Biology subjects

Kimura, S.

Publications and source records attributed to Kimura, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Cell-specialized chloroplast signaling orchestrates photosynthetic and extracellular reactive oxygen species for stress responses

Cellular responses to abiotic stress involve multiple signals including secondary messengers such as reactive oxygen species (ROS) and Ca2+, phytohormones such as abscisic acid (ABA) and chloroplast-to-nucleus retrograde signals such as 3-phosphoadenosine 5-phosphate (PAP). Mechanism(s) by which these messengers, produced in different subcellular compartments, intersect for cell regulation remain enigmatic. Previously we showed that the chloroplast retrograde signal PAP, similar to ABA, induces an increase in ROS levels in guard cells (Pornsiriwong et al, 2017). Here we demonstrate a mechanistic link enabling ABA and PAP to coordinate both chloroplast and plasma membrane ROS production. In whole leaves, PAP alters various ROS-related processes including plasmodesmal permeability as well as responses to ozone and the bacterial elicitor flg22, but mainly initiates processes that quench ROS during oxidative stress. Conversely, we show in guard cells, both PAP and ABA induce an increase in ROS levels in both chloroplasts via photosynthetic electron transport, and the apoplast via the RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). Both subcellular ROS sources were necessary for ABA- and PAP-mediated stomatal closure. However, PAP signaling diverges from ABA by activating RBOHD, instead of RBOHF, for apoplastic ROS mediated stomatal closure. We identified three calcium-dependent protein kinases (CPKs) as the post-translational activators of RBOHD-mediated ROS production. CPK13, CPK32, and CPK34 were transcriptionally induced by PAP and concurrently activate RBOHD and the slow anion channel SLAC1 by phosphorylating two Serine (S) residues, including S120 which is also targeted by the core ABA signaling kinase OPEN STOMATA 1 (OST1). Consequently, overexpression of the PAP-induced CPKs rescues stomatal closure in ost1. Our data identify stomatal chloroplasts, to be nodes in the multifaceted cellular stress response networks as they are both sources and mediators of ROS and retrograde signals such PAP. Thus, chloroplasts are not just mediators of photosynthesis in response to, for example, excess light, but can serve as critical nodes in the multifaceted cellular stress response networks in specialized cells via retrograde signals, providing support to the concept of sensory plastids. Significance StatementThe chloroplast is an environmental sensor for stresses such as excess light and drought via the activation of photosynthetic-mediated retrograde signals. However, how does it function in specialized cells for which carbon fixation is secondary? Here we show the chloroplast is an important node to coordinate multiple plant signaling pathways in response to stresses such as drought. The chloroplast retrograde signal 3-phosphoadenosine 5-phosphate (PAP) plays multiple roles in reactive oxygen species (ROS) signaling and homeostasis. While PAP suppresses ROS in photosynthetic tissue, PAP instead induces guard cell ROS in chloroplasts and extracellular space to induce stomatal closure. We decipher how PAP-induced proteins activate both extracellular ROS production and anion channels for stomatal closure, thus providing a mechanism by which chloroplasts provide a strategic complement to canonical hormonal pathways in regulating plant physiological responses in specialized cells.

plant biology↗

bioRxiv2023

No-go decay dampens zinc finger protein mRNAs in vertebrates

Quality control of translation is crucial for maintaining cellular and organismal homeostasis. Obstacles in translation elongation induce ribosome collision, which is monitored by multiple sensor mechanisms in eukaryotes. The E3 ubiquitin ligase Znf598 recognizes collided ribosomes, triggering ribosome-associated quality control (RQC) to rescue stalled ribosomes and no-go decay (NGD) to degrade stall-prone mRNAs. However, the impact of RQC and NGD on maintaining the translational homeostasis of endogenous mRNAs has remained unclear. In this study, we investigated the endogenous substrate mRNAs of NGD during the maternal-to-zygotic transition (MZT) of zebrafish development. RNA-Seq analysis of zebrafish znf598 mutant embryos revealed that Znf598 downregulates mRNAs encoding the C2H2-type zinc finger domain (C2H2-ZF) during the MZT. Reporter assays and disome profiling indicated that ribosomes stall and collide while translating tandem C2H2-ZFs, leading to mRNA degradation by Znf598. We further showed that mRNAs encoding C2H2-ZF were degraded in a ZNF598-dependent manner in HEK293T cells. Our results suggest that NGD maintains the quality of the translatome by mitigating the risk of ribosome collision at the abundantly present C2H2-ZF sequences in the vertebrate genome.

