Differential molecular mechanisms of substrate recognition by selenium methyltransferases, INMT and TPMT, in selenium detoxification and excretion.

It is known that the recommended dietary allowance of selenium (Se) is dangerously close to its tolerable upper intake level. Se is detoxified and excreted in urine as trimethylselenonium ion (TMSe) when the amount ingested exceeds the nutritional level. Recently, we demonstrated that the production of TMSe requires two methyltransferases: thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). In this study, we investigated the substrate recognition mechanisms of INMT and TPMT in the Se-methylation reaction. Examination of the Se-methyltransferase activities of two paralogs of INMT, namely, nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase, revealed that only INMT exhibited Se-methyltransferase activity. Consistently, molecular dynamics simulations demonstrated that dimethylselenide was preferentially associated with the active center of INMT. Using the fragment molecular orbital method, we identified hydrophobic residues involved in the binding of dimethylselenide to the active center of INMT. The INMT-L164R mutation resulted in a deficiency in Se- and N-methyltransferase activities. Similarly, TPMT-R152, which occupies the same position as INMT-L164, played a crucial role in the Se-methyltransferase activity of TPMT. Our findings suggest that TPMT recognizes negatively charged substrates, whereas INMT recognizes electrically neutral substrates in the hydrophobic active center embedded within the protein. These observations explain the sequential requirement of the two methyltransferases in producing TMSe.

Identification of Tellurium Metabolite in Broccoli Using Complementary Analyses of Inorganic and Organic Mass Spectrometry.

Tellurium (Te) is a chalcogen element like sulfur and selenium. Although it is unclear whether Te is an essential nutrient in organisms, unique Te metabolic pathways have been uncovered. We have previously reported that an unknown Te metabolite (UKTe) was observed in plants exposed to tellurate, a highly toxic Te oxyanion, by liquid chromatography-inductively coupled plasma mass spectrometer (LC-ICP-MS). In the present study, we detected UKTe in tellurate-exposed broccoli (Brassica oleracea var. italica) by LC-ICP-MS and identified it as gluconic acid-3-tellurate (GA-3Te) using electrospray ionization mass spectrometer with quadrupole-Orbitrap detector and tandem MS analysis, the high-sensitivity and high-resolution mass spectrometry for organic compounds. We also found that GA-3Te was produced from one gluconic acid and one tellurate molecule by direct complexation in an aqueous solution. GA-3Te was significantly less toxic than tellurate on plant growth. This study is the first to identify the Te metabolite GA-3Te in plants and will contribute to the investigation of tellurate detoxification pathways. Moreover, gluconic acid, a natural and biodegradable organic compound, is expected to be applicable to eco-friendly remediation strategies for tellurate contamination.

Iron-induced NCOA4 condensation regulates ferritin fate and iron homeostasis.

Iron is not only essential but also a toxic trace element. Under iron repletion, ferritin maintains cellular iron homeostasis by storing iron to avoid iron toxicity. Under iron depletion, the ferritin-specific autophagy adaptor NCOA4 delivers ferritin to lysosomes via macroautophagy to enable cells to use stored iron. Here, we show that NCOA4 also plays crucial roles in the regulation of ferritin fate under iron repletion. NCOA4 forms insoluble condensates via multivalent interactions generated by the binding of iron to its intrinsically disordered region. This sequesters NCOA4 away from ferritin and allows ferritin accumulation in the early phase of iron repletion. Under prolonged iron repletion, NCOA4 condensates can deliver ferritin to lysosomes via a TAX1BP1-dependent non-canonical autophagy pathway, thereby preventing relative iron deficiency due to excessive iron storage and reduced iron uptake. Together, these observations suggest that the NCOA4-ferritin axis modulates intracellular iron homeostasis in accordance with cellular iron availability.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100 nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10 nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10 nm SiNPs significantly contributed to the production of reactive oxygen species.

Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

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.

Cadmium body burden and health effects after restoration of cadmium-polluted soils in cadmium-polluted areas in the Jinzu River basin.

