Proteasomes are large, multisubunit proteolytic complexes presenting multiple targets for therapeutic intervention. The 26S proteasome consists of a 20S proteolytic core and one or two 19S regulatory particles. The 20S core contains three types of active sites. Many structurally diverse inhibitors of these active sites, both natural product and synthetic, have been discovered in the last two decades. One, bortezomib, is used clinically for treatment of multiple myeloma, mantle cell lymphoma, and acute allograft rejection. Five more recently developed proteasome inhibitors are in trials for treatment of myeloma and other cancers. Proteasome inhibitors also have activity in animal models of autoimmune and inflammatory diseases, reperfusion injury, promote bone and hair growth, and can potentially be used as anti-infectives. In addition, inhibitors of ATPases and deubiquitinases of 19S regulatory particles have been discovered in the last decade.

A C‐terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site‐directed probe that irreversibly modifies a subset of Ub C‐terminal hydrolases (UCHs) and Ub‐specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [125I]UbVS labeling with N‐ethylmaleimide and Ub aldehyde, but also by genetic analysis. [125I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [125I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S‐associated USP14 with [125I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

IntroductionGaucher disease, a relatively common recessively inherited lysosomal storage disorder, is caused by a deficiency in the enzyme glucocerebrosidase, encoded by the GBA gene. 1 Deficient enzymatic activity of glucocerebrosidase results in the lysosomal accumulation of its substrate glucosylceramide, most prominently in macrophages. Three variants of Gaucher disease are generally distinguished based on the absence (type 1) or presence of central nervous system involvement 1 (types 2 and 3). In the much more common type 1 variant of Gaucher disease, glycosphingolipidladen macrophages, referred to as Gaucher cells, accumulate in the visceral tissues liver, spleen, and bone marrow, inducing a pleiotropic array of symptoms, including hepatosplenomegaly and pancytopenia. In addition, type 1 Gaucher patients often develop bone complications: bone pain and crises, avascular necrosis, and pathologic fractures. 1 Two different types of therapeutic intervention are available for type 1 patients. One relies on chronic intravenous administration of recombinant glucocerebrosidase, denoted enzyme replacement therapy (ERT). 2 Two recombinant enzyme preparations are now registered for ERT in type 1 Gaucher disease: imiglucerase (Cerezyme; Genzyme Corp) and velaglucerase alfa (Vpriv; Shire HGT). 3 A third enzyme, a plant-cellexpressed recombinant glucocerebrosidase, is under clinical development (Taliglucerase; Protalix/Pfizer). 3 The other therapeutic intervention is based on oral administration of the iminosugar N-butyldeoxinojirimycin (Miglustat; Zavesca, Actelion). 4 This compound is thought to effectively lower synthesis of the accumulating metabolite, glucosylceramide, by inhibiting its synthesizing enzyme, glucosylceramide synthase. 5 The clinical responses to ERT are fast and impressive, such as significant corrections in hepatosplenomegaly, improvement of hematologic parameters and reduction of bone marrow infiltration as seen by magnetic resonance imaging. 6 The response to miglustat treatment is less prominent, and its use is authorized for mildly to moderately affected patients who are unsuitable for ERT (EMA) or in whom ERT is not a therapeutic option (FDA). 7 Future use of such small compounds for treating patients with a neuronopathic course of Gaucher disease is appealing given their potential to penetrate the brain (in contrast to recombinant enzyme). 8 The availability of costly therapies has stimulated searches for plasma biomarkers that can assist in clinical management of individual patients. Several circulating protein markers for Gaucher cells have meanwhile been identified (for a review see Aerts et al 9 ). It has been demonstrated that the enzyme chitotriosidase 10 and the chemokine CCL18 11 are produced by Gaucher cells and secreted into the circulation. Both proteins are candidate biomarkers since their plasma concentrations are markedly increased in symptomatic type 1 Gaucher patients and vary This article contains a data supplement.The publication costs of this article were defrayed in part b...

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here, we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.