Biology subjects

Kato, T.

Publications and source records attributed to Kato, T..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Cryo-EM structures of Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae

The Na+-pumping NADH-ubiquinone oxidoreductase (Na+-NQR) couples electron transfer from NADH to ubiquinone with Na+-pumping, generating an electrochemical Na+ gradient that is essential for energy-consuming reactions in bacteria. Since Na+-NQR is exclusively found in prokaryotes, it is a promising target for highly selective antibiotics. However, the molecular mechanism of inhibition is not well-understood for lack of the atomic structural information about an inhibitor-bound state. Here we present cryo-electron microscopy structures of Na+- NQR from Vibrio cholerae with or without a bound inhibitor at 2.5- to 3.1-[A] resolution. The structures reveal the arrangement of all six redox cofactors including riboflavin, whose position has been under debate, and a newly assigned 2Fe-2SNqrD/E cluster located between the membrane embedded NqrD and NqrE subunits. A large part of the hydrophilic NqrF near the cytoplasmic membrane surface is barely visible in the density map, suggesting a high degree of flexibility. This flexibility may be responsible to reducing the long distance between the 2Fe- 2S centers in NqrF and NqrD/E, consistent with physiologically relevant electron transfer. Two different types of specific inhibitors (korormicin A and aurachin D-42) bind to the N-terminal region of NqrB, which is disordered in the absence of inhibitors. The current inhibitor-bound structures reasonably explain our previous biochemical findings obtained by different chemistry-based experiments. This study provides a definite foundation for understanding the function of Na+-NQR and the molecular mechanism of its specific inhibitors to support molecular design of new antibiotics targeting the enzyme.

biochemistry↗

bioRxiv2022

Complete sequencing of the mouse pseudoautosomal region, the most rapidly evolving 'chromosome'

The pseudoautosomal region (PAR) of mammalian sex chromosomes is a small region of sequence identity that allows pairing, crossover, recombination, and proper chromosome segregation during male meiosis. The structure of the mouse PAR is largely unknown. Here, we developed a new assembly method to robustly resolve repetitive sequences and employed highly accurate long-read sequencing data to reveal the entire PAR sequence. The PAR of the widely-used inbred strain C57BL/6J is [~]700 kb, comprising 10 protein- coding genes in a mass of complex repetitive sequences. A large segmental duplication exhibiting copy-number polymorphisms even among C57BL/6J littermates is present. High GC-content exons and short introns are common properties of PAR genes and are the consequence of maintaining gene function, while PAR is rapidly evolving. Elucidating the mouse PAR sequence completes the mouse euchromatic genome sequencing and enables the exploration of the function and evolution of the PAR using modern molecular genetic approaches.

genomics↗

bioRxiv2022

Oral administration of S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and accelerates recovery from clinical aspects of COVID-19

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622, a novel inhibitor of SARS-CoV-2 main protease (Mpro, also known as 3C-like protease), decreases viral load and ameliorates the disease severity in SARS-CoV-2-infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to sub-micromolar concentrations in cells. Oral administration of S-217622 demonstrated eminent pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern (VOCs), including the highly pathogenic Delta variant and the recently emerged Omicron variant. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase II/III clinical trial, possesses remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

microbiology↗

bioRxiv2022

Distinctiveness and continuity in transcriptome and connectivity in the anterior-posterior axis of the paraventricular nucleus of thalamus

The paraventricular nucleus of the thalamus (PVT) projects axons to multiple areas and mediates a wide range of behaviors. Heterogeneity of functions and axonal projections in PVT have been reported, but what cell types exist in PVT and how different they are have not been addressed. We applied single-cell RNA sequencing to depict transcriptomic characteristics of mouse PVT neurons. The transcriptome of PVT neurons had a continuous distribution with the largest variance corresponding to the anterior-posterior axis. Although the single-cell transcriptome classified PVT neurons into four types, transcriptomic and histological analyses showed their continuity. Similarly, anterior and posterior subpopulations had nearly non-overlapping axon projection patterns, while another population showed intermediate patterns. In addition, they responded differently to appetite-related neuropeptides, and their chemogenetic activation showed opposing effects in food consumption. Our studies showed contrasts and continuity of PVT neurons underlying their function as a behavior-modulating hub.

