Yasunori Fukumoto

Activation of the ATR-dependent DNA damage response (ATR-DDR) is well characterized; however, the molecular mechanisms underlying its maintenance and inactivation remain largely elusive. Rhino is the least understood component of ATR-DDR. Structural modeling and binding free energy calculations revealed structural remodeling involving Rad17, Rad9–Hus1–Rad1 (9–1–1), and Rhino during ATR-DDR progression. Biochemical and computational analyses revealed the competitive binding of Rad17 and Rhino to the 9–1–1 complex, suggesting a structural transition from the Rad17–9–1–1 complex to the Rhino–9–1–1 complex. The presence of two conserved KYxxL+ motifs in Rhino suggests that it bridges the two 9–1–1 complexes. This enables the polymerization of multiple 9–1–1 complexes through Rhino and explains the long-standing discrepancy between the conventional model and experimental observations of Rad17 and Rad9 foci. Furthermore, structural analysis of the Rad9 C-terminal tail revealed its ability to compete with both Rhino and Rad17, leading to disassembly of the checkpoint complex and providing a mechanism for checkpoint inactivation. Quantum chemical calculations revealed comparable binding free energies for intermediate complexes. These observations suggest that the Rad17–9–1–1–Rhino complex undergoes energetically equivalent structural transitions, providing a mechanistic basis for the sequential progression of ATR-DDR.

Rare earth elements, comprising 17 elements including 15 lanthanides, are essential components in numerous high-tech applications. While physicochemical methods are commonly employed to remove toxic heavy metals (e.g., cadmium and mercury) from industrial wastewater, biological approaches offer increasingly attractive alternatives. Biomining, which utilizes microorganisms to extract valuable metals from ores and industrial wastes, and bioremediation, which leverages microorganisms to adsorb and transport metal ions into cells via active transport, provide eco-friendly solutions for resource recovery and environmental remediation. In this study, we investigated the potential of three recently identified lanthanide-binding proteins—SPL2, lanpepsy, and lanmodulin—for applications in these areas using single-cell inductively coupled plasma mass spectrometry (scICP-MS). Our results demonstrate that SPL2 exhibits superior characteristics for lanthanide and cadmium bioremediation. Heterologous expression of a cytosolic fragment of SPL2 in bacteria resulted in high expression levels and solubility. Single-cell ICP-MS analysis revealed that these recombinant bacteria accumulated lanthanum, cobalt, nickel, and cadmium, effectively sequestering lanthanum and cadmium from the culture media. Furthermore, SPL2 expression conferred enhanced bacterial tolerance to cadmium exposure. These findings establish SPL2 as a promising candidate for developing recombinant bacterial systems for heavy metal bioremediation and rare earth element biomining.

Tellurium (Te) is a metalloid widely used in various industries. However, its toxicological impact on humans is poorly understood. In this study, we investigated the role of two methyltransferases, thiopurine S‑methyltransferase (TPMT) and indolethylamine N‑methyltransferase (INMT), in the methylation of tellurite, an inorganic Te oxyanion. The products of the reaction of Te compounds catalyzed by recombinant human TPMT and/or INMT were analyzed by liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (LC-ICP-MS) and gas chromatography mass spectrometry (GC-MS). We found that TPMT catalyzes the methylation of non-methylated Te and methanetellurol to generate dimethyltelluride. On the other hand, INMT catalyzes the methylation of methanetellurol and dimethyltelluride to produce trimethyltelluronium ion, a metabolite excreted into animal urine. We conclude that TPMT and INMT are cooperatively responsible for the detoxification of Te oxyanions through methylation to form trimethyltelluronium ions.

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

The nucleolus is a non-membranous structure in the nucleus and forms around ribosomal DNA repeats. It plays a major role in ribosomal biogenesis through the transcription of ribosomal DNA and regulates mRNA translation in response to cellular stress including DNA damage. Rad17 is one of the proteins that initiate and maintain the activation of the ATR pathway, one of the major DNA damage checkpoints. We have recently reported that the central basic domain of Rad17 contains a nuclear localization signal and that the nuclear translocation of Rad17 promotes its proteasomal degradation. Here, we show that the central basic domain contains the nucleolar localization signal as well as the nuclear localization signal. The nucleolar localization signal overlaps with the nuclear localization signal and is capable of transporting an exogenous protein into the nucleolus. Phosphomimetic mutations of the central basic domain inhibit nucleolar accumulation, suggesting that the post-translational modification sites regulate the nucleolar localization. Nucleolar accumulation of Rad17 is promoted by proteasome inhibition and UV irradiation. Our data show the nucleolar localization of Rad17 and suggest a possible role of Rad17 in the nucleolus upon UV irradiation.