Biology subjects

Chiba, Y.

Publications and source records attributed to Chiba, Y..

16 recordsLinked to original sources

bioRxiv2025

Direct carbon monoxide fixation via the bacterial and archaeal Wood-Ljungdahl pathways

The Wood-Ljungdahl (WL) pathway, which is widely distributed in both archaea and bacteria, is an ancient carbon fixation pathway from CO2. CO2 fixation proceeds via two branches of pathway: progressive reduction to the methyl group in the methyl branch and one-step reduction to CO in the carbonyl branch. In the final step of the pathway, the methyl group, CO, and CoA are combined into a carbon monoxide dehydrogenase (CODH)/acetyl-CoA synthase (ACS) complex to form acetyl-CoA. Here, we show direct CO fixation to the carbonyl group of acetyl-CoA in both archaeal and bacterial WL pathways under hydrogenogenic growth conditions using 13C tracer-based metabolomics. A combination of metabolomics and proteomics suggested that the hydrogenotrophically grown Thermodesulfatator indicus and Archaeoglobus sp. strain MCR cells, directly fixed CO using free-form ACS in the carbonyl branch with relatively low CO availability. In contrast, carboxydotrophically grown Archaeoglobus cells utilize the CODH/ACS complex for CO2 fixation rather than CO fixation. Direct CO fixation by free-form ACS is more advantageous for conserving reduced ferredoxin compared with the thermodynamically challenged CO2 reduction by CODH. These findings provide further insight into the origin and evolution of the most ancient inorganic carbon fixation pathway and geochemical cycles on early Earth.

microbiology↗

bioRxiv2025

Molecular evolution of the Wood-Ljungdahl pathway and the reductive glycine pathway in Thermodesulfobacteriota

Carbon fixation is a fundamental metabolic process that sustains ecosystems, yet its origins and evolutionary history remain largely unresolved. In this study, we focused on the Wood-Ljungdahl (WL) pathway, which is considered one of the most ancient carbon fixation pathways and the reductive glycine (rGly) pathway, which shares several reactions with the WL pathway. The evolutionary scenario of the two carbon fixation pathways was inferred in the phylum Thermodesulfobacteriota, which includes microorganisms that operate either the WL pathway or the rGly pathway for autotrophic growth. The timing of gene gain and loss events was inferred by gene presence/absence analyses for both pathways, together with phylogenetic analyses of their key enzymes. Our results suggested that the common ancestor of Thermodesulfobacteriota possessed all genes encoding key enzymes of both pathways; formate dehydrogenase, the carbon monoxide dehydrogenase/acetyl-CoA synthase complex and the glycine cleavage system. Furthermore, analyses of complete gene sets for the WL and rGly pathway, together with downstream genes required for amino acid biosynthesis, supported the possibility that the common ancestor of this phylum had been capable of autotrophic growth through these carbon fixation pathways. Then, multiple lineages have lost the WL and rGly pathway genes independently during subsequent evolution. Gene replacements also occurred in the glycine cleavage system by regaining genes by horizontal gene transfer. These results suggest that carbon fixation pathways in extant organisms in the phylum Thermodesulfobacteriota arose through a combination of vertical inheritance, gene loss, and horizontal gene transfer.

bioinformatics↗

bioRxiv2025

Impact of acetate on CO2 fixation pathways in thermophilic and hydrogenotrophic bacteria

The Wood-Ljungdahl (WL) pathway and reductive tricarboxylic acid (rTCA) cycle are the dominant chemolithotrophic CO2 fixation pathways in bacteria inhabiting aphotic geothermal and deep-sea hydrothermal ecosystems. However, the activity of these bacterial metabolic systems in ecosystems with available organic carbons remains unclear. Here, we examined the impact of extracellular acetate on the CO2-fixation pathways of three thermophilic hydrogen-oxidizing and non-acetogenic bacteria using 13C tracer-based metabolomics. Under chemolithoautotrophic conditions, Thermodesulfatator indicus and Hydrogenobacter thermophilus fixed CO2 through the WL pathway and rTCA cycle, respectively, whereas Thermovibrio ammonificans, which has been suggested to operate both of these pathways, exhibited significant CO2 fixation through only the rTCA cycle. Under chemolithomixotrophic conditions with acetate, H. thermophilus and T. ammonificans assimilated both CO2 and acetate via the rTCA cycle. In contrast, acetate suppressed CO2 fixation through the WL pathway in T. indicus and was used as the primary carbon source under chemolithomixotrophic conditions. These results suggest that the contribution of the WL pathway for CO2 fixation might be overestimated in ecosystems where acetate is available. Moreover, the present findings indicate that simultaneous CO2 fixation through both the WL pathway and rTCA cycle in a cell, which has been proposed as a possible metabolic strategy for CO2-fixation in ancestral life, is not advantageous in extant microorganisms.

