Biology subjects

Kobayashi, S.

Publications and source records attributed to Kobayashi, S..

31 records · Page 2Linked to original sources

bioRxiv2023

Structure of the human Bre1 complex bound to the nucleosome

Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To reveal the structural basis of H2BK120 ubiquitination and its regulation, we determined the cryo-EM structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in both orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the relationships between Bre1 proteins and cancer and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.

biochemistry↗

bioRxiv2023

Inhibitory effect of Ephedra herba on human norovirus infection in human intestinal organoids

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases worldwide with public health concern, yet no antiviral therapies have been developed. In this study, we aimed to screen crude drugs, which are components of Japanese traditional medicine, Kampo" to see their effects on HuNoV infection using a reproducible HuNoV cultivation system, stem-cell derived human intestinal organoids/enteroids (HIOs). Among the 23 crude drugs tested, Ephedra herba significantly inhibited HuNoV infection in HIOs. A time-of-drug addition experiment indicated that this crude drug likely targets post-entry step for the inhibition. To our knowledge, this is the first anti-HuNoV inhibitor screen targeting crude drugs, and Ephedra herba was identified as a novel inhibitor candidate that merits further study.

microbiology↗

bioRxiv2023

Stereoinversion via alcohol dehydrogenases enables complete catabolism of β-1-type lignin-derived aromatic isomers

Sphingobium sp. SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened {beta}-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was heretofore unknown. Here we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the C alcohol groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the C carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by C-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of {beta}-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from C-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four {beta}-1-type stereoisomers through successive oxidation and reduction reactions of the C alcohol group of threo-DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCEIn many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with {beta}-1 linkages. Therefore, elucidating the catabolic system of {beta}-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. In this work, we found that threo-DGPD was converted by successive stereoselective oxidation and reduction at the C-position by multiple alcohol dehydrogenases to erythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native {beta}-1-type dimer catabolic system.

microbiology↗

bioRxiv2022

Morphogenesis of bullet-shaped rabies virus particles requires a functional interplay between the viral matrix protein and ESCRT-I component TSG101

Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host ESCRT (endosomal sorting complex required for transport) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection, and specifically the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a siRNA screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late-domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. ImportanceEnveloped-viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT (endosomal sorting complex required for transport) complexes. Some enveloped-viruses utilize their late (L)-domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped-virions, but the underlying molecular mechanisms involved remain elusive. Herein, we showed that TSG101, one of ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.

microbiology↗

bioRxiv2022

Enterococcal linear plasmids adapt to Enterococcus faecium and spread within multidrug-resistant clades.

Antimicrobial resistance (AMR) of bacterial pathogens, including enterococci, is a global concern, and plasmids are crucial for spreading and maintaining AMR genes. Plasmids with linear topology were recently identified in clinical multidrug-resistant enterococci. The enterococcal linear-form plasmids, such as pELF1, confer resistance to clinically important antimicrobials, including vancomycin; however, little information exists about their epidemiological and physiological effects. In this study, we identified several lineages of enterococcal linear plasmids that are structurally conserved and occur globally. pELF1-like linear plasmids show plasticity in acquiring and maintaining AMR genes, often via transposition with the mobile genetic element IS1216E. This linear plasmid family has several characteristics enabling long-term persistence in the bacterial population, including high horizontal self-transmissibility, low-level transcription of plasmid-encoded genes, and a moderate effect on the Enterococcus faecium genome alleviating fitness cost and promoting vertical inheritance. Combined with its broad host range, the linear plasmid is an important factor in the spread and maintenance of AMR genes among enterococci.

microbiology↗

bioRxiv2022

Deep learning-based approach for the characterization and quantification of histopathology in mouse models of colitis