molecular biology↗

bioRxiv2023

A tRNA modification in Mycobacterium tuberculosis facilitates optimal intracellular growth

Diverse chemical modifications fine-tune the function and metabolism of tRNA. Although tRNA modification is universal in all kingdoms of life, profiles of modifications, their functions, and physiological roles have not been elucidated in most organisms including the human pathogen, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To identify physiologically important modifications, we surveyed the tRNA of Mtb, using tRNA sequencing (tRNA-seq) and genome-mining. Homology searches identified 23 candidate tRNA modifying enzymes that are predicted to create 16 tRNA modifications across all tRNA species. Reverse transcription-derived error signatures in tRNA-seq predicted the sites and presence of 9 modifications. Several chemical treatments prior to tRNA-seq expanded the number of predictable modifications. Deletion of Mtb genes encoding two modifying enzymes, TruB and MnmA, eliminated their respective tRNA modifications, validating the presence of modified sites in tRNA species. Furthermore, the absence of mnmA attenuated Mtb growth in macrophages, suggesting that MnmA-dependent tRNA uridine sulfation contributes to Mtb intracellular growth. Our results lay the foundation for unveiling the roles of tRNA modifications in Mtb pathogenesis and developing new therapeutics against tuberculosis.

microbiology↗

bioRxiv2023

SARS-CoV-2 Omicron XBB.1.5 may be a cautionary variant by in silico study

In this research, we aimed to predict the relative risk of the recent new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the basis of our previous research. We first performed molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to determine the binding affinities to human cells of three new variants of SARS-CoV-2: Omicron BQ.1, XBB, and XBB.1.5 We then investigated the three variants to discover the evolutionary distance of the spike protein gene (S gene) from the Wuhan, Omicron BA.1, and Omicron BA.4/5 variants, to understand the changes in the S gene. The results indicated that the XBB.1.5 variant had the highest binding affinity of the spike protein with ACE2 and the longest evolutionary distance of the S gene. This in silico evidence suggested that the XBB.1.5 variant may produce infections that spread more widely and faster than can infections of preexisting variants.

bioinformatics↗

bioRxiv2022

Peptidyl tRNA hydrolase is required for robust prolyl-tRNA turnover in Mycobacterium tuberculosis

Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacterial species. We developed a sequencing-based strategy called Cu-tRNAseq to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtbs susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB.

microbiology↗

bioRxiv2022

PNPO-PLP Axis Senses Prolonged Hypoxia by Regulating Lysosomal Activity

Oxygen is critical for all metazoan organisms on the earth and impacts various biological processes in physiological and pathological conditions. While oxygen sensing systems inducing acute hypoxic response, including HIF pathway, have been identified, those operating in prolonged hypoxia remain to be elucidated. Here, we show that pyridoxine 5-phosphate oxidase (PNPO) that catalyzes bioactivation of vitamin B6 serves as an oxygen sensor and regulates lysosomal activity in macrophages. Decline of PNPO activity under prolonged hypoxia reduced an active form of vitamin B6, pyridoxal 5-phosphate (PLP), and inhibited lysosomal activity, leading to the augmentation of inflammatory response of macrophages. The PNPO-PLP axis creates a distinct layer of oxygen sensing, which gradually turns down and up the PLP-dependent metabolism according to prolonged changes in oxygen availability.

biochemistry↗

bioRxiv2022

Cleavage of α-1,4-Glycosidic Linkages by the Glycosylphosphatidylinositol-Anchored α-Amylase AgtA Decreases the Molecular Weight of Cell Wall α-1,3-Glucan in Aspergillus oryzae

Aspergillus fungi contain -1,3-glucan with a low proportion of -1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored -amylases are conserved in Aspergillus fungi. The GPI-anchored -amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall -1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall -1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall -1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial -1,3-glucan. Furthermore, rAoAgtA cleaved 3--maltotetraosylglucose with a structure similar to the predicted boundary structure between the -1,3-glucan main chain and a short spacer composed of -1,4-linked glucose residues in cell wall -1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the -1,4-glycosidic bonds of 3--maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall -1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight (MW) of cell wall -1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored -amylases can degrade the spacer -1,4-glycosidic linkages in cell wall -1,3-glucan before its insolubilization, and this spacer cleavage decreases the MW of cell wall -1,3-glucan in vivo.

