Biology subjects

Kume, K.

Publications and source records attributed to Kume, K..

13 recordsLinked to original sources

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↗

bioRxiv2023

Beacon-based sleep status and physical activity monitoring in humans.

One out of every five people in Japan is dissatisfied with their sleep and various diseases caused by lack of exercise have been pointed out, but there are few effective remedies for these problems. In this study, we aimed to develop a simple method for measuring behavioral sleep patterns and physical activity using a beacon accelerometer wirelessly connected with a smartphone. A sleep prediction model was created comparing the data obtained from the accelerometer with the sleep status data obtained by a previously validated sleep monitoring system. The Random Forest model was able to classify sleep and wakefulness with a 97.4% and 85.4% precision, respectively, which were comparable to those of conventional acceleration-based sleep monitoring devices. Additionally, the same data acquisition method was used to classify exercise intensity into seven levels and a high correlation (r=0.813, p<0.0001) was found when comparing the classified exercise intensity to metabolic equivalent (MET) values. This suggests that the proposed method can be used for accurate measurement of both behavioral sleep and physical activity classifying over a long period of time.

physiology↗

bioRxiv2023

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling

Initiation of B-cell receptor (BCR)1 signaling, and subsequent antigen-encounter in germinal centers2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL)4 and lymphoma5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells6-9, the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKC{delta} and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKC{delta}10-12, SHIP113,14 and SHP114, 15,16, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKC{delta}-mediated phosphorylation of CD25 on its cytoplasmic tail (S268). Genetic rescue experiments identified CD25-S268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca2+-oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

immunology↗

bioRxiv2023

Targeted engagement of β-catenin-Ikaros complexes in refractory B-cell malignancies

In most cell types, nuclear {beta}-catenin functions as prominent oncogenic driver and pairs with TCF7-family factors for transcriptional activation of MYC. Surprisingly, B-lymphoid malignancies not only lacked expression and activating lesions of {beta}-catenin but critically depended on GSK3{beta} for effective {beta}-catenin degradation. Our interactome studies in B-lymphoid tumors revealed that {beta}-catenin formed repressive complexes with lymphoid-specific Ikaros factors at the expense of TCF7. Instead of MYC-activation, {beta}-catenin was essential to enable Ikaros-mediated recruitment of nucleosome remodeling and deacetylation (NuRD) complexes for transcriptional repression of MYC. To leverage this previously unrecognized vulnerability of B-cell-specific repressive {beta}-catenin-Ikaros-complexes in refractory B-cell malignancies, we examined GSK3{beta} small molecule inhibitors to subvert {beta}-catenin degradation. Clinically approved GSK3{beta}-inhibitors that achieved favorable safety prof les at micromolar concentrations in clinical trials for neurological disorders and solid tumors were effective at low nanomolar concentrations in B-cell malignancies, induced massive accumulation of {beta}-catenin, repression of MYC and acute cell death. Preclinical in vivo treatment experiments in patient-derived xenografts validated small molecule GSK3{beta}-inhibitors for targeted engagement of lymphoid-specific {beta}-catenin-Ikaros complexes as a novel strategy to overcome conventional mechanisms of drug-resistance in refractory malignancies. HIGHLIGHTSO_LIUnlike other cell lineages, B-cells express nuclear {beta}-catenin protein at low baseline levels and depend on GSK3{beta} for its degradation. C_LIO_LIIn B-cells, {beta}-catenin forms unique complexes with lymphoid-specific Ikaros factors and is required for Ikaros-mediated tumor suppression and assembly of repressive NuRD complexes. C_LIO_LICRISPR-based knockin mutation of a single Ikaros-binding motif in a lymphoid MYC superenhancer region reversed {beta}-catenin-dependent Myc repression and induction of cell death. C_LIO_LIThe discovery of GSK3{beta}-dependent degradation of {beta}-catenin as unique B-lymphoid vulnerability provides a rationale to repurpose clinically approved GSK3{beta}-inhibitors for the treatment of refractory B-cell malignancies. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/532152v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1b70e4dorg.highwire.dtl.DTLVardef@10bfcc8org.highwire.dtl.DTLVardef@b10eorg.highwire.dtl.DTLVardef@1683a3e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIAbundant nuclear {beta}-catenin C_LIO_LI{beta}-catenin pairs with TCF7 factors for transcriptional activation of MYC C_LIO_LIB-cells rely on efficient degradation of {beta}-catenin by GSK3{beta} C_LIO_LIB-cell-specific expression of Ikaros factors C_LI Unique vulnerability in B-cell tumors: O_LIGSK3{beta}-inhibitors induce nuclear accumulation of {beta}-catenin. C_LIO_LI{beta}-catenin pairs with B-cell-specific Ikaros factors for transcriptional repression of MYC C_LI

