Biology subjects

Ishikawa, K.

Publications and source records attributed to Ishikawa, K..

28 records · Page 2Linked to original sources

bioRxiv2022

Genetic differentiation of water penny beetles may be associated with the formation process of the ancient Lake Biwa.

BackgroundWater flow is one of several factors that determine the habitats of aquatic insects. The conversions of stagnant (lentic) and running (lotic) water are physiologically and ecologically constrained. In general, aquatic insect species only occur in one of these environments. Though Eubrianax ramicornis (Coleoptera: Psephenidae) usually resides in rivers, however, it is also known to occur near the shores of Lake Biwa in Japan. Here, we investigated the genetic differences between the two Eubrianax species of Lake Biwa and its inflowing rivers, and other regions of Japan for the first time. Lake Biwa is an ancient lake and about four million years old. Though over 60 endemic species were described from Lake Biwa, they are mostly fish and molluscs, with only two species of aquatic insects. There are few reports of endemic aquatic insects in other ancient lakes, too. Discussion of the endemism of Eubrianax and its promoting factors is expected to provide insights into the evolution of aquatic insects in ancient lakes. MethodsWe surveyed and morphologically identified psephenid larvae in Lake Biwa and its inflowing rivers during the summers of 2017 and 2018 (Hayashi, 2009: Hayashi & Sota, 2009). We conducted phylogenetic analyses of two regions of COI and estimated the ages of population divergence based on fossil records of Eubrianax beetles in Lake Biwa as well as those of previously reported populations in other regions. We also analyzed two COI regions of the E. ramicornis haplotypes in Lake Biwa. ResultsFour psephenid species were identified among the samples collected in Lake Biwa and its inflowing rivers. Of these, E. ramicornis was collected only at the Lake Biwa shore whereas its congeneric E. granicollis was collected only from the rivers inflowing the lake. The phylogenetic analyses showed that E. ramicornis of Lake Biwa were clearly differentiated from those in previously reported regions and this divergence was estimated to have occurred 1.40 MYA. At that time, there was no lake at the location of present-day Lake Biwa. It is thought that rivers and their dammed flood plains to the east, with gravel and sand supplied by the rising Suzuka Mountains further to the east. Such an environments is preferable for E. ramicornis and they were thought to have become isolated from the surrounding rivers during the formation of Lake Biwa. On the contrary, there was no genetic differentiation between the E. granicollis population at the Lake Biwa inflow rivers and those elsewhere. Hence, E. granicollis might have migrated to the Lake Biwa watershed later than E. ramicornis and maintained gene flow over a wide area. The genetic differentiation of E. ramicornis reported here for the first time suggests that a species with a wide distribution may rather be evolving endemic to Lake Biwa. Reports on the endemism of aquatic insects in ancient lakes are scarce. This study is expected to bring new perspectives to the study of insect evolution in ancient lakes.

ecology↗

bioRxiv2022

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

neuroscience↗

bioRxiv2022

Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ, a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. RNA sequence analysis revealed that rpmJ knockout decreased the expression of synthetic genes for iron-sulfur cluster proteins, which are toxic targets for zinc. These findings suggest that knocking out RpmJ causes zinc resistance by decreasing zinc targets and lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance.

microbiology↗

bioRxiv2022

Knockout of ykcB, a putative glycosyltransferase, leads to vancomycin resistance in Bacillus subtilis

Vancomycin resistance of gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused vancomycin resistance in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to {beta}-lactams, and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had: i) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose, ii) a decreased amount of lipoteichoic acid, and iii) decreased biofilm formation ability. These phenotypes and vancomycin resistance were abolished by knockout of ykcC, a ykcB-operon partner involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin resistance in both wild-type B. subtilis and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to resistance to vancomycin, decreased biofilm formation ability, and increased pathogenicity to silkworms. IMPORTANCEAlthough vancomycin is effective against gram-positive bacteria, vancomycin-resistant bacteria is a major public health concern. While the vancomycin resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well-studied, they remain unclear in other gram-positive bacteria. In the present study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin resistance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in gram-positive bacteria.

microbiology↗

bioRxiv2022

Apoplastic CBM1-interacting proteins bind conserved carbohydrate binding module 1 motifs in fungal hydrolases to counter pathogen invasion

When infecting plants, fungal pathogens secrete cell wall degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsCBMIP binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsCBMIP to various CBM1-containing enzymes, suggesting it has a general role in inhibiting the catalytic activities of fungal enzymes. OsCBMIP is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown of OsCBMIP reduced rice defense against M. oryzae, demonstrating that inhibition of CBM1-containing fungal enzymes by OsCBMIP is crucial for rice defense. We also identified additional CBMIP-related proteins from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism. SummaryPlants have evolved various activity-inhibiting proteins as a defense against fungal cell wall degrading enzymes (CWDEs), but how plants counteract the function of fungal enzymes containing carbohydrate binding modules (CBMs) remains unknown. Here, we demonstrate that OsCBMIP, a member of the cysteine-rich repeat secretion protein family, interacts with fungal CBM1. OsCBMIP binding to CBM1 of a blast fungal xylanase blocks access to cellulose, resulting in the inhibition of xylanase enzymatic activity. Our study provides significant insights into plant countermeasure against CWDEs in the apoplastic space during plant-fungal pathogen interactions. It also reveals a molecular function of the DUF26 domain widely distributed in plant proteins.

