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Sample records for 26s proteasome inhibition

  1. Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition

    PubMed Central

    Dambacher, Corey M; Worden, Evan J; Herzik, Mark A; Martin, Andreas; Lander, Gabriel C

    2016-01-01

    The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11, a deubiquitinase within the ‘lid’ sub-complex. Prior to the lid’s incorporation into the proteasome, Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins. Here we present the atomic model of the isolated lid sub-complex, as determined by cryo-electron microscopy at 3.5 Å resolution, revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit, Rpn5. Through mutagenesis of specific residues, we describe the network of interactions that are required to stabilize this inhibited state. These results provide significant insight into the intricate mechanisms of proteasome assembly, outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme, which ultimately activates the deubiquitinase for substrate degradation. DOI: http://dx.doi.org/10.7554/eLife.13027.001 PMID:26744777

  2. Structure characterization of the 26S proteasome

    PubMed Central

    Kim, Ho Min; Yu, Yadong; Cheng, Yifan

    2010-01-01

    In all eukaryotic cells, 26S proteasome plays an essential role in the process of ATP-dependent protein degradation. In this review, we focus on structure characterization of the 26S proteasome. Although the progress towards a high-resolution structure of the 26S proteasome has been slow, the recently solved structures of various proteasomal subcomplexes have greatly enhanced our understanding of this large machinery. In addition to having an ATP-dependent proteolytic function, the 26S proteasome is also involved in many non-proteolytic cellular activities, which are often mediated by subunits in its 19S regulatory complex. Thus, we include a detailed discussion of the structures of 19S subunits, including proteasomal ATPases, ubiquitin receptors, deubiquitinating enzymes and subunits that contain PCI domain. PMID:20800708

  3. Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    PubMed Central

    Li, Sheng; Yang, Li-juan; Wang, Ping; He, Yu-jiao; Huang, Jun-mei; Liu, Han-wei; Shen, Xiao-fei; Wang, Fei

    2016-01-01

    Background Type I interferons (IFN-α/β) have broad and potent immunoregulatory and antiproliferative activities. However, it is still known whether the dietary flavonoids exhibit their antiviral and anticancer properties by modulating the function of type I IFNs. Objective This study aimed at determining the role of apigenin, a dietary plant flavonoid abundant in common fruits and vegetables, on the type I IFN-mediated inhibition of cancer cell viability. Design Inhibitory effect of apigenin on human 26S proteasome, a known negative regulator of type I IFN signaling, was evaluated in vitro. Molecular docking was conducted to know the interaction between apigenin and subunits of 26S proteasome. Effects of apigenin on JAK/STAT pathway, 26S proteasome-mediated interferon receptor stability, and cancer cells viability were also investigated. Results Apigenin was identified to be a potent inhibitor of human 26S proteasome in a cell-based assay. Apigenin inhibited the chymotrypsin-like, caspase-like, and trypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Results from computational modeling of the potential interactions of apigenin with the chymotrypsin site (β5 subunit), caspase site (β1 subunit), and trypsin site (β2 subunit) of the proteasome were consistent with the observed proteasome inhibitory activity. Apigenin enhanced the phosphorylation of signal transducer and activator of transcription proteins (STAT1 and STAT2) and promoted the endogenous IFN-α-regulated gene expression. Apigenin inhibited the IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Apigenin also sensitized the inhibitory effect of IFN-α on viability of cervical carcinoma HeLa cells. Conclusion These results suggest that apigenin potentiates the inhibitory effect of IFN-α on cancer cell viability by activating JAK/STAT signaling pathway through inhibition of 26S proteasome

  4. 26S Proteasome: Hunter and Prey in Auxin Signaling.

    PubMed

    Kong, Xiangpei; Zhang, Liangran; Ding, Zhaojun

    2016-07-01

    Auxin binds to TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALLING F-BOX proteins (TIR1/AFBs) and promotes the degradation of Aux/IAA transcriptional repressors. The proteasome regulator PROTEASOME REGULATOR1 (PTRE1) has now been shown to be required for auxin-mediated repression of 26S proteasome activity, thus providing new insights into the fine-tuning of the homoeostasis of Aux/IAA proteins and auxin signaling. PMID:27246455

  5. Characterization of the 26S proteasome network in Plasmodium falciparum

    PubMed Central

    Wang, Lihui; Delahunty, Claire; Fritz-Wolf, Karin; Rahlfs, Stefan; Helena Prieto, Judith; Yates, John R.; Becker, Katja

    2015-01-01

    In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system. PMID:26639022

  6. Phosphorylation of ATPase subunits of the 26S proteasome.

    PubMed

    Mason, G G; Murray, R Z; Pappin, D; Rivett, A J

    1998-07-01

    The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [32P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.

  7. Assembly, Structure and Function of the 26S proteasome

    PubMed Central

    Bedford, Lynn; Paine, Simon; Sheppard, Paul W.; Mayer, R. John; Roelofs, Jeroen

    2010-01-01

    The 26S proteasome is a large multi-protein complex involved in the regulated degradation of ubiquitinated proteins in the cell. The 26S proteasome has been shown to control an increasing number of essential biochemical mechanisms of the cellular lifecycle including DNA synthesis, repair, transcription, translation and cell signal transduction. Concurrently, it is increasingly seen that malfunction of the ubiquitin proteasome system contributes to the pathogenesis of disease. The recent identification of four molecular chaperones, in addition to five previously identified chaperones, have provided mechanistic insight into how this cellular megastructure is assembled in the cell. These data, together with new insights into the structure and function of the proteasome, provide a much better understanding of this complex protease. PMID:20427185

  8. Structural analysis of the 26S proteasome by cryoelectron tomography.

    PubMed

    Nickell, Stephan; Mihalache, Oana; Beck, Florian; Hegerl, Reiner; Korinek, Andreas; Baumeister, Wolfgang

    2007-02-01

    The 26S proteasome is the key enzyme of intracellular protein degradation in eukaryotic cells. It is a multisubunit complex of 2.5 MDa confining the proteolytic action to an inner compartment with tightly controlled access. Structural studies of this intriguing molecular machine have been hampered by its intrinsic instability and its dynamics. Here we have used an unconventional approach to obtain a three-dimensional structure of the holocomplex uncompromised by preparation-induced alterations and unbiased by any starting model. We have performed a tomographic reconstruction, followed by averaging over approx. 150 individual reconstructions, of Drosophila 26S proteasomes suspended in a thin layer of amorphous ice.

  9. The 26S Proteasome Complex: An Attractive Target for Cancer Therapy

    PubMed Central

    Frankland-Searby, Sarah; Bhaumik, Sukesh R.

    2011-01-01

    The 26S proteasome complex engages in an ATP-dependent proteolytic degradation of a variety of oncoproteins, transcription factors, cell cycle specific cyclins, cyclin-dependent kinase inhibitors, ornithine decarboxylase, and other key regulatory cellular proteins. Thus, the proteasome regulates either directly or indirectly many important cellular processes. Altered regulation of these cellular events is linked to the development of cancer. Therefore, the proteasome has become an attractive target for the treatment of numerous cancers. Several proteasome inhibitors that target the proteolytic active sites of the 26S proteasome complex have been developed and tested for anti-tumor activities. These proteasome inhibitors have displayed impressive anti-tumor functions by inducing apoptosis in different tumor types. Further, the proteasome inhibitors have been shown to induce cell cycle arrest, and inhibit angiogenesis, cell-cell adhesion, cell migration, immune and inflammatory responses, and DNA repair response. A number of proteasome inhibitors are now in clinical trials to treat multiple myeloma and solid tumors. Many other proteasome inhibitors with different efficiencies are being developed and tested for anti-tumor activities. Several proteasome inhibitors currently in clinical trials have shown significantly improved anti-tumor activities when combined with other drugs such as histone deacetylase (HDAC) inhibitors, Akt (protein kinase B) inhibitors, DNA damaging agents, Hsp90 (heat shock protein 90) inhibitors, and lenalidomide. The proteasome inhibitor bortezomib is now in the clinic to treat multiple myeloma and mantle cell lymphoma. Here, we discuss the 26S proteasome complex in carcinogenesis and different proteasome inhibitors with their potential therapeutic applications in treatment of numerous cancers. PMID:22037302

  10. Dss1 Is a 26S Proteasome Ubiquitin Receptor

    PubMed Central

    Paraskevopoulos, Konstantinos; Kriegenburg, Franziska; Tatham, Michael H.; Rösner, Heike I.; Medina, Bethan; Larsen, Ida B.; Brandstrup, Rikke; Hardwick, Kevin G.; Hay, Ronald T.; Kragelund, Birthe B.; Hartmann-Petersen, Rasmus; Gordon, Colin

    2014-01-01

    Summary The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked by K63 and K48. Atomic resolution data show that Dss1 is disordered and binds ubiquitin by binding sites characterized by acidic and hydrophobic residues. The complementary binding region in ubiquitin is composed of a hydrophobic patch formed by I13, I44, and L69 flanked by two basic regions. Mutations in the ubiquitin-binding site of Dss1 cause growth defects and accumulation of ubiquitylated proteins. PMID:25306921

  11. PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes

    PubMed Central

    Haddock, Christopher J.; Blomenkamp, Keith; Gautam, Madhav; James, Jared; Mielcarska, Joanna; Gogol, Edward; Teckman, Jeffrey; Skowyra, Dorota

    2014-01-01

    Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome

  12. PiZ mouse liver accumulates polyubiquitin conjugates that associate with catalytically active 26S proteasomes.

    PubMed

    Haddock, Christopher J; Blomenkamp, Keith; Gautam, Madhav; James, Jared; Mielcarska, Joanna; Gogol, Edward; Teckman, Jeffrey; Skowyra, Dorota

    2014-01-01

    Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome

  13. Comparative resistance of the 20S and 26S proteasome to oxidative stress.

    PubMed Central

    Reinheckel, T; Sitte, N; Ullrich, O; Kuckelkorn, U; Davies, K J; Grune, T

    1998-01-01

    Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without

  14. Toward an integrated structural model of the 26S proteasome.

    PubMed

    Förster, Friedrich; Lasker, Keren; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2010-08-01

    The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation.

  15. Toward an Integrated Structural Model of the 26S Proteasome*

    PubMed Central

    Förster, Friedrich; Lasker, Keren; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2010-01-01

    The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation. PMID:20467039

  16. An atomic structure of the human 26S proteasome.

    PubMed

    Huang, Xiuliang; Luan, Bai; Wu, Jianping; Shi, Yigong

    2016-09-01

    We report the cryo-EM structure of the human 26S proteasome at an average resolution of 3.5 Å, allowing atomic modeling of 28 subunits in the core particle (CP) and 18 subunits in the regulatory particle (RP). The C-terminal residues of Rpt3 and Rpt5 subunits in the RP can be seen inserted into surface pockets formed between adjacent α subunits in the CP. Each of the six Rpt subunits contains a bound nucleotide, and the central gate of the CP α-ring is closed despite RP association. The six pore 1 loops in the Rpt ring are arranged similarly to a spiral staircase along the axial channel of substrate transport, which is constricted by the pore 2 loops. We also determined the cryo-EM structure of the human proteasome bound to the deubiquitinating enzyme USP14 at 4.35-Å resolution. Together, our structures provide a framework for mechanistic understanding of eukaryotic proteasome function.

