Biology subjects

Ito, K.

Publications and source records attributed to Ito, K..

At least 109 records · Page 6Linked to original sources

bioRxiv2020

Rrp1 translocase and ubiquitin ligase activities restrict the genome destabilising effects of Rad51 in fission yeast

Rad51 is the key protein in homologous recombination DNA repair and has important roles during DNA replication. Auxiliary factors regulate Rad51 activity to facilitate productive, and prevent inappropriate, recombination that could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in counteracting Rad51 activity, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 activity in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 activity. Additionally, we demonstrate that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.

genetics↗

bioRxiv2020

Loss of CNOT9 begets impairment in gastrulation leading to embryonic lethality

The multi-subunit eukaryotic CCR4-NOT complex imparts gene expression control primarily via messenger RNA (mRNA) decay. Here, we present the role of subunit CNOT9 in target mRNA decay during embryonic development. CNOT9 null mice appear normal by the onset of gastrulation (E7.0), however, exhibit growth and differentiation defects accompanied by extensive cell death by embryonic day 9.5 (E9.5). Sox-2 Cre conditional CNOT9 knockout mice show almost identical phenotype with brief delay in onset and progression, suggesting defects to be epiblast-dominant. Among various identified targets, we show that Lefty2 mRNA expression is post-transcriptionally regulated by CNOT9. Lefty2 3-UTR containing mRNA has significantly higher stability in cells expressing mutant form of CNOT9, relative to cells expressing wild-type CNOT9. In addition, CNOT9 primarily localizes within the cytoplasm and bridges interactions between the CCR4-NOT complex and miRNA-RISC complex in gastrulating embryos.

developmental biology↗

bioRxiv2020

Rhythmic temperature control in a homeothermic plant: A field study of thermogenic Japanese skunk cabbage, Symplocarpus renifolius

The spadices of skunk cabbage (Symplocarpus renifolius) can generate heat and maintain an internal temperature of about 23{degrees}C, even when the ambient air temperature drops below freezing. In this study, we continuously measured the spadix temperatures of skunk cabbage together with ambient air temperatures during March 2019 in their natural habitat in northern Honshu on Japans main island. Using Fourier transform we showed that the temperature of the thermogenic spadix oscillates over a period of approximately 60 min. Our data suggest that Japanese skunk cabbage possesses a novel timekeeping mechanism that may be associated with a homeothermic control of this plant.

plant biology↗

bioRxiv2020

A flexible target-specific anti-infection therapeutic platform that can be applied to different microbial species

The emergence of new microbial pathogens, including drug-resistant strains, complicates treatment, thereby threatening global health. We demonstrated a photoimmuno-antimicrobial strategy (PIAS) that eliminated antibody-targets using a photo-activated anti-pathogen antibody generating mechanical stress that damaged the targets binding sites. PIAS is effective against many pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), the fungal pathogen Candida albicans, and viral particles irrespective of their species or drug-resistance status. Animal experiments demonstrated that PIAS saved mice from fatal infections; microbiome and histochemical analyses indicated no apparent effect on normal host microflora and tissues in PIAS-treated mice. Resistance to PIAS was not observed during the eight years of this study. As a new type of anti-infection therapy, PIAS may contribute to advances in anti-infection strategies.

microbiology↗

bioRxiv2020

3D Models of glycosylated SARS-CoV-2 spike protein suggest challenges and opportunities for vaccine development

Here we have generated 3D structures of glycoforms of the spike (S) glycoprotein from SARS-CoV-2, based on reported 3D structures and glycomics data for the protein produced in HEK293 cells. We also analyze structures for glycoforms representing those present in the nascent glycoproteins (prior to enzymatic modifications in the Golgi), as well as those that are commonly observed on antigens present in other viruses. These models were subjected to molecular dynamics (MD) simulation to determine the extent to which glycan microheterogeneity impacts the antigenicity of the S glycoprotein. Lastly, we have identified peptides in the S glycoprotein that are likely to be presented in human leukocyte antigen (HLA) complexes, and discuss the role of S protein glycosylation in potentially modulating the adaptive immune response to the SARS-CoV-2 virus or to a related vaccine. The 3D structures show that the protein surface is extensively shielded from antibody recognition by glycans, with the exception of the ACE2 receptor binding domain, and also that the degree of shielding is largely insensitive to the specific glycoform. Despite the relatively modest contribution of the glycans to the total molecular weight (17% for the HEK293 glycoform) the level of surface shielding is disproportionately high at 42%.

