SummaryGenetic, epidemiologic, and biochemical evidence suggests that predisposition to Alzheimer’s disease (AD) may arise from altered cholesterol metabolism, although the molecular pathways that may link cholesterol to AD phenotypes are only partially understood. Here, we perform a phenotypic screen for pTau accumulation in AD-patient iPSC-derived neurons and identify cholesteryl esters (CE), the storage product of excess cholesterol, as upstream regulators of Tau early during AD development. Using isogenic induced pluripotent stem cell (iPSC) lines carrying mutations in the cholesterol-binding domain of APP or APP null alleles, we found that while CE also regulate Aβ secretion, the effects of CE on Tau and Aβ are mediated by independent pathways. Efficacy and toxicity screening in iPSC-derived astrocytes and neurons showed that allosteric activation of CYP46A1 lowers CE specifically in neurons and is well tolerated by astrocytes. These data reveal that CE independently regulate Tau and Aβ and identify a druggable CYP46A1-CE-Tau axis in AD.

Drugs that increase 26S proteasome activity have potential therapeutic applications in the treatment of neurodegenerative diseases. A chemical genetics screen of over 2,750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay revealed more than ten compounds that increase proteasome activity, with the p38 MAPK inhibitor PD169316 being one of the most potent ones. Genetic and chemical inhibition of either p38 MAPK, its upstream regulators, ASK1 and MKK6, and downstream target, MK2, enhance proteasome activity. Chemical activation of the 26S proteasome increases PROTAC-mediated and ubiquitin-dependent protein degradation and decreases the levels of both overexpressed and endogenous α-synuclein, without affecting the overall protein turnover. In addition, survival of cells overexpressing toxic α-synuclein assemblies is increased in the presence of p38 MAPK inhibitors. These findings highlight the potential of activation of 26S proteasome activity and that this can be achieved through multiple mechanisms by distinct molecules.

Ubiquitin is a 76-amino acid regulatory protein involved in many important cellular processes. Ubiquitin can be attached to other proteins at either a lysine residue or to the N-terminus by the consecutive actions of E1, E2, and E3 enzymes. Ubiquitin can also be attached to itself, resulting in poly-ubiquitin chains. Ubiquitination affects substrate proteins in different ways, for example by resulting in degradation of the substrate protein by the 26S proteasome. Ubiquitination can be reversed by deubiquitinating enzymes, which either trim or remove ubiquitin chains from proteins. Many proteins involved in either the ubiquitination, deubiquitination or degradation of proteins are implicated in human diseases and are currently under investigation as potential drug targets.

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The ubiquitin proteasome system is an attractive pharmacological target for the treatment of cancer. The proteasome inhibitor bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma but is associated with substantial adverse effects and the occurrence of resistance, underscoring the continued need for novel proteasome inhibitors. In this study, bortezomib and the novel proteasome inhibitor delanzomib were compared for their ability to inhibit proteasome activity using both fluorogenic substrates and a recently developed fluorescent proteasome activity probe. Bortezomib and delanzomib were equipotent in inhibiting distinct subunits of the proteasome in a panel of cell lines in vitro. In a preclinical multiple myeloma model, both inhibitors inhibited the proteasome in normal tissues to a similar extent. Tumor proteasome activity was inhibited to a significantly higher extent by delanzomib (60%) compared to bortezomib (32%). In addition, delanzomib was able to overcome bortezomib resistance in vitro. The present findings demonstrate that proteasome activity probes can accurately monitor the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models and can be used as a diagnostic approach to predict resistance against treatment with proteasome inhibitors. Furthermore, the data presented here provide rationale for further clinical development of delanzomib.

Proteasomes are critical proteases in the cell responsible for the turnover of many cytoplasmic and nuclear proteins. They are essential for many cellular processes and various diseases are associated with their malfunctioning. Proteasome activity depends on the nature of the catalytic subunits, as well as the interaction with associated proteasome regulators. Here we describe various fluorescence-based methods to study proteasome function, highlighting the use of activity-based probes to study proteasome localization, dynamics, and activity in living cells.

Infected or transformed cells must present peptides derived from endogenous proteins on MHC class I molecules to be recognized and targeted for elimination by Ag-specific cytotoxic T cells. In the first step of peptide generation, proteins are degraded by the proteasome. In this study, we investigated the role of the ubiquitin-specific protease 14 (Usp14), a proteasome-associated deubiquitinase, in direct Ag presentation using a ligand-stabilized model protein expressed as a self-antigen. Chemical inhibition of Usp14 diminished direct presentation of the model antigenic peptide, and the effect was especially pronounced when presentation was restricted to the defective ribosomal product (DRiP) form of the protein. Additionally, presentation specifically from DRiP Ags was diminished by expression of a catalytically inactive form of Usp14. Usp14 inhibition did not appreciably alter protein synthesis and only partially delayed protein degradation as measured by a slight increase in the half-life of the model protein when its degradation was induced. Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP-derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.

Deubiquitinating enzymes (DUBs) are of interest as potential new targets for pharmacological intervention. Active-site-directed probes can be used for the accurate profiling of DUB activity as well as the identification of DUBs and DUB inhibitor selectivity. Previously, active-site directed DUB probes have been obtained using intein-based methods that have inherent limitations. Total chemical synthesis of ubiquitin allows for easy incorporation of different tags, such as fluorescent reporters, affinity tags, and cleavable linkers. Here, we describe the total chemical synthesis of a fluorescent active-site directed DUB probe, which facilitates fast, in-gel detection of active DUBs and circumvents the use of Western blot analysis. In addition, an in-gel activity-based DUB profiling assay is described in detail, in which the fluorescent DUB probe is used to visualize active DUBs in cell lysates. Finally, an inhibition assay is described in which the fluorescent probe is used to determine the specificity and potency of a small molecule DUB inhibitor.