  17. Insights into the molecular architecture of the 26S proteasome.

    PubMed

    Nickell, Stephan; Beck, Florian; Scheres, Sjors H W; Korinek, Andreas; Förster, Friedrich; Lasker, Keren; Mihalache, Oana; Sun, Na; Nagy, István; Sali, Andrej; Plitzko, Jürgen M; Carazo, Jose-Maria; Mann, Matthias; Baumeister, Wolfgang

    2009-07-21

    Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the "base" part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the "wobbling" model that was previously proposed to explain the role of the regulatory complex in opening the gate in the alpha-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.

  18. Insights into the molecular architecture of the 26S proteasome

    PubMed Central

    Nickell, Stephan; Beck, Florian; Scheres, Sjors H. W.; Korinek, Andreas; Förster, Friedrich; Lasker, Keren; Mihalache, Oana; Sun, Na; Nagy, István; Sali, Andrej; Plitzko, Jürgen M.; Carazo, Jose-Maria; Mann, Matthias; Baumeister, Wolfgang

    2009-01-01

    Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the “base” part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the “wobbling” model that was previously proposed to explain the role of the regulatory complex in opening the gate in the α-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry. PMID:19581588

  19. Viruses and the 26S proteasome: hacking into destruction.

    PubMed

    Banks, Lawrence; Pim, David; Thomas, Miranda

    2003-08-01

    The discovery that the human papillomavirus E6 oncoprotein could direct the ubiquitination and degradation of the p53 tumour suppressor at the 26S proteasome was the beginning of a new view on virus-host interactions. A decade later, a plethora of viral proteins have been shown to direct host-cell proteins for proteolytic degradation. These activities are required for various aspects of the virus life-cycle from entry, through replication and enhanced cell survival, to viral release. As with oncogenes and cell-cycle control, the study of apparently simple viruses has provided a wealth of information on the function of a whole class of cellular proteins whose function is arguably as important as that of the kinases: the ubiquitin-protein ligases.

  20. Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

    PubMed

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

    2011-02-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

  1. Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13*

    PubMed Central

    Berko, Dikla; Herkon, Ora; Braunstein, Ilana; Isakov, Elada; David, Yael; Ziv, Tamar; Navon, Ami; Stanhill, Ariel

    2014-01-01

    The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps. PMID:24429290

  2. Characterization of the Brain 26S Proteasome and its Interacting Proteins

    PubMed Central

    Tai, Hwan-Ching; Besche, Henrike; Goldberg, Alfred L.; Schuman, Erin M.

    2010-01-01

    Proteasome-mediated proteolysis is important for synaptic plasticity, neuronal development, protein quality control, and many other processes in neurons. To define proteasome composition in brain, we affinity purified 26S proteasomes from cytosolic and synaptic compartments of the rat cortex. Using tandem mass spectrometry, we identified the standard 26S subunits and a set of 28 proteasome-interacting proteins that associated substoichiometrically and may serve as regulators or cofactors. This set differed from those in other tissues and we also found several proteins that associated only with either the cytosolic or the synaptic proteasome. The latter included the ubiquitin-binding factor TAX1BP1 and synaptic vesicle protein SNAP-25. Native gel electrophoresis revealed a higher proportion of doubly-capped 26S proteasome (19S-20S-19S) in the cortex than in the liver or kidney. To investigate the interplay between proteasome regulation and synaptic plasticity, we exposed cultured neurons to glutamate receptor agonist NMDA. Within 4 h, this agent caused a prolonged decrease in the activity of the ubiquitin-proteasome system as shown by disassembly of 26S proteasomes, decrease in ubiquitin-protein conjugates, and dissociation of the ubiquitin ligases UBE3A (E6-AP) and HUWE1 from the proteasome. Surprisingly, the regulatory 19S particles were rapidly degraded by proteasomal, not lysosomal degradation, and the dissociated E3 enzymes also degraded. Thus the content of proteasomes and their set of associated proteins can be altered by neuronal activity, in a manner likely to influence synaptic plasticity and learning. PMID:20717473

  3. DBC2 resistance is achieved by enhancing 26S proteasome-mediated protein degradation.

    PubMed

    Collado, Denise; Yoshihara, Takashi; Hamaguchi, Masaaki

    2007-08-31

    Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.

  4. Purification and characterization of 26S proteasomes from human and mouse spermatozoa.

    PubMed

    Tipler, C P; Hutchon, S P; Hendil, K; Tanaka, K; Fishel, S; Mayer, R J

    1997-12-01

    We purified by fractionation on 10-40% glycerol gradients, 26S proteasomes from normal human spermatozoa. These proteasomes, which participate in the ATP-dependent degradation of ubiquitinated proteins, share a similar sedimentation coefficient to those purified from other human tissues. Fluorogenic peptide assays reveal they have chymotrypsin, trypsin and peptidyl-glutamyl-like peptide hydrolysing activities; the chymotrypsin activity is ablated by the specific 26S proteasome inhibitor MG132. Confirmation that these large proteases are 26S proteasomes is provided by detection of the 20S proteasome subunits HC2, XAPC7, RN3 and Z and regulatory ATPases MSS1, TBP1, SUG1 and SUG2 by Western analyses with monoclonal antisera. These antigens are found only in the gradient fractions enriched in proteolytic activities. We have also shown that, although mature spermatozoa from mice have considerably reduced amounts of a ubiquitin-conjugating enzyme (E2) and ubiquitin-protein conjugates in comparison with less mature germ cells, they retain relatively high values of 26S proteasome activity. This suggests that proteasomes may have further roles to play in normal sperm physiology.

  5. DNA damage modulates interactions between microRNAs and the 26S proteasome

    PubMed Central

    Tsimokha, Anna S; Kulichkova, Valentina A.; Karpova, Elena V.; Zaykova, Julia J.; Aksenov, Nikolai D; Vasilishina, Anastasia A.; Kropotov, Andrei V.; Antonov, Alexey; Barlev, Nikolai A.

    2014-01-01

    26S proteasomes are known as major non-lysosomal cellular machines for coordinated and specific destruction of ubiquitinylated proteins. The proteolytic activities of proteasomes are controlled by various post-translational modifications in response to environmental cues, including DNA damage. Besides proteolysis, proteasomes also associate with RNA hydrolysis and splicing. Here, we extend the functional diversity of proteasomes by showing that they also dynamically associate with microRNAs (miRNAs) both in the nucleus and cytoplasm of cells. Moreover, DNA damage induced by an anti-cancer drug, doxorubicin, alters the repertoire of proteasome-associated miRNAs, enriching the population of miRNAs that target cell cycle checkpoint regulators and DNA repair proteins. Collectively, these data uncover yet another potential mode of action for proteasomes in the cell via their dynamic association with microRNAs. PMID:25004448

  6. Structure of the human 26S proteasome at a resolution of 3.9 Å

    PubMed Central

    Schweitzer, Andreas; Aufderheide, Antje; Rudack, Till; Beck, Florian; Pfeifer, Günter; Plitzko, Jürgen M.; Sakata, Eri; Schulten, Klaus; Förster, Friedrich; Baumeister, Wolfgang

    2016-01-01

    Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing. PMID:27342858

  7. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  8. Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii.

    PubMed

    Gogliettino, Marta; Balestrieri, Marco; Riccio, Alessia; Facchiano, Angelo; Fusco, Carmela; Palazzo, Vincenzo Cecere; Rossi, Mosè; Cocca, Ennio; Palmieri, Gianna

    2016-01-01

    Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its 'uncommon' ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery.

  9. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators.

  10. Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution

    PubMed Central

    Bohn, Stefan; Beck, Florian; Sakata, Eri; Walzthoeni, Thomas; Beck, Martin; Aebersold, Ruedi; Förster, Friedrich; Baumeister, Wolfgang; Nickell, Stephan

    2010-01-01

    The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 Å by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the α-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high “activity” surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex. PMID:21098295

  11. Automated cryoelectron microscopy of "single particles" applied to the 26S proteasome.

    PubMed

    Nickell, Stephan; Beck, Florian; Korinek, Andreas; Mihalache, Oana; Baumeister, Wolfgang; Plitzko, Jürgen M

    2007-06-19

    The 26S proteasome is a large molecular machine with a central role in intracellular protein degradation in eukaryotes. The 2.5 MDa complex, which is built from two copies each of more than 30 different subunits, is labile and prone to dissociation into subcomplexes. Hence it is difficult if not impossible, to obtain structurally homogeneous preparations and, as a consequence, it is very cumbersome to obtain large numbers of images of the holocomplex. In this communication, we describe an automated procedure for the acquisition of large data sets of cryoelectron micrographs. The application of this procedure to the 26S proteasome from Drosophila has allowed us to determine the three-dimensional structure of the complex to a resolution of 2.9 nm and the prospects for further improvements are good.

  12. Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution.

    PubMed

    Bohn, Stefan; Beck, Florian; Sakata, Eri; Walzthoeni, Thomas; Beck, Martin; Aebersold, Ruedi; Förster, Friedrich; Baumeister, Wolfgang; Nickell, Stephan

    2010-12-01

    The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 Å by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the α-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high "activity" surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex.

  13. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

    PubMed Central

    Lasker, Keren; Förster, Friedrich; Bohn, Stefan; Walzthoeni, Thomas; Villa, Elizabeth; Unverdorben, Pia; Beck, Florian; Aebersold, Ruedi; Sali, Andrej; Baumeister, Wolfgang

    2012-01-01

    The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates. PMID:22307589

  14. Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii

    PubMed Central

    Gogliettino, Marta; Balestrieri, Marco; Riccio, Alessia; Facchiano, Angelo; Fusco, Carmela; Palazzo, Vincenzo Cecere; Rossi, Mosè; Cocca, Ennio; Palmieri, Gianna

    2016-01-01

    Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its ‘uncommon’ ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery. PMID:26933238

  15. Near-atomic resolution structural model of the yeast 26S proteasome

    PubMed Central

    Beck, Florian; Unverdorben, Pia; Bohn, Stefan; Schweitzer, Andreas; Pfeifer, Günter; Sakata, Eri; Nickell, Stephan; Plitzko, Jürgen M.; Villa, Elizabeth; Baumeister, Wolfgang; Förster, Friedrich

    2012-01-01

    The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits. PMID:22927375

  16. Near-atomic resolution structural model of the yeast 26S proteasome.

    PubMed

    Beck, Florian; Unverdorben, Pia; Bohn, Stefan; Schweitzer, Andreas; Pfeifer, Günter; Sakata, Eri; Nickell, Stephan; Plitzko, Jürgen M; Villa, Elizabeth; Baumeister, Wolfgang; Förster, Friedrich

    2012-09-11

    The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.

  17. Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition

    PubMed Central

    van Baarsel, Eric D.; Metz, Patrick J.; Fisch, Kathleen; Widjaja, Christella E.; Kim, Stephanie H.; Lopez, Justine; Chang, Aaron N.; Geurink, Paul P.; Florea, Bogdan I.; Overkleeft, Hermen S.; Ovaa, Huib; Bui, Jack D.; Yang, Jing; Chang, John T.

