Biology subjects

Sugiura, M.

Publications and source records attributed to Sugiura, M..

14 recordsLinked to original sources

bioRxiv2026

Infection-related images reduce cortical edge metastability: a specification-curve analysis of a block-design fMRI study

Cues of infection evoke disgust, attentional capture, withdrawal and shifts in social orientation, a set of responses attributed to the behavioral immune system. Responses this varied have to be coordinated, as the effects of inflammation are, so they should be visible in whole-cortex coupling. We predicted that infection-related images hold cortical dynamics in a narrower repertoire, reducing cortical edge metastability, the temporal variability of mean cofluctuation across parcel pairs. In two fMRI runs, 44 adults viewed alternating 18-s blocks of infection-related and neutral images; the primary test was specified before we looked at the data. That test gave Cohen's d = -0.31 (permutation p = .042), and 263 of 480 specifications were significant, all negative (joint p < .001). With global signal regression, the synchrony-based reduction survived removal of the stimulus-locked response, detrending, and motion, white-matter and cerebrospinal-fluid regression (d = -0.47), recurred in the second run (d = -0.44), and was smaller on strictly alternating blocks (d = -0.36). Without global signal regression, the pre-specified effect did not survive white-matter and cerebrospinal-fluid regression. Individual-difference predictions were not supported, and participants' contrasts did not replicate across runs. Infection-related images reduce the variability of residual global cofluctuation at the group level; whether this is specific to pathogen cues remains open.

neuroscience↗

bioRxiv2026

Multi-modal ion-transport function of potassium channelrhodopsins

Light-gated K+ channelrhodopsins (KCRs) are recognized as an ideal neuronal silencing tool in optogenetics. KCRs contain functionally important residues for a light-driven H+ pump bacteriorhodopsin, but their roles have been unclear. Utilizing electrophysiological methods, we demonstrate that KCRs function as a light-driven H+ pump in the absence of K+. Transport experiments using pH electrodes further elucidated the function of the H+-pump, which operates even in the presence of K+. The hypothesis that the H+ pump exerts a blocking effect on K+ transport in KCRs has been postulated. Furthermore, we ascertained that there was conversion of KCRs from the H+ pump into the H+ channel by light, yet this conversion was never reversed. However, the addition of K+ resulted in the formation of the K+ channel. KCRs exhibit three distinct functional states, H+ pump, H+ channel, and K+ channel. These multifaceted functions are modulated by environmental ions and light.

biophysics↗

bioRxiv2025

Structural basis for color tuning and passive ion conductance in red-shifted pump-fold channelrhodopsin ChR024

ChR024 is a cation-conducting channelrhodopsin with a red-shifted absorption spectrum, recently discovered from a previously unidentified clade through machine-learning-guided gene mining. Due to its unique position in the phylogenetic tree and distinctive spectral properties, it serves as an important model for elucidating rhodopsin evolution and for engineering optogenetic tools with enhanced performance, but its structure and function remain poorly understood. Here, we present cryo-electron microscopy structures of ChR024 in detergent micelles and lipid nanodiscs at resolutions of 3.22 and 2.45 [A], respectively. The structures reveal an architecture strikingly similar to that of ion-pumping rhodopsins, even more so than other pump-like channelrhodopsins such as ChRmine. Structural, theoretical, and spectroscopic analyses uncover a unique color-tuning mechanism, in which charged residues close and distant from the retinal chromophore cooperatively modulate the pKa of the Schiff base counterion and thereby determine the absorption maximum wavelength. Finally, comparative structural analysis of channel- and pump-type rhodopsins, combined with electrophysiology, provides insights into the molecular boundary between these two functional classes and demonstrates the conversion of an outward proton pump into a light-gated channel. Together, these findings illuminate diverse mechanisms of color tuning and functional specification within the rhodopsin family, paving the way for the rational engineering of next-generation optogenetic tools.

biophysics↗

bioRxiv2025

Differential effects of the D1/S264V mutation in Photosystem II with either PsbA1 or PsbA3 on QB, non-heme Iron, and the associated hydrogen-bond network

