Biology subjects

Yokoyama, K.

Publications and source records attributed to Yokoyama, K..

15 recordsLinked to original sources

bioRxiv2026

Mycothiazole enables structural characterization of entire ubiquinone-accessing tunnel in mitochondrial respiratory complex I

Mitochondrial NADH-ubiquinone (UQ) oxidoreductase (respiratory complex I) reduces UQ within its long tunnel-like cavity, which is coupled to triggering proton translocation at the membrane domain. Although mammalian complex I adopts distinct "open" and "closed" conformations, the architecture of the UQ-accessing tunnel in the open conformation remains poorly defined owing to partial disorder of the UQ catalytic region near Fe-S cluster N2 in the tunnel. Here, we applied cryo-electron microscopy (EM) single particle analysis to determine the structures of complex I in bovine heart submitochondrial particles in the presence or absence of a mycothiazole-type inhibitor. Preceding photoaffinity labeling experiments using a synthetic photoreactive derivative ([125I]pMT) revealed that mycothiazole specifically labels the 49-kDa and ND1 subunits, both of which form a part of the UQ-accessing tunnel. Cryo-EM analysis demonstrated that bound mycothiazole reorganizes and stabilizes the UQ catalytic region in both open and closed conformations, enabling the first complete structural modeling of the entire UQ-accessing tunnel in the open conformation. Unexpectedly, in the presence of bound mycothiazole, loops of the PSST subunit forming the catalytic region in the tunnel adopted closed conformation-like features, whereas open conformation-like features were determined for the transmembrane helices of ND1 and ND6 subunits. The present study revealed that bound mycothiazole can reorganize the UQ catalytic region in the UQ-accessing tunnel and lead to a hybrid conformational state beyond the canonical open-versus-closed conformational model.

biochemistry↗

bioRxiv2025

Upregulation of the lncRNA XACT sustains pluripotency, blocks lineage specification, and drives germ cell tumor-like transcriptional programs in human pluripotent stem cells

Long noncoding RNAs (lncRNAs) represent a vast class of regulatory transcripts and are spatiotemporally controlled, yet only a few have been functionally implicated in human development. Here, we identify the X-linked lncRNA XACT, abundantly but transiently expressed during early human embryogenesis, as a critical regulator of pluripotency, lineage specification, and cancer-like states. In human pluripotent stem cells (hPSCs), XACT overexpression--but not depletion--sustains self-renewal without exogenous factors and prevents lineage commitment. Mechanistically, XACT upregulation drives hyper-elevation of the core pluripotency factors OCT4 and NANOG at the protein level by repressing their 3' untranslated regions (UTRs). XACT overexpression confers context-dependent states: in standard hPSC medium it promotes a naive-like program, whereas in the absence of exogenous factors it drives transcriptomic states resembling testicular germ cell tumors, linking misregulation of a developmentally restricted lncRNA to tumorigenic potential. In hPSC-based models of post-implantation development, XACT expression normally declines, whereas its sustained expression disrupts embryonic progression, while depletion has little effect. Finally, transcriptomic analysis of post-implantation human embryos showed that XACT levels correlate positively with pluripotency-associated gene networks. Together, these findings establish XACT as a potent, human-specific modulator of pluripotency and early embryogenesis, and suggest that its aberrant upregulation may underlie both developmental failure and germ cell tumorigenesis.

cell biology↗

bioRxiv2025

In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane.

To understand how membrane protein complexes function within biological membranes, it is essential to determine their structure in their natural membrane environment. Here, we employed cryoEM structure analysis to elucidate the structures of ATP synthase FoF1 and respiratory Supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. On SMPs, the majority of FoF1 was identified as dimers bound by the regulatory factor dimeric IF1. In addition, a tetrameric structure formed by association of FoF1 IF1 dimers and with a linear arrangement of the F1 head were also identified. These structures induced a steep membrane curvature, indicating the presence of a structure on SMPs similar to that found on the tips of mitochondrial cristae. High-resolution structures of the respiratory complexes were also determined, and sub-class structures of both CI and CIII2 were resolved. Most SCs were of the CI1CIII2CIV3 structure, although the presence of the CI2CIII2CIV6 mega complex was also identified. Our study enabled rapid in situ structural determination of SCs and FoF1 ATP synthase from small amount of membrane fractions, paving the way for elucidation of the molecular basis of metabolic disorders and mitochondrial diseases at the level of higher-order architecture.

biochemistry↗

bioRxiv2025

Efficient gene disruption with CRISPR-Cas3 in human T cells

The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease (GVHD) risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.