BACKGROUND: Itai-itai disease is caused by environmental cadmium (Cd) pollution in the Jinzu River basin in Japan. To reduce the Cd contamination of rice, soil restoration of paddy fields was carried out. We evaluated the effect of soil restoration on the health status of residents of the former Cd-polluted area. METHODS: Participants were 1,030 men and 944 women who lived in the area of restoration of Cd-polluted rice paddies. First morning urine was collected and urinary Cd, ß2-microglobulin (ß2MG), and N-acetyl-ß-D-glucosaminidase (NAG) levels were measured. Associations among age, years of residence before and after soil restoration, and urinary Cd, ß2MG, and NAG levels were evaluated by multiple regression analysis. RESULTS: The geometric mean (interquartile range) of urinary Cd (µg/g Cr) was 1.00 (0.58-1.68) in men and 1.67 (1.02-2.91) in women. The geometric means of urinary ß2MG (µg/g Cr) and NAG (U/g Cr) were 174.6 (92.6-234.2) and 1.47 (0.72-3.14) in men, and 217.6 (115.3-28.7) and 1.48 (0.73-2.96) in women, respectively. Urinary Cd, ß2MG, and NAG were significantly positively correlated (p < 0.01 all). Age and duration of residence in the Cd-polluted area before soil restoration were independently associated with urinary Cd, ß2MG, and NAG. Among the 916 participants who had resided in the area before the soil restoration, urinary Cd concentrations were significantly higher, thus by 1.03-fold (95% CI, 1.01-1.04) in men and 1.03-fold (95% CI, 1.01-1.05) in women, when the years of residence before soil restoration by each 5-years increment. By contrast, urinary Cd concentrations were significantly lower, thus 0.97-fold (95% CI, 0.96-0.99) lower in men and 0.97-fold (95% CI, 0.95-0.99) lower in women, by each 5-year increment of residence after soil restoration. A similar association was observed for urinary ß2MG concentration, and no significant association was observed for urinary NAG levels in men or women. CONCLUSIONS: Cd exposure and associated renal tubular dysfunction in residents of a former Cd-polluted area were influenced by Cd exposure from the environment prior to soil restoration. Soil restoration in Cd-polluted areas reduced the Cd exposure of local residents.

Nuclear translocation promotes proteasomal degradation of human Rad17 protein through the N-terminal destruction boxes.

The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230-270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.

Detection of Histidine-Tagged Protein in Escherichia coli by Single-Cell Inductively Coupled Plasma-Mass Spectrometry.

We have developed a rapid and precise quantification method for a histidine (His)-tagged recombinant protein produced in Escherichia coli (E. coli) by single-cell inductively coupled plasma-mass spectrometry (SC-ICP-MS). Plasmid vector containing enhanced green fluorescent protein (EGFP) or red fluorescent protein (mCherry) gene fused with His-tag was transformed into E. coli. The transformed E. coli was exposed to nickel (Ni) chloride or cobalt (Co) chloride for labeling His-tag with the Ni or Co ion. Then, E. coli was analyzed by SC-ICP-MS to determine the amount of EGFP or mCherry protein on the basis of the signal of Ni or Co bound to His-tag. By comparing Ni and Co contents in E. coli expressing His-tagged mCherry with those in nontagged mCherry, the specific binding of Co to His-tag was more clearly detected than that of Ni. The Co contents were increased until 6 h after the protein induction, and this observation was coincident with the increases in fluorescence intensity of EGFP or mCherry measured by a flow cytometer. However, the Co contents were decreased for EGFP and kept at a constant level for mCherry from 6 to 24 h despite the continuous increase in the fluorescence intensity through incubation. The fluorescent proteins were mainly recovered in the insoluble fraction 24 h after the induction. This can be explained by the fact that the overexpressed fluorescent proteins with His-tag are transferred into inclusion bodies, which hampers the binding of the fluorescent proteins to the Co ion. SC-ICP-MS can be a useful technique to precisely quantify soluble recombinant proteins in E. coli without the extraction and purification process.

Evaluation of Post-Mortem Interaction between Hemoglobin and Oxime-Type Carbamate Pesticides.

