Biology subjects

Kitagawa, Y.

Publications and source records attributed to Kitagawa, Y..

8 recordsLinked to original sources

bioRxiv2026

Augmenting Radiation Sensitivity by Targeting PAR-Dependent Replication Fork Vulnerability in IDH-Mutant Glioma

Mutations in isocitrate dehydrogenase 1 (IDH1) drive the early stages of gliomagenesis while simultaneously imposing replication stress that creates targetable vulnerabilities. Using both in vitro and in vivo models, we show that inhibition of poly(ADP-ribose) glycohydrolase (PARG) induces a poly(ADP-ribose) (PAR)-dependent augmentation of radiosensitivity in IDH1-mutant glioma cells. Metabolic repletion of NAD+ fails to rescue this effect, indicating that the vulnerability cannot be explained solely by NAD+ depletion. Instead, PARG inhibition profoundly alters replication fork progression and S-phase kinetics in IDH1-mutant cells. Mechanistically, ionizing radiation preferentially activates replication fork-associated damage response proteins DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and X-ray repair cross-complementing protein 1 (XRCC1) in IDH1-mutant cells, a response partially reversed by pharmacologic inhibition of mutant IDH1. Importantly, pharmacologic inhibition of DNA-PKcs with AZD7648 during irradiation disrupts fork-associated repair signaling and markedly enhances cytotoxicity in IDH1-mutant glioma models. Together, these findings identify a PAR-dependent replication fork vulnerability that can be therapeutically exploited to selectively enhance radiosensitivity in IDH1-mutant gliomas. Statement of significanceIDH-mutant gliomas harbor intrinsic replication stress yet lack targeted radiosensitization strategies. We identify a PAR-dependent replication fork vulnerability in which disruption amplifies radiation cytotoxicity by deregulating S-phase fork signaling. Pharmacologic DNA-PKcs inhibition exploits this dependency, providing a genotype-selective approach to enhance radiotherapy in IDH-mutant glioma.

cancer biology↗

bioRxiv2024

Protonation/deprotonation-driven switch for the redox stability of low-potential ferredoxin

Ferredoxin is a small iron-sulfur protein and acts as an electron carrier. Low-potential ferredoxins harbor [4Fe-4S] cluster(s), which play(s) a crucial role as the redox center. Low-potential ferredoxins are able to cover a wide range of redox potentials (-700 to -200 mV); however, the mechanisms underlying the factors which control the redox potential are still enigmatic. Here, we determined the neutron structure of ferredoxin from Bacillus thermoproteolyticus, and experimentally revealed the exact hydrogen-bonding network involving the [4Fe-4S] cluster. The density functional theory calculations based on the hydrogen-bonding network revealed that protonation states of the sidechain of Asp64 close to the [4Fe-4S] cluster critically affected the stability of the reduced state in the cluster. These findings provide the first identification of the intrinsic control factor of redox potential for the [4Fe-4S] cluster in low-potential ferredoxins.

biochemistry↗

bioRxiv2022

Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19-convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

microbiology↗

bioRxiv2022

An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants

As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA. 1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.

microbiology↗

bioRxiv2022

SMN promotes mitochondrial metabolic maturation during myogenesis by regulating the MYOD-miRNA axis.

