An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination in cultured neurons

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The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control.A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag.We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates.We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14.The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases.Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant.We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome.Finally, we show that IU1-47 stimulates autophagic flux in primary neurons.In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.The ubiquitin-proteasome system (UPS) 4 is the major cellular pathway responsible for selective protein degradation within the eukaryotic cell (1-4).Proteins destined for elimination typically carry covalent ubiquitin modifications, which can serve as recognition signals for the proteasome as well as for selective autophagy.The formation of a substrate-bound ubiquitin chain requires the activity of three classes of enzymes: ubiquitin-activating enzyme (E1), which activates the carboxyl terminus of ubiquitin; a ubiquitin-conjugating enzyme (E2), which receives ubiquitin from E1, forming a thioester adduct; and a ubiquitin-protein ligase (E3), which binds substrates and guides the donation of ubiquitin from the E2.The serial addition of ubiquitin to a substrate through this pathway, often forming ubiquitin chains, generates substrates of progressively higher affinity for the proteasome and thus more rapid rates of degradation.The proteasome recognizes substrates via specific ubiquitin receptors within its 19-subunit regulatory particle.Subsequently, in an ATP-dependent process, the proteasome translocates the substrate into the 28-subunit core particle to be degraded (2,5,6).Integral subunits of the proteasome are complemented by a variety of proteins that associate with it in a salt-sensitive manner and that are not stoichiometric within the complex (7).These proteasome-associated proteins regulate protein degradation in diverse ways; they include both inhibitors and activators of the proteasome as well as factors that alter its specificity.Among these factors, a deubiquitinating enzyme (DUB) known

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