microbiology↗

bioRxiv2024

Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3

DNA methylation maintenance is essential for cell fate inheritance. In differentiated cells, this involves orchestrated actions of DNMT1 and UHRF1. In mice, the high-affinity binding of DPPA3 to the UHRF1 PHD finger regulates UHRF1 chromatin dissociation and cytosolic localization, which is required for oocyte maturation and early embryo development. However, the human DPPA3 ortholog functions during these stages remain unclear. Here, we report the structural basis for human DPPA3 binding to the UHRF1 PHD finger. The conserved human DPPA3 85VRT87 motif binds to the acidic surface of UHRF1 PHD finger, whereas mouse DPPA3 binding additionally utilizes two unique -helices. The binding affinity of human DPPA3 for the UHRF1 PHD finger was weaker than that of mouse DPPA3. Consequently, human DPPA3, unlike mouse DPPA3, failed to inhibit UHRF1 chromatin binding and DNA remethylation in Xenopus egg extracts effectively. Our data provide novel insights into the distinct function and structure of human DPPA3.

biochemistry↗

bioRxiv2023

CDCA7 is a hemimethylated DNA adaptor for the nucleosome remodeler HELLS

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome, characterized by hypomethylation at heterochromatin. The unique zinc-finger domain, zf-4CXXC_R1, of CDCA7 is widely conserved across eukaryotes but is absent from species that lack HELLS and DNA methyltransferases, implying its specialized relation with methylated DNA. Here we demonstrate that zf-4CXXC_R1 acts as a hemimethylated DNA sensor. The zf-4CXXC_R1 domain of CDCA7 selectively binds to DNA with a hemimethylated CpG, but not unmethylated or fully methylated CpG, and ICF disease mutations eliminated this binding. CDCA7 and HELLS interact via their N-terminal alpha helices, through which HELLS is recruited to hemimethylated DNA. While placement of a hemimethylated CpG within the nucleosome core particle can hinder its recognition by CDCA7, cryo-EM structure analysis of the CDCA7-nucleosome complex suggests that zf-4CXXC_R1 recognizes a hemimethylated CpG in the major groove at linker DNA. Our study provides insights into how the CDCA7-HELLS nucleosome remodeling complex uniquely assists maintenance DNA methylation.

molecular biology↗

bioRxiv2023

Rechargeable Biomineral Induced by Sulfate Reducing Bacterium Cupidesulfovibrio sp. HK-II

A black precipitate produced by a sulfate reducing bacterium Cupidesulfovibrio sp. strain HK was investigated with multidisciplinary methods. X-ray diffraction (XRD) analysis revealed that the black precipitate was mackinawite. Cyclic voltammetry analysis showed the obvious redox peaks, and the biogenic mackinawite exhibited rechargeable properties. XRD analyses showed that the form of the rechargeable biogenic mackinawite (RBM-II) was changed by discharge and recharge treatments: Field-emission transmission electron microscope analyses revealed that lepidocrocite and amorphous iron oxide were appeared from mackinawite on discharged condition, and the three kinds of minerals were intermingled via the rechargeable treatments. Physicochemical parameters were changed regularly under the treatments, suggesting that discharge would be occurred by iron oxidation and sulfur reduction, and vice versa. These results indicated that dynamics of sulfur is important key process in rechargeable mechanism, supporting that a part of mackinawite was transformed to lepidocrocite and iron oxides, and vice versa. Microbial fuel cells (MFCs) equipped with lactate, strain HK-II and anode including RBM-II consumed lactate even under opened circuit conditions, after which MFCs generated higher current density at re-closed circuit conditions. These results demonstrated that the biogenic mackinawite is one of rechargeable materials and would play important roles in geomicrobiological reactions and biotechnology.

bioengineering↗

bioRxiv2023

Diverse Phosphoserine Phosphatases Exhibit Maximum Activity at an Intermediate Binding Affinity in Accord with the Sabatier Principle of Catalysis