The role of the D1/S264 residue and the role of its environment in the proton-coupled electron transfer reaction on the acceptor side of Photosystem II were investigated. To this end, D1/S264V mutants were constructed in the thermophilic cyanobacterium Thermosynechococcus elongatus, with D1 being either PsbA1 or PsbA3. The PSII mutants were investigated using EPR spectroscopy, thermoluminescence, (time-resolved) absorption changes measurements, and oximetry. While the mutation had minor effects in PsbA1-PSII, the S264V mutation in PsbA3-PSII had significant consequences: i) thermoluminescence data show inefficient electron transfer from QA- to QB; ii) re-oxidation of QA- was slowed, by at least a factor of 10; iii) the herbicides inhibit weakly O2 evolution; iv) no Fe2+QB- EPR signal was detected in dark-adapted PSII; instead, v) a large Fe3+ signal was present with vi) modified EPR properties; vii) no QA-Fe2+QB- biradical signal was observed after illumination at 198 K following a flash illumination, confirming the inefficient formation of QB-; viii) either no proton uptake coupled to non-heme iron reduction occurred or with a very slow rate compared to PsbA3-PSII; ix) changes were noted in the electrochromic response associated with QA- formation; and x) increased production of singlet oxygen, both with and without herbicides. The S264V mutation in PsbA3-PSII leads to a significant decrease in the energy gap between the QA-QB and QAQB- states. The effects listed above are discussed regarding the differences between PsbA1-PSII and PsbA3-PSII as those related to the sulfoquinovosyldiacylglycerol, the water molecules and the H-bond network.

biophysics↗

bioRxiv2025

Covalent anti-HIV compound that induces HIV-1 capsid multimerization and degradation decomposes the viral core

Covalent inhibitors form strong, essentially irreversible interactions with their targets, offering enhanced structural selectivity within the target cavity and prolonged target engagement even at low doses. Here, we report that ACAi-001, an HIV-1 capsid (CA)-targeting compound identified through in silico screening of the hydrophobic cavity in the CA N-terminal domain (CA-NTD), inhibits HIV-1 replication in cell-based assays. ACAi-001 covalently binds CA to induce aberrant CA multimerization and degradation in vitro, a pharmacological property distinct from that of existing CA inhibitors such as lenacapavir. This mechanism was confirmed by Western blotting (WB), size exclusion chromatography (SEC), differential scanning fluorimetry (DSF), and liquid chromatography-mass spectrometry (LC-MS). These results suggest that ACAi-001, with two putative covalent interactions in its chemical structure, binds directly to Ser16 in the N-terminal domain targeting cavity in addition to Cys198 or Cys218 in the C-terminal domain of CA. The treatment of viral particles with ACAi-001 results in the loss of intact, conical HIV-1 cores, as directly visualized by transmission electron microscopy (TEM), establishing the HIV-1 core as the direct target of ACAi-001. These findings demonstrated that ACAi-001 may be a promising next-generation anti-HIV-1 CA inhibitor with unique mechanisms of action, offering new insight into HIV-1 core assembly and disassembly.

microbiology↗

bioRxiv2024

Investigation of electrochromic band-shifts in the Soret region induced by the formation of TyrD, TyrZ, and QA- in Photosystem II

The effects of TyrD*, TyrZ*, and QA*- radical formation on the absorption spectrum in the Soret region were studied in Mn-depleted Photosystem II at pH 8.6 (in order to be in the TyrD state after dark adaptation). Flash-induced difference spectra were recorded in several PSII samples from: i) Thermosynechococcus vestitus (formerly T. elongatus), ii) Synechocystis sp. PCC 6803, iii) Chroococcidiopsis thermalis PCC 7203 grown under far-red light, and iv) Acaryochloris marina. In the case of T. vestitus, mutants D1/H198Q, D1/T1789H, D2/I178H, and D2/Y160F, with PsbA1/Q130 instead of PsbA3/E130, were also studied for possible contributions from PD1, ChlD1, ChlD2, and PheD1, respectively. For a possible contribution from PD2, the D2/H197A mutant was studied in S. 6803. While PD1 is clearly the species whose spectrum is blue-shifted by [~]3nm in the presence of QA*-, as has already been well documented in the literature, the species whose spectra shift upon the formation of TyrD* and TyrZ* remain to be clearly identified, as they appear different from PD1, PD2, PheD1, ChlD1, and ChlD2, as concluded by the lack of different light-induced difference spectra in the mutants listed above. Although we cannot rule out a weak effect, considering the accuracy of the experiments, it is proposed that other pigments, such as antenna Chl and/or Car, near the reaction center are involved. Additionally, it is shown that: i) there is no proton release into the bulk upon the oxidation of TyrD at pH 8.6, and ii) the rearrangement of the electrostatic environment of the pigments involved in the light-induced difference spectra in the samples studied, upon the formation of TyrD*, TyrZ*, and QA*-, likely occurs differently from both a kinetic and structural perspective.

