Yusaku Nakabeppu

Inosine triphosphate (ITP) is a noncanonical nucleotide. Human cells possess inosine triphosphate pyrophosphatase (ITPA), and ITPA deficiency causes ITP accumulation within cells, leading to the onset of “Developmental and Epileptic Encephalopathy 35.” Two potential pathways for ITP biosynthesis have been proposed: i) oxidative deamination of the adenine base in adenosine triphosphate or ii) a two-step phosphorylation reaction of inosine monophosphate (IMP), an intermediate in the de novo synthesis of purine nucleotides. However, the mechanisms responsible for ITP biosynthesis in mammals remain unclear. In this study, using an Itpa knockout (KO) mouse cell line, we identified guanylate kinase 1 ( Guk1 ) as a responsible gene for inosine accumulation in RNA. In Itpa -KO/ Guk1 knockdown (KD) cell lines, Guk1 mRNA expression levels were positively correlated with the amount of inosine in RNA, most of which is incorporated during RNA synthesis in ITPA-deficient cells. The transient expression of human GUK1 in Itpa -KO/ Guk1 -KD mouse cells significantly increased inosine levels in RNA. Recombinant human GUK1 phosphorylates IMP into inosine diphosphate. Furthermore, in brain-specific Itpa -KO mice, Guk1 haploinsufficiency resulted in a significant reduction in inosine in cortical RNA and an extended lifespan. Thus, our results suggest that GUK1 is probably an enzyme responsible for ITP biosynthesis although the K m of GUK1 for IMP is very high in vitro.

8-oxo-7,8-dihydroguanine (8-oxoG), the most frequent form of oxidative-DNA-base lesion caused by ROS, is recognized and repaired by 8-oxoguanine DNA glycosylase 1 (OGG1) through base excision repair (BER) pathway. Beyond its role in DNA repair, OGG1 has been shown to promote transcriptional activation of proinflammatory mediators and contribute to both acute and chronic lung inflammation. However, pioneering studies have shown an anti-inflammation role for OGG1 in inflammatory bowel disease (IBD), but its underlying molecular mechanism remains unclear. In the present study, we unveiled that OGG1 plays an important role in the differentiation of inducible regulatory T cells (iTregs). Binding of OGG1 to 8-oxoG facilitated the recruitment of Smad3 to the Foxp3 promoter, leading to the transcriptional activation. Moreover, OGG1 binding promoted demethylation of CpG sites in the conserved noncoding sequence 2 (CNS2) region of Foxp3 by decreasing Dnmt1 occupancy and enhancing recruitment of Tet1/2. Notably, the S326C variant—a naturally occurring polymorphism in humans—was more effective than the wild-type protein in promoting iTreg differentiation and showed a negative correlation with IBD incidence. Furthermore, treatment with O8, a selective OGG1 inhibitor that blocks base excision activity without affecting substrate binding, significantly alleviated IBD in a mouse model, suggesting a promising therapeutic strategy. Together, these findings extend the understanding of OGG1’s epigenetic role in transcriptional regulation and highlight its protective function in inflammatory diseases, potentially shaped by aerobic evolution.

8-oxoguanine (8-oxoGua) is one of the most frequent forms of oxidative DNA base lesions, repaired by 8-oxoguanine DNA glycosylase 1 (OGG1) via base excision repair (BER) pathway to maintain genome fidelity. The human allelic variant hOGG1 ^S326C , prevalent in Caucasians and Asians, has been regarded as a susceptibility factor for various diseases, yet its pathogenic mechanism remains elusive. In this study, we demonstrate that Ogg1 ^S326C/S326C mice exhibit increased and sustained airway inflammation compared with wild-type (WT) Ogg1 ^S326/S326 mice. Mechanistically, in response to inflammatory stimulation, OGG1S326C undergoes reactive oxygen species-induced dimerization, which impairs its base excision function, but prolongs its association with promoter-embedded substrate(s), leading to an increase in NF-κB’ DNA occupancy, subsequently the excessive expression of proinflammatory cytokines and chemokines, and the exacerbated lung inflammation. In contrast, Serine at position 326 in WT -OGG1 is constitutively phosphorylated by CDK4. To fulfill the requirement for its function in transcriptional regulation, the phosphorylated OGG1 needs to undergo dephosphorylation to rescue DNA binding ability. In this scenario, OGG1S326C lacks this phosphorylation site, disrupting this regulatory cycle. Notably, administration of a small molecule inhibitor of OGG1 prevents OGG1S326C from binding to DNA and significantly decreases gene expression and inflammatory responses. Our findings elucidate a molecular basis for the increased disease susceptibility of individuals carrying the hOGG1 ^S326C variant and propose the therapeutic potential of OGG1 inhibitors in mitigating inflammation-driven pathologies.

Oxidative stress–induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO₃; an oxidizing agent)–containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. AllMutyh^−/−mice treated with >0.1% KBrO₃developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis ofMutyh^−/−/rpsLtransgenic mice revealed that G:C > T:A transversion was the only mutation type correlated positively with KBrO₃dose and tumor incidence. These mutations preferentially occurred at 5′G in GG and GAA sequences inrpsL. This characteristic mutation pattern was also observed in the genomic region ofMutyh^−/−tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers,ApcorCtnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

Somatic hypermutation (SHM) is necessary for Ab diversification and involves error-prone DNA repair of activation-induced cytidine deaminase–induced lesions in germinal center (GC) B cells but can also cause genomic instability. GC B cells express low levels of the DNA repair protein apurinic/apyrimidinic (AP) endonuclease (APE)1 and high levels of its homolog APE2. Reduced SHM in APE2-deficient mice suggests that APE2 promotes SHM, but these GC B cells also exhibit reduced proliferation that could impact mutation frequency. In this study, we test the hypothesis that APE2 promotes and APE1 suppresses SHM. We show how APE1/APE2 expression changes in primary murine spleen B cells during activation, impacting both SHM and class-switch recombination (CSR). High levels of both APE1 and APE2 early after activation promote CSR. However, after 2 d, APE1 levels decrease steadily with each cell division, even with repeated stimulation, whereas APE2 levels increase with each stimulation. When GC-level APE1/APE2 expression was engineered by reducing APE1 genetically (apex1+/−) and overexpressing APE2, bona fide activation-induced cytidine deaminase–dependent VDJH4 intron SHM became detectable in primary B cell cultures. The C terminus of APE2 that interacts with proliferating cell nuclear Ag promotes SHM and CSR, although its ATR-Chk1–interacting Zf-GRF domain is not required. However, APE2 does not increase mutations unless APE1 is reduced. Although APE1 promotes CSR, it suppresses SHM, suggesting that downregulation of APE1 in the GC is required for SHM. Genome-wide expression data compare GC and cultured B cells and new models depict how APE1 and APE2 expression and protein interactions change during B cell activation and affect the balance between accurate and error-prone repair during CSR and SHM.

Normal morphology in other organs.

Slight increase of APE2 expression in mouse PTCs after carboplatin and oxaliplatin treatment.

Table S1: MS/MS analyses of APE2 pull-down proteins in 293T cells

Slight increase of APE2 expression in mouse PTCs after carboplatin and oxaliplatin treatment.

Table S1: MS/MS analyses of APE2 pull-down proteins in 293T cells