microbiology↗

bioRxiv2022

The chromosome-level genome of Water-Wisteria establishes an ecological plant model and provides insight into the molecular mechanisms underlying heterophylly in amphibious plants

Heterophylly is a phenomenon in which an individual plant dramatically changes its leaf shape in response to the surrounding environment. Hygrophila difformis (Acanthaceae), also known as water wisteria, has recently emerged as a model plant to study heterophylly because of its striking leaf shape variation in response to various ecological factors. Under submerged conditions, H. difformis develops complex leaves and in terrestrial conditions it develops simple leaves. Here, we sequenced and assembled the chromosome-level genome of triploid H. difformis (scaffold N50: 60.43 Mb, genome size: 871.92 Mb), which reveals 36,099 predicted protein-coding genes distributed over 15 pseudochromosomes. H. difformis diverged from its relatives during the Oligocene climate-change period and expanded the gene families related to its amphibious lifestyle. Genes involved in environmental stimuli, leaf development, and other pathways are differentially expressed in submerged and terrestrial conditions, possibly modulating morphological and physiological acclimation to changing environments. We confirmed that auxin plays a role in the heterophylly of H. difformis. Finally, we discovered candidate genes that respond to different environmental conditions and elucidated the role of LATE MERISTEM IDENTITY 1 (LMI1) in heterophylly. Our study establishes H. difformis as a model for studying the interconnections between ecological adaptation and plant morphological features.

plant biology↗

bioRxiv2022

SARS-CoV-2 Omicron BA.2.75 variant may be much more infective than preexisting variants

ObjectivesIn our previous research, we developed a mathematical model via molecular simulation analysis to predict the infectivity of seven SARS-CoV-2 variants. In this report, we aimed to predict the relative risk of the recent new variants of SARS-CoV-2 as based on our previous research. MethodsWe subjected Omicron BA.4/5 and BA.2.75 variants of SARS-CoV-2 to the analysis to determine the absolute evolutionary distance of the spike protein gene (S gene) of the variants from the Wuhan variant so as to appreciate the changes in the spike protein. We performed the molecular docking simulation analyses of the spike proteins with human angiotensin-converting enzyme 2 (ACE2) to understand the docking affinities of these variants. We then compared the evolutionary distances and the docking affinities of these variants with those of the seven variants that we had analyzed in our previous research. ResultsThe evolutionary distances of the S gene in BA.4/5 and BA.2.75 from the Wuhan variant were longer than those of the other variants. BA.2.75 had the highest docking affinity of the spike protein with ACE2 (ratio per Wuhan variant). ConclusionThe important results from this analysis are the following: BA.2.75 has both the highest docking affinity and the longest evolutionary distance of the S gene. These results suggest that BA.2.75 infection can spread farther than can infections of preexisting variants.

bioinformatics↗

bioRxiv2022

Development of a versatile high-throughput mutagenesis assay with multiplexed short read NGS using DNA-barcoded supF shuttle vector library amplified in non-SOS E. coli

A forward mutagenesis assay using the supF gene has been widely employed for the last several decades in studies addressing mutation frequencies and mutation spectra associated with various intrinsic and environmental mutagens. In this study, by using a supF shuttle vector and non-SOS-induced Escherichia coli with short read Next Generation Sequencing (NGS) technology, we present an advanced method for the study of mutations, which is simple, versatile, and cost-effective. We demonstrate the performance of our newly developed assay via pilot experiments with UV-irradiation, the results from which emerge more relevant than expected. The NGS data obtained from samples of the indicator E. coli grown on titer plates provides mutation frequency and spectrum data, and uncovers obscure mutations that cannot be detected by a conventional supF assay. Furthermore, a very small amount of NGS data from selection plates reveals the almost full spectrum of mutations in each specimen and offers us a novel insight into the mechanisms of mutagenesis, despite them being considered already well-known. We believe that the method presented here will contribute to future opportunities for research on mutagenesis, DNA repair, and cancer.

molecular biology↗

bioRxiv2022

Chromosome-level genome assembly of Rorippa aquatica revealed its allotetraploid origin and mechanisms of heterophylly upon submergence

