小椋 康光

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.

Elemental analysis at the single-cell level is an emerging technique in the field of inductively coupled plasma mass spectrometry (ICP-MS). In comparison to the analysis of cell suspensions by fast time-resolved analysis, single-cell sampling by laser ablation (LA) allows the discriminatory analysis of single cells according to their size and morphology. In this study, we evaluated the changes in elemental contents in a single cell through differentiation of rat adrenal pheochromocytoma into neuron-like cells by LA-ICP-MS. The contents of seven essential minerals were increased about 2-3 times after the differentiation. In addition, we evaluated the degree of differentiation at the single-cell level by means of imaging cytometry after immunofluorescence staining of microtubule-associated protein 2 (Map2), a neuron-specific protein. The fluorescence intensities of Alexa Fluor 488-conjugated secondary antibody against the anti-Map2 primary antibody showed large variations among the cells after the onset of differentiation. Although the average fluorescence intensity was increased through the differentiation, there were still less-matured neuron-like cells that exhibited a lower fluorescence intensity after 5 days of differentiation. Since a positive correlation between the fluorescence intensity and the cell size in area was found, we separately measured the elemental contents in the less-matured smaller cells and well-matured larger cells by LA-ICP-MS. The larger cells had higher elemental contents than the smaller cells, indicating that the essential minerals are highly required at a later stage of differentiation.

Copper is an essential trace element that participates in many biological processes through its unique redox cycling between cuprous (Cu+) and cupric (Cu2+) oxidation states. To elucidate the biological functions of copper, chemical biology tools that enable selective visualization and detection of copper ions and proteins in copper-rich environments are required. Herein, we describe the design of Cu+-responsive reagents based on a conditional protein labeling strategy. Upon binding Cu+, the probes generated quinone methide via oxidative bond cleavage, which allowed covalent labeling of surrounding proteins with high Cu+ selectivity. Using gel- and imaging-based analyses, the best-performing probe successfully detected changes in the concentration of labile Cu+ in living cells. Moreover, conditional proteomics analysis suggested intramitochondrial Cu+ accumulation in cells undergoing cuproptosis. Our results highlight the power of Cu+-responsive protein labeling in providing insights into the molecular mechanisms of Cu+ metabolism and homeostasis.