Inflammatory bowel disease (IBD) is a chronic immune-mediated disease of the gastrointestinal tract. While therapies exist, response can be limited within the patient population. As such, researchers have studied mouse models of colitis to further understand its pathogenesis and identify new treatment targets. Although bench methods like flow cytometry and RNA-sequencing can characterize immune responses with single-cell resolution, whole murine colon specimens are processed at once. Given the simultaneous presence of colonic regions that are involved or uninvolved with abnormal histology, processing whole colons may lead to a loss of spatial context. Detecting these regions in hematoxylin and eosin (H&E)-stained colonic tissues offers the downstream potential of quantifying immune populations in areas with and without disease involvement by immunohistochemistry on serially sectioned slides. This could provide a complementary, spatially-aware approach to further characterize populations identified by other methods. However, detection of such regions requires expert interpretation by pathologists and is a tedious process that may be difficult to perform consistently across experiments. To this end, we have trained a deep learning model to detect Involved and Uninvolved regions from H&E-stained colonic slides across controls and three mouse models of colitis - the dextran sodium sulfate (DSS) chemical induction model, the recently established intestinal epithelium-specific, inducible Klf5{Delta}IND (Villin-CreERT2;Klf5fl/fl) genetic model, and one that combines both induction methods. The trained classifier allows for extraction of Involved colonic regions across mice to cluster and identify histological classes. Here, we show that quantification of Involved and Uninvolved image patch classes in swiss rolls of colonic specimens can be utilized to train a linear determinant analysis classifier to distinguish between mouse models. Such an approach has the potential for revealing histological links and improving synergy between various colitis mouse model studies to identify new therapeutic targets and pathophysiological mechanisms.

immunology↗

bioRxiv2022

Prox1 dynamically regulates downstream targets and chromatin accessibility during venous to lymphatic endothelial cell transdifferentiation in the embryo

During development, the lymphatic vasculature forms as a second, new vascular network derived from blood vessels. The transdifferentiation of embryonic venous endothelial cells (VECs) into lymphatic endothelial cells (LECs) is the first step in this process. Specification, differentiation and maintenance of LEC fate are all driven by the transcription factor Prox1, yet downstream mechanisms remain to be elucidated. We present a single cell transcriptomic atlas of lymphangiogenesis in zebrafish revealing new markers and hallmarks of LEC differentiation over four developmental stages. We further profile single cell transcriptomic and chromatin accessibility changes in zygotic prox1a mutants that are undergoing a VEC-LEC fate reversion during differentiation. Using maternal and zygotic prox1a/prox1b mutants, we determine the earliest transcriptomic changes directed by Prox1 during LEC specification. This work altogether reveals new transcriptional targets and regulatory regions of the genome downstream of Prox1 in LEC maintenance, as well as showing that Prox1 specifies LEC fate primarily by limiting blood vascular and hematopoietic fate. This extensive single cell resource provides new mechanistic insights into the enigmatic role of Prox1 and the control of LEC differentiation in development.

developmental biology↗

bioRxiv2021

SIPA1L1/SPAR1 is a non-PSD protein involved in GPCR signaling

SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the postsynaptic density (PSD)-95/N-methyl-D-aspartate receptor (NMDA-R)-complex. However, contrary to this view, our super-resolution and immunoelectron microscopic analyses demonstrate that SIPA1L1 is mainly localized to general submembranous and cytoplasmic regions in neurons, but scarcely to PSD. Our screening for native interactors of SIPA1L1 identified spinophilin and neurabin-1, regulators of G protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to 2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.

neuroscience↗

bioRxiv2020

Identification and classification of innexin gene transcripts in the central nervous system of the terrestrial slug Limax valentianus

In invertebrates, innexin is involved in the formation of single-cell membrane channels and intercellular gap junction channels. Generally, there are multiple isoforms of innexin family proteins in various animal species, which enable the precise regulation of channel function. In molluscan species, the sequence information of innexins is still limited and the sequences have not been classified. This study examined the innexin transcripts expressed in the central nervous system of the terrestrial slug Limax valentianus and identified 16 transcripts of 12 innexin isoforms, including the splicing variants. To examine the function of molluscan innexin isoforms, phylogenetic analysis was performed using the innexin sequences of molluscan species. Next, the phosphorylation, N-glycosylation, and S-nitrosylation sites in the isoforms were predicted to characterize the innexin isoforms. Further, 16 circular RNA sequences of nine innexin isoforms were identified in the central nervous system of Limax. The identification and classification of the gene transcripts of molluscan innexins provided novel insights for understanding the regulatory mechanism of innexins in the central nervous system.

molecular biology↗

bioRxiv2020

A biomimetic five-module chimeric antigen receptor (5MCAR) designed to target and eliminate antigen-specific T cells