The ability to respond to environmental variability is essential for living systems, especially to sessile organisms such as plants. The amphibious plant Rorippa aquatica exhibits a drastic type of phenotypic plasticity known as heterophylly, a phenomenon where leaf form is altered in response to the surrounding environment. Although heterophylly has been studied in various plant species, its molecular mechanism has not been fully elucidated. To establish the genetic basis and analyze the evolutionary processes responsible for heterophylly, we assembled the chromosome-level genome of R. aquatica by combining data from Illumina short-read sequencing, PacBio long-read sequencing, and High-throughput Chromosome Conformation Capture (Hi-C) sequencing technologies. Fine-scale comparative chromosome painting and chromosomal genomics revealed that allopolyploidization and subsequent post-polyploid descending dysploidy occurred during R. aquatica speciation. The genomic information above was the basis for the transcriptome analyses to examine the mechanisms involved in heterophylly, especially in response to the submerged condition, which uncovered that the ethylene and blue light signaling pathways participate in regulating heterophylly under submerged conditions. The assembled R. aquatica reference genome provides novel insights into the molecular mechanisms and evolution of heterophylly.

plant biology↗

bioRxiv2022

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis

Lateral roots (LRs) are critical to rhizosphere development in plants. Although the molecular mechanisms by which auxin regulates LR development has been studied extensively, many additional regulatory systems are thought to be involved. Based on the expression analysis of LTPG1 and 2, we found that they were specifically expressed at the developing LR primordium (LRP), and the number of LRs were reduced in these mutants. Because LTPG is a protein that transports Very Long Chain Fatty Acids (VLCFAs), we hypothesized that VLCFAs regulated LR development. We revealed that late LRP development was stunted when VLCFA levels were reduced in the synthetic mutant, kcs1-5. In this study, we established a method to analyze the LRP development stages with high temporal resolution using a deep neural network. We identified a VLCFA responsible transcription factor, MYB93, from transcriptome analysis of kcs1-5. Interestingly, MYB93 showed a carbon chain length-specific expression response after treatment of VLCFA. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. Our results indicated that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression.

plant biology↗

bioRxiv2022

Prediction of infectivity of SARS-CoV2: Mathematical Model with Docking Simulation analysis between Spike Protein and ACE2

Variants of a coronavirus (SARS-CoV-2) have been spreading in a global pandemic. Improved understanding of the infectivity of future new variants is important so that effective countermeasures against them can be quickly undertaken. In our research reported here, we aimed to predict the infectivity of SARS-CoV-2 by using a mathematical model with molecular simulation analysis, and we used phylogenetic analysis to determine the evolutionary distance of the spike protein gene (S gene) of SARS-CoV-2. We subjected the six variants and the wild type of spike protein and human angiotensin-converting enzyme 2 (ACE2) to molecular docking simulation analyses to understand the binding affinity of spike protein and ACE2. We then utilized regression analysis of the correlation coefficient of the mathematical model and the infectivity of SARS-CoV-2 to predict infectivity. The evolutionary distance of the S gene correlated with the infectivity of SARS-CoV-2 variants. The coefficient of the mathematical model obtained with results of molecular docking simulation also correlated with the infectivity of SARS-CoV-2 variants. These results suggest that the data from the docking simulation for the receptor binding domain of variant spike proteins and human ACE2 were valuable for prediction of SARS-CoV-2 infectivity. In addition, we developed a mathematical model for prediction of SARS-CoV-2 variant infectivity by using binding affinity obtained via molecular docking and the evolutionary distance of the S gene.

bioinformatics↗

bioRxiv2022

Sequential action of a tRNA base editor in conversion of cytidine to pseudouridine

Post-transcriptional RNA editing modulates gene expression in a condition-dependent fashion. We recently discovered C-to-{Psi} editing in Vibrio cholerae tRNA. Here, we characterize the biogenesis, regulation, and functions of this previously undescribed RNA editing process. We show that an enzyme, TrcP, mediates the editing of C-to-U followed by the conversion of U to {Psi}, consecutively. AlphaFold-2 predicts that TrcP consists of two globular domains, including a novel cytidine deaminase and a pseudouridylase, along with a long helical domain. The latter domain tethers tRNA substrates during both the C-to-U editing and pseudouridylation, likely enabling a substrate channeling mechanism for efficient catalysis all the way to the terminal product. C-to-{Psi} editing both requires and suppresses other modifications, creating an interdependent network of modifications in the tRNA anticodon loop that facilitates coupling of tRNA modification states to iron availability. Our findings provide mechanistic insights into an RNA editing process that likely promotes environmental adaptation.