Oxime-type carbamate pesticides having an oxime moiety such as aldicarb, butocarboxim, methomyl, oxamyl, and thiofanox are widely used and have been detected in many fatal cases of accidental exposure or suicide. In forensic toxicology, the accurate determination of blood pesticide concentration is obligatory to prove death by oxime-type carbamate pesticide poisoning. However, the fatal pesticide concentration in blood at autopsy differs from that at the time of death. In this study, we found that oxime-type carbamate pesticides were decomposed by Hb in a temperature-dependent fashion. The mechanism underlying methomyl, aldicarb, oxamyl, and thiofanox decomposition involves the formation of adducts with the amino acids in Hb. With regard to butocarboxim, its decomposition involves the oxidation of the free form and the formation of adducts with the amino acids in Hb. The mass spectra obtained by liquid chromatography quadrupole time-of-flight mass spectrometry revealed that carbamylated amino acid adducts such as Wcar-adduct and Vcar-adduct were formed in Hb solution incubated with methomyl, aldicarb, oxamyl, and thiofanox, whereas alkylated amino acid adducts such as Walkyl-adduct were formed in Hb solution incubated with butocarboxim. These results indicate that aldicarb, butocarboxim, methomyl, oxamyl, and thiofanox are post-mortem changed by Hb.

Rad17 Translocates to Nucleolus upon UV Irradiation through Nucleolar Localization Signal in the Central Basic Domain.

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.

Recent advances in copper analyses by inorganic mass spectrometry.

Copper (Cu) participates in the biological redox reaction in the body, and its deficiency is fatal to the body. At the same time, Cu is extremely toxic when it exists in excess. Thus, the body has to tightly and spatiotemporally regulate the concentration of Cu within a physiological range by several groups of Cu-regulating proteins. However, entire mechanisms underlying the maintenance of Cu homeostasis in body and cells have not fully understood. It is necessary to analyze Cu itself in a body and in a cell to reveal the Cu homeostasis. In this review, recent advances in the analytical techniques to understand the Cu metabolism such as speciation, imaging and single-cell analysis of Cu were highlighted.

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5-ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5-ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5-ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

Post-Mortem Changes of Methomyl in Blood with Hemoglobin.

Methomyl, (E,Z)-methyl N-{[(methylamino)carbonyl]oxy}ethanimidothioate, is a widely used pesticide that has been detected in many fatal cases of accidental exposure or suicide. Forensic toxicologists have been baffled that the blood methomyl concentration in persons who have died of methomyl poisoning is much lower than the expected concentration in blood. In this study, we speculated two mechanisms underlying the insufficient recovery of methomyl in blood. First, methomyl is decomposed by serum albumin as esterase. Second, methomyl is bound to a specific blood protein, resulting in insufficient recovery in the free form. However, human serum albumin does not show esterase activity for the decomposition of methomyl. On the contrary, specific methomyl hemoglobin adducts have been detected by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS). The mass spectra indicated that methomyl was specifically bound to tryptophan (W), tyrosine (Y), and valine (V) residues in hemoglobin. The amounts of W- and V-adducts dose-dependently increased in vitro when the methomyl concentration was lower than the lethal concentration. In addition, the W-adduct was detected in blood sampled from an autopsied subject who died of intentional methomyl ingestion, suggesting that the W-adduct could be used as a biomarker of methomyl poisoning. We were able to estimate the amount of methomyl ingested on the basis of the amount of the W-adduct.

Formation Mechanism and Toxicological Significance of Biogenic Mercury Selenide Nanoparticles in Human Hepatoma HepG2 Cells.

It is widely recognized that the toxicity of mercury (Hg) is attenuated by the simultaneous administration of selenium (Se) compounds in various organisms. In this study, we revealed the mechanisms underlying the antagonistic effect of sodium selenite (Na2SeO3) on inorganic Hg (Hg2+) toxicity in human hepatoma HepG2 cells. Observations by transmission electron microscopy indicated that HgSe (tiemannite) granules of up to 100 nm in diameter were accumulated in lysosomal-like structures in the cells. The HgSe granules were composed of a number of HgSe nanoparticles, each measuring less than 10 nm in diameter. No accumulation of HgSe nanoparticles in lysosomes was observed in the cells exposed to chemically synthesized HgSe nanoparticles. This suggests that intracellular HgSe nanoparticles were biologically generated from Na2SeO3 and Hg2+ ions transported into the cells and were not derived from HgSe nanoparticles formed in the extracellular fluid. Approximately 85% of biogenic HgSe remained in the cells at 72 h post culturing, indicating that biogenic HgSe was hardly excreted from the cells. Moreover, the cytotoxicity of Hg2+ was ameliorated by the simultaneous exposure to Na2SeO3 even before the formation of insoluble HgSe nanoparticles. Our data confirmed for the first time that HepG2 cells can circumvent the toxicity of Hg2+ through the direct interaction of Hg2+ with a reduced form of Se (selenide) to form HgSe nanoparticles via a Hg-Se soluble complex in the cells. Biogenic HgSe nanoparticles are considered the ultimate metabolite in the Hg detoxification process.