    2016-01-01

    The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy. PMID:26930717

  18. Tyrosine Nitration of PA700 Activates the 26S Proteasome to Induce Endothelial Dysfunction in Mice With Angiotensin II–Induced Hypertension

    PubMed Central

    Xu, Jian; Wang, Shuangxi; Wu, Yong; Song, Ping; Zou, Ming-Hui

    2010-01-01

    The ubiquitin-proteasome system has been implicated in oxidative stress–induced endothelial dysfunction in cardiovascular diseases. However, the mechanism by which oxidative stress alters the ubiquitin-proteasome system is poorly defined. The present study was conducted to determine whether oxidative modifications of PA700, a 26S proteasome regulatory subunit, contributes to angiotensin II (Ang II)–induced endothelial dysfunction. Exposure of human umbilical vein endothelial cells to low concentrations of Ang II, but not vehicle, for 6 hours significantly decreased the levels of tetrahydro-L-biopterin (BH4), an essential cofactor of endothelial NO synthase, which was accompanied by a decrease in GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis. In addition, Ang II increased both tyrosine nitration of PA700 and the 26S proteasome activity, which were paralleled by increased coimmunoprecipitation of PA700 and the 20S proteasome. Genetic inhibition of NAD(P)H oxidase or administration of uric acid (a peroxynitrite scavenger) or NG-nitro-L-arginine methyl ester (nonselective NO synthase inhibitor) significantly attenuated Ang II–induced PA700 nitration, 26S proteasome activation, and reduction of GTP cyclohydrolase I and BH4. Finally, Ang II infusion in mice decreased the levels of both BH4 and GTP cyclohydrolase I and impaired endothelial-dependent relaxation in isolated aortas, and all of these effects were prevented by the administration of MG132, a potent inhibitor for 26S proteasome. We conclude that Ang II increases tyrosine nitration of PA700 resulting in accelerated GTP cyclohydrolase I degradation, BH4 deficiency, and consequent endothelial dysfunction in hypertension. PMID:19597039

  19. Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

    PubMed

    Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection.

  20. Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the '26 S' multienzyme complex.

    PubMed Central

    Seelig, A; Kloetzel, P M; Kuehn, L; Dahlmann, B

    1991-01-01

    On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:1741750

  1. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.

    PubMed

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

  2. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato

    PubMed Central

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

  3. ATP-DEPENDENT STEPS IN THE BINDING OF UBIQUITIN CONJUGATES TO THE 26S PROTEASOME THAT COMMIT TO DEGRADATION

    PubMed Central

    Peth, Andreas; Uchiki, Tomoaki; Goldberg, Alfred L.

    2010-01-01

    Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high affinity binding of ubiquitin chains, but in their absence ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2-4 fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP) to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded. PMID:21095592

  4. The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death

    PubMed Central

    Livneh, Ido; Cohen-Kaplan, Victoria; Cohen-Rosenzweig, Chen; Avni, Noa; Ciechanover, Aaron

    2016-01-01

    The 26S proteasome is a large, ∼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms. PMID:27444871

  5. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    PubMed Central

    Förster, Friedrich; Lasker, Keren; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-01-01

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners. PMID:19653995

  6. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome.

    PubMed

    Förster, Friedrich; Lasker, Keren; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  7. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    SciTech Connect

    Foerster, Friedrich; Lasker, Keren; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  8. Convergence of the 26S proteasome and the REVOLUTA pathways in regulating inflorescence and floral meristem functions in Arabidopsis.

    PubMed

    Zhang, Zhenzhen; Wang, Hua; Luo, Dexian; Zeng, Minhuan; Huang, Hai; Cui, Xiaofeng

    2011-01-01

    The 26S proteasome is a large multisubunit proteolytic complex, regulating growth and development in eukaryotes by selective removal of short-lived regulatory proteins. Here, it is shown that the 26S proteasome and the transcription factor gene REVOLUTA (REV) act together in maintaining inflorescence and floral meristem (IM and FM) functions. The characterization of a newly identified Arabidopsis mutant, designated ae4 (asymmetric leaves1/2 enhancer4), which carries a mutation in the gene encoding the 26S proteasome subunit, RPN2a, is reported. ae4 and rev have minor defects in phyllotaxy structure and meristem initiation, respectively, whereas ae4 rev demonstrated strong developmental defects. Compared with the rev single mutant, an increased percentage of ae4 rev plants exhibited abnormal vegetative shoot apical and axillary meristems. After flowering, ae4 rev first gave rise to a few normal-looking flowers, and then flowers with reduced numbers of all types of floral organs. In late reproductive development, instead of flowers, the ae4 rev IM produced numerous filamentous structures, which contained cells seen only in the floral organs, and then carpelloid organs. In situ hybridization revealed that expression of the WUSCHEL and CLAVATA3 genes was severely down-regulated or absent in the late appearing ae4 rev primordia, but the genes were strongly expressed in top-layer cells of inflorescence tips. Double mutant plants combining rev with other 26S proteasome subunit mutants, rpn1a and rpn9a, resembled ae4 rev, suggesting that the 26S proteasome might act as a whole in regulating IM and FM functions.

  9. Mouse homologue of yeast Prp19 interacts with mouse SUG1, the regulatory subunit of 26S proteasome

    SciTech Connect

    Sihn, Choong-Ryoul; Cho, Si Young; Lee, Jeong Ho; Lee, Tae Ryong; Kim, Sang Hoon . E-mail: shkim@khu.ac.kr

    2007-04-27

    Yeast Prp19 has been shown to involve in pre-mRNA splicing and DNA repair as well as being an ubiquitin ligase. Mammalian homologue of yeast Prp19 also plays on similar functional activities in cells. In the present study, we isolated mouse SUG1 (mSUG1) as binding partner of mouse Prp19 (mPrp19) by the yeast two-hybrid system. We confirmed the interaction of mPrp9 with mSUG1 by GST pull-down assay and co-immunoprecipitation assay. The N-terminus of mPrp19 including U-box domain was associated with the C-terminus of mSUG1. Although, mSUG1 is a regulatory subunit of 26S proteasome, mPrp19 was not degraded in the proteasome-dependent pathway. Interestingly, GFP-mPrp19 fusion protein was co-localized with mSUG1 protein in cytoplasm as the formation of the speckle-like structures in the presence of a proteasome inhibitor MG132. In addition, the activity of proteasome was increased in cells transfected with mPrp19. Taken together, these results suggest that mPrp19 involves the regulation of protein turnover and may transport its substrates to 26S proteasome through mSUG1 protein.

  10. The 26S proteasome is a multifaceted target for anti-cancer therapies

    PubMed Central

    Grigoreva, Tatyana A; Tribulovich, Vyacheslav G.; Garabadzhiu, Alexander V.; Melino, Gerry; Barlev, Nickolai A.

    2015-01-01

    Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications. PMID:26295307

  11. Black tea polyphenols inhibit tumor proteasome activity.

    PubMed

    Mujtaba, Taskeen; Dou, Q Ping

    2012-01-01

    Tea is a widely consumed beverage and its constituent polyphenols have been associated with potential health benefits. Although black tea polyphenols have been reported to possess potent anticancer activities, the effect of its polyphenols, theaflavins on the tumor's cellular proteasome function, an important biological target in cancer prevention, has not been carefully studied. Here black tea extract (T5550) enriched in theaflavins inhibited the chymotrypsin-like (CT) activity of the proteasome and proliferation of human multiple myeloma cells in a dose-dependent manner. Also an isolated theaflavin (TF-1) can bind to, and inhibit the purified 20S proteasome, accompanied by suppression of tumor cell proliferation, suggesting that the tumor proteasome is an important target whose inhibition is at least partially responsible for the anticancer effects of black tea.

  12. Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly

    PubMed Central

    Sun, Peng; Feng, Li-xing; Zhang, Dong-mei; Liu, Miao; Liu, Wang; Mi, Tian; Wu, Wan-ying; Jiang, Bao-hong; Yang, Min; Hu, Li-hong; Guo, De-an; Liu, Xuan

    2016-01-01

    Aim: Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells. Methods: A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis. Results: The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome β1 subunit and no binding affinity to the β2 and β5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome β1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the β1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity. Conclusion: BF211 inhibits proteasome activity in A549 cells by decreasing β1 subunit expression and disrupting proteasome assembly

  13. Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome.

    PubMed

    Gillette, Thomas G; Kumar, Brajesh; Thompson, David; Slaughter, Clive A; DeMartino, George N

    2008-11-14

    The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.

  14. Structural Determinants Involved in the Regulation of CXCL14/BRAK Expression by the 26S Proteasome

    PubMed Central

    Peterson, Francis C.; Thorpe, Jeffery A.; Harder, Adam; Volkman, Brian F.; Schwarze, Steven R.

    2006-01-01

    The chemokine CXCL14/BRAK participates in immune surveillance by recruiting dendritic cells. CXCL14 gene expression is altered in a number of cancers, but protein expression levels have not been investigated. Here we report that CXCL14 protein can be expressed in primary epithelial cells, however in several immortalized and cancer cell lines this protein is targeted for polyubiquitylation and proteasomal degradation. We determined the NMR structure of CXCL14 to identify motifs controlling its expression. CXCL14 adopts the canonical chemokine tertiary fold but contains a unique five amino acid insertion (41VSRYR45) relative to other CXC chemokines. Deletion or substitution of key residues within this insertion prevented proteasomal degradation. Furthermore, we defined a 15 amino acid fragment of CXCL14 that is sufficient to induce proteasomal degradation. This study elucidates a post-translational mechanism for the loss of CXCL14 in cancer and a novel mode of chemokine regulation. PMID:16987528

  15. cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins.

    PubMed

    Lokireddy, Sudarsanareddy; Kukushkin, Nikolay Vadimovich; Goldberg, Alfred Lewis

    2015-12-29

    Although rates of protein degradation by the ubiquitin-proteasome pathway (UPS) are determined by their rates of ubiquitination, we show here that the proteasome's capacity to degrade ubiquitinated proteins is also tightly regulated. We studied the effects of cAMP-dependent protein kinase (PKA) on proteolysis by the UPS in several mammalian cell lines. Various agents that raise intracellular cAMP and activate PKA (activators of adenylate cyclase or inhibitors of phosphodiesterase 4) promoted degradation of short-lived (but not long-lived) cell proteins generally, model UPS substrates having different degrons, and aggregation-prone proteins associated with major neurodegenerative diseases, including mutant FUS (Fused in sarcoma), SOD1 (superoxide dismutase 1), TDP43 (TAR DNA-binding protein 43), and tau. 26S proteasomes purified from these treated cells or from control cells and treated with PKA degraded ubiquitinated proteins, small peptides, and ATP more rapidly than controls, but not when treated with protein phosphatase. Raising cAMP levels also increased amounts of doubly capped 26S proteasomes. Activated PKA phosphorylates the 19S subunit, Rpn6/PSMD11 (regulatory particle non-ATPase 6/proteasome subunit D11) at Ser14. Overexpression of a phosphomimetic Rpn6 mutant activated proteasomes similarly, whereas a nonphosphorylatable mutant decreased activity. Thus, proteasome function and protein degradation are regulated by cAMP through PKA and Rpn6, and activation of proteasomes by this mechanism may be useful in treating proteotoxic diseases.