immunology↗

bioRxiv2020

Transcriptional down-regulation of metabolic genes by Gdown1 ablation induces quiescent cell re-entry into the cell cycle

Liver regeneration and metabolism are highly interconnected. Here, we show that hepatocyte-specific ablation of RNA polymerase II (Pol II)-associated Gdown1 leads to down-regulation of highly expressed genes involved in plasma protein synthesis and metabolism, a concomitant cell cycle re-entry associated with induction of cell cycle-related genes (including cyclin D1). and up-regulation of p21 through activation of p53 signaling. In the absence of p53, Gdown1-deficient hepatocytes show a severe dysregulation of cell cycle progression, with incomplete mitoses, and a pre-malignant-like transformation. Mechanistically, Gdown1 is associated with elongating Pol II on the highly expressed genes and its ablation leads to reduced Pol II recruitment to these genes, suggesting that Pol II redistribution may facilitate hepatocyte re-entry into the cell cycle. These results establish an important physiological function for a Pol II regulatory factor (Gdown1) in the maintenance of normal liver cell transcription through constraints on cell cycle re-entry of quiescent hepatocytes.

molecular biology↗

bioRxiv2020

Input connectivity reveals additional heterogeneity of dopaminergic reinforcement in Drosophila

Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control [1]. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions [2, 3]. Here we used a recent electron microscope volume of the fly brain [4] to reconstruct the fine anatomy of individual DANs within three MB compartments. We find the 20 DANs of the {gamma}5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within {gamma}5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-{gamma}5 DAN subtype {gamma}5(fb) receives direct recurrent input from {gamma}5{beta}2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from MBONs. We similarly reconstructed the single aversively reinforcing PPL1-{gamma}1pedc DAN. The {gamma}1pedc DAN inputs mostly differ from those of {gamma}5 DANs and they cluster onto distinct dendritic branches, presumably separating its established roles in aversive reinforcement and appetitive motivation [5, 6]. Tracing also identified neurons that provide broad input to {gamma}5, {beta}2a and {gamma}1pedc DANs suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal further complexity of dopaminergic reinforcement circuits between and within MB compartments. HighlightsO_LINanoscale anatomy reveals additional subtypes of rewarding dopaminergic neurons. C_LIO_LIConnectomics reveals extensive input specificity to subtypes of dopaminergic neurons. C_LIO_LIAxon morphology implies dopaminergic neurons provide subcompartment-level function. C_LIO_LIUnique dopaminergic subtypes serve aversive memory extinction and sugar learning. C_LI

neuroscience↗

bioRxiv2019

Arabidopsis ROOT PHOTOTROPISM2 is a Light-Dependent Dynamic Modulator of Phototropin1

Arabidopsis thaliana phototropin1 (phot1) is a blue-light photoreceptor, i.e. a blue-light-activated Ser/Thr-protein kinase that mediates various light responses including phototropism. Phot1 functions in hypocotyl phototropism dependent on the light induction of ROOT PHOTOTROPISM2 (RPT2) proteins within a broad range of blue light intensities. It is not yet known however how RPT2 contributes to the photosensory adaptation of phot1 to high intensity blue light and the second positive phototropism. We here show that RPT2 suppresses the activity of phot1. Yeast two-hybrid analysis indicated RPT2 binding to the LOV1 (light, oxygen or voltage sensing 1) domain of phot1 required for its high photosensitivity. Our biochemical analyses revealed that RPT2 inhibits the autophosphorylation of phot1, suggesting that it suppresses the photosensitivity and/or kinase activity of phot1 through the inhibition of LOV1 function. We found for the first time that RPT2 proteins are degraded via a ubiquitin-proteasome pathway when phot1 is inactive and stabilized under blue-light conditions in a phot1-dependent manner. We propose that RPT2 is a molecular rheostat that maintains a moderate activation level of phot1 under any light intensity conditions.

plant biology↗

bioRxiv2019

Population-specific and transethnic genome-wide analyses reveal distinct and shared genetic risks of coronary artery disease