neuroscience↗

bioRxiv2022

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane {beta} sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

biochemistry↗

bioRxiv2022

Discovery of S-217622, a Non-Covalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in millions of deaths and threatens public health and safety. Despite the rapid global spread of COVID-19 vaccines, effective oral antiviral drugs are urgently needed. Here, we describe the discovery of S-217622, the first oral non-covalent, non-peptidic SARS-CoV-2 3CL protease inhibitor clinical candidate. S-217622 was discovered via virtual screening followed by biological screening of an in-house compound library, and optimization of the hit compound using a structure-based drug-design strategy. S-217622 exhibited antiviral activity in vitro against current outbreaking SARS-CoV-2 variants and showed favorable pharmacokinetic profiles in vivo for once-daily oral dosing. Furthermore, S-217622 dose-dependently inhibited intrapulmonary replication of SARS-CoV-2 in mice, indicating that this novel non-covalent inhibitor could be a potential oral agent for treating COVID-19.

biochemistry↗

bioRxiv2021

Interplay of adherens junctions and matrix proteolysis determines the invasive pattern and growth of squamous cell carcinoma

Cancers, such as squamous cell carcinoma, frequently invade as multicellular units. However, these invading units can be organized in a variety of ways, ranging from thin discontinuous strands to thick pushing collectives. Here we employ an integrated experimental and computational approach to identify the factors that determine the mode of collective cancer cell invasion. We find that matrix proteolysis is linked to the formation of wide strands, but has little effect on the maximum extent of invasion. Cell-cell junctions also favour wide strands, but our analysis also reveals a requirement for cell-cell junctions for efficient invasion in response to uniform directional cues. Unexpectedly, the ability to generate wide invasive strands is coupled to the ability to grow effectively when surrounded by ECM in 3D assays. Combinatorial perturbation of both matrix proteolysis and cell-cell adhesion demonstrates that the most aggressive cancer behaviour, both in terms of invasion and growth, is achieved at high levels of cell-cell adhesion and high levels of proteolysis. Contrary to expectation, cells with canonical mesenchymal traits - no cell-cell junctions and high proteolysis - exhibit reduced growth and lymph node metastasis. Thus, we conclude that the ability of squamous cell carcinoma cells to invade effectively is also linked to their ability to generate space for proliferation in confined contexts. These data provide an explanation for the apparent advantage of retaining cell-cell junctions in SCC.

cancer biology↗

bioRxiv2021

Oscillatory population-level activity of dorsal raphe serotonergic neurons sculpts sleep structure

Dorsal raphe (DR) 5-HT neurons are involved in regulating sleep-wake transitions. Previous studies demonstrated that single-unit activity of DR 5-HT neurons is high during wakefulness, decreases during non-rapid eye movement (NREM) sleep, and ceases during rapid eye movement (REM) sleep. However, characteristics of the population-level activity of DR 5-HT neurons, which can influence the entire brain, are largely unknown. Here we measured population activities of 5-HT neurons in male and female mouse DR across the sleep-wake cycle by a ratiometric fiber photometry system. We found a slow oscillatory activity of compound intracellular Ca2+ signals during NREM sleep. The trough of concave 5-HT activity increased along with sleep progression, but the 5-HT activity level always returned to that seen in wake periods. When the trough reached the minimum level and remained there, REM sleep initiated. We also found a unique coupling of the oscillatory 5-HT activity and EEG power fluctuation, suggesting that EEG fluctuation is a proxy for 5-HT activity. Optogenetic activation of 5-HT neurons during NREM sleep triggered a high EMG power and induced wakefulness. Optogenetic inhibition induced REM sleep or sustained NREM with an EEG power increase and EEG fluctuation. These manipulations demonstrated a causal role of DR 5-HT neurons in sculpting sleep-wake structure. We also observed EEG fluctuations in human males during NREM sleep, implicating the existence of 5- HT oscillatory activity in humans. We propose that NREM sleep is not a monotonous state, but that it is dynamically regulated by the oscillatory population activity of DR 5- HT neurons. Significant statementPrevious studies have demonstrated single-cell 5-HT neuronal activity across sleep- wake conditions; however, population-level activities of these neurons are largely unknown. We monitored dorsal raphe (DR) 5-HT population activity using a fiber photometry system in mice and demonstrated that activity was highest during wakefulness, and lowest during rapid eye movement (REM) sleep. Surprisingly, during non-REM (NREM) sleep, the 5-HT population activity decreased with an oscillatory pattern, coinciding with EEG fluctuations. We examined the causal role of these 5-HT neuron activities by optogenetics and found that DR 5-HT neurons sculpted sleep-wake conditions by influencing EEG and EMG patterns. We found similar EEG fluctuations in a human sleep EEG study, suggesting the presence of oscillatory 5-HT neuron activity during NREM across species.