  16. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    SciTech Connect

    Samaras, Susan E.; Chen, Billy; Koch, Stephen R.; Sawyer, Douglas B.; Lim, Chee Chew; Davidson, Jeffrey M.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. Black-Right-Pointing-Pointer Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. Black-Right-Pointing-Pointer Differential degradation appears related to nuclear vs. sarcolemmal localization. Black-Right-Pointing-Pointer Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

  17. Shikonin Exerts Antitumor Activity via Proteasome Inhibition and Cell Death Induction in vitro and in vivo

    PubMed Central

    Yang, Huanjie; Zhou, Ping; Huang, Hongbiao; Chen, Di; Ma, Ningfang; Cui, Cindy Qiuzhi; Shen, Shouxing; Dong, Weihua; Zhang, Xiaoyan; Lian, Wen; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2009-01-01

    Dysregulation of the ubiquitin-proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell-killing activity, and results from a clinical study using a shikonin-containing mixture demonstrated its safety and efficacy for the treatment of late-stage lung cancer. In the present study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C1 and C4 of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin-like activity of purified 20S proteasome (IC50 12.5 μmol/L) and tumor cellular 26S proteasome (IC50 between 2-16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC-3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB-α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC-3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin, and inhibition of the proteasome activity by shikonin contributes to its anti-tumor property. PMID:19165859

  18. Primary proteasome inhibition results in cardiac dysfunction

    PubMed Central

    Herrmann, Joerg; Wohlert, Christine; Saguner, Ardan M.; Flores, Ana; Nesbitt, Lisa L.; Chade, Alejandro; Lerman, Lilach O.; Lerman, Amir

    2013-01-01

    Aims The proteasome prevents the intracellular accumulation of proteins and its impairment can lead to structural and functional alterations, as noted for the coronary vasculature in a previous study. Utilizing the same model, this study was designed to test the hypothesis that chronic proteasome inhibition (PSI) also leads to structural and functional changes of the heart. Methods and results Female domestic pigs were randomized to a normal diet without (N) or with twice-weekly subcutaneous injections of the proteasome inhibitor MLN-273 (0.08 mg/kg, N + PSI, n = 5 each group). In vivo data on cardiac structure and function as well as myocardial perfusion and microvascular permeability response to adenosine and dobutamine were obtained by electron beam computed tomography after 11 weeks. Subsequent ex vivo myocardial analyses included immunoblotting, immunostaining, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling), Masson trichrome, and Congo red staining. Compared with N, an increase in LV mass was observed in N + PSI (106.5 ± 16.4 g vs. 183.1 ± 24.2 g, P < 0.05). The early to late diastolic filling ratio was increased in N + PSI vs. N (3.5 ± 0.6 vs. 1.8 ± 0.1, P < 0.05). The EF tended to be lower (46 ± 12% and 53 ± 9%, respectively) and cardiac output was significantly lower in N + PSI than in N (2.9 ± 1.1 vs. 4.7 ± 1.1 L/min, P < 0.05). Tissue analyses demonstrated an accumulation of proteasome substrates, apoptosis, and fibrosis in the PSI group. Compared with N, the myocardial perfusion response was reduced and microvascular permeability was increased in N + PSI. Conclusion The current study demonstrates that chronic proeasome inhibition affects the cardiovascular system, leading to functional and structural alteration of the heart consistent with a hypertrophic–restrictive cardiomyopathy phenotype. PMID:23616520

  19. The RPT2 Subunit of the 26S Proteasome Directs Complex Assembly, Histone Dynamics, and Gametophyte and Sporophyte Development in Arabidopsis[W

    PubMed Central

    Lee, Kwang-Hee; Minami, Atsushi; Marshall, Richard S.; Book, Adam J.; Farmer, Lisa M.; Walker, Joseph M.; Vierstra, Richard D.

    2011-01-01

    The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly. PMID:22158466

  20. The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis.

    PubMed

    Lee, Kwang-Hee; Minami, Atsushi; Marshall, Richard S; Book, Adam J; Farmer, Lisa M; Walker, Joseph M; Vierstra, Richard D

    2011-12-01

    The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly.

  1. Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis

    SciTech Connect

    Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu; Kim, Ki-Jeong; Park, Chang-Jin; Kim, Young-Jin; Park, Ohkmae K.; Paek, Kyung-Hee . E-mail: khpaek95@korea.ac.kr

    2006-12-15

    Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P{sub 0} interaction, but not during compatible TMV-P{sub 1.2} interaction. When CaRPN7::GFP fusion protein was targeted in onion cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.

  2. Salt stress-induced disassembly of Arabidopsis cortical microtubule arrays involves 26S proteasome-dependent degradation of SPIRAL1.

    PubMed

    Wang, Songhu; Kurepa, Jasmina; Hashimoto, Takashi; Smalle, Jan A

    2011-09-01

    The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress-induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.

  3. Could inhibition of the proteasome cause mad cow disease?

    PubMed

    Hooper, Nigel M

    2003-04-01

    The proteasome is the cellular machinery responsible for the degradation of normal and misfolded proteins. Inhibitors of the proteasome are being evaluated as therapeutic agents and recent work suggests that such inhibition might promote the neurotoxic properties of the prion protein (the causative agent of mad cow disease) and its conformational conversion to the infectious form, thus raising the question as to whether proteasome inhibitors might facilitate the development of prion diseases. PMID:12679058

  4. Could inhibition of the proteasome cause mad cow disease?

    PubMed

    Hooper, Nigel M

    2003-04-01

    The proteasome is the cellular machinery responsible for the degradation of normal and misfolded proteins. Inhibitors of the proteasome are being evaluated as therapeutic agents and recent work suggests that such inhibition might promote the neurotoxic properties of the prion protein (the causative agent of mad cow disease) and its conformational conversion to the infectious form, thus raising the question as to whether proteasome inhibitors might facilitate the development of prion diseases.

  5. CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

    PubMed Central

    Yokota, K; Kagawa, S; Shimizu, Y; Akioka, H; Tsurumi, C; Noda, C; Fujimuro, M; Yokosawa, H; Fujiwara, T; Takahashi, E; Ohba, M; Yamasaki, M; DeMartino, G N; Slaughter, C A; Toh-e, A; Tanaka, K

    1996-01-01

    The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome. Images PMID:8816993

  6. The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acid signaling.

    PubMed

    Smalle, Jan; Kurepa, Jasmina; Yang, Peizhen; Emborg, Thomas J; Babiychuk, Elena; Kushnir, Sergei; Vierstra, Richard D

    2003-04-01

    The 26S proteasome is an essential protease complex responsible for removing most short-lived intracellular proteins, especially those modified with polyubiquitin chains. We show here that an Arabidopsis mutant expressing an altered RPN10 subunit exhibited a pleiotropic phenotype consistent with specific changes in 26S proteasome function. rpn10-1 plants displayed reduced seed germination, growth rate, stamen number, genetic transmission through the male gamete, and hormone-induced cell division, which can be explained partially by a constitutive downregulation of the key cell cycle gene CDKA;1. rpn10-1 also was more sensitive to abscisic acid (ABA), salt, and sucrose stress and to DNA-damaging agents and had decreased sensitivity to cytokinin and auxin. Most of the phenotypes can be explained by a hypersensitivity to ABA, which is reflected at the molecular level by the selective stabilization of the short-lived ABA-signaling protein ABI5. Collectively, these results indicate that RPN10 affects a number of regulatory processes in Arabidopsis likely by directing specific proteins to the 26S proteasome for degradation. A particularly important role may be in regulating the responses to signals promulgated by ABA.

  7. Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

    PubMed Central

    Hampton, R Y; Gardner, R G; Rine, J

    1996-01-01

    3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the

  8. Pyrrolidine dithiocarbamate and zinc inhibit proteasome-dependent proteolysis.

    PubMed

    Kim, Insook; Kim, Chul Hoon; Kim, Joo Hee; Lee, Jinu; Choi, Jun Jeong; Chen, Zheng Ai; Lee, Min Goo; Chung, Kwang Chul; Hsu, Chung Y; Ahn, Young Soo

    2004-08-01

    Proteasomes play important roles in a variety of cellular processes such as cell cycle progression, signal transduction and immune responses. Proteasome activity is important in maintaining rapid turnover of short-lived proteins, as well as preventing accumulation of misfolded or damaged proteins. Alteration in ubiquitin-proteasome function may be detrimental to its crucial role in maintaining cellular homeostasis. Here, we have found that treatment of pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, resulted in the accumulation of several proteasome substrates including p53 and p21 in HeLa cells. The PDTC effect was due to an extended half-life of these proteins through the mobilization of zinc. PDTC and/or zinc also increased fluorescence intensity of Ub(G76V)-GFP fusion protein that is degraded rapidly by the ubiquitin-proteasome system. Treatment of cells with zinc induced formation of ubiquitinated inclusions in the centrosome, a histological marker of proteasome inhibition. Western blotting showed zinc-induced increase in laddering bands of polyubiquitin-conjugated proteins. In vitro study, zinc inhibited the ubiquitin-independent proteasomal degradations of p21 and alpha-synuclein. These results suggest that zinc may modulate cell functions through its action on the turnover of proteins that are susceptible to proteasome-dependent proteolysis. PMID:15242777

  9. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

    PubMed

    Choy, Milly M; Zhang, Summer L; Costa, Vivian V; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-11-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue. PMID:26565697

  10. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection

    PubMed Central

    Costa, Vivian V.; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-01-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue. PMID:26565697

  11. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

    PubMed

    Choy, Milly M; Zhang, Summer L; Costa, Vivian V; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-11-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.

  12. Inhibition of Tumor Proteasome Activity by Gold Dithiocarbamato Complexes via both Redox-Dependent and –Independent Processes

    PubMed Central

    Milacic, Vesna; Ronconi, Luca; Fan, Yuhua; Bi, Caifeng; Fregona, Dolores; Dou, Q Ping

    2013-01-01

    We have previously reported on a gold(III) complex, namely [AuBr2(DMDT)] (N,N-dimethyldithiocarbamate) showing potent in vitro and in vivo growth inhibitory activities toward human cancer cells and identifying the cellular proteasome as one of the major targets. However, the importance of the oxidation state of the gold center and the involved mechanism of action has yet to be established. Here we show that both gold(III)- and gold(I)-dithiocarbamato species, namely [AuBr2(ESDT)] (AUL12) and [Au(ESDT)]2 (AUL15), could inhibit the chymotrypsin-like activity of purified 20S proteasome and 26S proteasome in human breast cancer MDA-MB-231 cells, resulting in accumulation of ubiquitinated proteins and proteasome target proteins, and induction of cell death, but at significantly different levels. Gold(I) and gold(III) compounds-mediated proteasome inhibition and cell death induction were completely reversed by the addition of a reducing agent, dithiothreitol or N-acetyl-l-cysteine, suggesting the involvement of redox processes. Furthermore, treatment of MDA-MB-231 cells with gold(III) compound (AUL12), but not the gold(I) analogue (AUL15), resulted in the production of significant level of reactive oxygen species. Our study provides strong evidence that the cellular proteasome is an imporant target of both gold(I) and gold(III) dithiocarbamates, but distinct cellular mechanisms of action are responsible for their different overall effect. PMID:19911377

  13. Cellular and computational studies of proteasome inhibition and apoptosis induction in human cancer cells by amino acid Schiff base–copper complexes

    PubMed Central

    Zuo, Jian; Bi, Caifeng; Fan, Yuhua; Buac, Daniela; Nardon, Chiara; Daniel, Kenyon G.; Dou, Q. Ping

    2013-01-01

    Proliferation and apoptosis pathways are tightly regulated in a cell by the ubiquitin–proteasome system (UPS) and alterations in the UPS may result in cellular transformation or other pathological conditions. Indeed, the proteasome is often found to be overactive in cancer cells. It has also been found that cancer cells are more sensitive to proteasome inhibition than normal cells, and therefore proteasome inhibitors are pursued as antitumor drugs. The use of the proteasome inhibitor Bortezomib for treatment of multiple myeloma and mantle cell lymphoma has proved this principle. Recent studies have suggested that copper complexes can inhibit proteasome activity and induce apoptosis in some human cancer cells. However, the involved molecular mechanism is unknown. In this study, we investigated the biological activities of four amino acid Schiff base–copper(II) complexes by using human breast (MDA-MB-231 and MCF-7) and prostate (PC-3) cancer cells. The complexes C1 and C3, but not their counterparts C2 and C4, inhibit the chymotrypsin-like activity of purified 20S proteasome and human cancer cellular 26S proteasome, cause accumulation of proteasome target proteins Bax and IκB-α, and induce growth inhibition and apoptosis in concentration- and time-dependent manners. Docking analysis shows that C1, but not C2 has hydrophobic, pi–pi, pi–cation and hydrogen bond interactions with the proteasomal chymotrypsin-like pocket and could stably fit into the S3 region, leading to specific inhibition. Our study has identified the mechanism of action of these copper complexes on inhibiting tumor cell proteasome and suggested their great potential as novel anticancer agents. PMID:23142973

  14. Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A*

    PubMed Central

    Svozil, Julia; Hirsch-Hoffmann, Matthias; Dudler, Robert; Gruissem, Wilhelm; Baerenfaller, Katja

    2014-01-01

    As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures

  15. Reply to Vangala et al.: Complete inhibition of the proteasome reduces new proteasome production by causing Nrf1 aggregation.