To elucidate the genetics of coronary artery disease (CAD) in the Japanese population, we conducted a large-scale genome-wide association study (GWAS) of 168,228 Japanese (25,892 cases and 142,336 controls) with genotype imputation using a newly developed reference panel of Japanese haplotypes including 1,782 CAD cases and 3,148 controls. We detected 9 novel disease-susceptibility loci and Japanese-specific rare variants contributing to disease severity and increased cardiovascular mortality. We then conducted a transethnic meta-analysis and discovered 37 additional novel loci. Using the result of the meta-analysis, we derived a polygenic risk score (PRS) for CAD, which outperformed those derived from either Japanese or European GWAS. The PRS prioritized risk factors among various clinical parameters and segregated individuals with increased risk of long-term cardiovascular mortality. Our data improves clinical characterization of CAD genetics and suggests the utility of transethnic meta-analysis for PRS derivation in non-European populations.

genetics↗

bioRxiv2019

Real-time tracking reveals the catalytic process of Rad51-driven DNA strand exchange

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for second donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA family recombinases.

molecular biology↗

bioRxiv2019

ATP Binding Cassette Proteins ABCG37 and ABCG33 are required for potassium-independent cesium uptake in Arabidopsis roots

Radiocesium, accumulated in the soil by nuclear accidents is a major environmental concern. The transport process of cesium (Cs+) is tightly linked to the indispensable plant nutrient potassium (K+) as they both belong to the group I alkali metal with similar chemical properties. Most of the transporters that had been characterized to date as Cs+ transporters are directly or indirectly linked to K+. Using a combinatorial approach of physiology, genetics, cell biology and root uptake assay, here we identified two ATP-Binding Cassette (ABC) proteins, ABCG37 and ABCG33 as facilitators of Cs+ influx. The gain-of-function mutant of ABCG37 (abcg37-1) showed hypersensitive response to Cs+-induced root growth inhibition, while the double knock out mutant of ABCG33 and ABCG37 (abcg33-1abcg37-2) showed resistance. Single loss-of-function mutant of ABCG33 and ABCG37 did not show any alteration in Cs+ response. Short term uptake experiment with radioactive Cs+ revealed reduced Cs+ uptake in abcg33-1abgc37-2 compared with wild type in presence or absence of K+. Potassium response and content were unaffected in the double mutant background confirming that Cs+ uptake by ABCG33 and ABCG37 is independent of K+. Collectively, this work identified two ABC proteins as new Cs+ influx carriers, which act redundantly and independent of K+ uptake pathway.

plant biology↗

bioRxiv2019

Large scale genome-wide association study in a Japanese population identified 45 novel susceptibility loci for 22 diseases

INTRODUCTORY PARAGRAPHThe overwhelming majority of participants in current genetic studies are of European ancestry1-3, limiting our genetic understanding of complex disease in non-European populations. To address this, we aimed to elucidate polygenic disease biology in the East Asian population by conducting a genome-wide association study (GWAS) with 212,453 Japanese individuals across 42 diseases. We detected 383 independent signals in 331 loci for 30 diseases, among which 45 loci were novel (P < 5 x 10-8). Compared with known variants, novel variants have lower frequency in European populations but comparable frequency in East Asian populations, suggesting the advantage of this study in discovering these novel variants. Three novel signals were in linkage disequilibrium (r2 > 0.6) with missense variants which are monomorphic in European populations (1000 Genomes Project) including rs11235604(p.R220W of ATG16L2, a autophagy-related gene) associated with coronary artery disease. We further investigated enrichment of heritability within 2,868 annotations of genome-wide transcription factor occupancy, andidentified 378 significant enrichments across nine diseases (FDR < 0.05) (e.g. NF-{kappa}B for immune-related diseases). This large-scale GWAS in a Japanese population provides insights into the etiology of common complex diseases and highlights the importance of performing GWAS in non-European populations.

genetics↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗

bioRxiv2019

Induced RPB1 depletion reveals a direct gene-specific control of RNA Polymerase III function by RNA Polymerase II

Increasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

molecular biology↗

bioRxiv2019

Transethnic meta-analysis of genome-wide association studies identifies three new loci and characterizes population-specific differences for coronary artery disease

