Akimori Wada

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

UV-Sensitive Photoreceptor Protein OPN5 in Humans and Mice

A variety of animal species utilize the ultraviolet (UV) component of sunlight as their environmental cues, whereas physiological roles of UV photoreception in mammals, especially in human beings, remain open questions. Here we report that mouse neuropsin (OPN5) encoded by the Opn5 gene exhibited an absorption maximum (λmax) at 380 nm when reconstituted with 11-cis-retinal. Upon UV-light illumination, OPN5 was converted to a blue-absorbing photoproduct (λmax 470 nm), which was stable in the dark and reverted to the UV-absorbing state by the subsequent orange light illumination, indicating its bistable nature. Human OPN5 also had an absorption maximum at 380 nm with spectral properties similar to mouse OPN5, revealing that OPN5 is the first and hitherto unknown human opsin with peak sensitivity in the UV region. OPN5 was capable of activating heterotrimeric G protein Gi in a UV-dependent manner. Immuno-blotting analyses of mouse tissue extracts identified the retina, the brain and, unexpectedly, the outer ears as the major sites of OPN5 expression. In the tissue sections of mice, OPN5 immuno-reactivities were detected in a subset of non-rod/non-cone retinal neurons as well as in the epidermal and muscle cells of the outer ears. Most of these OPN5-immuno-reactivities in mice were co-localized with positive signals for the alpha-subunit of Gi. These results demonstrate the first example of UV photoreceptor in human beings and strongly suggest that OPN5 triggers a UV-sensitive Gi-mediated signaling pathway in the mammalian tissues.

Two isoforms of chicken melanopsins show blue light sensitivity

Melanopsin is a vertebrate non‐visual opsin and functions as a circadian photoreceptor in mammalian retinas. Here we found the expression of two kinds of melanopsin genes in the chicken pineal gland and identified the presence of five isoforms derived from these two genes. Reconstitution of the recombinant proteins with 11‐cis‐retinal revealed that at least two of these melanopsin protein isoforms can function as blue‐sensitive photopigments with absorption maxima at 476–484 nm. These values are consistent with maximal sensitivities of action spectra determined from the physiological and behavioral studies on mammalian melanopsins. The melanopsin isoforms found in this study may function as pineal circadian photoreceptors.

Reagent-Controlled Oxidative Aromatization in Iodocyclization: Switchable Access to Dihydropyrazoles and Pyrazoles

Switchable access to dihydropyrazoles and pyrazoles has been developed from common hydrazides by reagent-controlled iodocyclization. Controlling the oxidative aromatization in iodocyclization for heterocycles is reported for the first time, and this methodology maximally utilizes the dual nature of iodine.

Iodocyclization of Ethoxyethyl Ethers to Alkynes: A Broadly Applicable Synthesis of 3-Iodobenzo[b]furans

A wide variety of 3-iodobenzo[b]furans were readily prepared by iodocyclization of 2-alkynyl-1-(1-ethoxyethoxy)benzenes with the I(coll)2PF6-BF3 x OEt2 combination. Aryl-, vinylic-, and alkyl-substituted alkynes undergo iodocyclization in good to excellent yields. The mechanism of the reaction is also discussed.

Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats.

Background: Menadione is an intermediate in phylloquinone to menaquinone-4 conversion in mammals. Results: Menadione is released from phylloquinone in the intestine and converted to menaquinone-4 in tissues after being reduced. Conclusion: Menadione is a catabolic product of phylloquinone and circulating precursor of tissue menaquinone-4. Significance: Determining how phylloquinone is metabolized in the body is crucial for understanding vitamin K biology. Mice have the ability to convert dietary phylloquinone (vitamin K1) into menaquinone-4 (vitamin K2) and store the latter in tissues. A prenyltransferase enzyme, UbiA prenyltransferase domain-containing 1 (UBIAD1), is involved in this conversion. There is evidence that UBIAD1 has a weak side chain cleavage activity for phylloquinone but a strong prenylation activity for menadione (vitamin K3), which has long been postulated as an intermediate in this conversion. Further evidence indicates that when intravenously administered in mice phylloquinone can enter into tissues but is not converted further to menaquinone-4. These findings raise the question whether phylloquinone is absorbed and delivered to tissues in its original form and converted to menaquinone-4 or whether it is converted to menadione in the intestine followed by delivery of menadione to tissues and subsequent conversion to menaquinone-4. To answer this question, we conducted cannulation experiments using stable isotope tracer technology in rats. We confirmed that the second pathway is correct on the basis of structural assignments and measurements of phylloquinone-derived menadione using high resolution MS analysis and a bioassay using recombinant UBIAD1 protein. Furthermore, high resolution MS and 1H NMR analyses of the product generated from the incubation of menadione with recombinant UBIAD1 revealed that the hydroquinone, but not the quinone form of menadione, was an intermediate of the conversion. Taken together, these results provide unequivocal evidence that menadione is a catabolic product of oral phylloquinone and a major source of tissue menaquinone-4.

