaceans and pinnipeds) and sea snakes in two fundamental ways: (1) pseudogenization of opsins in marine mammals; and (2) large blue shifts of LWS pigments in sea snakes. It may be possible to explain these two differences at the level of photoreceptor cell composition given that cone and rod cells both exist in mammals whereas only cone cells exist in fully-aquatic sea snakes. We hypothesize that the differences in photoreceptor cell compositions may have differentially affected the evolution of opsins in divergent amniote lineages.

Alcohol Dehydrogenase Activity Converts 3″-Hydroxy-geranylhydroquinone to an Aldehyde Intermediate for Shikonin and Benzoquinone Derivatives in Lithospermum erythrorhizon.

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

A dearomative ipso-iodocyclization/desymmetrization sequence leading to optically active tricyclic piperazine scaffolds.

Dearomative ipso-iodocyclization of 4-(1-ethoxyethoxy)-N-propargylanilines leading to 1-azaspiro[4.5]deca-3,6,9-trien-8-ones has been developed. This reaction is characterized by the yield of fewer toxic byproducts and is conducted by a more user-friendly protocol compared to other reported methods. The synthetic application of these spiro products was demonstrated by transformation of the iodo component into other functionalities. The desymmetrization of the resulting cyclohexadienones was also achieved by the diastereoselective aza-Michael addition of internal amino acid moieties to yield tricyclic piperazine scaffolds with two newly formed contiguous chiral centers.

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants.

In optogenetics, red-shifted channelrhodopsins (ChRs) are eagerly sought. We prepared six kinds of new chromophores with one double bond inserted into the polyene side chain of retinal (A1) or 3,4-didehydroretinal (A2), and examined their binding efficiency with opsins (ReaChR and ChrimsonR). All analogs bound with opsins to afford new ChRs. Among them, A2-10ex (an extra double bond is inserted at the C10-C11 position of A2) showed the greatest red-shift in the absorption spectrum of ChrimsonR, with a maximum absorbance at 654 nm (67 nm red-shifted from that of A1-ChrimsonR). Moreover, a long-wavelength spectral boundary of A2-10ex-ChrimsonR was extended to 756 nm, which reached into the far-red region (710-850 nm).

Carotenoid Profiling of a Red Seaweed Pyropia yezoensis: Insights into Biosynthetic Pathways in the Order Bangiales.

Carotenoids are natural pigments that contribute to light harvesting and photo-protection in photosynthetic organisms. In this study, we analyzed the carotenoid profiles, including mono-hydroxy and epoxy-carotenoids, in the economically valuable red seaweed Pyropia yezoensis, to clarify the detailed biosynthetic and metabolic pathways in the order Bangiales. P. yezoensis contained lutein, zeaxanthin, α-carotene, and ß-carotene, as major carotenoids in both the thallus and conchocelis stages. Monohydroxy intermediate carotenoids for the synthesis of lutein with an ε-ring from α-carotene, α-cryptoxanthin (ß,ε-caroten-3'-ol), and zeinoxanthin (ß,ε-caroten-3-ol) were identified. In addition, ß-cryptoxanthin, an intermediate in zeaxanthin synthesis from ß-carotene, was also detected. We also identified lutein-5,6-epoxide and antheraxanthin, which are metabolic products of epoxy conversion from lutein and zeaxanthin, respectively, by LC-MS and ¹H-NMR. This is the first report of monohydroxy-carotenoids with an ε-ring and 5,6-epoxy-carotenoids in Bangiales. These results provide new insights into the biosynthetic and metabolic pathways of carotenoids in red seaweeds.

Pinopsin evolved as the ancestral dim-light visual opsin in vertebrates.

Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.

Silyl Group-Directed 6-exo-dig Iodocyclization of Homopropargylic Carbamates and Amides.

Iodocyclization of silyl group-substituted homopropargylic carbamates and amides proceeded via 6-exo-dig mode to afford 6-vinylene-4,5-dihydro-1,3-oxazines in moderate to quantitative yields. This is the first report for silyl group-solely directed iodocyclization of alkynes utilizing the ß-silyl effect. Under these mild reaction conditions, various functionalities such as secondary alcohol, acetal, urea, and sulfide were tolerated.

Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues.

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.