cancer biology↗

bioRxiv2022

Functionally conserved Pkd2, mutated in autosomal dominant polycystic kidney disease, localizes to the endoplasmic reticulum and regulates cytoplasmic calcium homeostasis in fission yeast

Mutations in PKD1 or PKD2 genes lead to autosomal dominant polycystic kidney disease (ADPKD) that is the most frequent family inherited renal disorder. These genes encode polycystin-1/PC-1 and polycycstin-2/PC-2, respectively. Although the genetic basis of ADPKD is well established, the crucial functions of polycystins underlying onset and development of cyst formation remain elusive. Fission yeast Schizosaccharomyces pombe has a single polycystin homolog, Pkd2, which is essential for cell growth. In this study, the truncation analyses of Pkd2 reveal that Pkd2 localizes to not only the plasma membrane but also the endoplasmic reticulum (ER) and regulates cytoplasmic calcium signaling in fission yeast. Internal transmembrane domains within Pkd2 are sufficient for these processes. Surprisingly, more than half of Pkd2 is not required for cell viability. Cytoplasmic calcium levels are mainly regulated through C-terminus of Pkd2. Importantly, human Pkd2 also localizes to the ER and furthermore, fully complements the loss of fission yeast Pkd2. As the functions of polycystin-2 are conserved, fission yeast provides a suitable model to study the mechanism of ADPKD as well as polycystins.

cell biology↗

bioRxiv2022

Interneurons of fan-shaped body promote arousal in Drosophila

Sleep is required to maintain physiological functions and is widely conserved across species. To understand the sleep-regulatory mechanisms, sleep-regulating genes and neuronal circuits are studied in various animal species. In the sleep-regulatory neuronal circuits in Drosophila melanogaster, the dorsal fan-shaped body (dFB) is a major sleep-promoting region. However, other sleep-regulating neuronal circuits were not well identified. We recently found a novel sleep-regulatory circuit consisting of arousal-promoting T1 dopamine neurons and protocerebral bridge (PB) neurons innervating the ventral part of the FB, which we named "the PB-FB pathway". However, the post-synaptic target of the PB-FB pathway was still unknown. To identify it, we performed anterograde tracing, immunohistochemistry, and Ca2+ imaging analysis and found that the PB-FB pathway projects to FB interneurons, also known as pontine neurons. Besides, we found that cholinergic pontine neurons promote arousal. Moreover, we indicated that pontine neurons form an anatomical connection with sleep-promoting dFB neurons. Together, we showed that pontine neurons receive excitatory signals from the PB-FB pathway and cholinergic pontine neurons promote arousal. These results completed one of the output pathways from the PB-FB pathway.

neuroscience↗

bioRxiv2022

The regulation of circadian rhythm by insulin signaling in Drosophila

Circadian rhythm is well conserved across species and relates to numerous biological functions. Circadian misalignment impairs metabolic function. Insulin signaling is a key modulator of metabolism in the fruit fly as well as mammals and its defects cause metabolic disease. Daily diet timing affects both circadian rhythmicities of behavior and metabolism. However, the relationship between circadian clock and insulin signaling is still elusive. Here, we report that insulin signaling regulates circadian rhythm in Drosophila melanogaster. We found the insulin receptor substrate mutant, chico1, showed a shorter free-running circadian period. The knockdown of insulin receptor (InR), or another signaling molecule downstream of InR, dp110, or the expression of a dominant-negative form of InR resulted in the shortening of the circadian period and diminished its amplitude. The impairment of insulin signaling both in all neurons and restricted circadian clock neurons altered circadian period length, indicating the insulin signaling plays a role in the regulation of circadian rhythm in clock cells. Among 3 insulin-like ligands expressed in the brain, dilp5 showed the largest effect on circadian phenotype when deleted. These results suggested that insulin signaling contributes to the robustness of the circadian oscillation and coordinates metabolism and behavior. HighlightsO_LIInsulin receptor substrate mutant, chico1, displayed circadian rhythm phenotype. C_LIO_LIPan-neuronal inhibition of insulin receptor signaling shortened circadian cycle. C_LIO_LIInhibition of insulin signaling only in clock neurons altered circadian cycle. C_LIO_LIDilp5 is a major insulin receptor ligand for circadian effects. C_LI

