Biology subjects

Ozaki, S.

Publications and source records attributed to Ozaki, S..

7 recordsLinked to original sources

bioRxiv2025

Dynamic DnaA-DnaB interactions at oriC coordinate the loading and coupled translocation of two DnaB helicases for bidirectional replication

Bidirectional replication is a conserved principle requiring the coordinated loading and activation of two replicative helicases at the origin. In Escherichia coli, the initiator protein DnaA constructs a higher-order initiation complex at the origin oriC which locally unwinds the DNA, and recruits DnaB helicase-DnaC loader complexes to the unwound region. We previously demonstrated that the two DnaA subcomplexes formed on oriC bind a specific DNA strand of the unwound origin, and tether individual DnaB-DnaC complexes via stable interactions between DnaA domain I and DnaB. A low-affinity DnaA-DnaB interaction mediated by DnaA domain III His136 is essential for DnaB-dependent origin unwinding. Here, we identified DnaB Thr86 as the critical residue mediating this low-affinity interaction. Structural modelling suggests that Thr86 is surface-exposed near the DNA entry site of DnaB. Functional analyses revealed that DnaB Thr86 was specifically required for DnaB loading onto the DnaA-bound strand of the unwound oriC. Furthermore, this strand-specific DnaB loading was required for enabling translocation of the opposing DnaB helicase loaded on the DnaA-free strand. Our findings define a novel regulatory mechanism of strand-specific helicase loading, mediated by the low-affinity DnaA-DnaB interactions, which ensures the coordinated loading and translocation of DnaB helicases for bidirectional replication from oriC.

molecular biology↗

bioRxiv2024

Primosomal protein PriC rescues replication initiation stress by bypassing the DnaA-DnaB interaction step for DnaB helicase loading at oriC

In Escherichia coli, replisome and replication fork assembly is initiated by DnaB helicase loading at the chromosomal origin oriC via its interactions with the DnaA initiator and the DnaC helicase loader. Upon replication fork arrest, the replisome including DnaB dissociates from the stalled fork. Replication fork progression is rescued by primosomal protein PriA- or PriC-dependent pathway in which PriA and PriC promote reloading of DnaB in different mechanisms. However, the mechanism responsible for rescue of blocked replication initiation at oriC remains unclear. Here, we found that PriC rescued blocked replication initiation in cells expressing an initiation-specific DnaC mutant, in mutant cells defective in DnaA-DnaB interactions, and in cells containing truncated oriC sequence variants. PriC rescued DnaB loading at oriC even in the absence of Rep helicase, a stimulator of the PriC-dependent replication fork restart pathway. These and results of in vitro reconstituted assays concordantly suggest that this initiation-specific rescue mechanism provides a bypass of the DnaA-DnaB interaction for DnaB loading by PriC-promoted loading of DnaB to the unwound oriC region. These findings expand understanding of mechanisms sustaining the robustness of replication initiation and specific roles for PriC in the genome maintenance.

microbiology↗

bioRxiv2021

Digital control of c-di-GMP in E. coli balances population-wide developmental transitions and phage sensitivity

Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness.

microbiology↗

bioRxiv2020

Spatial heterogeneity of glioblastoma cells reveals sensitivity to NAD+ depletion at tumor edge

Even after total resection of glioblastoma core lesions by surgery and aggressive post-surgical treatments, life-threatening tumors inevitably recur. A characteristic obstacle in effective treatment is high intratumoral heterogeneity, both longitudinally and spatially. Recurrence occurs predominantly at the brain parenchyma-tumor core interface, a region termed tumor edge. Given the difficulty of accessing it surgically, the composition of the tumor edge, harboring both cancerous and non-cancerous cells, remains largely unknown. Here, to identify phenotypic diversity among heterogeneous glioblastoma core and edge lesions, we uncovered the existence of three phenotypically-distinct clonal subpopulations within individual tumors from glioblastoma patients. Clones from the tumor core shared the same phenotype, exclusively generating tumor-core cells. In contrast, two distinct clonal subtypes were identified at the tumor edge: one generated only edge-lesion cells and the other expanded more broadly to establish both edge- and core-lesions. Using multiple xenograft experimental models in mouse brains, tumor edge development was found to require that both somatic and tumor cells express the NADase CD38, combinedly elevating glioblastoma malignancy. In vitro data suggested that intracellular NADase activity at the edge was provoked through intercellular communication between edge clones and normal astrocytes. Systemic treatment of tumor-bearing mice with 78c, a small-molecule CD38 inhibitor, attenuated the formation of glioblastoma edge lesions, suggesting its clinical potential to pharmacologically eliminate tumor-edge lesions. Collectively, these findings provide novel phenotypic and mechanistic insights into clonal heterogeneity within glioblastoma, particularly in the surgically unresectable, currently understudied tumor edge.