neuroscience↗

bioRxiv2021

Epoxidized graphene grid for high-throughput high-resolution cryoEM structural analysis

Many specimens suffer from low particle density and/or preferred orientation in cryoEM specimen grid preparation, making data collection and structure determination time consuming. We developed an epoxidized graphene grid (EG-grid) that effectively immobilizes protein particles by applying an oxidation reaction using photoactivated ClO2* and further chemical modification. The particle density and orientation distribution are both dramatically improved, having enabled us to reconstruct the density map of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), at 1.99 and 2.16 [A] resolution from only 504 and 241 micrographs, respectively. A low concentration sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 [A] resolution density map of SARS-CoV-2 spike protein from 1,163 micrographs. The density maps of V1-ATPase, {beta}-galactosidase, and apoferritin were also reconstructed at 3.03, 1.81, and 1.29 [A] resolution, respectively. These results indicate that the EG-grid will be a powerful tool for high-throughput cryoEM data collection to accelerate high-resolution structural analysis of biological macromolecules.

molecular biology↗

bioRxiv2021

Structural insights into human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

molecular biology↗

bioRxiv2021

Structural snapshots of V/A-ATPase reveal a new paradigm for rotary catalysis

V/A-ATPase is a motor protein that shares a common rotary catalytic mechanism with FoF1 ATP synthase. When powered by ATP hydrolysis, the V1 moiety rotates the central rotor against the A3B3 hexamer, composed of three catalytic AB dimers adopting different conformations (ABopen, ABsemi, and ABclosed). Here we have determined the atomic models of 18 catalytic intermediates of the V1 moiety of V/A-ATPase under different reaction conditions by single particle Cryo-EM, which revealed that the rotor does not rotate immediately after binding of ATP to the V1. Instead, three events proceed simultaneously with the 120{degrees} rotation of the shaft: hydrolysis of ATP in ABsemi, zipper movement in ABopen by the binding ATP, and unzipper movement in ABclosed with release of both ADP and Pi. This indicates the unidirectional rotation of V/A-ATPase by a ratchet-like mechanism owing to ATP hydrolysis in ABsemi, rather than the power stroke model proposed previously for F1-ATPase.

biophysics↗

bioRxiv2021

Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution

Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound X-ray structures of an Asgard tubulin from hydrothermal-living Odinarchaeota (OdinTubulin). The GTP-bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the "+" end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolution intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.

evolutionary biology↗

bioRxiv2021

The kinesin KIF4 mediates HBV/HDV entry through regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP C4 cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection and suppressed both wild-type HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 regains the surface localization of NTCP in these cells. Furthermore, immunofluorescence (IF) revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study showed that KIF4 physically binds to NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent suppression of HBV infection in HepG2-hNTCP and primary human hepatocytes (PXB) (Bexarotene, IC50 1.89 {+/-} 0.98 M). Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates that target HBV and HDV entry phases. Author SummaryUnderstanding HBV/HDV entry machinery and the mechanism by which NTCP (HBV/HDV entry receptor) surface expression is regulated is crucial to develop antiviral entry inhibitors. We found that NTCP surface transport is mainly controlled by the motor kinesin KIF4. Surprisingly, KIF4 was negatively regulated by RXR receptors through FOXM1-mediated suppression This study not only mechanistically correlated the role of RXR receptors in regulating HBV/HDV entry but also suggested a novel approach to develop therapeutic rexinoids for preventing HBV and/or HDV infections in important clinical situations, such as in patients undergoing liver transplantation or those who are at a high risk of HBV infection and unresponsive to HBV vaccination.