T cells express clonotypic T cell receptors (TCRs) that recognize peptide antigens in the context of class I or II MHC molecules (pMHCI/II). These receptor modules associate with three signaling modules (CD3{gamma}{varepsilon}, {delta}{varepsilon}, and {zeta}{zeta}), and work in concert with a coreceptor module (either CD8 or CD4), to drive T cell activation in response to pMHCI/II. Here we describe a first generation biomimetic 5-module chimeric antigen receptor (5MCAR). We show that: (i) chimeric receptor modules built with the ectodomains of pMHCII assemble with CD3 signaling modules into complexes that redirect cytotoxic T lymphocyte (CTL) specificity and function in response to the clonotypic TCRs of pMHCII-specific CD4+ T cells; and, (ii) surrogate coreceptor modules enhance the function of these complexes. Furthermore, we demonstrate that adoptively transferred 5MCAR-CTLs can mitigate type I diabetes by targeting autoimmune CD4+ T cells in NOD mice. This work provides a framework for the construction of biomimetic 5MCARs that can be used as tools to study the impact of particular antigen-specific T cells in immune responses, and may hold potential for ameliorating diseases mediated by pathogenic T cells.

bioengineering↗

bioRxiv2020

Ameliorative effect of a quinazoline-based bromodomain inhibitor, CN210, on experimentally-induced Crohn's disease-like murine ileitis via inhibition of inflammatory cytokine expression

Inhibitors of bromodomain and extra-terminal motif proteins ("BET inhibitors") are emerging epigenetic therapeutics that exert their effects by suppressing the expression of genes that drive cancer and inflammation. The present study examined the anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, which also inhibits bromodomains of two paralogous histone acetyltransferases (HATs), such as cAMP-responsive element binding protein-binding protein (CBP) and p300. To assess its effectiveness against inflammatory bowel disease (IBD), CN210 was tested in an experimentally-induced murine Crohns disease (CD)-like ileitis model. Ileitis was induced in mice by subcutaneous administration of indomethacin and CN210 was given orally 30 min before and 24 h after the indomethacin administration. Whilst indomethacin produced severe ileitis accompanied by an increase in ileal mucosal myeloperoxidase (MPO) activity, administration of CN210 reduced the severity of ileitis in a dose-dependent manner and suppressed the MPO activity. Similarly, upregulation of inflammatory cytokines was observed in ileitis mucosa after indomethacin injection but this response was significantly attenuated by CN210 administration. To further characterize the effects of CN210 on inflammatory pathways, monocyte/macrophage-like cell line RAW264 treated with lipopolysaccharide (LPS) was exposed to CN210. CN210 significantly attenuated the expression of inflammatory cytokines and reversed the activation of NF-{kappa}B and MAP kinases induced by LPS. These findings suggest that CN210 ameliorates indomethacin-induced ileitis due at least in part to the inhibition of inflammatory cytokine expression via attenuation of NF-{kappa}B and MAP kinase pathways, thus representing a new mode of therapy for CD.

pharmacology and toxicology↗

bioRxiv2019

Long read sequencing reveals a novel class of structural aberrations in cancers: identification and characterization of cancerous local amplifications

Here we report identification of a new class of local structural aberrations in lung cancers. The whole-genome sequencing of cell lines using a long read sequencer, PromethION, demonstrated that typical cancerous mutations, such as point mutations, large deletions and gene fusions can be detected also on this platform. Unexpectedly, we revealed unique structural aberrations consisting of complex combinations of local duplications, inversions and micro deletions. We further analyzed and found that these mutations also occur in vivo, even in key cancer-related genes. These mutations may elucidate the molecular etiology of patients for whom causative cancerous events and therapeutic strategies remain elusive.

genomics↗

bioRxiv2018

Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population

The etiology of pancreatic cancer remains largely unknown. Here, we report the results of a meta-analysis of three genome-wide association studies (GWASs) comprising 2,039 pancreatic cancer cases and 32,592 controls, the largest sample size in the Japanese population. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0x10-8) and 4 suggestive loci (P<1.0x10-6) for pancreatic cancer. Of these risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (odds ratio (OR)=1.46, 95% CI=1.29-1.66, P=4.28x10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant predicted to be highly deleterious. Additionally, the gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified GP2 gene variants were pleiotropic for multiple traits, including type 2 diabetes, hemoglobin A1c (HbA1c) levels, and pancreatic cancer. Mendelian randomization analyses corroborated causality between HbA1c and pancreatic cancer. These findings suggest that GP2 gene variants are associated with pancreatic cancer susceptibility in the Japanese population, prompting further functional characterization of this locus.

genetics↗