A unified framework to rationalize enzymatic activity is essential to understand cellular function and metabolic evolution. Recent studies have shown that the activity of several hydrolases is maximized when the substrate binding affinity (Michaelis-Menten constant: Km) is neither too strong nor too weak. This is because an intermediate Km resolves the trade-off between Km and kcat, in accord with the Sabatier principle of artificial catalysis. However, it remains unclear whether this concept is applicable to enzymes in general, especially for those which catalyze the same reaction but have evolved under different selection pressures due to the phylogeny or physiology of the host organism. Here, we demonstrate that the activity of 10 distinct wild-type phosphoserine phosphatases (PSP) exhibits a maximum at an intermediate binding affinity (Km {approx} 0.5 mM), indicating that they also follow the Sabatier principle. Furthermore, by considering not only Km but also the equilibrium rate constant [Formula] of each enzyme, we have succeeded in rationalizing the PSP activity quantitatively. [Formula] is the rate constant of product release (ES [->] E + P) in the absence of any driving force, and a large [Formula] allows kcat to be increased without increasing Km. Although the traditional Sabatier principle considers only the binding affinity (Km), we show that the additional contribution of [Formula] drastically improves the consistency between experiments and theory. Our expanded framework which quantitatively explains the activity of phylogenetically and physiologically diverse enzymes with respect to their physicochemical parameters may lead to the rational design of highly active enzymes.

biochemistry↗

bioRxiv2023

Universal Design Principle to Enhance Enzymatic Activity using the Substrate Affinity

Design principles to improve enzymatic activity are essential to promote energy-material conversion using biological systems. For more than a century, the Michaelis-Menten equation has provided a fundamental framework of enzymatic activity. However, there is still no concrete guideline on how the parameters should be optimized to enhance enzymatic activity. Here, we demonstrate that tuning the Michaelis-Menten constant (Km) to the substrate concentration (S) maximizes enzymatic activity. This guideline (Km = S) was obtained by applying the Bronsted (Bell)-Evans-Polanyi (BEP) principle of heterogeneous catalysis to the Michaelis-Menten equation, and is robust even with mechanistic deviations such as reverse reactions and inhibition. Furthermore, Km and S are consistent to within an order of magnitude over an experimental dataset of approximately 1000 wild-type enzymes, suggesting that even natural selection follows this principle. The concept of an optimum Km offers the first quantitative guideline towards improving enzymatic activity which can be used for highthroughput enzyme screening.

biochemistry↗

bioRxiv2022

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing {beta}-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

immunology↗

bioRxiv2022

Structural basis for activation of DNMT1

DNMT1 is an essential enzyme that maintains genomic DNA methylation, and its function is regulated by mechanisms that are not yet fully understood. Here, we report the cryo-EM structure of human DNMT1 bound to its two natural activators: hemimethylated DNA and ubiquitinated histone H3. We find that a hitherto unstudied linker, between the RFTS and CXXC domains, plays a key role for activation. It contains a conserved -helix which engages a crucial "Toggle" pocket, displacing a previously described inhibitory linker, and allowing the DNA recognition helix to spring into the active conformation. This is accompanied by large-scale reorganization of the inhibitory RFTS and CXXC domains, allowing the enzyme to gain full activity. Our results therefore provide a mechanistic basis for the activation of DNMT1, with consequences for basic research and drug design.

biochemistry↗

bioRxiv2022

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced - helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

biochemistry↗

bioRxiv2022

The termination of UHRF1-dependent PAF15 ubiquitin signaling is regulated by USP7 and ATAD5

UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.

molecular biology↗

bioRxiv2022

Thoracic crop formation is spatiotemporally coordinated with flight muscle histolysis during claustral colony foundation of a Lasius japonicus queen

In a majority of ants, a newly mated queen independently founds a colony and claustrally raises her first brood without foraging outside the nest. During claustral independent colony foundation (ICF) in several ants, the esophagus of the founding queen expands and develops into a thoracic crop, which is then filled with a liquid substrate for larval feeding. It has been suggested that these substrates are converted from her body reserves (e.g., histolyzed flight muscles) or redistributed from a gastral crop. Here, we examined thoracic crop development in Lasius japonicus during claustral ICF. The foundresses claustrally fed their larvae from week 2 to 5 after ICF onset, and the first worker emerged at week 6. The development proceeded as follows: in week 0, foundress dorsal esophagus wall was pleated and thickened. Then, from week 2 to 5, the esophagus expanded dorsally toward where flight muscles had been present, following flight muscle histolysis. Gastral crop expansion followed esophagus expansion. Thus, thoracic crop formation may be spatiotemporally coordinated with flight muscle histolysis in Lasius japonicus queens, and similar developmental regulations might be common in other claustral ICF ants.

zoology↗

bioRxiv2020

HPF1-dependent PARP activation promotes LIG3-XRCC1-mediated backup pathway of Okazaki fragment ligation