animal behavior and cognition↗

bioRxiv2021

Phylogenomics invokes the clade housing Cryptista, Archaeplastida, and Microheliella maris.

As-yet-undescribed branches in the tree of eukaryotes are potentially represented by some of "orphan" protists (unicellular micro-eukaryotes), of which phylogenetic affiliations have not been clarified in previous studies. By clarifying the phylogenetic positions of orphan protists, we may fill the previous gaps in the diversity of eukaryotes and further uncover the novel affiliation between two (or more) major lineages in eukaryotes. Microheliella maris was originally described as a member of the phylum Heliozoa, but a pioneering large-scale phylogenetic analysis failed to place this organism within the previously described species/lineages with confidence. In this study, we analyzed a 319-gene alignment and demonstrated that M. maris represents a basal lineage of one of the major eukaryotic lineages, Cryptista. We here propose a new clade name "Pancryptista" for Cryptista plus M. maris. The 319-gene analyses also indicated that M. maris is a key taxon to recover the monophyly of Archaeplastida and the sister relationship between Archaeplastida and Pancryptista, which is collectively called as "CAM clade" here. Significantly, Cryptophyceae tend to be attracted to Rhodophyta depending on the taxon sampling (ex., in the absence of M. maris and Rhodelphidia) and the particular phylogenetic "signal" most likely hindered the stable recovery of the monophyly of Archaeplastida in previous studies. We hypothesize that many cryptophycean genes (including those in the 319-gene alignment) recombined partially with the homologous genes transferred from the red algal endosymbiont during secondary endosymbiosis and bear a faint phylogenetic affinity to the rhodophytan genes.

evolutionary biology↗

bioRxiv2021

Genome evolution of a non-parasitic secondary heterotroph, the diatom Nitzschia putrida

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a non-photosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms revealed that a combination of genes loss, the horizontal acquisition of genes involved in organic carbon degradation, a unique secretome and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a non-parasitic, free-living secondary heterotroph.

genomics↗

bioRxiv2020

Protocerebral bridge neurons that regulate sleep in Drosophila melanogaster

The central complex is one of the major brain regions that control sleep in Drosophila, but the circuitry details of sleep regulation have yet to be elucidated. Here, we show a novel sleep-regulating neuronal circuit in the protocerebral bridge (PB) of the central complex. Activation of the PB interneurons labeled by the R59E08-Gal4 and the PB columnar neurons in the R52B10-Gal4 promoted sleep and wakefulness, respectively. A targeted GFP reconstitution across synaptic partners (t-GRASP) analysis demonstrated synaptic contacts between these two groups of sleep-regulating PB neurons. Furthermore, we found that activation of a pair of dopaminergic (DA) neurons projecting to the PB (T1 DA neurons) decreased sleep. The wake-promoting T1 DA neurons and the sleep-promoting PB interneurons formed close associations. Dopamine 2-like receptor (Dop2R) knockdown in the sleep-promoting PB interneurons increased sleep. These results indicated that the neuronal circuit in the PB regulated by dopamine signaling mediates sleep-wakefulness.