    PubMed

    Sha, Zhe; Goldberg, Alfred L

    2016-09-26

    An important adaptation of cells to proteasome inhibition is the induction of new proteasomes via the transcription factor Nrf1 [1,2], which is produced as a precursor bound to the endoplasmic reticulum (ER) through its amino terminus. Nrf1 was reported to require proteolytic processing to enter the nucleus [3]. Increased proteasome production is induced by low concentrations of proteasome inhibitors that reduce proteolysis by <50%. Surprisingly, in earlier studies we found that proteasome induction and Nrf1 processing to its shorter form (which we estimated to be 75 kDa [2]) were suppressed by high concentrations of inhibitors that markedly reduce proteasome activity [4]. This unusual bimodal concentration dependence implied that some proteasome function was necessary for Nrf1 processing. Because we found that Nrf1 processing also required ubiquitin conjugation [2], we previously proposed that Nrf1 processing is catalyzed by partially inhibited proteasomes [2]. However, Vangala et al.[5] present compelling evidence that conversion of the ER-bound Nrf1 to the shorter form, which they describe as 110 kDa, is independent of proteasomes and is not blocked by high concentrations of proteasome inhibitors. Therefore, we investigated the basis for these differing results. Here we report that we and Vangala et al. have studied the same processed form of Nrf1, the actual molecular weight of which appears to be 90-95 kDa. We confirm our earlier finding [2] that high concentrations of proteasome inhibitors suppress proteasome induction and accumulation of processed Nrf1 in soluble lysates. However, we now show that the inhibitors do so not by blocking Nrf1 processing, but instead by causing the processed Nrf1 to aggregate. Therefore, Nrf1 must be cleaved by a non-proteasomal endoprotease that we show requires ubiquitination. Finally, we provide evidence supporting the recent report that Ddi1/Ddi2 is the critical protease [6,7]. PMID:27676298

  16. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

    SciTech Connect

    Schlax, Peter E.; Zhang Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T. Glen . E-mail: tlawson@bates.edu

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

  17. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

    PubMed

    Schlax, Peter E; Zhang, Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T Glen

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination. PMID:17150238

  18. Induction of tumor cell apoptosis by taurine Schiff base copper complex is associated the with inhibition of proteasomal activity

    PubMed Central

    ZHANG, XIA; BI, CAIFENG; FAN, YUHUA; CUI, QIUZHI; CHEN, DI; XIAO, YAN; DOU, Q. PING

    2013-01-01

    Schiff bases have been intensively investigated due to their antibacterial and antitumor properties. Copper is a cofactor essential for the tumor angiogenesis processes, whereas other transition metals are not. Consistently, high serum or tissue levels of copper were found in many types of human cancer including breast, prostate, colon, lung, and brain, supporting the idea that copper could be used as a novel selective target for cancer therapies. In the current study we hypothesize that a synthetic taurine Schiff base copper complex (Compound 1) could suppress tumor cell growth via the direct inhibition of proteasome activity. Compound 1 potently inhibits the activity of purified 20S and 26S proteasome in human breast cancer MDA-MB-231 and leukemia Jurkat T cells. Inhibition of tumor cellular proteasomal activity by Compound 1 results in the accumulation of ubiquitinated protein and the proteasome target proteins p27 and Bax, followed by the induction of apoptosis. Our results strongly suggest that taurine Schiff base copper complexes, as potent proteasome inhibitors, have great potential to be developed into novel anticancer drugs. PMID:18949390

  19. Proteasome inhibition and its therapeutic potential in multiple myeloma

    PubMed Central

    Chari, Ajai; Mazumder, Amitabha; Jagannath, Sundar

    2010-01-01

    Due to an unmet clinical need for treatment, the first in class proteasome inhibitor, bortezomib, moved from drug discovery to FDA approval in multiple myeloma in an unprecedented eight years. In the wake of this rapid approval arose a large number of questions about its mechanism of action and toxicity as well as its ultimate role in the treatment of this disease. In this article, we briefly review the preclinical and clinical development of the drug as the underpinning for a systematic review of the large number of clinical trials that are beginning to shed some light on the full therapeutic potential of bortezomib in myeloma. We conclude with our current understanding of the mechanism of action of this agent and a discussion of the novel proteasome inhibitors under development, as it will be progress in these areas that will ultimately determine the true potential of proteasome inhibition in myeloma. PMID:21116326

  20. Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression, but Reduces Induced Pluripotent Stem Cell Colony Formation

    PubMed Central

    Floyd, Elizabeth Z.; Staszkiewicz, Jaroslaw; Power, Rachel A.; Kilroy, Gail; Kirk-Ballard, Heather; Barnes, Christian W.; Strickler, Karen L.; Rim, Jong S.; Harkins, Lettie L.; Gao, Ru; Kim, Jeong

    2015-01-01

    Abstract There is ample evidence that the ubiquitin–proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. PMID:25826722

  1. Chronic aspirin via dose-dependent and selective inhibition of cardiac proteasome possibly contributed a potential risk to the ischemic heart.

    PubMed

    Tan, Chunjiang; Chen, Wenlie; Wu, Yanbin; Lin, Jiumao; Lin, Ruhui; Tan, Xuerui; Chen, Songming

    2013-08-01

    Impaired cardiac proteasome has been reported in ischemic heart and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it's unclear whether it affects cardiac proteasome functions. Myocardial infarction (MI), sham or normal male SD rats were injected intraperitoneally with high (300 mg/kg), low (5 mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated human ventricular myocytes. Dose-related increases in heart and ventricular weight, and impaired cardiac functions, were found more exacerbated in the aspirin-treated MI rat hearts than the saline-treated MI counterparts. The activity of 26S, 20S and 19S declined by about 30%, or the 20S proteasome subunits β5, β2 and β1 decreased by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats (P<0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, β5 by 30% and 12%, and β1 by 40% and 30%, respectively, and the lost activity was correlated with the compromised cardiac functions or the decreased cell viability. The dose-related and selective inhibition of 26S and 20S proteasome, or the 20S proteasome subunits β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. The impaired cardiac proteasome, enhanced by chronic aspirin treatment, attenuated the removal of oxidative and ubiquitinated proteins, and chronic aspirin treatment via selective and dose-dependent inhibition of cardiac proteasome possibly constituted a potential risk to ischemic heart.

  2. Inhibition of Interjacent Ribonucleic Acid (26S) Synthesis in Cells Infected by Sindbis Virus

    PubMed Central

    Scheele, Christina M.; Pfefferkorn, E. R.

    1969-01-01

    The interrelationship of viral ribonucleic acid (RNA) and protein synthesis in cells infected by Sindbis virus was investigated. When cultures were treated with puromycin early in the course of infection, the synthesis of interjacent RNA (26S) was preferentially inhibited. A similar result was obtained by shifting cells infected by one temperature-sensitive mutant defective in RNA synthesis from the permissive (29 C) to the nonpermissive (41.5 C) temperature. Under both conditions, the viral RNA produced appeared to be fully active biologically. Once underway, the synthesis of viral RNA in wild-type Sindbis infections did not require concomitant protein synthesis. PMID:5817400

  3. The multiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover.

    PubMed

    van Nocker, S; Sadis, S; Rubin, D M; Glickman, M; Fu, H; Coux, O; Wefes, I; Finley, D; Vierstra, R D

    1996-11-01

    The 26S proteasome is an essential proteolytic complex that is responsible for degrading proteins conjugated with ubiquitin. It has been proposed that the recognition of substrates by the 26S proteasome is mediated by a multiubiquitin-chain-binding protein that has previously been characterized in both plants and animals. In this study, we identified a Saccharomyces cerevisiae homolog of this protein, designated Mcb1. Mcb1 copurified with the 26S proteasome in both conventional and nickel chelate chromatography. In addition, a significant fraction of Mcb1 in cell extracts was present in a low-molecular-mass form free of the 26S complex. Recombinant Mcb1 protein bound multiubiquitin chains in vitro and, like its plant and animal counterparts, exhibited a binding preference for longer chains. Surprisingly, (delta)mcb1 deletion mutants were viable, grew at near-wild-type rates, degraded the bulk of short-lived proteins normally, and were not sensitive to UV radiation or heat stress. These data indicate that Mcb1 is not an essential component of the ubiquitin-proteasome pathway in S.cerevisiae. However, the (delta)mcb1 mutant exhibited a modest sensitivity to amino acid analogs and had increased steady-state levels of ubiquitin-protein conjugates. Whereas the N-end rule substrate, Arg-beta-galactosidase, was degraded at the wild-type rate in the (delta)mcb1 strain, the ubiquitin fusion degradation pathway substrate, ubiquitin-Pro-beta-galactosidase, was markedly stabilized. Collectively, these data suggest that Mcb1 is not the sole factor involved in ubiquitin recognition by the 26S proteasome and that Mcb1 may interact with only a subset of ubiquitinated substrates.

  4. ARS5 is a component of the 26S proteasome complex and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis

    PubMed Central

    Sung, Dong-Yul; Kim, Tae-Houn; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2010-01-01

    Summary A forward genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 is the strongest arsenate and arsenite resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and the arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knockout mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by over expression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild type Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in wild type and this arsenic-induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5 compared to WT suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background. PMID:19453443

  5. Proteasome inhibition induces both antioxidant and hb f responses in sickle cell disease via the nrf2 pathway.

    PubMed

    Pullarkat, Vinod; Meng, Zhuo; Tahara, Stanley M; Johnson, Cage S; Kalra, Vijay K

    2014-01-01

    Oxidant stress is implicated in the manifestations of sickle cell disease including hemolysis and vascular occlusion. Strategies to induce antioxidant response as well as Hb F (α2γ2) have the potential to ameliorate the severity of sickle cell disease. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) is a transcription factor that regulates antioxidant enzymes as well as γ-globin transcription. The Nrf2 in the cytoplasm is bound to its adapter protein Keap-1 that targets Nrf2 for proteasomal degradation, thereby preventing its nuclear translocation. We examined whether inhibiting the 26S proteasome using the clinically applicable proteasome inhibitors bortezomib and MLN 9708 would promote nuclear translocation of Nrf2, and thereby induce an antioxidant response and as well as Hb F in sickle cell disease. Proteasome inhibitors induced reactive oxygen species (ROS) and thereby increased Nrf2-dependent antioxidant enzyme transcripts, elevated cellular glutathione (GSH) levels and γ-globin transcripts as well as Hb F levels in the K562 cell line and also in erythroid burst forming units (BFU-E) generated from peripheral blood mononuclear cells of sickle cell disease patients. These responses were abolished by siRNA-mediated knockdown of Nrf2. Proteasome inhibitors, especially newer oral agents such as MLN9708 have the potential to be readily translated to clinical trials in sickle cell disease with the dual end points of antioxidant response and Hb F induction.