Systematic study of the selenium fractionation in human plasma from a cancer prevention trial using HPLC hyphenated to ICP-MS and ESI-MS/MS.

This work represents the first systematic speciation study of selenium (Se) in plasma from subjects participating in a pilot study for a cancer prevention trial (PRECISE). This involved supplementation of elderly British and Danish individuals with selenised yeast for 6 months and 5 years, respectively, at 100, 200, and 300 µg Se/day or placebo. Speciation data was obtained for male plasma using HPLC-ICP-MS and HPLC-ESI-MS/MS. With the proposed strategy, approximately 1.5 mL of plasma was needed to determine total Se concentration and the fractionation of Se in high molecular weight (HMW) and low molecular weight (LMW) pools, and for quantification and identification of small Se species. For the first time, Se-methyl-selenocysteine (MSC) and methyl-2-acetamido-2deoxy1-seleno-ß-D-galactopyranoside (Selenosugar-1) were structurally confirmed in plasma after supplementation with selenised yeast within the studied range. Determination of selenomethionine (SeMet) incorporated non-specifically into albumin (SeALB) was achieved by HPLC-ICP-MS after hydrolysis. By subtracting this SeMet concentration from the total Se in the HMW pool, the concentration of Se incorporated into selenoproteins was calculated. Results from the speciation analysis of the free Se metabolite fraction (5% of total plasma Se) suggest a significant increase in the percentage of Se (as SeMet plus Selenosugar-1) of up to 80% of the total Se in the LMW fraction after 6 months of supplementation. The Se distribution in the HMW fraction reflects a significant increase in SeALB with Se depletion from selenoproteins, which occurs most significantly at doses of over 100 µg Se/day after 5 years. The results of this work will inform future trial design. Graphical abstract.

Distributions of mercury and selenium in rats ingesting mercury selenide nanoparticles.

Mercury (Hg) is one of the most toxic environmental pollutants, and is biocondensed via the food chain. Selenium (Se) is an essential element that possesses an antagonistic property towards Hg in vivo. The antagonistic property is explained by the assumption that Hg and Se directly interact to form HgSe nanoparticles (HgSe NPs) in organs. It is presumed that the toxic effects of HgSe NPs are lower than that of ionic Hg; however, no precise evaluation has been conducted so far. In the present study, we evaluated the distribution of HgSe NPs ingested in Se-deficient rats. The recovery of serum selenoproteins from a deficient level was not observed in rats orally administered HgSe NPs. In addition, the excretion of Hg and Se via urine was not observed. Interestingly, the biosynthesis of selenoproteins and urinary selenometabolites would have required the production of selenide through the degradation of HgSe NPs. Therefore, it seems that selenide and Hg are not released from HgSe NPs in vivo. The administration of HgSe NPs did not increase Hg and Se concentrations in organs, and almost all HgSe NPs were recovered in feces, indicating no or low bioaccessibility of HgSe NPs even in Se-deficient rats. These results suggest that HgSe NPs are biologically inert and do not become a secondary environmental pollutant of Hg.

Quantitative Elemental Analysis of a Single Cell by Using Inductively Coupled Plasma-Mass Spectrometry in Fast Time-Resolved Analysis Mode.

The elemental composition of a single yeast, green alga, or red blood cell (RBC) was precisely determined by using inductively coupled plasma-mass spectrometry (ICP-MS) operating in fast time-resolved analysis (TRA) mode. The technique is known as single-cell (SC)-ICP-MS. Phosphorus, sulfur, magnesium, zinc, and iron were detected in the three types of cell. The elemental composition of yeast and green alga obtained by SC-ICP-MS was consistent with results obtained from conventional ICP-MS measurements following acid digestion of the cells. Slight differences were found in the measured values between SC-ICP-MS and the conventional ICP-MS results for RBC. However, the SC-ICP-MS results for S and Fe in RBC were closer to the estimated values for these elements that were calculated from the level of hemoglobin in RBCs. The data suggest that SC-ICP-MS is suitable for the analysis of various cell types, namely, fungus, plant, and animal cells.

Production of a Urinary Selenium Metabolite, Trimethylselenonium, by Thiopurine S-Methyltransferase and Indolethylamine N-Methyltransferase.

