Biology subjects

Tsukamoto, H.

Publications and source records attributed to Tsukamoto, H..

5 recordsLinked to original sources

bioRxiv2024

Characterization and engineering of a blue-sensitive, Gi/o-biased, and bistable ciliary opsin from a fan worm

Ciliary opsins have been identified not only in vertebrates but also in invertebrates. An invertebrate ciliary opsin was recently identified in the fan worm Acromegalomma interruptum (formerly named Megalomma interrupta); however, its spectral and signaling characteristics are unknown. In the present study, we characterized the spectral properties and light-induced cellular signaling properties of the opsin (AcrInvC-opsin). AcrInvC-opsin showed an absorption maximum at 464 nm and upon blue-light absorption, the spectrum was red-shifted by approximately 50 nm. The two states are inter-convertible by illumination with blue and orange light. Blue light illumination of AcrInvC-opsin caused specific coupling with Gi, sustained Gi dissociation, decreased intracellular cAMP levels, and activation of GIRK channels. The cellular responses by the activated opsin were partially terminated by orange light illumination. These light-dependent responses indicate that the InvC-opsin is a typical bistable pigment wherein the resting and activated states can be inter-converted by visible light illumination. We also attempted to modulate the spectral and functional properties of AcrInvC-opsin using site-directed mutagenesis. Substitution of Ser-94 with Ala caused little spectral shift in the resting state but a further red-shift of [~]10 nm in the activated state, indicating that the absorption spectra of the two states were tuned differently. In contrast, the S94A substitution did not significantly affect the light-dependent signaling properties of AcrInvC-opsin. Because AcrInvC-opsin is a blue-sensitive, Gi/o-biased, and bistable pigment, it has the potential to serve as an optical control tool to specifically and reversibly regulate Gi/o-dependent signaling pathways by visible light.

biophysics↗

bioRxiv2024

Meta-omic insights into active bacteria mediating N2O mitigation and dissimilatory nitrate reduction to ammonium in an ammonia recovery bioreactor

Shifting from ammonia removal to recovery is the current strategy in wastewater treatment management. We recently developed a microaerophilic activated sludge (MAS) system for retaining ammonia while removing organic carbon with minimal N2O emissions. A comprehensive understanding of nitrogen metabolisms in the MAS system is essential to optimize system performance. Here, we employed metagenomics and metatranscriptomics analyses to characterize the microbial community structure and activity during the transition from a microaerophilic to an aerobic condition. A hybrid approach of high-quality Illumina short reads and Nanopore long reads recovered medium-to high-quality 98 non-redundant metagenome-assembled genomes (MAGs) from the MAS communities. The suppressed bacterial ammonia monooxygenase (amoA) expression was upregulated after shifting from a microaerophilic to an aerobic condition. The 73 MAGs (>74% of the total) from 11 bacterial phyla harbored genes encoding proteins involved in nitrate respiration; 39 MAGs ([~]53%) carried N2O reductase (nosZ) genes with the predominance of clade II nosZ (31 MAGs), and 24 MAGs ([~]33%) possessed nitrite reductase (ammonia forming) genes (nrfA). Clade II nosZ and nrfA genes exhibited the highest and second-highest expressions among nitrogen metabolism genes, indicating robust N2O consumption and ammonification. Non-denitrifying clade II nosZ bacteria, Cloacibacterium spp., in the most abundant and active phylum Bacteroioda, were likely major N2O sinks. Elevated dissolved oxygen (DO) concentration inhibited clade II nosZ expression but not nrfA expression, potentially switching phenotypes from N2O reduction to ammonification. Collectively, the multi-omics analysis illuminated vital bacteria responsible for N2O reduction and ammonification in microaerophilic and aerobic conditions, facilitating high-performance ammonia recovery.

ecology↗

bioRxiv2024

Molecular basis underlying specific interaction of mammalian melanopsins with an antagonist AA92593