Lipophilic amines as potent inhibitors of N-acylethanolamine-hydrolyzing acid amidase.

N-Acylethanolamines (NAEs) including N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine are endogenous lipid mediators. These molecules are degraded to the corresponding fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH) or NAE-hydrolyzing acid amidase (NAAA). Lipophilic amines, especially pentadecylamine (2c) and tridecyl 2-aminoacetate (11b), were found to exhibit potent NAAA inhibitory activities (IC(50)=5.7 and 11.8μM), with much weaker effects on FAAH. These simple structures would provide a scaffold for further improvement in NAAA inhibitory activity.

Correlation between nuptial colors and visual sensitivities tuned by opsins leads to species richness in sympatric Lake Victoria cichlid fishes.

Reproductive isolation that prevents interspecific hybridization between closely related coexisting species maintains sympatric species diversity. One of the reproductive isolations is mate choice based on color signals (breeding color perceived by color vision). This is well known in several animal taxa, yet little is known about its genetic and molecular mechanism. Lake Victoria cichlid fishes are thought to be an example of sympatric species diversity. In the species inhabiting different light environments in rocky shore, speciation by sensory drive through color signals has been proposed by analyses of the long wavelength-sensitive (LWS) opsin gene and the male nuptial coloration. However, the genetic and molecular mechanism of how diversity of sympatric species occurring in the same habitat is maintained remains unknown. To address this issue, we determined nucleotide sequences of eight opsins of six sympatric species collected from a sandy-muddy shore--an ideal model system for studying sympatric species. Among eight opsins, the LWS and RH1 alleles were diversified and one particular allele is dominant or fixed in each species, and we propose that this is due to natural selection. The functions of their LWS alleles were also diversified as shown by absorption measurements of reconstituted visual pigments. To analyze the relationship between nuptial coloration and the absorption of LWS pigments, we systematically evaluated and defined nuptial coloration. We showed that the coloration was species specific with respect to hue and significantly differentiated by the index values of hue (dominant wavelength: λ(d)). The λ(d) value of the male nuptial coloration correlated with the absorption of LWS pigments from all the species, suggesting that reproductive isolation through mate choice using color signals may prevent sympatric interspecific hybridization, thereby maintaining the species diversity in sympatric species in Lake Victoria.

Iodocyclization of Hydroxylamine Derivatives Based on the Control of Oxidative Aromatization Leading to 2,5-Dihydroisoxazoles and Isoxazoles

An efficient method for the synthesis of 2,5-dihydroisoxazoles and isoxazoles using iodocyclization of N-alkoxycarbonyl O-propargylic hydroxylamines has been developed. 2,5-Dihydro-4-iodoisoxazole underwent the cross-coupling reactions without aromatization to afford polyfunctionalized 2,5-dihydroisoxazoles. This process was applied to the preparation of valdecoxib and its 2,5-dihydro-derivative.

Steric constraint in the primary photoproduct of sensory rhodopsin II is a prerequisite for light-signal transfer to HtrII.

Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all- trans to 13- cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C 14-D stretching vibration of the retinal chromophore at 2244 cm (-1) upon formation of the K state and interpreted that a steric constraint occurs at the C 14D group in SRII K. Here, we identify the counterpart of the C 14D group as Thr204, because the C 14-D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C 14-HOOP (hydrogen out-of-plane) vibration at 864 cm (-1). Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm (-1) bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C 14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C 14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.