Protein homeostasis or proteostasis is a fundamental cellular property that encompasses the dynamic balancing of processes in the proteostasis network (PN). Such processes include protein synthesis, folding, and degradation in both non-stressed and stressful conditions. The role of the PN in neurodegenerative disease is well-documented, where it is known to respond to changes in protein folding states or toxic gain-of-function protein aggregation. Dual-specificity phosphatases have recently emerged as important participants in maintaining balance within the PN, acting through modulation of cellular signaling pathways that are involved in neurodegeneration. In this review, we will summarize recent findings describing the roles of dual-specificity phosphatases in neurodegeneration and offer perspectives on future therapeutic directions.
We have developed a pair of cell-based reporter gene assays to quantitatively measure γ-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor γ-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-ΔE (NΔE; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress γ-secretase cleavage of APP-C99 in CG cells, but not NΔE in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for γ-secretase modulators, and these responses are in stark contrast to the pan-inhibition of γ-secretase induced by DAPT. Our studies provide direct evidence that γ-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.
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