cancer biology↗

bioRxiv2020

Brain aging-dependent glioma traits reversible by NAD+/BDNF-mediated neuronal reactivation

The rise in aging population worldwide is increasing death from cancer, including glioblastoma. Here, we explore the impact of brain aging on glioma tumorigenesis. We find that glioblastoma in older patients and older mice displayed reduced neuronal signaling, including a decline of NTRK-like family member 6 (SLITRK6), a receptor for neurotrophic factor BDNF. This reduction was linked to the systemic decline of nicotinamide adenine dinucleotide (NAD+) with aging, as old mice exposed to young blood via parabiosis or supplemented with the NAD+ precursor NMN (nicotinamide mononucleotide) reverted phenotypically to young-brain responses to glioma, with reactivated neuronal signaling and reduced death from tumor burden. Interestingly, the phenotypic reversal by NMN was largely absent in old mice undergoing parabiosis with BDNF+/- young mice and in BDNF+/- mice undergoing tumor challenge, supporting the notion that the lower NAD+-BDNF signaling in the aging brain aggravated glioma tumorigenesis. We propose that the aging-associated decline in brain NAD+ worsens glioma outcomes at least in part by decreasing neuronal/synaptic activity and increasing neuroinflammation.

cancer biology↗

bioRxiv2020

Tumor Edge-to-Core Transition Promotes Malignancy in Primary-to-Recurrent Glioblastoma Progression in a PLAGL1/CD109-mediated mechanism

BackgroundGlioblastoma remains highly lethal due to its inevitable recurrence. This recurrence is found locally in most cases, indicating that post-surgical tumor-initiating cells (TICs) accumulate at tumor edge. These edge TICs then generate recurrent tumors harboring new core lesions. Here, we investigated the clinical significance of the edge-to-core transition (ECT) signature causing glioblastoma recurrence and sought to identify central mediators for ECT. MethodsFirst, we examined the association of the ETC-related expression changes and patient outcome in matched primary and recurrent samples (n=37). Specifically, we tested whether the combined decrease of the edge TIC marker PROM1 (CD133) with the increase of the core TIC marker CD109 representing ECT during the primary-to-recurrence progression indicates poorer patient outcome. We then investigated the specific molecular mediators that trigger tumor recurrence driven by the ECT signature. Subsequently, the functional and translational significance of the identified molecule was validated within our patient-derived tumor edge-TIC models in vitro and in vivo. ResultsPatients exhibiting a CD133down/CD109up signature during recurrence representing ECT displayed a strong association with poorer progression-free survival and overall survival among all tested patients. Differential gene expression identified that PLAGL1 was tightly correlated with the core TIC marker CD109 and was linked to a shorter survival of glioblastoma patients. Experimentally, forced PLAGL1 overexpression enhanced, while its knockdown reduced, the glioblastoma edge-derived tumor growth in vivo and subsequent mouse survival, suggesting its essential role in the ECT-mediated glioblastoma development. ConclusionsECT is likely an ongoing lethal process in primary glioblastoma contributing to its recurrence partly in a PLAGL1/CD109-mediated mechanism. Key PointsO_LIECT is a pathobiological process contributing to glioblastoma lethality C_LIO_LIThe CD133down/CD109up signature is a novel prognostic molecular biomarker in ECT C_LIO_LIPLAGL1 regulates growth of edge-located tumor-initiating cells C_LI Importance of the StudyVery few studies have sought to longitudinally characterize the transition of molecular landscapes from primary to recurrent glioblastoma. Post-surgical edge-located TICs are presumably the predominant source of tumor recurrence, yet this cellular subpopulation in glioblastoma remains largely uncharacterized. This study evaluates the significance of glioblastoma edge-derived core transition (ECT) for tumor recurrence in the primary-recurrent paired sample set. We elucidate a prognostically-significant shift in molecular and cellular phenotypes associated with ECT in the CD133down/CD109up group. Moreover, our results provide clinical and experimental evidence that PLAGL1 is a mediator of glioblastoma ECT and its subsequent tumor development by the direct transcriptional regulation of the core TIC marker CD109.

cancer biology↗

bioRxiv2020

Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producer cells assists de novo infection by enhancing viral gene expression

Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, secrete extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communications. However, the roles of exosomes in viral infection remain unclear. Here we screened viral proteins to identify those responsible for the exosome-mediated upregulation of Epstein-Barr virus (EBV) infection. We found BGLF2 protein encapsulated in exosomes, which were released from EBV-infected cells. BGLF2 protein is a tegument protein that exists the space between the envelope and the nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby assisting the EBV infection. In summary, the EBV tegument protein BGLF2 is encapsulated not only encapsulated in viral particles, but also in exosomes secreted from infected cells. Therefore, BGLF2 may play a crucial role in establishing EBV latent infection.

microbiology↗