Inhibition of proteasome deubiquitinating activity as a novel cancer therapy

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.CancerfondenRadiumhemmets forskningsfonderVetenskapsrådetStrategiska forskningsstiftelsenVinnovaEuropean Union CHEMORES, Frame program 6 (LSHC-CT-2007-037665)Swedish Children Cancer SocietyAccepte

Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation

Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimer's disease often accumulate UBB+1, a mutant ubiquitin carrying a 19–amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB+1 is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB+1 may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins

Functional studies of the ubiquitin-proteasome system using GFP-based reporters

Ubiquitin-dependent proteasomal degradation is of paramount importance for cellular processes such as cell cycle progression, transcriptional regulation, apoptosis and disposal of misfolded and aberrant proteins. Moreover the ubiquitin-proteasome system is the main producer of peptides used by major histocompatibility complex class 1 for antigen presentation. Considering its critical involvement in these cellular processes, it is not surprising that aberrant ubiquitin-proteasome- dependent degradation is implicated in human disorders. The aim of this thesis has been to develop reporter systems for quantitative and functional analysis of this system in cells and to use these reporters to gain insight into the interactions between viral and cellular proteins and the ubiquitin- proteasome system. The green fluorescent protein (GFP) was chosen as a reporter for degradation and was in the initial studies modified by the addition of degradation signals that transformed the GFP into a substrate of the proteasome. Upon inhibition of the system the GFP reporters accumulate and the level of obstruction can be monitored by the fluorescence. The GFP reporters were initially characterised and used for studies in cellular systems but more recently we developed a transgenic mouse model constitutively expressing one of the reporters. We used the GFP reporters and new site-specific inhibitors to elucidate the individual contribution of the different proteasomal active sites. Our study demonstrated that the catalytic activities are of unequal importance for degradation. Furthermore this study indicated the presence of a non-catalytic modifier site that regulates the activity of the proteolytic sites through binding of peptides. The role of a cellular and a viral repetitive sequence on proteolysis was also investigated using the GFP reporters. The Epstein-Barr virus derived Gly-Ala repeat functions as a stabilising domain. We concluded that it protects a protein from degradation in a lengthdependent manner. Another repetitive sequence with a length-dependent effect is the poly-GIn repeat, which is expanded in several proteins involved in neurodegeneration. We showed that in contrast to the Gly- Ala repeat this repeat stabilises proteins through the formation of insoluble aggregates. Another protein associated with neurodegeneration, and that also colocalises with different aggregates involved in such diseases, is the transcript mutant of ubiquitin, UBB+1. With the use of the GFP reporters we demonstrated that UBB+1 is a substrate of the proteasome and is ubiquitinated in a specific manner and this causes all inhibition of the ubiquitin-proteasome system. It remains to be resolved if this inhibitory activity contributes to neuropathogenesis. Together these studies shed some light on the roles of the ubiquitin-proteasome system in normal and pathologic conditions. The cellular and transgenic animal models presented in this thesis will be important tools for ftirther studies on this intriguing proteolytic system

The ubiquitin specific protease-4 (USP4) interacts with the S9/Rpn6 subunit of the proteasome

The proteasome is the major non-lysosomal proteolytic machine in cells that, through degradation of ubiquitylated substrates, regulates virtually all cellular functions. Numerous accessory proteins influence the activity of the proteasome by recruiting or deubiquitylating proteasomal substrates, or by maintaining the integrity of the complex. Here we show that the ubiquitin specific protease (USP)-4, a deubiquitylating enzyme with specificity for both Lys48 and Lys63 ubiquitin chains, interacts with the S9/Rpn6 subunit of the proteasome via an internal ubiquitin-like (UBL) domain. S9/Rpn6 acts as a molecular clamp that holds together the proteasomal core and regulatory sub-complexes. Thus, the interaction with USP4 may regulate the structure and function of the proteasome or the turnover of specific proteasomal substrates. © 2012 Elsevier Inc

