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  • No effect on the A production by secretase was observed

    2019-07-11

    No effect on the Aβ production by γ-secretase was observed at 50μM, the highest effective concentration used in cultured BPTES by the study of Flajolet et al. IC261 did also not exert a modulatory effect on the production of Aβ, Aβ and Aβ species in the cell-free assay at this concentration. γ-Secretase activity was also not affected at 150μM IC261, however, γ-secretase inhibition was observed at 250μM. This may be due to the resemblance of IC261 to known γ-secretase inhibitors derived from NSAIDs, which are characterized by ICies around 100–300μM, for example, ., , Consistent with previous results,, presence of the highly potent GSI L-685,458 (structure see Supplementary data) at 0.5μM completely blocked γ-secretase activity in this assay. The cellular activity of IC261 and its activity in cell-free assays is inconsistent with an exclusively CK1δ mediated effect on Aβ secretion via γ-secretase inhibition but an influence of other CK1 isoforms, for example, CK1ε or by β-secretase could not be excluded. Another potential mechanism, a Notch mediated effect, was excluded by Flajolet et al. The cell-free assay is neither subject to CK1δ or CK1ε dependent regulation, thus a pleiotropic effect is suggested for the interference of Aβ secretion by IC261: (a) cellular γ-secretase inhibition may occur at high concentration of IC261 or through a metabolic activation of IC261. It is not clear, however, whether the interference of IC261 with γ-secretase activity at high concentration (in the cell-free assay at 250μM, i.e., five times above the concentration used by Flajolet et al. in the cellular assay) does reflect enzyme inhibition. The observed reduction in γ-secretase activity could also be due to a damage of the lipid environment by this particular compound in the in vitro assays; (b) upstream modulation of Aβ metabolism by an unidentified mode of action, which does not necessarily involve CK1δ at low concentrations, which is also confirmed by Flajolet et al. investigation. Sunitinib, a promiscuous kinase inhibitor, was selected for structural similarity to IC261 and submitted to the assay panel. It reduces Aβ-secretion in the cellular assay. However, Sunitinib did not display an effect on Aβ generation in the cell-free assay suggesting that an unidentified kinase may be involved in Aβ-secretion in the cellular assay. Even a subtle variation of the IC261 substitution pattern resulted in complete loss of CK1 inhibition but still exerts Aβ lowering capability (e.g.,
    Reversible posttranslational modification by phosphorylation is widely known to fine-tune and extend protein functions by introducing an acidic group, which can lead to altered ligand binding and may allosterically affect functional centers , . When target proteins reside at different cellular locations, a site-specific and reversible modification can physiologically BPTES be highly relevant even if only small fractions of the target protein are affected. This reasoning encourages thorough analyses of protein families by exploring their capacity to be kinase substrates. One approach is testing recombinant substrates in kinase assays combined with detailed product analysis, which enables detection of phosphorylation events even at a low level. We here focus on the adhesion/growth-regulatory galectins, a class of potent effectors known for their ability to bind distinct carbohydrate determinants of cellular glycoconjugates , . Galectin-1 (Gal-1), to give a clinically relevant example, is the key component of tumor suppressor p16-induced anoikis of pancreatic carcinoma cells . Produced on free ribosomes, these lectins can be intracellular (cytoplasmic and/or nuclear), secreted by a nonconventional route, and reimported for further functions (e.g., in glycoconjugate trafficking) , , , , , . Of note, their range of binding partners goes beyond glycans. Intracellularly, galectins can target distinct sets of physiological counterreceptors in a carbohydrate-independent manner. The example of the Gal-1/oncogenic H-Ras interplay reveals involvement of protein regions different from the binding pocket for glycan ligands . In close connection to another tumor suppressor (i.e., p53), galectin-7 (Gal-7) in nuclei/cytoplasm is pro-apoptotic, functioning upstream of JNK activation and cytochrome release and by bcl-2 binding , , . As homodimeric lectins, Gal-1 and Gal-7 are able to engage cell surface glycans to trigger apoptosis via outside-in signaling in activated T cells .