microbiology↗

bioRxiv2021

Hyperconcentrated Mucus Unifies Submucosal Gland and Superficial Airway Dysfunction in Cystic Fibrosis

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway CF model revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Utilizing proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, proteomics analyses of CF sputum revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and within SMGs of the CF lung.

pathology↗

bioRxiv2021

The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines

mRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

microbiology↗

bioRxiv2021

Flexible recruitments of fundamental muscle synergies in the trunk and lower limbs for highly variable movements and postures

The extent to which muscle synergies represent the neural control of human behavior remains unknown. Here, we tested whether certain sets of muscle synergies that are fundamentally necessary across behaviors exist. We measured the electromyographic activities of 26 muscles including bilateral trunk and lower limb muscles during 24 locomotion, dynamic and static stability tasks, and extracted the muscle synergies using non-negative matrix factorization. Our results showed that 13 muscle synergies that may have unique functional roles accounted for almost all 24 tasks by combinations of single and/or merging of synergies. Therefore, our results may support the notion of the low dimensionality in motor outputs, in which the central nervous system flexibly recruits fundamental muscle synergies to execute diverse human behaviors. Further studies using manipulations of the central nervous system and/or neural recording are required the neural representation with such fundamental components of muscle synergies.

neuroscience↗

bioRxiv2021

Change in the bladder function of rats with focal cerebral infarction induced by photochemically-induced thrombosis

The photochemically-induced thrombosis(photothrombosis) method can create focal cerebral infarcts anywhere in the relatively superficial layers of the cerebrum; it is easy to implement and minimally invasive. Taking advantage of this versatility, we aimed to establish a new rat model of urinary frequency with focal cerebral infarction, which was characterized by its simplicity, nonlethal nature, and high reproducibility. The prefrontal cortex and the anterior cingulate cortex, which are urinary centers, were targeted for focal cerebral infarction, and urinary parameters were measured by cystometrogram. Cystometric analysis indicated that micturition intervals significantly shortened in photothrombosis-treated rats compared with those in the sham operative group on Days 1 and 7 (P < 0.01), but prolonged after 14 days, with no difference between the two groups. Immunopathological evaluation showed an accumulation of activated microglia, followed by an increase in reactive astrocytes at the peri-infarct zone after photothrombotic stroke. Throughout this study, all postphotothrombosis rats showed cerebral infarction in the prefrontal cortex and anterior cingulate cortex; there were no cases of rats with fatal cerebral infarction. This model corresponded to the clinical presentation, in that the micturition status changed after stroke. In conclusion, this novel model combining nonlethality and high reproducibility may be a suitable model of urinary frequency after focal cerebral infarction.

physiology↗

bioRxiv2021

Chained structure of dimeric F1-like ATPase in Mycoplasma mobile gliding machinery

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called chains that are each comprised of 17 repeating protein units called particles. These proteins include homologs of the catalytic - and {beta}-subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOBs 1660 (-subunit homolog), 1670 ({beta}-subunit homolog), 1630, 1620, and 4530, and showed ATP hydrolyzing activity. The 2D structure, with dimensions of 35 and 26 nm, showed a hexameric ring dimer about 12 nm in diameter, resembling F1-ATPase catalytic ({beta})3. We isolated the F1-like ATPase unit, which is composed of MMOBs 1660, 1670, and 1630. Furthermore, we isolated the chain and analyzed the 3D structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31 nm intervals. An atomic model of F1-ATPase catalytic ({beta})3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCEF1Fo- ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria, and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

microbiology↗