neuroscience↗

bioRxiv2020

Signaling input from divergent pathways subverts malignant B-cell transformation

Malignant transformation typically involves multiple genetic lesions whose combined activity gives rise to cancer1. Our analysis of 1,148 patient-derived B-cell leukemia (B-ALL) samples revealed that individual mutations did not promote leukemogenesis unless they converged on one single oncogenic pathway characteristic for the differentiation status of these transformed B cells. Specifically, we show here the JAK/STAT5 signaling pathway supports the developmental stage-specific expansion of pro-B ALL whereas the ERK-pathway that of pre-B ALL. Mutations that were not aligned with the central oncogenic driver would activate divergent pathways and subvert malignant transformation. Oncogenic lesions in B-ALL frequently mimic survival and proliferation signals downstream of cytokine receptors (through activation of STAT5)2-7 or the pre-B cell receptor (through activation of ERK)8-13. STAT5- (372 cases) and ERK- (386 cases) activating lesions were frequently found but only co-occurred in [~]3% (37) of cases (P=2.2E-16). Single-cell mutation and phosphoprotein analyses revealed that even in these rare cases, oncogenic STAT5- or ERK-activation were mutually exclusive and segregated to competing clones. STAT5 and ERK engaged opposing biochemical and transcriptional programs orchestrated by MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway came at the expense of the principal oncogenic driver and reversed malignant transformation. Conversely, Cre-mediated deletion of divergent pathway components triggered leukemia-initiation and accelerated development of fatal disease. Thus, persistence of divergent signaling pathways represents a powerful barrier to malignant transformation while convergence on one principal driver defines a key event during leukemia-initiation. Proof-of-concept studies in patient-derived B-ALL cells revealed that pharmacological reactivation of suppressed divergent circuits strongly synergized with direct inhibition of the principal oncogenic driver. Hence, pharmacological reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to deepen treatment responses and to overcome drug-resistance. Current treatment approaches for drug-resistant cancer are focused on drug-combinations to suppress the central oncogenic driver and multiple alternative pathways14-17. Here, we introduce a concept based on inhibition of the principal driver combined with pharmacological reactivation of divergent pathways.

cancer biology↗

bioRxiv2020

Lgr5-mediated restraint of β-catenin is essential for B-lymphopoiesis and leukemia-initiation

Upon productive immunoglobulin gene rearrangement, expression of a functional pre-B cell receptor (pre-BCR) initiates positive selection of pre-B cells, clonal expansion and self-renewal1-2. Studying mechanisms driving this first wave of B-lymphopoiesis, we identified the G-protein coupled receptor Lgr5 as an essential initiator of positive selection. Lgr5 was extensively studied as determinant of stem cell populations in multiple tissues3-6, but not in B-cells. While undetectable throughout the hematopoietic system, positively selected pre-B cells were marked with a sharp peak of Lgr5 expression. Conditional deletion of Lgr5 preceding the pre-BCR checkpoint induced negative selection and complete abortion of B-cell development. Proteomic studies of Lgr5-ablation revealed massive (>250-fold) accumulation of {beta}-catenin and suppression of MYC. Lgr5-deficient pre-B cells fully recovered by concurrent {beta}-catenin-deletion, demonstrating a central role of Lgr5-mediated restraint of {beta}-catenin at the pre-BCR checkpoint. In other cell types, {beta}-catenin/TCF4 complexes drive transcriptional activation of MYC7-9. Instead of TCF4, proximity-based interactome studies in pre-B cells identified the B-lymphoid transcription factors IKZF1 and IKZF310-11 as {beta}-catenin-binding partners, which had the opposite effect and caused transcriptional repression of MYC. On positively selected pre-B cells, Lgr5 prevented accumulation of {beta}-catenin and formation of complexes with IKZF1 and IKZF3, which relieved transcriptional repression of MYC. Activating {beta}-catenin-mutations are common throughout all main types of cancer7-8, but were conspicuously absent in pre-B leukemia (B-ALL). Like pre-B cells, B-ALL cells were uniquely sensitive to genetic and pharmacological {beta}-catenin hyperactivation, which recapitulated the effects of Lgr5-deletion and compromised colony formation and leukemia-initiation. A new LGR5 antibody-drug conjugate targeted leukemia-initiating cells in patient-derived B-ALL and achieved long-term disease-control. Likewise, small molecule hyperactivation of {beta}-catenin selectively killed B-ALL but not other cell types. Hence, Lgr5-mediated restraint of {beta}-catenin activation is essential for B-lymphopoiesis and revealed an unexpected vulnerability that can be leveraged for the treatment of drug-resistant B-ALL.

cancer biology↗

bioRxiv2019

Barthelonids represent a deep-branching Metamonad clade with mitochondrion-related organelles generating no ATP.

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) was used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching Metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. Strain PAP020 is most likely incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected the putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.

evolutionary biology↗