BackgroundGenome-wide association studies (GWAS) provided many biological insights into coronary artery disease (CAD), but these studies were mainly performed in Europeans. GWAS in diverse populations have the potential to advance our understanding of CAD.\n\nMethods and ResultsWe conducted two GWAS for CAD in the Japanese population, which included 12,494 cases and 28,879 controls, and 2,808 cases and 7,261 controls, respectively. Then, we performed transethnic meta-analysis using the results of the CARDIoGRAMplusC4D 1000 Genomes meta-analysis with UK Biobank. We identified 3 new loci on chromosome 1q21 (CTSS), 10q26 (WDR11-FGFR2), and 11q22 (RDX-FDX1). Quantitative trait locus analyses suggested the association of CTSS and RDX-FDX1 with atherosclerotic immune cells. Tissue/cell type enrichment analysis showed the involvement of arteries, adrenal glands and fat tissues in the development of CAD. Finally, we performed tissue/cell type enrichment analysis using East Asian-frequent and European-frequent variants according to the risk allele frequencies, and identified significant enrichment of adrenal glands in the East Asian-frequent group while the enrichment of arteries and fat tissues was found in the European-frequent group. These findings indicate biological differences in CAD susceptibility between Japanese and Europeans.\n\nConclusionsWe identified 3 new loci for CAD and highlighted the genetic differences between the Japanese and European populations. Moreover, our transethnic analyses showed both shared and unique genetic architectures between the Japanese and Europeans. While most of the underlying genetic bases for CAD are shared, further analyses in diverse populations will be needed to elucidate variations fully.

genetics↗

bioRxiv2019

Rad51 Interaction Analysis Reveals a Functional Interplay Among Recombination Auxiliary Factors

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. Here, we present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a widely conserved auxiliary factor. NMR and site-specific crosslinking experiments revealed two distinct sites within the intrinsically disordered N-terminus of Sfr1 that cooperatively bind to Rad51. Although disruption of this binding severely impaired Rad51 stimulation in vitro, interaction mutants did not show any defects in DNA repair. Unexpectedly, in the absence of the Rad51 paralogs Rad55-Rad57, which constitute another auxiliary factor complex, these interaction mutants were unable to promote DNA repair. Our findings provide molecular insights into Rad51 stimulation by Swi5-Sfr1 and suggest that, rather than functioning in an independent subpathway of HR as was previously proposed, Rad55-Rad57 facilitates the recruitment of Swi5-Sfr1 to Rad51.

molecular biology↗

bioRxiv2019

ResQ, a release factor-dependent ribosome rescue factor in the Gram-positive bacterium Bacillus subtilis

Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named ResQ. Genetic analysis shows that B. subtilis requires the function of either trans-translation or ResQ for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-EM characterization demonstrates that ResQ binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although ResQ is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

molecular biology↗

bioRxiv2019

Self-body recognition through a mirror: Easing spatial-consistency requirements for rubber hand illusion

Typical experiments to induce the rubber hand illusion (RHI) require experimental participants to gaze at a fake hand while tactile stimuli are provided to both the fake and hidden actual hands in a synchronous manner. However, under such conditions, postural and apparent disagreement between a seen fake hand and hidden actual hand prevents illusory body ownership. Provided that humans recognize mirror images as copies of the real world in spite of their spatial uncertainties or incongruence, the sensory disagreement may be accepted in RHI settings if using a mirror to show a fake hand. The present study performed two experiments to reveal how self-body recognition of a fake hand feature via mirror affects the RHI. These experiments were conducted in an RHI environment involving voluntary hand movements to investigate not only body ownership but also agency. The first experiment (Experiment 1) examined whether illusory ownership of a fake hand seen in a mirror could be induced. Then, we examined whether the RHI using a mirror image allows disagreement in orientation between the rubber and actual hands (Experiment 2). Subjective evaluations using a RHI questionnaire demonstrated that evoked embodiment of the rubber hand was stronger in the presence of a mirror than in the absence of it (Experiment 1) and that participants experienced the RHI even if the actual and rubber hands were incongruent in terms of orientation (45 {degrees}; Experiment 2). No significant difference was found in the change of perceived finger location (proprioceptive drift) between these experiments. These findings suggest that the use of a mirror masks subtle spatial incongruency or degrades the contribution of visual cues for spatial recognition and facilitates multisensory integration for bodily illusions.

neuroscience↗