The proteasome is an intracellular multicatalytic protease involved in the cell cycle regulation, signaling response, antigen presentation and apoptosis. Since proteasome inhibitors promote cell death by apoptosis, they have been proposed as new anti-tumoral drugs. Terrein, a secondary metabolite secreted by the fungus Aspergillus terreus, was firstly described in 1935. In the present work we report that terrein isolated through the screening for inhibitors of the 20S proteasome showed inhibitory effect upon both chymotrypsin- and trypsin-like activities of the multicatalytic core particle, the 20S proteasome. Despite of the high inhibitory concentration determined in vitro, that verified by incubating cells (fibroblasts and a pulmonary tumor cell line) in the presence of terrein was 4-fold lower indicating the proteasome as a selective intracellular target. Moreover, terrein promoted apoptotic cell death on both fibroblasts and pulmonary tumor cell line tested. Although terrein concentrations (mM range) necessary to elicit apoptosis in the cellular models herein tried were high when compared to those (μM and nM range) of other inhibitors recently described, its chemical structure is not correlated to any other inhibitor reported thus far. Therefore...

Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together...

OBJECTIVE: We have previously demonstrated that proteasome activity is higher in bone marrow from patients with chronic myeloid leukemia (CML) than normal controls. This study investigates whether there is any relationship between Bcr-Abl expression and proteasome activity. MATERIALS AND METHODS: Fluorogenic substrate assays and an activity-based probe were used to profile proteasome activity in CML cell-line models and the effect of the proteasome inhibitor BzLLLCOCHO on these cell-line models and primary CML cells was investigated. RESULTS: We have demonstrated that oncogenic transformation by BCR-ABL is associated with an increase in proteasome proteolytic activity. Furthermore, small interfering RNA targeted against BCR-ABL reduces proteasome activity. In addition, we have found that Bcr-Abl-positive cells are more sensitive than Bcr-Abl-negative cells to induction of apoptosis by the proteasome inhibitor BzLLLCOCHO, and that sequential addition of imatinib followed by BzLLLCOCHO has an additive effect on the induction of apoptosis in Bcr-Abl-positive cells. Finally, we demonstrate that cell lines that become resistant to imatinib remain sensitive to proteasome inhibition. CONCLUSION: This is the first time that a direct relationship has been demonstrated between BCR-ABL transformation and the enzymatic activity of the proteasome. Our results suggest that the proteasome might provide a useful therapeutic target in CML...

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional...

The 26S proteasome has emerged over the past decade as an attractive therapeutic target in the treatment of cancers. Here, we report new tripeptide aldehydes that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new specific proteasome inhibitors demonstrated high potency and specificity for sarcoma cells, with therapeutic windows superior to those observed for benchmark proteasome inhibitors, MG132 and Bortezomib. Constraining the peptide backbone into the β-strand geometry, known to favor binding to a protease, resulted in decreased activity in vitro and reduced anticancer activity. Using these new proteasome inhibitors, we show that the presence of an intact p53 pathway significantly enhances cytotoxic activity, thus suggesting that this tumor suppressor is a critical downstream mediator of cell death following proteasomal inhibition.; Paul M. Neilsen, Ashok D. Pehere, Kathleen I. Pishas, David F. Callen, and Andrew D. Abell

Gegenstand dieser experimentellen Dissertation ist es, synergistische zytotoxische Effekte von Protease- und Proteasominhibitoren mit anderen Substanzgruppen und insbesondere mit bereits als Medikament verfügbaren Substanzen am Modell einer AML-Zelllinie sowie bei primären AML-Blasten zu identifizieren und zu charakterisieren. Mit Hilfe der von einem kovalent bindenden Proteasominhibitor abgeleiteten Affinitätssonde DansylAhx3L3VS (DALVS) werden in dieser Arbeit erstmals selektiv die aktiven Proteasomuntereinheiten in vitalen malignen humanen hämatopoetischen Zellen dargestellt. Die selektive Wirkung von Bortezomib auf die beta1/beta5 katalytischen Untereinheiten des Proteasoms konnten bestätigt werden. Da postuliert wurde, dass auch der HIV-Proteaseinhibitor Ritonavir eine Proteasom-inhibierende Aktivität hat (und dieses Medikament damit potentiell sehr interessant für die Therapie hämatologischer Neoplasien sein könnte), wurde der Effekt von Ritonavir auf AML-Zelllinien sowie primäre AML-Blasten mittels Affinitätsmarkierung untersucht. Dabei konnte gezeigt werden, dass Ritonavir in den klinisch relevanten Konzentrationen die Proliferation von AML-Zelllinien und primären humanen Blasten zwar hemmt, jedoch nicht über eine Proteasominhibition sondern vielmehr (ähnlich wie Bortezomib) zu einer der Proteasominhibition nachgeschalteten Aktivierung der Unfolded Protein Response (UPR) und ER-Stress führt...