biochemistry↗

bioRxiv2021

Skeletal muscle releases extracellular vesicles with distinct protein and miRNA signatures that accumulate and function within the muscle microenvironment

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since the skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish SkM-EVs from others, whether the skeletal muscle releases EVs in vivo and how much the skeletal muscle-derived EVs (SkM-EVs) account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings reveal that the skeletal muscle releases exosome-like small EVs with distinct protein and miRNA profiles in vivo and that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

physiology↗

bioRxiv2021

Prospective isolation of mouse and human hematopoietic stem cells using Plexin domain containing 2

Numerous strategies exist to isolate hematopoietic stem cells (HSCs) using complex combinations of markers and flow cytometry. However, robust identification of HSCs using imaging techniques is substantially more challenging which has prompted the recent development of HSC reporter mice. To date, none of the molecules used in these reporters have been useful for human HSC identification. Here we report that PLXDC2 is a useful marker for both mouse and human HSCs. Using a green fluorescent protein (GFP) knock-in at the Plxdc2 locus in mice (hereafter denoted as Plxdc2-GFP), we showed that Plxdc2-GFP is highly expressed in HSCs with 1 in 2.8 Plxdc2-GFP+CD150+ cells giving long-term multi-lineage reconstitution in transplantation. Moreover, we developed a novel human PLXDC2 antibody and showed that human PLXDC2+ HSCs have stronger long-term multilineage reconstitution ability compared with PLXDC2- HSCs in a xenograft model. Thus, our study identifies PLXDC2 as a highly relevant molecule in HSC identification, potentially allowing greater purity and live in vivo tracking of these cells. SummaryTo date, few molecules are available for isolation of HSCs across species. The present study shows that PLXDC2 is a highly useful molecule for isolation of HSCs, which works across mouse and human.

cell biology↗

bioRxiv2021

The two-component response regulator OrrA confers dehydration tolerance by regulating anaKa expression in the cyanobacterium Anabaena sp. strain PCC 7120

The aquatic cyanobacterium Anabaena sp. strain PCC 7120 exhibits dehydration tolerance. The regulation of gene expression in response to dehydration is crucial for the acquisition of dehydration tolerance, but the molecular mechanisms underlying dehydration responses remain unknown. In this study, the functions of the response regulator OrrA in the regulation of salt and dehydration responses were investigated. Disruption of orrA abolished or diminished the induction of hundreds of genes in response to salt stress and dehydration. Thus, OrrA is a principal regulator of both stress responses. In particular, OrrA plays a crucial role in dehydration tolerance because an orrA disruptant completely lost the ability to regrow after dehydration. Moreover, in the OrrA regulon, anaKa encoding a protein of unknown function was revealed to be indispensable for dehydration tolerance. OrrA and AnaK are conserved among the terrestrial cyanobacteria, suggesting their conserved functions in dehydration tolerance in cyanobacteria.

microbiology↗

bioRxiv2021

Regulation of suberin biosynthesis and Casparian strip development in the root endodermis by two plant auxins

The biological function of the auxin phenylacetic acid (PAA) is not well characterized in plants. Although some aspects of its biology; transport, signaling and metabolism have recently been described. Previous work on this phytohormone has suggested that PAA behaves in an identical manner to IAA (indole-3-acetic acid) in promoting plant growth, yet plants require greater concentrations of PAA to elicit the same physiological responses. Here we show that normalized PAA treatment results in the differential expression of a unique list of genes, suggesting that plants can respond differently to the two auxins. This is further explored in endodermal barrier regulation where the two auxins invoke striking differences in the deposition patterns of suberin. We further show that auxin acts antagonistically on Casparian strip (CS) formation as it can circumvent the CS transcriptional machinery to repress CS related genes. Additionally, altered suberin biosynthesis reduces endogenous levels of PAA and CS deficiency represses the biosynthesis of IAA and the levels of both auxins. These findings implicate auxin as a regulator of endodermal barrier formation and highlight a novel role for PAA in root development and differentiation.

plant biology↗