  6. BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation.

    PubMed

    Xu, Dongqing; Jiang, Yan; Li, Jigang; Lin, Fang; Holm, Magnus; Deng, Xing Wang

    2016-07-01

    BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub. PMID:27325768

  7. Proteolytic activity of the 26S proteasome is required for the meiotic resumption, germinal vesicle breakdown, and cumulus expansion of porcine cumulus-oocyte complexes matured in vitro.

    PubMed

    Yi, Young-Joo; Nagyova, Eva; Manandhar, Gaurishankar; Procházka, Radek; Sutovsky, Miriam; Park, Chang-Sik; Sutovsky, Peter

    2008-01-01

    The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 microM MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 microM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 microM). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 microM MG132 and 10 microM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 microM MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin

  8. Selective increase of in vivo firing frequencies in DA SN neurons after proteasome inhibition in the ventral midbrain.

    PubMed

    Subramaniam, Mahalakshmi; Kern, Beatrice; Vogel, Simone; Klose, Verena; Schneider, Gaby; Roeper, Jochen

    2014-09-01

    The impairment of protein degradation via the ubiquitin-proteasome system (UPS) is present in sporadic Parkinson's disease (PD), and might play a key role in selective degeneration of vulnerable dopamine (DA) neurons in the substantia nigra pars compacta (SN). Further evidence for a causal role of dysfunctional UPS in familial PD comes from mutations in parkin, which results in a loss of function of an E3-ubiquitin-ligase. In a mouse model, genetic inactivation of an essential component of the 26S proteasome lead to widespread neuronal degeneration including DA midbrain neurons and the formation of alpha-synuclein-positive inclusion bodies, another hallmark of PD. Studies using pharmacological UPS inhibition in vivo had more mixed results, varying from extensive degeneration to no loss of DA SN neurons. However, it is currently unknown whether UPS impairment will affect the neurophysiological functions of DA midbrain neurons. To answer this question, we infused a selective proteasome inhibitor into the ventral midbrain in vivo and recorded single DA midbrain neurons 2 weeks after the proteasome challenge. We found a selective increase in the mean in vivo firing frequencies of identified DA SN neurons in anesthetized mice, while those in the ventral tegmental area (VTA) were unaffected. Our results demonstrate that a single-hit UPS inhibition is sufficient to induce a stable and selective hyperexcitability phenotype in surviving DA SN neurons in vivo. This might imply that UPS dysfunction sensitizes DA SN neurons by enhancing 'stressful pacemaking'.

  9. The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design.

    PubMed

    Schrader, Jil; Henneberg, Fabian; Mata, Ricardo A; Tittmann, Kai; Schneider, Thomas R; Stark, Holger; Bourenkov, Gleb; Chari, Ashwin

    2016-08-01

    The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics. PMID:27493187

  10. Zinc ionophores pyrithione inhibits herpes simplex virus replication through interfering with proteasome function and NF-κB activation.

    PubMed

    Qiu, Min; Chen, Yu; Chu, Ying; Song, Siwei; Yang, Na; Gao, Jie; Wu, Zhiwei

    2013-10-01

    Pyrithione (PT), known as a zinc ionophore, is effective against several pathogens from the Streptococcus and Staphylococcus genera. The antiviral activity of PT was also reported against a number of RNA viruses. In this paper, we showed that PT could effectively inhibit herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). PT inhibited HSV late gene (Glycoprotein D, gD) expression and the production of viral progeny, and this action was dependent on Zn(2+). Further studies showed that PT suppressed the expression of HSV immediate early (IE) gene, the infected cell polypeptide 4 (ICP4), but had less effect on another regulatory IE protein, ICP0. It was found that PT treatment could interfere with cellular ubiquitin-proteasome system (UPS), leading to the inhibition of HSV-2-induced IκB-α degradation to inhibit NF-κB activation and enhanced promyelocytic leukemia protein (PML) stability in nucleus. However, PT did not show direct inhibition of 26S proteasome activity. Instead, it induced Zn(2+) influx, which facilitated the dysregulation of UPS and the accumulation of intracellular ubiquitin-conjugates. UPS inhibition by PT caused disruption of IκB-α degradation and NF-κB activation thus leading to marked reduction of viral titer. PMID:23867132

  11. The activation sequence of cellular protein handling systems after proteasomal inhibition in dopaminergic cells

    PubMed Central

    Xiong, Rui; Siegel, David; Ross, David

    2013-01-01

    Dysfunction of protein handling has been implicated in many neurodegenerative diseases and inhibition of the ubiquitin-proteasome system (UPS) has been linked to the formation of protein aggregates and proteinopathies in such diseases. While proteasomal inhibition could trigger an array of downstream protein handling changes including up-regulation of heat shock proteins (HSPs), induction of molecular chaperones, activation of the ER stress/unfolded protein response (UPR), autophagy and aggresome formation, little is known of the relationship of proteasomal inhibition to the sequence of activation of these diverse protein handling systems. In this study we utilized the reversible proteasome inhibitor MG132 and examined the activity of several major protein handling systems in the immortalized dopaminergic neuronal N27 cell line. In the early phase (up to 6 hours after proteasomal inhibition), MG132 induced time-dependent proteasomal inhibition which resulted in stimulation of the UPR, increased autophagic flux and stimulated heat shock protein response as determined by increased levels of phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2α), C/EBP homologous protein (CHOP)/GADD153, turnover of autophagy related microtubule-associated protein 1 light chain 3 (LC3) and increased levels of Hsp70 respectively. After prolonged proteasomal inhibition induced by MG132, we observed the formation of vimentin-caged aggresome-like inclusion bodies. A recovery study after MG132-induced proteasomal inhibition indicated that the autophagy-lysosomal pathway participated in the clearance of aggresomes. Our data characterizes the relationship between proteasome inhibition and activation of other protein handling systems. These data also indicated that the induction of alternate protein handling systems and their temporal relationships may be important factors that determine the extent of accumulation of misfolded proteins in cells as a result of

  12. APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome

    PubMed Central

    Palumbo, Rosanna; Gogliettino, Marta; Cocca, Ennio; Iannitti, Roberta; Sandomenico, Annamaria; Ruvo, Menotti; Balestrieri, Marco; Rossi, Mosè; Palmieri, Gianna

    2016-01-01

    The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH–proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21Waf1, and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies. PMID:27669226

  13. APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

    PubMed

    Palumbo, Rosanna; Gogliettino, Marta; Cocca, Ennio; Iannitti, Roberta; Sandomenico, Annamaria; Ruvo, Menotti; Balestrieri, Marco; Rossi, Mosè; Palmieri, Gianna

    2016-01-01

    The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies. PMID:27669226

  14. Inhibition of the purified 20S proteasome by non-heme iron complexes

    PubMed Central

    Prakash, Jai; Schmitt, Sara M.; Dou, Q. Ping; Kodanko, Jeremy J.

    2013-01-01

    Polypyridyl pentadentate ligands N4Py (1) and Bn-TPEN (2), along with their respective iron complexes, have been investigated for their ability to inhibit the purified 20S proteasome. Results demonstrated that the iron complexes of both ligands are potent inhibitors of the 20S proteasome (IC50 = 9.2 μM for [FeII(OH2)(N4Py)]2+ (3) and 4.0 μM for [FeII(OH2)(Bn-TPEN)]2+ (4)). Control experiments showed that ligand 1 or FeII alone showed no inhibition, whereas 2 was moderately active (IC50 = 96 μM), suggesting that iron, when bound to these ligands, plays a key role in proteasome inhibition. Results from time-dependent inactivation studies suggest different modes of action for the iron complexes. Time-dependent decay of proteasome activity was observed upon incubation in the presence of 4, which accelerated in the presence of DTT, suggesting reductive activation of O2 and oxidation of the 20S proteasome as a mode of action. In contrast, loss of 20S proteasome activity was not observed with 3 over time, suggesting inhibition through direct binding of the iron complex to the enzyme. Inhibition of the 20S proteasome by 4 was not blocked by reactive oxygen species scavengers, consistent with a unique oxidant being responsible for the time-dependent inhibition observed. PMID:22170477

  15. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate

    SciTech Connect

    Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.

    2006-01-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

  16. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

    PubMed Central

    Radhakrishnan, Senthil K; den Besten, Willem; Deshaies, Raymond J

    2014-01-01

    Proteasome inhibition elicits an evolutionarily conserved response wherein proteasome subunit mRNAs are upregulated, resulting in recovery (i.e., ‘bounce-back’) of proteasome activity. We previously demonstrated that the transcription factor Nrf1/NFE2L1 mediates this homeostatic response in mammalian cells. We show here that Nrf1 is initially translocated into the lumen of the ER, but is rapidly and efficiently retrotranslocated to the cytosolic side of the membrane in a manner that depends on p97/VCP. Normally, retrotranslocated Nrf1 is degraded promptly by the proteasome and active species do not accumulate. However, in cells with compromised proteasomes, retrotranslocated Nrf1 escapes degradation and is cleaved N-terminal to Leu-104 to yield a fragment that is no longer tethered to the ER membrane. Importantly, this cleavage event is essential for Nrf1-dependent activation of proteasome gene expression upon proteasome inhibition. Our data uncover an unexpected role for p97 in activation of a transcription factor by relocalizing it from the ER lumen to the cytosol. DOI: http://dx.doi.org/10.7554/eLife.01856.001 PMID:24448410

  17. Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer.

    PubMed

    Steg, Adam D; Burke, Mata R; Amm, Hope M; Katre, Ashwini A; Dobbin, Zachary C; Jeong, Dae Hoon; Landen, Charles N

    2014-08-30

    The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of α-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance. PMID:25216523

  18. Transcriptional upregulation of BAG3 upon proteasome inhibition

    SciTech Connect

    Wang Huaqin Liu Haimei; Zhang Haiyan; Guan Yifu; Du Zhenxian

    2008-01-11

    Proteasome inhibitors exhibit antitumoral activity against malignancies of different histology. Emerging evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, thus it seems plausible that activation of survival signaling cascades might compromise their antitumoral effects. Bcl-2-associated athanogene (BAG) family proteins are characterized by their property of interaction with a variety of partners involved in modulating the proliferation/death balance, including heat shock proteins (HSP), Bcl-2, Raf-1. In this report, we demonstrated that BAG3 is a novel antiapoptotic molecule induced by proteasome inhibitors in various cancer cells at the transcriptional level. Moreover, we demonstrated that BAG3 knockdown by siRNA sensitized cancer cells to MG132-induced apoptosis. Taken together, our results suggest that BAG3 induction might represents as an unwanted molecular consequence of utilizing proteasome inhibitors to combat tumors.