plant biology↗

bioRxiv2021

Structural analysis and construction of thermostable antifungal chitinase

Chitin is a biopolymer of N-acetyl-D-glucosamine with {beta}-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the {beta}-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzymes thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual applicable conditions. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. Based on the crystal structure and sequence alignment among other chitinases, we rationally introduced proline residues, a disulfide bond, and salt bridge in the chitinase using protein engineering methods. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. IMPORTANCEWe solved the crystal structure of the chitinase from Gazyumaru (Ficus microcarpa) latex exhibiting antifungal activity. Furthermore, we demonstrated that the thermostable mutant enzyme with a melting temperature (Tm) 6.9 {degrees}C higher than wild type (WT) and a half-life at 60{degrees}C that is 15 times longer than WT was constructed through 10 amino acid substitutions, including five proline residues substitutions, making disulfide bonding, and building a salt bridge network in the enzyme. These mutations do not affect its high antifungal activity and chitinase activity, and the principle for the construction of the thermostable chitinase was well explained by its crystal structure. Our results provide a useful strategy to enhance the thermostability of this enzyme family and to use the thermostable mutant as a seed for antifungal agents for practical use.

biochemistry↗

bioRxiv2020

Small molecule targeting r(UGGAA)n disrupts RNA foci and alleviates disease phenotype in Drosophila model

Synthetic small molecules modulating RNA structure and function have therapeutic potential for RNA diseases. Here we report our discovery that naphthyridine carbamate dimer (NCD) targets disease-causing r(UGGAA)n repeat RNAs in spinocerebellar ataxia type 31 (SCA31). Structural analysis of the NCD-UGGAA/UGGAA complex by nuclear magnetic resonance (NMR) spectroscopy clarified the mode of binding that recognizes four guanines in UGGAA/UGGAA pentad by hydrogen bonding with four naphthyridine moieties of two NCD molecules. Biological studies show that NCD disrupts naturally occurring RNA foci built on r(UGGAA)n repeat RNA known as nuclear stress bodies (nSBs) by interfering with RNA-protein interactions resulting in the suppression of nSBs-mediated splicing event. Feeding NCD to larvae of the Drosophila model of SCA31 alleviates disease phenotype induced by toxic r(UGGAA)n repeat RNA. These studies demonstrated that small molecules targeting toxic repeat RNAs are a promising chemical tool for studies on repeat expansion diseases.

biochemistry↗

bioRxiv2020

SNF1-related protein kinase 2 directly regulate group C Raf-like protein kinases in abscisic acid signaling

ABSTRUCTA phytohormone abscisic acid (ABA) has a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) mediates ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as an interacting protein with SnRK2. A series of reverse genetic and biochemical analyses revealed that Raf36 negatively regulates ABA responses and is directly phosphorylated by SnRK2s. In addition, we found that Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. Comparative phosphoproteomic analysis using Arabidopsis wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results reveal a novel subsection of ABA-responsive phosphosignaling pathways branching from SnRK2.

plant biology↗

bioRxiv2019

Exploring the Complexity of Protein-Level Dosage Compensation that Fine-Tunes Stoichiometry of Multiprotein Complexes

Proper control of gene expression levels upon various perturbations is a fundamental aspect of cellular robustness. Protein-level dosage compensation is one mechanism buffering perturbations to stoichiometry of multiprotein complexes through accelerated proteolysis of unassembled subunits. Although N-terminal acetylation- and ubiquitin-mediated proteasomal degradation by the Ac/N-end rule pathway enables selective compensation of excess subunits, it is unclear how dominant this pathway contributes to stoichiometry control. Here we report that dosage compensation depends only partially on the Ac/N-end rule pathway. Our analysis of genetic interactions between 18 subunits and 10 quality control factors in budding yeast demonstrated that multiple E3 ubiquitin ligases and N-acetyltransferases are involved in dosage compensation. We find that N-acetyltransferases-mediated compensation is not simply predictable from N-terminal sequence despite their sequence specificity for N-acetylation. We also find that the compensation of Pop3 and Bet4 is due in large part to a minor N-acetyltransferase NatD. Furthermore, canonical NatD substrates histone H2A/H4 were compensated even in its absence, suggesting N-acetylation-independent compensation. Our study reveals the complexity and robustness of the stoichiometry control system.

genetics↗

bioRxiv2018

Integrative Transcription Start Site Analysis and Physiological Phenotyping Reveal Torpor-Specific Expressions in Mouse Skeletal Muscle

Mice enter an active hypometabolic state, called daily torpor, when they experience a lowered caloric intake under cool ambient temperature (TA). During torpor, the oxygen consumption rate (VO2) drops to less than 30% of the normal rate without harming the body. This safe but severe reduction in metabolism is attractive for various clinical applications; however, the mechanism and molecules involved are unclear. Therefore, here we systematically analyzed the expression landscape of transcription start sites (TSS) in mouse skeletal muscles under various metabolic states to identify torpor-specific transcription patterns. We analyzed the soleus muscles from 38 mice in torpid, non-torpid, and torpor-deprived conditions, and identified 287 torpor-specific promoters. Furthermore, we found that the transcription factor ATF3 was highly expressed during torpor deprivation and that the ATF3-binding motif was enriched in torpor-specific promoters. Our results demonstrate that the mouse torpor has a distinct hereditary genetic background and its peripheral tissues are useful for studying active hypometabolism.

physiology↗