Eukaryotic cells contain a major intracellular proteolytic activity known as the proteasome. The proteasome is a strongly conserved cylindrical structure of high molecular weight (650 kDa, -20 S) and desmonstrates multiple endopeptidase activities. The general structural, biochemical and genetic features of the proteasonie are conserved from archaebacteria through yeast to humans. This structure fulfills an essential role by functioning as the proteolytic core of a 26 S multienzyme complex responsible for the energydependent degradation of ubiquitinated proteins. The bulk of intracellular proteolysis appears to be through the ubiquitin-dependent pathway. Incorporation of the proteasome into the 26 S niultienzyme complex appears to confer both a specificity for ubiquitinated proteins as well as a nieans to tightly regulate proteolytic activity. Thus, one function of the proteasome is required for the degradation of either abnormal or certain regulatory proteins by the ubiquitin pathway. Proteasoine subunits appear to be encoded by a related gene family as defined by extensive sequence similarities. The gene products are confined to either of two general classes: a-type which appear to be structural and 8-type which may be catalytic. Genes encoding at least two proteasome subunits map to the Ma-jor Histocompatibility Complex. Accurnulating evidence points to the proteasome (or a specialized form) participatirig in the cytosolic degradation of these viral proteins upon cellular infection. Through a previously unforseen mechanisin. it appears that the products from the digestion of the viral proteins may be rescued from further digestion to amino acids and shuttled from the cytoplasm through the endoplasniic reticulum to the cell surface where they serve as antigenic peptides for recognition by the immune system. The proteasome may have been recruited by the immune system to serve as the cytosolic activity responsible for generating these antigenic peptides. The proteasome may function in the ubiquitindependent degradation of not only certain self-proteins but may fulfill a second essential role in the degradation of proteins originating from viral infection

A recent model of Parkinson’s disease (PD) suggests that the neuropathological, behavioural and cognitive symptoms progress in stages. There is substantial evidence for a prodromal stage of PD, during which time pre-motor symptoms develop. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a risk factor for developing PD and may be part of the pre-motor stage. In both disorders, neuropathological α-synuclein aggregates are thought to be a direct cause of the resulting symptoms. One model has shown that in rats, proteasome inhibition produced by systemic exposure to environmental toxins results in α-synuclein pathology and motor behaviour dysfunction that mimics the progression of PD in humans. The present study examined the hypothesis that the systemic proteasome inhibition model would produce pre-Parkinsonian RBD-like pathology in rats. It was expected that sleep disturbances would be seen prior to behavioural disturbances in rats treated systemically with PSI (a proteasome inhibitor). Following baseline sleep recording and training on the inclined beam-traverse task, rats were injected with PSI (a proteasome inhibitor) or ethanol (control), 6 times over 2 wk. Sleep recording over 8 wk and behavioural testing over 16 wk provided no evidence of sleep disturbances or motor dysfunction. Post-mortem immunohistochemical analyses of brain tissue provided no evidence of PSI-associated α-synuclein aggregates in the locus coeruleus...

Ubiquitin proteasome pathway is an important cellular pathway that affects the fate of almost all intracellular proteins. Misregulation of this pathway has been found to be associated with a broad range of human diseases, such as cancer, neurodegenerative diseases, as well as viral infections. Ubiquitin recognition by the proteasome is of central importance to this pathway. So far, two proteasome subunits, Rpn10 and Rpn13, have been identified as ubiquitin receptors. An alternative pathway is mediated by shuttling factors. In yeast, three shuttling factors, known as UBL-UBA proteins, have been found. A UBL receptor activity of the proteasome has been attributed to Rpn1. However, yeast cell mutated all five proteasomal ubiquitin receptors is still viable. To identify the additional proteasomal ubiquitin receptor in cells, I first obtained and characterized a new Rpn13 mutant allele. This Rpn13 mutant completely abolished its ubiquitin binding activity, and functionally resembles a null allele. Rpn13 substrate pool has also been sought in this mutant cells. In the second part of this dissertation, I reported a novel ubiquitin binding site on proteasomal subunit Rpn1. With the help of NMR analysis, Rpn1's ubiquitin and UBL binding surfaces were resolved at high resolution and found to substantially overlap. A specific Rpn1 mutation that disrupts both ubiquitin and UBL binding while not compromising the folding of Rpn1 was obtained. This mutant allele shows a pleiotropic proteasomal defect in vivo. Moreover...

Aims: b-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition. Results: We report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of α-subunit of eukaryotic initiation factor 2 phosphorylation. Innovation: The findings establish that different modes of proteasome inhibition result in distinct cellular responses...

This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.celrep.2015.06.061; [Graphical abstract - see article] Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome can also bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here, we identify that pure homotypic lysine-11 linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell cycle regulator, cyclin B1. Thus, while heterotypic lysine-11 linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrates the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.; This work is supported by a Wellcome Trust Senior Clinical Research Fellowship to JAN (102770/Z/13/Z)...