  19. Molecular study on copper-mediated tumor proteasome inhibition and cell death.

    PubMed

    Xiao, Yan; Chen, Di; Zhang, Xia; Cui, Qiuzhi; Fan, Yuhua; Bi, Caifeng; Dou, Q Ping

    2010-07-01

    The metal ion copper is a cofactor essential for maintaining normal biological and physical functions in human beings. High copper levels have been found in variety of tumor tissues and are involved in tumor angiogenesis processes. The ubiquitin-proteasome system plays an important role in cell growth and apoptosis and has been shown as a novel target for cancer therapy. We previously reported that some organic copper complexes can inhibit the proteasomal chymotrypsin-like activity and induce apoptosis in human cancer cells and xenograft models. In the current study, we investigated the effect of oxidation status of copper, Cu(I) or Cu(II), on inhibition of proteasome activity, induction of apoptosis, and induction of reactive oxygen species (ROS) in human cancer cells. We report four major findings here: i) both Cu(I) and Cu(II) could inhibit the chymotrypsin-like activity of purified 20S proteasome, but Cu(I) was more potent than Cu(II), ii) purified 20S proteasome protein was able to reduce Cu(II) to Cu(I), suggesting that Cu(I) is the oxidation status of copper that directly reacts with the proteasome, iii) when complexed with the copper ligand neocuproine, Cu(I) showed higher ability to induce ROS production in cancer cells, compared with Cu(II), iv) addition of a ROS scavenger in the cancer cell culture-blocked copper-induced ROS generation, but did not overcome copper-mediated proteasome-inhibitory and cell death-inducing events, demonstrating the ROS-independent proteasome-inhibitory property of copper complexes.

  20. The Proteasome Is a Molecular Target of Environmental Toxic Organotins

    PubMed Central

    Shi, Guoqing; Chen, Di; Zhai, Guangshu; Chen, Marina S.; Cui, Qiuzhi Cindy; Zhou, Qunfang; He, Bin; Dou, Q. Ping; Jiang, Guibin

    2009-01-01

    Background Because of the vital importance of the proteasome pathway, chemicals affecting proteasome activity could disrupt essential cellular processes. Although the toxicity of organotins to both invertebrates and vertebrates is well known, the essential cellular target of organotins has not been well identified. We hypothesize that the proteasome is a molecular target of environmental toxic organotins. Objectives Our goal was to test the above hypothesis by investigating whether organotins could inhibit the activity of purified and cellular proteasomes and, if so, the involved molecular mechanisms and downstream events. Results We found that some toxic organotins [e.g., triphenyltin (TPT)] can potently and preferentially inhibit the chymotrypsin-like activity of purified 20S proteasomes and human breast cancer cellular 26S proteasomes. Direct binding of tin atoms to cellular proteasomes is responsible for the observed irreversible inhibition. Inhibition of cellular proteasomes by TPT in several human cell lines results in the accumulation of ubiquitinated proteins and natural proteasome target proteins, accompanied by induction of cell death. Conclusions The proteasome is one of the molecular targets of environmental toxic organotins in human cells, and proteasome inhibition by organotins contributes to their cellular toxicity. PMID:19337512

  1. Proteasome Inhibition Promotes Parkin-Ubc13 Interaction and Lysine 63-Linked Ubiquitination

    PubMed Central

    Ng, Xiao-Hui; Henry-Basil, Adeline; Sim, Roy W. X.; Tan, Jeanne M. M.; Chai, Chou; Lim, Kah-Leong

    2013-01-01

    Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination (which is usually uncoupled from the proteasome) may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired. PMID:24023840

  2. Physiological levels of ATP Negatively Regulate Proteasome Function

    PubMed Central

    Huang, Hongbiao; Zhang, Xiaoyan; Li, Shujue; Liu, Ningning; Lian, Wen; McDowell, Emily; Zhou, Ping; Zhao, Canguo; Guo, Haiping; Zhang, Change; Yang, Changshan; Wen, Guangmei; Dong, Xiaoxian; Lu, Li; Ma, Ningfang; Dong, Weihua; Dou, Q. Ping; Wang, Xuejun; Liu, Jinbao

    2010-01-01

    Intracellular protein degradation by the ubiquitin-proteasome system is ATP-dependent and the optimal ATP concentration to activate proteasome function in vitro is ~100 μM. Intracellular ATP levels are generally in the low millimolar range but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50–100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated, the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly up-regulate proteasome activity upon ATP reduction under stress conditions. PMID:20805844

  3. Enzyme inhibition by hydroamination: design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor.

    PubMed

    Trivella, Daniela B B; Pereira, Alban R; Stein, Martin L; Kasai, Yusuke; Byrum, Tara; Valeriote, Frederick A; Tantillo, Dean J; Groll, Michael; Gerwick, William H; Moore, Bradley S

    2014-06-19

    Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,β-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.

  4. Enzyme inhibition by hydroamination: Design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor

    PubMed Central

    Trivella, Daniela B. B.; Pereira, Alban R.; Stein, Martin L.; Kasai, Yusuke; Byrum, Tara; Valeriote, Frederick A.; Tantillo, Dean J.; Groll, Michael; Gerwick, William H.; Moore, Bradley S.

    2014-01-01

    Summary Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. Here we report a new mechanism of proteasome inhibition by hydroamination involving alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction resulting in a stable 6-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,β-unsaturated amide-based inhibitors, is partially reversible and gives insight to the design of proteasome inhibitors for cancer chemotherapy. PMID:24930969

  5. Proteasome inhibition enhances the killing effect of BikDD gene therapy.

    PubMed

    Sun, Ye; Ponz-Sarvise, Mariano; Chang, Shih-Shin; Chang, Wei-Chao; Chen, Chung-Hsuan; Hsu, Jennifer L; Hung, Mien-Chie

    2015-01-01

    BikDD, a phosphorylation-mimic mutant of pro-apoptotic protein Bik, elicits strong apoptosis in cancer cells when introduced via an expression platform termed VP16-GAL4-WPRE integrated systemic amplifier (VISA) under the control of a cancer-specific promoter both in vitro and in vivo. C-VISA-BikDD expression plasmid encapsulated in liposomes is currently in the process to initiate a phase I clinical trial for pancreatic cancer. In this study, we report a potential combination approach of BikDD with proteasome inhibitors on the basis of our findings that exogenously expressed BikDD protein undergoes proteasome-mediated degradation via both ubiquitin-dependent and -independent pathways. Inhibition of proteasome increases the protein stability of BikDD, enhancing the apoptotic effect of BikDD. Hence, high proteasome activity may be a mechanism by which intrinsic and acquired resistance occurs in BikDD gene therapy, and a combination therapy with current clinically approved proteasome inhibitor may overcome resistance. PMID:25901200

  6. Inhibition of chymotryptic-like standard proteasome activity exacerbates doxorubicin-induced cytotoxicity in primary cardiomyocytes.

    PubMed

    Spur, Eva-Margarete; Althof, Nadine; Respondek, Dorota; Klingel, Karin; Heuser, Arnd; Overkleeft, Hermen S; Voigt, Antje

    2016-04-15

    The anthracycline doxorubicin (DOX) is a potent anticancer agent for multiple myeloma (MM). A major limitation of this drug is the induction of death in cardiomyocytes leading to heart failure. Here we report on the role of the ubiquitin-proteasome system (UPS) as a critical surveillance pathway for preservation of cell vitality counteracting DOX treatment. Since in addition to DOX also suppression of proteasome activity is a rational therapeutic strategy for MM, we examined how small molecular compounds with clinically relevant proteasome subunit specificity affect DOX cytotoxicity. We found that during DOX-treatment, the activity of the β5 standard proteasome subunit is crucial for limiting off-target cytotoxicity in primary cardiomyocytes. In contrast, we demonstrate that the β5 equivalent LMP7 of the immunoproteasome represents a safe target for subunit-specific inhibitors in DOX-exposed cardiomyocytes. Neither inhibition of LMP7 in primary cardiomyocytes nor genetic ablation of LMP7 in heart tissue influenced the development of DOX cardiotoxicity. Our results indicate that as compared to compounds like carfilzomib, which target both the β5 standard proteasome and the LMP7 immunoproteasome subunit, immunoproteasome-specific inhibitors with known anti-tumor capacity for MM cells might be advantageous for reducing cardiomyocyte death, when a combination therapy with DOX is envisaged.

  7. Proteasome inhibition improves the muscle of laminin α2 chain-deficient mice.

    PubMed

    Carmignac, Virginie; Quéré, Ronan; Durbeej, Madeleine

    2011-02-01

    Muscle atrophy, a significant characteristic of congenital muscular dystrophy with laminin α2 chain deficiency (also known as MDC1A), occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) plays a key role in protein degradation in skeletal muscle cells. In order to identify new targets for drug therapy against MDC1A, we have investigated whether increased proteasomal degradation is a feature of MDC1A. Using the generated dy(3K)/dy(3K) mutant mouse model of MDC1A, we studied the expression of members of the ubiquitin-proteasome pathway in laminin α2 chain-deficient muscle, and we treated dy(3K)/dy(3K) mice with the proteasome inhibitor MG-132. We show that members of the UPS are upregulated and that the global ubiquitination of proteins is raised in dystrophic limb muscles. Also, phosphorylation of Akt is diminished in diseased muscles. Importantly, proteasome inhibition significantly improves the dystrophic dy(3K)/dy(3K) phenotype. Specifically, treatment with MG-132 increases lifespan, enhances locomotive activity, enlarges muscle fiber diameter, reduces fibrosis, restores Akt phosphorylation and decreases apoptosis. These studies promote better understanding of the disease process in mice and could lead to a drug therapy for MDC1A patients.

  8. Nuclear effects of ethanol-induced proteasome inhibition in liver cells

    PubMed Central

    Bardag-Gorce, Fawzia

    2009-01-01

    Alcohol ingestion causes alteration in several cellular mechanisms, and leads to inflammation, apoptosis, immunological response defects, and fibrosis. These phenomena are associated with significant changes in the epigenetic mechanisms, and subsequently, to liver cell memory. The ubiquitin-proteasome pathway is one of the vital pathways in the cell that becomes dysfunctionial as a result of chronic ethanol consumption. Inhibition of the proteasome activity in the nucleus causes changes in the turnover of transcriptional factors, histone modifying enzymes, and therefore, affects epigenetic mechanisms. Alcohol consumption has been associated with an increase in histone acetylation and a decrease in histone methylation, which leads to gene expression changes. DNA and histone modifications that result from ethanol-induced proteasome inhibition are key players in regulating gene expression, especially genes involved in the cell cycle, immunological responses, and metabolism of ethanol. The present review highlights the consequences of ethanol-induced proteasome inhibition in the nucleus of liver cells that are chronically exposed to ethanol. PMID:19291815

  9. Inhibition of 19S proteasome-associated deubiquitinases by metal-containing compounds.

    PubMed

    Liu, Ningning; Huang, Hongbiao; Dou, Q Ping; Liu, Jinbao

    2015-01-01

    Copper and gold complexes have clinical activity in several diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is associated with targeting the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. Here we discuss metal DUB inhibitors in treating cancer and other diseases. (from Editor). Several copper and gold complexes have clinical activity in treating some human diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is tightly associated with their ability to target and inhibit the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. In this article we review small molecule inhibitors of DUBs and 19S proteasome-associated DUBs. We then describe and discuss the ubique nature of CuPT and auranofin, which is inhibition of 19S proteasome-associated UCHL5 and USP14. We finally suggest the potential to develop novel, specific metal-based DUB inhibitors for treating cancer and other diseases. PMID:26097878

  10. Proteasome inhibition by new dual warhead containing peptido vinyl sulfonyl fluorides.

    PubMed

    Brouwer, Arwin J; Herrero Álvarez, Natalia; Ciaffoni, Adriano; van de Langemheen, Helmus; Liskamp, Rob M J

    2016-08-15

    The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long lasting ties. We have introduced peptido sulfonyl fluorides for this purpose. Tuning the reactivity of this electrophilic trap may be crucial for modulating the biological action. Here we describe incorporation of a vinyl moiety into a peptido sulfonyl fluoride backbone, which should lead to a combined attack of the proteasome active site threonine on the double bond and the sulfonyl fluoride. Although this led to strong proteasome inhibitors, in vitro studies did not unambiguously demonstrate the formation of the proposed seven-membered ring structure. Possibly, formation of a seven-membered covalent adduct with the proteosomal active site threonine can only be achieved within the context of the enzyme. Nevertheless, this dual warhead concept may provide exclusive possibilities for duration and selectivity of proteasome inhibition. PMID:27316540

  11. Inhibition of 19S proteasome-associated deubiquitinases by metal-containing compounds

    PubMed Central

    Liu, Ningning; Huang, Hongbiao; Ping Dou, Q.; Liu, Jinbao

    2015-01-01

    Copper and gold complexes have clinical activity in several diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is associated with targeting the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. Here we discuss metal DUB inhibitors in treating cancer and other diseases. (from Editor). Several copper and gold complexes have clinical activity in treating some human diseases including cancer. Recently, we have reported that the anti-cancer activity of copper (II) pyrithione CuPT and gold (I) complex auranofin is tightly associated with their ability to target and inhibit the 19S proteasome-associated deubiquitinases (DUBs), UCHL5 and USP14. In this article we review small molecule inhibitors of DUBs and 19S proteasome-associated DUBs. We then describe and discuss the ubique nature of CuPT and auranofin, which is inhibition of 19S proteasome-associated UCHL5 and USP14. We finally suggest the potential to develop novel, specific metal-based DUB inhibitors for treating cancer and other diseases PMID:26097878

  12. Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay.