The ubiquitin specific protease-4 (USP4) interacts with the S9/Rpn6 subunit of the proteasome

The proteasome is the major non-lysosomal proteolytic machine in cells that, through degradation of ubiquitylated substrates, regulates virtually all cellular functions. Numerous accessory proteins influence the activity of the proteasome by recruiting or deubiquitylating proteasomal substrates, or by maintaining the integrity of the complex. Here we show that the ubiquitin specific protease (USP)-4, a deubiquitylating enzyme with specificity for both Lys48 and Lys63 ubiquitin chains, interacts with the S9/Rpn6 subunit of the proteasome via an internal ubiquitin-like (UBL) domain. S9/Rpn6 acts as a molecular clamp that holds together the proteasomal core and regulatory sub-complexes. Thus, the interaction with USP4 may regulate the structure and function of the proteasome or the turnover of specific proteasomal substrates. © 2012 Elsevier Inc

An N-terminal SIAH-interacting motif regulates the stability of the ubiquitin specific protease (USP)-19

The Ubiquitin Specific Protease-19 (USP19) regulates cell cycle progression and is involved in the cellular response to different types of stress, including the unfolded protein response (UPR), hypoxia and muscle atrophy. Using the unique N-terminal domain as bait in a yeast-two hybrid screen we have identified the ubiquitin ligases Seven In Absentia Homolog (SIAH)-1 and SIAH2 as binding partners of USP19. The interaction is mediated by a SIAH-consensus binding motif and promotes USP19 ubiquitylation and proteasome-dependent degradation. These findings identify USP19 as a common substrate of the SIAH ubiquitin ligases. © 2013 Elsevier Inc

An N-terminal SIAH-interacting motif regulates the stability of the ubiquitin specific protease (USP)-19

The Ubiquitin Specific Protease-19 (USP19) regulates cell cycle progression and is involved in the cellular response to different types of stress, including the unfolded protein response (UPR), hypoxia and muscle atrophy. Using the unique N-terminal domain as bait in a yeast-two hybrid screen we have identified the ubiquitin ligases Seven In Absentia Homolog (SIAH)-1 and SIAH2 as binding partners of USP19. The interaction is mediated by a SIAH-consensus binding motif and promotes USP19 ubiquitylation and proteasome-dependent degradation. These findings identify USP19 as a common substrate of the SIAH ubiquitin ligases. © 2013 Elsevier Inc

Epstein-Barr Virus Encodes Three Bona Fide Ubiquitin-Specific Proteases▿ ‡

Manipulation of the ubiquitin proteasome system (UPS) is emerging as a common theme in viral pathogenesis. Some viruses have been shown to encode functional homologs of UPS enzymes, suggesting that a systematic identification of these products may provide new insights into virus-host cell interactions. Ubiquitin-specific proteases, collectively known as deubiquitinating enzymes (DUBs), regulate the activity of the UPS by hydrolyzing ubiquitin peptide or isopeptide bonds. The prediction of viral DUBs based on sequence similarity with known enzymes is hampered by the diversity of viral genomes. In this study sequence alignments, pattern searches, and hidden Markov models were developed for the conserved C- and H-boxes of the known DUB families and used to search the open reading frames (ORFs) of Epstein-Barr virus (EBV), a large gammaherpesvirus that has been implicated in the pathogenesis of a broad spectrum of human malignancies of lymphoid and epithelial cell origin. The searches identified a limited number of EBV ORFs that contain putative DUB catalytic domains. DUB activity was confirmed by functional assays and mutation analysis for three high scoring candidates, supporting the usefulness of this bioinformatics approach in predicting distant homologues of cellular enzymes

The ER-resident ubiquitin-specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic-reticulum-associated degradation (ERAD). Although the involvement of membrane-associated ubiquitin-conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin-specific protease (USP)19. We show that USP19 is a tail-anchored ubiquitin-specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T-cell receptor-α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non-catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane-anchored DUB involved in the turnover of ERAD substrates