Melanopsin functions in intrinsically photosensitive retinal ganglion cells of mammals to regulate circadian clock and pupil constriction. The opsinamide AA92593 has been reported to specifically inhibit mouse and human melanopsin functions as a competitive antagonist against retinal; however, the molecular mechanisms underlying its specificity have not been resolved. In this study, we attempted to identify amino acid residues responsible for the specific interaction of AA92593 with mammalian melanopsins. Our cell-based assays confirmed that AA92593 effectively inhibited the light-induced cellular responses of mammalian melanopsins, but not those of non-mammalian vertebrate and invertebrate melanopsins. These results suggest that amino acid residues specifically conserved among mammalian melanopsins are important for the antagonistic effect of AA92593, and we noticed Phe-94, Ser-188, and Ser-269 as candidate residues. Substitutions of these residues reduced the antagonistic effect of AA92593. We conducted docking and molecular dynamics simulations based on the AlphaFold-predicted melanopsin structure. The simulations indicated that Phe-94, Ser-188, and Ser-269 are located at the AA92593-binding site, and additionally identified Trp-189 and Leu-207 interacting with the antagonist. Substitutions of Trp-189 and Leu-207 affected the antagonistic effect of AA92593. Furthermore, substitutions of these amino acid residues converted AA92593-insensitive melanopsins susceptible to the antagonist. Based on experiments and molecular simulations, five amino acid residues, at positions 94, 188, 189, 207, and 269, were found to be responsible for the specific interaction with AA92593 in mammalian melanopsins.

biochemistry↗

bioRxiv2023

A self-inactivating invertebrate opsin with resistance to retinal depletion optically drives biased signaling toward Gβγ-dependent ion channel modulation

Animal opsins, light-sensitive G protein-coupled receptors (GPCRs), have been utilized for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Ga and G{beta}{gamma} subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Ga-, G{beta}{gamma}-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Ga and G{beta}{gamma}. Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for G{beta}{gamma}-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between G{beta}{gamma}-dependent and Ga-dependent responses. The opsin-induced transient Gi/o activation preferably causes activation of the kinetically-fast G{beta}{gamma}-dependent GIRK channels rather than slower Gi/o-dependent adenylyl cyclase inhibition. Although similar G{beta}{gamma}-biased signaling properties were observed in a selfinactivating vertebrate visual pigment, Platynereis c-opsin1 needs fewer retinal molecules to evoke cellular responses. Furthermore, the G{beta}{gamma}-biased signaling properties of Platynereis c-opsinl are enhanced by genetically fused with RGS8 protein which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for G{beta}{gamma}-dependent ion channel modulation.

biophysics↗

bioRxiv2022

Microaerophilic activated sludge system for ammonia recovery from high-strength nitrogenous wastewater: Performance and microbial communities

A transition to ammonia recovery from wastewater has started; however, a technology for sustainable nitrogen retention in the form of ammonia is still in development. This study validated a microaerophilic activated sludge (MAS) system to efficiently retain ammonia from high-strength nitrogenous wastewater. The MAS is based on conventional activated sludge (CAS) with aerobic and settling compartments. Low dissolved oxygen (DO) concentrations (<0.1 mg/L) and short solid retention times (SRTs) (<5 d) eliminated nitrifying bacteria. The two parallel MASs were successfully operated for 300 d and had ammonia retention of 101.7 {+/-} 24.9% and organic carbon removal of 85.5 {+/-} 8.9%. The MASs mitigated N2O emissions with an emission factor of <0.23%, much lower than the default value of CAS (1.6%). A short-term step-change test demonstrated that N2O indicated the initiation of nitrification and the completion of denitrification in the MAS. The parallel MASs had comparable microbial diversity, promoting organic carbon oxidation while inhibiting ammonia-oxidizing microorganisms (AOMs), as revealed by 16S rRNA gene amplicon sequencing, qPCR of functional genes, and fluorescent in situ hybridization of {beta}-Proteobacteria AOB. The microbial analyses also uncovered that filamentous bacteria were positively correlated with effluent turbidity. Together, controlling DO and SRT achieved successful ammonia retention, mainly by suppressing AOM activity. This process represents a new nitrogen management paradigm. SynopsisMoving from nitrogen removal to nitrogen recovery is critical for establishing a sustainable society. We provided proof-of-the-concept for a novel ammonia retention technology by retrofitting an activated sludge system.

microbiology↗