biophysics↗

bioRxiv2024

Kinetics of reformation of an S0 state capable of progressing to an S1 state after the O2 release by Photosystem II

The active site for water oxidation in Photosystem II (PSII) comprises a Mn4CaO5 cluster adjacent to a redox-active tyrosine residue (TyrZ). During the water-splitting process, the enzyme transitions through five sequential oxidation states (S0 to S4), with O2 evolution occurring during the S3TyrZ* to S0TyrZ transition. Chloride also plays a role in this mechanism. Using PSII from Thermosynechococcus vestitus, where Ca and Cl were replaced with Sr and Br to slow the S3TyrZ* to S0TyrZ + O2 transition (t1/2 [~] 5 ms at room temperature), it was observed that the recovery of a S0 state, defined as the state able to progress to S1, exhibits similar kinetics (t1/2 [~] 5 ms). This suggests that in CaCl-PSII, the reformation of the functional S0 state directly follows the S3TyrZ* to S0TyrZ + O2 transition, with no additional delay required for the insertion of a new substrate water molecule (O5) and associated protons.

biochemistry↗

bioRxiv2024

Contributions of PD1 and PD2 to the difference spectrum in the Soret region in Photosystem II

Flash-induced absorption changes in the Soret region, which originate from the [PD1PD2]+ state, the chlorophyll cation radical formed upon Photosystem II (PSII) excitation, were investigated in Mn-depleted Photosystem II. In wild-type PSII from Thermosynechococcus elongatus, the [PD1PD2]+-minus-[PD1PD2] difference spectrum shows a main negative feature at 434 nm and a smaller negative feature at 446 nm [Boussac et al. Photosynth Res (2023), https://doi.org/10.1007/s11120-023-01049-3]. While the main feature at 434 nm is associated with PD1+ formation, the origin of the dip at 446 nm remains to be identified. For that, we have compared the [PD1PD2]+-minus-[PD1PD2] difference spectra from the PsbA3/H198Q PSII mutant in T. elongatus and D2/H197A PSII mutant in Synechocystis sp. PCC 6803 with their respective wild type strains. By modifying the PD1 axial ligand with the H198Q mutation in the D1 protein in T. elongatus, the contribution at 434 nm was shifted to 431 nm, while the contribution at 446 nm was hardly affected. In Synechocystis sp. PCC 6803, by modifying the PD2 axial ligand with the H197A mutation in the D2 protein, the contribution at 446 nm was downshifted by [~] 3 nm to [~] 443 nm, while the main contribution at 432 nm was only slightly shifted upwards to 433 nm. This result suggests that the bleaching seen at 446 nm involves PD2. This could reflects a change in the [PD1+PD2]{longleftrightarrow}[PD1PD2+] equilibrium or a more complex mechanism.

biophysics↗

bioRxiv2022

Structural basis for ion selectivity in potassium-selective channelrhodopsins

The KCR channelrhodopsins are recently-discovered light-gated ion channels with high K+ selectivity, a property that has attracted broad attention among biologists- due to intense interest in creating novel inhibitory tools for optogenetics leveraging this K+ selectivity, and due to the mystery of how this selectivity is achieved in the first place. Indeed, the molecular and structural mechanism for K+ selectivity in KCRs has remained especially puzzling since these 7-transmembrane retinal-binding proteins completely lack structural similarity with known K+ channels, which generally coordinate K+ in a precisely symmetric conduction pathway formed by a tight interface among multiple small monomeric channel subunits (presumably not an accessible mechanism for the large KCR rhodopsin proteins). Here we present the cryo-electron microscopy structures of two KCRs from Hyphochytrium catenoides with distinct spectral properties for light absorption and channel actuation, HcKCR1, and HcKCR2, at resolutions of 2.6 and 2.5 [A], respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal in HcKCR2, explaining the large spectral difference between HcKCR1 and 2. Next, our combined structural, electrophysiological, computational, and spectroscopic analyses revealed a new solution to the challenging problem of K+-selective transport. KCRs indeed do not exhibit the canonical tetrameric K+ selectivity filter that specifically coordinates dehydrated K+; instead, single KCR monomers form a size exclusion filter using aromatic residues at the extracellular side of the pore which inhibits passage of bulky hydrated ions. This unique feature allows KCRs to function as K+ channels under relevant physiological conditions, providing not only a novel mechanism for achieving high K+ permeability ratios in biological ion channels, but also a framework for designing the next generation of inhibitory optogenetic tools. In BriefThe first structures of K+-selective channelrhodopsins (HcKCR1 and 2) are determined, revealing a K+ selectivity mechanism distinctly different from canonical K+ channels. HighlightsO_LIThe cryo-EM structures of K+-selective channelrhodopsins, HcKCR1 and 2, in nanodisc C_LIO_LIConditions under which naturally-occurring microbial rhodopsins have a 6-s-cis retinal C_LIO_LIIdentification of key residues for high K+ permeability ratios C_LIO_LIThe unique K+ selectivity mechanism of KCRs C_LI

