Variants of triggering receptor expressed on myeloid cells 2 (TREM2) are associated with an increased incidence of Alzheimer’s disease, as well as other neurodegenerative disorders. Using a newly developed, highly sensitive reporter cell model, consisting of Jurkat T cells stably overexpressing a reporter gene and a gene encoding TREM2DAP12 fusion protein, we show here that TREM2-dependent signal transduction in response to apoptotic Neuro2a cells is mediated by aminophospholipid ligands, phosphatidylserine and phosphatidylethanolamine, which are not exposed on the intact cell surface, but become exposed upon apoptosis. We also show that signal-transducing TREM2 ligands different from aminophospholipids, which appear to be derived from neurons, might be present in membrane fractions of mouse cerebral cortex. These results may suggest that TREM2 regulates microglial function by transducing intracellular signals from aminophospholipids on apoptotic cells, as well as unidentified ligands in the membranes of the cerebral cortex.

Zn(2+)-induced fluorescence enhancement of the TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine)-based ligand, N,N-bis(1-isoquinolylmethyl)-N',N'-bis(pyridylmethyl)ethylenediamine (N,N-1-isoBQBPEN, 1b), has been investigated. Upon Zn(2+) binding, 1b shows a fluorescence increase (ϕZn = 0.028) at 353 and 475 nm. The fluorescence enhancement at longer wavelengths is due to intramolecular excimer formation of two isoquinolines and is specific for Zn(2+); Cd(2+) induces very small fluorescence at 475 nm (ICd/IZn = 10%). The excimer formation of the [Zn(1b)](2+) complex in the excited state is supported by the time-dependent DFT calculation. Neither long-wavelength fluorescence nor excimer formation is observed in the Zn(2+) complex of N,N'-1-isoBQBPEN (2b). The quinoline analog N,N-BQBPEN (1a) exhibits similar but significantly smaller excimer formation. Thermodynamic and kinetic comparisons of Zn(2+) binding properties of ethylenediamine-based hexadentate ligands with pyridines and (iso)quinolines are comprehensively discussed.

Covalent ligands are generally filtered out of chemical libraries used for high-throughput screening, because electrophilic functional groups are considered to be pan-assay interference compounds (PAINS). Therefore, screening strategies that can distinguish true covalent ligands from PAINS are required. Hydrogen/deuterium−exchange mass spectrometry (HDX−MS) is a powerful tool for evaluating protein stability. Here, we report a covalent modifier screening approach using HDX−MS. In this study, HDX−MS was used to classify peroxisome proliferatoractivated receptor γ (PPARγ) and vitamin D receptor ligands. HDX−MS could discriminate the strength of ligand−protein interactions. Our HDX−MS screening method identified LT175 and nTZDpa, which can bind concurrently to the PPARγ ligand-binding domain (PPARγ−LBD) with synergistic activation. Furthermore, iodoacetic acid was identified as a novel covalent modifier that stabilizes the PPARγ−LBD.

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