cancer biology↗

bioRxiv2025

A planar dimer of bovine ATP synthase

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by antiparallel arrangement of two ATP synthase complexes linked by a straight conformation of the inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent subunit. These structural divergences suggest a specialized functional role and localization distinct from the canonical sharply membrane bending ATP synthase oligomers, providing structural support for a model of membrane curvature-driven division of labor within mitochondrial ATP synthase populations.

biophysics↗

bioRxiv2024

Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing

Erosion of X-chromosome inactivation (XCI) complicates disease and developmental modeling in female human pluripotent stem cells (hPSCs). Previous studies demonstrated that Cas9-mediated editing of the XIST promoter via non-homologous end joining (NHEJ) or homology-directed repair (HDR) with a selection cassette upstream of XIST can trigger DNA demethylation and XIST reactivation, restoring XCI. Here, we show that NHEJ-mediated XCI reacquisition is more stable during differentiation than HDR. We further developed a novel, efficient XIST reactivation method by combining TP53 inhibition with DNA methylation maintenance suppression during Cas9-mediated NHEJ, offering a robust approach to achieving stable XCI in female hPSCs for diverse applications.

cell biology↗

bioRxiv2023

How to control unpredictable others in court-net sports

Competition in sports does not involve a single solution because individuals aim to behave unpredictably, thereby preventing others from predicting their actions. This study determined how individuals in court net sports tried to control others unpredictable behavior, thereby addressing the gap of the lack of clarity about strategies employed by individuals in competitive situations. We employed a switching hybrid dynamics model, considering external inputs when analyzing individual behaviors. The study shows that skilled individuals, unlike intermediate players, exhibit greater regularity in their behavior, lead others to anticipate this regularity, and employ strategies to disrupt these expectations. This strategy exploits the principles of active human inference, implying that competition involves cooperation. We revealed this by analyzing both human decision-making and behavior in actual matches as discrete and continuous dynamical systems. This strategy helps policymakers adopt a new policy targeting cooperation with competitors, which would increase competitiveness in our daily lives.

biophysics↗

bioRxiv2022

Mechanism of ATP hydrolysis dependent rotation of ATP synthases

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central {gamma}-subunit proceeds in 120{degrees} steps against a surrounding 3{beta}3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic {beta} dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80{degrees} rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic {beta} dimers. The remaining 40{degrees} rotation of the complete 120{degrees} step is driven by completion of ATP hydrolysis at D{beta}D, and proceeds through three sub-steps (83{degrees}, 91{degrees}, 101{degrees}, and 120{degrees}) with three associated conformational intermediates. All sub-steps except for one between 91{degrees} and 101{degrees} associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40{degrees} rotation is largely driven by release of intramolecular strain accumulated by the 80{degrees} rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

biophysics↗

bioRxiv2022

Structural transition of the ground-state structure to steady-state structures by sequential binding of ATP to V/A-ATPase

V/A-ATPase is a rotary ATPase that shares a common rotary catalytic mechanism with FoF1 ATP synthase. Structural images of V/A-ATPase obtained by single-particle cryo-electron microscopy (cryo-EM) during ATP hydrolysis identified several intermediates, revealing the rotary mechanism under steady-state conditions. Here, we identified the cryo-EM structures of V/A-ATPase corresponding to short-lived initial intermediates during the activation of the ground state structure by time-resolving snapshot analysis. These intermediate structures provide insights into how the ground-state structure changes to the active, steady state through the sequential binding of ATP to its three catalytic sites. All the intermediate structures of V/A-ATPase adopt the same asymmetric structure, whereas the three catalytic dimers adopt different conformations. This is significantly different from the initial activation process of FoF1, where the overall structure of the F1 domain changes during the transition from a pseudo-symmetric to a canonical asymmetric structure. Our findings will enhance the future prospects for the initial activation processes of the enzymes with dynamical information, which contains unknown intermediate structures in their functional pathway.

biophysics↗

bioRxiv2022

A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics

Accurate quantitation of antibody is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

immunology↗

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↗

bioRxiv2020

The mechanical inhibition of the isolated Vo from V-ATPase for proton conductance

V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 moiety, with proton flow through the Vo membrane moiety, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo of eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and the isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo adopts the auto-inhibited form and how the holo complex prevents the formation of this inhibited Vo form. One Sentence SummaryCryo-EM structures of rotary V-ATPase reveal the ON-OFF switching mechanism of H+ translocation in the Vo membrane domain.

biophysics↗

bioRxiv2020

Accumulation of an unprecedented 5'-deoxyadenos-4'-yl radical unmasks the kinetics of the radical-mediated C-C bond formation step in MoaA catalysis