Selenium (Se) is an essential trace element in animals; however, the element can become highly toxic in excess amounts beyond the nutritional level. Although Se is mainly excreted into urine as a selenosugar within the nutritional level, excess amounts of Se are transformed as an alternative urinary metabolite, trimethylselenonium ion (TMSe). Se methylation appears to be an important metabolic process for the detoxification of excess Se; however, the biochemical mechanisms underlying the Se methylation have not been elucidated. In this study, we evaluated biochemical characteristics of two human methyltransferases for Se methylation, thiopurine S-methyltransferase (TPMT) and indolethylamine N-methyltransferase (INMT). The first methylation of Se, i.e., a nonmethylated to a monomethylated form, was specifically driven by TPMT, and INMT specifically mediated the third methylation, i.e., dimethylated to trimethylated form. The second methylation, i.e., a monomethylated to dimethylated form, was driven by either TPMT or INMT. Exogenous expression of TPMT, but not INMT, ameliorated the cytotoxicity of inorganic nonmethylated selenium salt, suggesting that only TPMT gave the cellular resistance against selenite exposure. TPMT was ubiquitously expressed in most mouse tissues and preferably expressed in the liver and kidneys, while INMT was specifically expressed in the lung and supplementally expressed in the liver and kidneys. Our results revealed that both TPMT and INMT cooperatively contributed to the TMSe production, enabling urinary excretion of Se and maintenance of homeostasis of this essential yet highly toxic trace element. Thus, TPMT and INMT can be recognized as selenium methyltransferases as a synonym.

Casein kinase 2 promotes interaction between Rad17 and the 9-1-1 complex through constitutive phosphorylation of the C-terminal tail of human Rad17.

An interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential for ATR-Chk1 signaling, which is one of the major DNA damage checkpoints. Recently, we showed that the polyanionic C-terminal tail of human Rad17 and the embedded conserved sequence iVERGE are important for the interaction with 9-1-1 complex. Here, we show that Rad17-S667 in the C-terminal tail is constitutively phosphorylated in vivo in a casein kinase 2-dependent manner, and the phosphorylation is important for 9-1-1 interaction. The serine phosphorylation of Rad17 could be seen in the absence of exogenous genotoxic stress, and was mostly abolished by S667A substitution. Rad17-S667 was also phosphorylated when the C-terminal tail was fused with EGFP, but the phosphorylation was inhibited by two casein kinase 2 inhibitors. Furthermore, interaction between Rad17 and the 9-1-1 complex was inhibited by the casein kinase 2 inhibitor CX-4945/Silmitasertib, and the effect was dependent on the Rad17-S667 residue, indicating that S667 phosphorylation is the only role of casein kinase 2 in the 9-1-1 interaction. Our data raise the possibility that the C-terminal tail of vertebrate Rad17 regulates ATR-Chk1 signaling through multi-site phosphorylation in the iVERGE.

Speciation of Selenium in Brown Rice Fertilized with Selenite and Effects of Selenium Fertilization on Rice Proteins.

Foliar Selenium (Se) fertilizer has been widely used to accumulate Se in rice to a level that meets the adequate intake level. The Se content in brown rice (Oryza sativa L.) was increased in a dose-dependent manner by the foliar application of sodium selenite as a fertilizer at concentrations of 25, 50, 75, and 100 g Se/ha. Selenite was mainly transformed to organic Se, that is, selenomethionine in rice. Beyond the metabolic capacity of Se in rice, inorganic Se also appeared. In addition, four extractable protein fractions in brown rice were analyzed for Se concentration. The Se concentrations in the glutelin and albumin fractions saturated with increasing Se concentration in the fertilizer compared with those in the globulin and prolamin fractions. The structural analyses by fluorescence spectroscopy, Fourier transform infrared spectrometry, and differential scanning calorimetry suggest that the secondary structure and thermostability of glutelin were altered by the Se treatments. These alterations could be due to the replacements of cysteine and methionine to selenocysteine and selenomethionine, respectively. These findings indicate that foliar fertilization of Se was effective in not only transforming inorganic Se to low-molecular-weight selenometabolites such as selenoamino acids, but also incorporating Se into general rice proteins, such as albumin, globulin glutelin, and prolamin, as selenocysteine and selenomethionine in place of cysteine and methionine, respectively.