Spinal muscular atrophy (SMA) is a congenital neuromuscular disease caused by the mutation or deletion of survival motor neuron 1 (SMN1) gene. Although the primary cause of progressive muscle atrophy in SMA has classically been considered the degeneration of motor neurons, recent studies have indicated a skeletal muscle-specific pathological phenotype such as impaired mitochondrial function and enhanced cell death. Here we found that the downregulation of SMN causes mitochondrial dysfunction and subsequent cell death in in vitro models of skeletal myogenesis with both a murine C2C12 cell line and human induced pluripotent stem cells. During myogenesis, SMN binds to the genome upstream of the transcriptional start sites of MYOD1 and microRNA (miR)-1 and -206. Accordingly, the loss of SMN downregulates these miRs, whereas supplementation of the miRs recovers the mitochondrial function, cell survival and myotube formation of SMN-deficient C2C12, indicating the SMN-miR axis is essential for myogenic metabolic maturation. Additionally, introduction of the miRs into ex vivo muscle stem cells derived from {Delta}7-SMA mice caused myotube formation and muscle contraction. In conclusion, our data revealed novel transcriptional roles of SMN during myogenesis, providing an alternative muscle-oriented therapeutic strategy for SMA patients. HighlightsO_LIReduced SMN causes mitochondrial dysregulation in myogenic cells. C_LIO_LIReduced SMN downregulates miR-1 and miR-206 expression in myogenic cells. C_LIO_LISMN protein binds to the genome upstream of MYOD1, miR-1 and miR-206. C_LIO_LImiR-1 and miR-206 are sufficient to improve skeletal muscle function in an SMA model. C_LI

cell biology↗

bioRxiv2021

Deeply hidden genome organization directly mediated by SATB1

Mammalian genomes are organized by multi-level folding, yet how this organization contributes to cell type-specific transcription remain unclear. A nuclear protein SATB1 forms a SATB1-rich subnuclear structure that resists high-salt extraction. SATB1 binds in the minor groove of double-stranded base-unpairing regions (BURs), genomic elements with high unwinding propensities. We uncovered that SATB1 establishes a two-tiered chromatin organization, one through indirect binding and another by direct binding of BURs. Published ChIP-seq datasets show SATB1 binding to highly accessible chromatin at enhancers and CTCF sites, but not to BURs. By employing urea ChIP-seq, which retains only directly bound protein:DNA complexes, we found that BURs, but not CTCF sites, are direct SATB1 binding targets genome-wide. BURs bound to the SATB1 nuclear substructure interact with accessible chromatin crossing multiple topologically associated domains (TADs). SATB1 is required for these megabase-scale interactions linked to cell type-specific gene expression. BURs are highly enriched within lamina associated domains (LADs), but some ([~]10%) are found in gene-rich accessible chromatin outside LADs as well. Only a subset of BURs is bound to SATB1 depending on cell type. Notably, despite the mutually exclusive SATB1-binding profiles uncovered by the two ChIP-seq methods, we found most peaks in both profiles are valid and require SATB1. Based on these and previous data, we propose that the SATB1 protein network forms a chromatin scaffold, providing an interface that connects accessible chromatin to a subnuclear architectural structure, thereby facilitating the three-dimensional organization linked to cell type-specific gene expression.

genomics↗

bioRxiv2021

Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain

In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic1,2. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.

immunology↗

bioRxiv2020

Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world because of severe symptoms and relatively high mortality. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate the efficacy of prophylactics and therapeutics in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in cynomolgus macaques until 28 days after virus inoculation in the present study. Cynomolgus macaques showed body temperature rises after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening clinical signs of disease corresponding that approximately 80% of human patients did not show a critical disease in COVID-19. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-{gamma} and interleukin (IL)-2 specifically for SARS-CoV-2 N protein were detected on day 14 in the macaque that showed viral pneumonia. On the other hand, in the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-{gamma} specifically for SARS-CoV-2 N protein increased on day 7 to day 14 prior to an increase in the number of T-lymphocytes that produced IL-2. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-{gamma} has a role in the elimination of SARS-CoV-2. Thus, because of the mild clinical signs of disease and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development. Author SummarySevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate their efficacy in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in a non-human primate model until 28 days after virus inoculation. Cynomolgus macaques showed a fever after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening symptoms. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-{gamma} and interleukin (IL)-2 specifically for SARS-CoV-2 protein were detected on day 14 in the macaque that showed viral pneumonia. In the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-{gamma} specifically for SARS-CoV-2 N protein increased on day 7 to day 14. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-{gamma} has a role in the elimination of SARS-CoV-2. Thus, because of the mild symptoms and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development.

microbiology↗