Radical S-adenosyl-L-methionine (SAM) enzymes catalyze various free radical-mediated reactions. In these enzymes, the rate-determining SAM cleavage kinetically masks all the subsequent steps. Due to this kinetic masking, detailed mechanistic characterization of radical transformations catalyzed by these enzymes is very difficult. Here, we report a successful kinetic characterization of the radical C-C bond formation catalyzed by a MoaA radical SAM enzyme. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterization, we found that MoaA accumulates a 5'-deoxyadenos-4'-yl radical (5'-dA-C4'*) under the turnover conditions, and forms (4'S)-5'-deoxyadenosine ((4'S)-5'-dA), which is a C-4' epimer of the naturally occurring (4'R)-5'-dA. Together with kinetic characterizations, these observations revealed the presence of a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from 5'-dA to transiently generate 5'-dA-C4'* that is subsequently reduced stereospecifically to yield (4'S)-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics, and determined the rate of the on-pathway 3',8-cyclization step as 2.7 {+/-} 0.7 s-1. Together with DFT calculations, this observation suggested that the 3',8-cyclization is accelerated by 6 [~] 9 orders of magnitude by MoaA. Potential contributions of the active-site amino acid residues, and their potential relationships with human Moco deficiency disease are discussed. This is the first determination of the magnitude of catalytic rate acceleration by a radical SAM enzyme, and provides the foundation for understanding how radical SAM enzymes achieve highly specific radical catalysis.

biochemistry↗

bioRxiv2019

Neuropeptide B mediates female sexual receptivity in medaka fish, acting in a female-specific but reversible manner

Male and female animals display innate sex-specific mating behaviors. Among vertebrates, teleosts are unique in that altering the adult sex steroid milieu can reverse sex-typical mating behaviors, suggesting sexual lability of their brains. In the teleost medaka, neuropeptide B (NPB) is expressed female-specifically in the brain nuclei implicated in mating behavior. Here, we demonstrate that NPB is a direct mediator of estrogen action on female mating behavior, acting in a female-specific but reversible manner. Analysis of regulatory mechanisms revealed that the female-specific expression of NPB is dependent on direct transcriptional activation by estrogen via an estrogen-responsive element and is reversed in response to changes in the adult sex steroid milieu. Behavioral studies of NPB knockouts revealed that female-specific NBP mediates female receptivity to male courtship. The female-specific NPB signaling identified herein is presumably a critical element of the neural circuitry underlying sexual dimorphism and lability of mating behaviors in teleosts.

neuroscience↗

bioRxiv2019

Higher seroprevalence of Entamoeba histolytica than that of HIV-1 at a voluntary counselling and testing centre in Tokyo

BackgroundAmebiasis, which is caused by Entamoeba histolytica, is a re-emerging public health issue owing to sexually transmitted infection (STI) in Japan. However, epidemiological data are quite limited. MethodologyTo reveal the relative prevalence of sexually transmitted E. histolytica infection to other STIs, we conducted a cross-sectional study at a voluntary counselling and testing (VCT) centre in Tokyo. Seroprevalence of E. histolytica was assessed according to positivity with an enzyme-linked immunosorbent assay for E. histolytica-specific IgG in serum samples collected from anonymous VCT clients. Principal FindingsAmong 2,083 samples, seropositivity for E. histolytica was 2.64%, which was higher than that for HIV-1 (0.34%, p < 0.001) and comparable to that for syphilis (rapid plasma reagin (RPR) 2.11%, p = 0.31). Positivity for Chlamydia trachomatis in urine by transcription-mediated amplification (TMA) was 4.59%. Seropositivity for E. histolytica was high among RPR-or Treponema pallidum hemagglutination (TPHA)-positive individuals and it was not different between clients with and without other STIs. Both seropositivity of E. histolytica and RPR were high among male clients. The seropositive rate for anti-E. histolytica antibody was positively correlated with age. TMA positivity for urine C. trachomatis was high among female clients and negatively correlated with age. Regression analysis identified that male sex, older age, and TPHA-positive results are independent risk factors of E. histolytica seropositivity. ConclusionsSeroprevalence of E. histolytica was 7.9 times higher than that of HIV-1 at a VCT centre in Tokyo, with a tendency to be higher among people at risk for syphilis infection. Author summaryAmebiasis caused by Entamoeba histolytica is an increasingly prevalent sexually transmitted infection (STI) in Japan; however, relative to other STIs, the prevalence of E. histolytica has not been fully assessed. We investigated the seropositivity of E. histolytica using serum samples from 2,083 clients of a voluntary counselling and testing centre in Tokyo. E. histolytica seroprevalence (2.64%) was 7.9 times higher than that of HIV-1 (0.31%) and the same as that of syphilis (rapid plasma reagin: 2.11%). Logistic regression analysis showed that E. histolytica seroprevalence tended to be higher among individuals who were male, older, and positive in Treponema pallidum hemagglutination. These results strongly suggest that public health interventions should be considered to control sexual transmission of E. histolytica infection, which is currently neglected in Japan.

microbiology↗