biophysics↗

bioRxiv2022

Absorption changes in Photosystem II in the Soret band region upon the formation of the chlorophyll cation radical +

Flash-induced absorption changes in the Soret region arising from the [PD1PD2]+ state, the chlorophyll cation radical formed upon light excitation of Photosystem II (PSII), were measured in Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrD is i) reduced before the first flash, and ii) oxidized before subsequent flashes. In wild-type PSII, when TyrD[bullet] is present, an additional signal in the [PD1PD2]+-minus-[PD1PD2] difference spectrum was observed when compared to the first flash when TyrD is not oxidized. The additional feature was "W-shaped" with troughs at 434 nm and 446 nm. This feature was absent when TyrD was reduced, but was present i) when TyrD was physically absent (and replaced by phenylalanine) or ii) when its H-bonding histidine (D2-His189) was physically absent (replaced by a Leucine). Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, seemed to require the native structural environment around the reduced TyrD and its H bonding partners to be present. We found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559 heme in the W-shaped difference spectrum. However, the use of a mutant of the PD2 axial His ligand, the D2-His197Ala, shows that the PD2 environment seems involved in the formation of "W-shaped" signal.

biophysics↗

bioRxiv2022

MITE infestation of germline accommodated by genome editing in Blepharisma

During a sophisticated developmental process, ciliates excise numerous internally eliminated sequences (IESs) from a germline genome copy, producing a functional somatic genome. Most IESs ultimately originate from transposons but homology is obscured by sequence decay. To obtain more representative perspectives on ciliate genome editing, we assembled forty thousand IESs of Blepharisma stoltei, from a much earlier-diverging lineage than existing models. Short IESs (< 115 bp) were largely non-repetitive, with a pronounced ~10 bp length periodicity, whereas longer IESs (max 7 kbp) were non-periodic and contained abundant interspersed repeats. Contrary to current models, the Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8000 copies) was a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We propose MITEs as an important and eventually self-limiting IES source. Rather than defending germline genomes against mobile elements, we argue that transposase domestication actually facilitates junk DNA accumulation.

genomics↗

bioRxiv2021

The Blepharisma stoltei macronuclear genome: towards the origins of whole genome reorganization

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout a copy of the ciliate germline genome are deleted during development of the streamlined somatic genome. Blepharisma represents one of the two earliest diverging ciliate classes, and, unusually, has dual pathways of somatic nuclear development, making it ideal for investigating the functioning and evolution of these processes. Here, we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mb), arranged as numerous alternative telomere-capped minichromosomes. This genome encodes eight PiggyBac transposase homologs liberated from transposons. All are subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We propose PiggyBac homologs were ancestral excisases that enabled evolution of extensive, natural genome editing.

genomics↗

bioRxiv2020

Structural insights into the mechanism of rhodopsin phosphodiesterase

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase (Rh-GC) and photosensitive adenylyl cyclase (PAC). Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The 2.6 [A] resolution crystal structure of the transmembrane domain revealed a new topology of rhodopsin, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the transmembrane and PDE domain (2.1 [A]) with their connecting linkers. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

biophysics↗

bioRxiv2019

High-throughput genotyping of a full voltage-gated sodium channel gene via genomic DNA using target capture sequencing and analytical pipeline MoNaS to discover novel insecticide resistance mutations

Insects voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein are known to confer insecticide resistance and are selected under insecticide pressure. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure of gene VGSC, it is usually challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes via annotated CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all homologous VGSC CDSs except tiny exons from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency by virtue of the high conservation of VGSC at the nucleotide level. Furthermore, we developed an automated bioinformatic analysis pipeline to genotype VGSC after capture sequencing--MoNaS (Mosquito Na+ channel mutation Search)--which conducts mapping of reads, variant calling, and variant annotation for nonsynonymous mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetics studies on resistance alleles with respect to the origin, selection, and migration of both clinically and agriculturally important insect pests.

genetics↗