Biology subjects

Igarashi, K.

Publications and source records attributed to Igarashi, K..

26 records · Page 2Linked to original sources

bioRxiv2021

Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

cell biology↗

bioRxiv2020

Crystal structure of GH43 exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium provides insights into the mechanism of bypassing side chains

Arabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases. In this study, we solved the apo and liganded structures of exo-{beta}-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the {beta}-1,6-linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.

molecular biology↗

bioRxiv2020

Thermostable mutants of glycoside hydrolase family 6 cellobiohydrolase from the basidiomycete Phanerochaete chrysosporium

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) is a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase PcCel6A from the fungus Phanerochaete chrysosporium, and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris. Comparison of the hydrolytic activities at 50 and 60 {degrees}C indicated that the thermal stability of PcCel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.

biochemistry↗

bioRxiv2019

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate cellular epigenetic states. This metabolism-epigenome link enables the sensitization of chromatin methylation to altered SAM abundance. However, a chromatin-wide understanding of the adaptive/responsive mechanisms that allow cells to actively protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear de novo mono-methylation of H3 Lys 9 (H3K9) at the expense of global losses in histone di- and tri-methylation. Under SAM-depleted conditions, de novo H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide the first evidence for active epigenetic adaptation and persistence to metabolic stress.

biochemistry↗

bioRxiv2019

A hydrogen dependent geochemical analogue of primordial carbon and energy metabolism

Hydrogen gas, H2, is generated in alkaline hydrothermal vents from reactions of iron containing minerals with water during a geological process called serpentinization. It has been a source of electrons and energy since there was liquid water on the early Earth, and it fuelled early anaerobic ecosystems in the Earths crust1-3. H2 is the electron donor for the most ancient route of biological CO2 fixation, the acetyl-CoA (or Wood-Ljungdahl) pathway, which unlike any other autotrophic pathway simultaneously supplies three key requirements for life: reduced carbon in the form of acetyl groups, electrons in the form of reduced ferredoxin, and ion gradients for energy conservation in the form of ATP4,5. The pathway is linear, not cyclic, it releases energy rather than requiring energy input, its enzymes are replete with primordial metal cofactors6,7, it traces to the last universal common ancestor8 and abiotic, geochemical organic syntheses resembling segments of the pathway occur in hydrothermal vents today9,10. Laboratory simulations of the acetyl-CoA pathways reactions include the nonenzymatic synthesis of thioesters from CO and methylsulfide11, the synthesis of acetate12 and pyruvate13 from CO2 using native iron or external electrochemical potentials14 as the electron source. However, a full abiotic analogue of the acetyl-CoA pathway from H2 and CO2 as it occurs in life has not been reported to date. Here we show that three hydrothermal minerals -- awaruite (Ni3Fe), magnetite (Fe3O4) and greigite (Fe3S4) -- catalyse the fixation of CO2 with H2 at 100 {degrees}C under alkaline aqueous conditions. The product spectrum includes formate (100 mM), acetate (100 M), pyruvate (10 M), methanol (100 M), and methane. With these simple catalysts, the overall exergonic reaction of the acetyl-CoA pathway is facile, shedding light on both the geochemical origin of microbial metabolism and on the nature of abiotic formate and methane synthesis in modern hydrothermal vents.

evolutionary biology↗

bioRxiv2019

Ferroptosis is programmed by the coordinated regulation of glutathione and iron metabolism by BACH1

Ferroptosis is an iron-dependent programmed cell death resulting from alterations of metabolic processes. However, its regulation and physiological significance remain to be elucidated. By analyzing transcriptional responses of murine embryonic fibroblasts exposed to the ferroptosis-inducer erastin, we found that a set of genes related to oxidative stress protection was induced upon ferroptosis. We further showed that the transcription factor BACH1 promoted ferroptosis by repressing the expression of a subset of erastin-inducible genes involved in the synthesis of glutathione or metabolism of intracellular labile iron, including Gclm, Gclc, Slc7a11, Hmox1, Fth1, Ftl1, and Slc40a1. Compared with wild-type mice, Bach1-/- mice showed resistance to myocardial infarction, the seriousness of which was palliated by the iron-chelator deferasirox, which suppressed ferroptosis. Our findings suggest that ferroptosis is programmed at the transcriptional level to induce genes combating labile-iron-induced oxidative stress and executed upon disruption of the balance between the transcriptional induction of protective genes and accumulation of iron-mediated damage. BACH1 is suggested to control the threshold of ferroptosis and to be a therapeutic target for palliating myocardial infarction.

cell biology↗

bioRxiv2019

The haploinsufficient tumor suppressor Tip60 negatively regulates the oncogenic Aurora B kinase

The Aurora kinases represent a group of serine/threonine kinases which are crucial regulators of mitosis. Dysregulated Aurora kinase B (AurkB) expression, stemming from genomic amplification, increased gene transcription or overexpression of its allosteric activators, is capable of initiating and sustaining malignant phenotypes. Although AurkB level in cells is well-orchestrated, studies that relate to its stability or activity, independent of mitosis, are lacking. We report that AurkB undergoes acetylation in vitro by lysine acetyltransferases (KATs) belonging to different families, namely by p300 and Tip60. The haploinsufficient tumor suppressor Tip60 acetylates two highly conserved lysine residues within the kinase domain of AurkB which not only impinges the protein stability but also its kinase activity. These results signify a probable outcome on the increase in \"overall activity\" of AurkB upon Tip60 downregulation, as observed under cancerous conditions. The present work, therefore, uncovers an important functional interplay between AurkB and Tip60, frailty of which may be an initial event in carcinogenesis.

biochemistry↗

bioRxiv2019

Aurora kinase A mediated phosphorylation of mPOU is critical for skeletal muscle differentiation

Aurora kinases are Ser/Thr-directed protein kinases which play pivotal roles in mitosis. Recent evidences highlight the importance of these kinases in non-mitotic biological events like skeletal myogenesis. Our earlier study identified POU6F1 (or mPOU) as a novel Aurora kinase A (AurkA) substrate. Here, we report that AurkA phosphorylates POU6F1 at Ser197 and inhibits its DNA binding ability. Delving into POU6F1 physiology, we find that the phospho-mimic (S197D) POU6F1 mutant exhibits enhancement, while wild type (WT) or phospho-deficient (S197A) mutant shows retardation in C2C12 myoblast differentiation. Interestingly, POU6F1 depletion phenocopies S197D-POU6F1 overexpression in the differentiation context. Collectively, our results signify mPOU as a negative regulator of skeletal muscle differentiation and strengthens the importance of AurkA in skeletal myogenesis.

cell biology↗