DNA Ligase 1 (LIG1) is known as the major DNA ligase responsible for Okazaki fragment joining. Recent studies have implicated LIG3 complexed with XRCC1 as an alternative player in Okazaki fragment joining in cases where LIG1 is not functional, although the underlying mechanisms are largely unknown. Here, using a cell-free system derived from Xenopus egg extracts, we demonstrated the essential role of PARP1-HPF1 in LIG3-dependent Okazaki fragment joining. We found that Okazaki fragments were eventually ligated even in the absence of LIG1, employing in its place LIG3-XRCC1 which was recruited onto chromatins. Concomitantly, LIG1 deficiency induces ADP-ribosylation of histone H3 in a PARP1-HPF1-dependent manner. The depletion of PARP1 or HPF1 resulted in a failure to recruit LIG3 onto chromatin and a subsequent failure in Okazaki fragment joining in LIG1-depleted extracts. Importantly, Okazaki fragments were not ligated at all when LIG1 and XRCC1 were co-depleted. Our results suggest that a unique form of ADP-ribosylation signalling promotes the recruitment of LIG3 on chromatins and its mediation of Okazaki fragment joining as a backup system for LIG1 perturbation.

molecular biology↗

bioRxiv2020

Discovery of divided RdRp sequences and a hitherto unknown genomic complexity in fungal viruses

By identifying variations in viral RNA genomes, cutting-edge metagenome technology has potential to reshape current concepts about the evolution of RNA viruses. This technology, however, cannot process low-homology genomic regions properly, leaving the true diversity of RNA viruses unappreciated. To overcome this technological limitation we applied an advanced method, Fragmented and Primer-Ligated Double-stranded (ds) RNA Sequencing (FLDS), to screen RNA viruses from 155 fungal isolates, which allowed us to obtain complete viral genomes in a homology-independent manner. We created a high-quality catalog of 19 RNA viruses (12 viral species) that infect Aspergillus isolates. Among them, nine viruses were not detectable by the conventional methodology involving agarose gel electrophoresis of dsRNA, a hallmark of RNA virus infections. Segmented genome structures were determined in 42% of the viruses. Some RNA viruses had novel genome architectures; one contained a dual methyltransferase domain and another had a separated RNA-dependent RNA polymerase (RdRp) gene. A virus from a different fungal taxon (Pyricularia) had an RdRp sequence that was separated on different segments, suggesting that a divided RdRp is widely present among fungal viruses, despite the belief that all RNA viruses encode RdRp as a single gene. These findings illustrate the previously hidden diversity and evolution of RNA viruses, and prompt reconsideration of the structural plasticity of RdRp. By highlighting the limitations of conventional surveillance methods for RNA viruses, we showcase the potential of FLDS technology to broaden current knowledge about these viruses. Author SummaryThe development of RNA-seq technology has facilitated the discovery of RNA viruses in all types of biological samples. However, it is technically difficult to detect highly novel viruses using RNA-seq. We successfully reconstructed the genomes of multiple novel fungal RNA viruses by screening host fungi using a new technology, FLDS. Surprisingly, we identified two viral species whose RNA-dependent RNA polymerase (RdRp) proteins were separately encoded on different genome segments, overturning the commonly accepted view of the positional unity of RdRp proteins in viral genomes. This new perspective on divided RdRp proteins should hasten the discovery of viruses with unique RdRp structures that have been overlooked, and further advance current knowledge and understanding of the diversity and evolution of RNA viruses.

microbiology↗

bioRxiv2020

Auditory experience prevents loss of the innate song preference as a selective cue in Drosophila

Auditory learning is a prerequisite step for acoustic communication learning, which was previously assumed to be restricted to animals with high levels of cognition, such as humans, cetaceans, and birds. How animals that rely on auditory learning for acoustic communication form sound preferences is not known. Fruit flies are a recently proposed novel animal model for studying experience-dependent auditory perceptual plasticity because of their ability to acquire song preferences via song exposure. Whether fruit flies have innate courtship song preferences, however, is unclear. Here we report that, similar to songbirds, fruit flies exhibit an innate preference for conspecific courtship songs. Maintenance of innate song preference requires song input, reminiscent of the song learning process in songbirds. Our findings also indicate that the response to conspecific and heterospecific songs manifests temporal and experience-dependent differentiation, which may underlie innate song preference and its plasticity. In addition, we find that flies have a robust ability to reacquire song preference during aging. Fruit flies thus offer a novel and simple approach for studying sound preference formation and its underlying mechanisms.

neuroscience↗