    PubMed

    Lee, Susan J; Levitsky, Konstantin; Parlati, Francesco; Bennett, Mark K; Arastu-Kapur, Shirin; Kellerman, Lois; Woo, Tina F; Wong, Alvin F; Papadopoulos, Kyriakos P; Niesvizky, Ruben; Badros, Ashraf Z; Vij, Ravi; Jagannath, Sundar; Siegel, David; Wang, Michael; Ahmann, Gregory J; Kirk, Christopher J

    2016-06-01

    While proteasome inhibition is a validated therapeutic approach for multiple myeloma (MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (ProCISE) assay to quantify proteasome subunit occupancy in samples from five phase I/II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15-56 mg/m(2) ), dose-dependent inhibition of c20S and i20S chymotrypsin-like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells (PBMCs): ≥75%]. A similar inhibition profile was observed in bone marrow-derived CD138(+) tumour cells. Carfilzomib-induced proteasome inhibition was durable, with minimal recovery in PBMCs after 24 h but near-complete recovery between cycles. Importantly, the ProCISE assay can be used to quantify occupancy of individual c20S and i20S subunits. We observed a relationship between MM patient response (n = 29), carfilzomib dose and occupancy of multiple i20S subunits, where greater occupancy was associated with an increased likelihood of achieving a clinical response at higher doses. ProCISE represents a new tool for measuring proteasome inhibitor activity in clinical trials and relating drug action to patient outcomes. PMID:27071340

  13. Proteasome inhibition in skeletal muscle cells unmasks metabolic derangements in type 2 diabetes.

    PubMed

    Al-Khalili, Lubna; de Castro Barbosa, Thais; Ostling, Jörgen; Massart, Julie; Cuesta, Pablo Garrido; Osler, Megan E; Katayama, Mutsumi; Nyström, Ann-Christin; Oscarsson, Jan; Zierath, Juleen R

    2014-11-01

    Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients, marking the existence of an insulin-resistant signature that was retained in cultured myotubes. In conclusion, BZ treatment further exacerbates insulin resistance and unmasks intrinsic features of metabolic disease in myotubes derived from type 2 diabetic patients. Our results highlight the existence of a confounding inherent abnormality in cellular protein dynamics in metabolic disease, which is uncovered through concurrent inhibition of the proteasome system.

  14. Mapping subunit contacts in the regulatory complex of the 26 S proteasome. S2 and S5b form a tetramer with ATPase subunits S4 and S7.

    PubMed

    Gorbea, C; Taillandier, D; Rechsteiner, M

    2000-01-14

    The 19 S regulatory complex (RC) of the 26 S proteasome is composed of at least 18 different subunits, including six ATPases that form specific pairs S4-S7, S6-S8, and S6'-S10b in vitro. One of the largest regulatory complex subunits, S2, was translated in reticulocyte lysate containing [(35)S]methionine and used to probe membranes containing SDS-polyacrylamide gel electrophoresis separated RC subunits. S2 bound to two ATPases, S4 and S7. Association of S2 with regulatory complex subunits was also assayed by co-translation and sedimentation. S2 formed an immunoprecipitable heterotrimer upon co-translation with S4 and S7. The non-ATPase S5b also formed a ternary complex with S4 and S7 and the three proteins assembled into a tetramer with S2. Neither S2 nor S5b formed complexes with S6'-S10b dimers or with S6-S8 oligomers. The use of chimeric ATPases demonstrated that S2 binds the NH(2)-terminal region of S4 and the COOH-terminal two-thirds of S7. Conversely, S5b binds the COOH-terminal two-thirds of S4 and to S7's NH(2)-terminal region. The demonstrated association of S2 with ATPases in the mammalian 19 S regulatory complex is consistent with and extends the recent finding that the yeast RC is composed of two subcomplexes, the lid and the base (Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cejka, Z., Baumeister, W., Fried, V. A., and Finley, D. (1998) Cell 94, 615-623).

  15. Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: Comparison with arsenite

    SciTech Connect

    Noreault-Conti, Trisha L.; Jacobs, Judith M.; Trask, Heidi W.; Wrighton, Steven A.; Sinclair, Jacqueline F.; Nichols, Ralph C. . E-mail: ralph.c.nichols@dartmouth.edu

    2006-12-15

    Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

  16. Anti-tumor activity of benzylideneacetophenone derivatives via proteasomal inhibition in prostate cancer cells.

    PubMed

    Lee, Yun-hee; Yun, Jaesuk; Jung, Jae-Chul; Oh, Seikwan; Jung, Young-Suk

    2016-05-01

    A number of some chalcone derivatives possess promising biological properties including anti-inflammation, anti-oxidant, and anti-tumor activity. Although it has been shown that some derivatives of chalcone induce apoptosis in different kinds of cancer cells, the involved mechanism of action is not well defined. The purpose of this study is to investigate the primary target of a benzylideneacetophenone derivative (JC3), which is a synthetic compound derived from the chalcone family, in human cancer, using prostate cancer cells as a working model. Herein, we show that JC3 inhibits proteasomal activity as indicated by both in vitro and in cell-based assays. Especially, the JC3-dimer was more potent than monomer in the aspect of proteasome inhibition, which induced apoptosis significantly in the prostate cancer cells. Owing to the critical roles of the proteasome in the biology of human tumor progression, invasion, and metastasis, these findings give an important clue for the development of novel anti-tumor agents. PMID:27348972

  17. A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition.

    PubMed

    Rodriguez, Karl A; Osmulski, Pawel A; Pierce, Anson; Weintraub, Susan T; Gaczynska, Maria; Buffenstein, Rochelle

    2014-11-01

    The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31years) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although Heat shock protein 72 kDa (HSP72) and Heat shock protein 40 kDa (Homolog of bacterial DNAJ1) (HSP40(Hdj1)) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging.

  18. Basic Leucine Zipper Protein Cnc-C Is a Substrate and Transcriptional Regulator of the Drosophila 26S Proteasome▿ †

    PubMed Central

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M.; Young, Patrick

    2011-01-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n’ collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis. PMID:21149573

  19. Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells

    PubMed Central

    Sun, Peng; Feng, Li-Xing; Liu, Miao; Hu, Li-Hong; Wu, Wan-Ying; Jiang, Bao-Hong; Yang, Min; Qu, Xiao-Bo; Guo, De-An; Liu, Xuan

    2015-01-01

    Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit. PMID:26512898

  20. Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells.

    PubMed

    Li, Dong; Lu, Yu; Sun, Peng; Feng, Li-Xing; Liu, Miao; Hu, Li-Hong; Wu, Wan-Ying; Jiang, Bao-Hong; Yang, Min; Qu, Xiao-Bo; Guo, De-An; Liu, Xuan

    2015-01-01

    Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit. PMID:26512898

  1. A leucine-supplemented diet restores the defective postprandial inhibition of proteasome-dependent proteolysis in aged rat skeletal muscle

    PubMed Central

    Combaret, Lydie; Dardevet, Dominique; Rieu, Isabelle; Pouch, Marie-Noëlle; Béchet, Daniel; Taillandier, Daniel; Grizard, Jean; Attaix, Didier

    2005-01-01

    We tested the hypothesis that skeletal muscle ubiquitin–proteasome-dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome-dependent proteolysis in incubated muscles from 8- and 22-month-old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22-month-old rats compared with 8-month-old animals, while the rate of ubiquitination was not altered. Furthermore, the down-regulation of in vitro proteasome-dependent proteolysis that prevailed in the postprandial state in 8-month-old rats was defective in 22-month-old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine-supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome-dependent proteolysis in 22-month-old animals, by down-regulating both rates of ubiquitination and proteasome activities. In Experiment 2, we verified that dietary leucine supplementation had long-lasting effects by comparing 8- and 22-month-old rats that were fed either a leucine-supplemented diet or an alanine-supplemented diet for 10 days. The inhibited in vitro proteolysis was maintained in the postprandial state in the 22-month-old rats fed the leucine-supplemented diet. Moreover, elevated mRNA levels for ubiquitin, 14-kDa ubiquitin-conjugating enzyme E2, and C2 and X subunits of the 20S proteasome that were characteristic of aged muscle were totally suppressed in 22-month-old animals chronically fed the leucine-supplemented diet, demonstrating an in vivo effect. Thus the defective postprandial down-regulation of in vitro proteasome-dependent proteolysis in 22-month-old rats was restored in animals chronically fed a leucine-supplemented diet. PMID:16195315

  2. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways

    SciTech Connect

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O.; Kang, Seok-Seong

    2011-01-14

    Research highlights: {yields} Distinct inclusion bodies are developed by inhibition of UPP and ALP. {yields} The inclusion bodies differ in morphology, localization and formation process. {yields} The inclusion bodies are distinguishable by the localization of TSC2. {yields} Inhibition of both UPP and ALP simultaneously induces those inclusion bodies. -- Abstract: Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

  3. The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells

    PubMed Central

    Lagadec, Chann; Vlashi, Erina; Frohnen, Patricia; Alhiyari, Yazeed; Chan, Mabel; Pajonk, Frank

    2014-01-01

    Cancer stem cells (CSCs) or tumor-initiating cells, similar to normal tissue stem cells, rely on developmental pathways, such as the Notch pathway, to maintain their stem cell state. One of the regulators of the Notch pathway is Musashi-1, a mRNA-binding protein. Musashi-1 promotes Notch signaling by binding to the mRNA of Numb, the negative regulator of Notch signaling, thus preventing its translation. Cancer stem cells have also been shown to down-regulate their 26S proteasome activity in several types of solid tumors, thus making them resistant to proteasome-inhibitors used as anti-cancer agents in the clinic. Interestingly, the Notch pathway can be inhibited by proteasomal degradation of the Notch intracellular domain (Notch-ICD), therefore down-regulation of the 26S proteasome activity can lead to stabilization of Notch-ICD. Here we present evidence that the down-regulation of the 26S proteasome in CSCs constitutes another level of control by which Musashi-1 promotes signaling through the Notch pathway and maintenance of the stem cell phenotype of this subpopulation of cancer cells. We demonstrate that Musashi-1 mediates the down-regulation of the 26S proteasome by binding to the mRNA of NF-YA, the transcriptional factor regulating 26S proteasome subunit expression, thus providing an additional route by which the degradation of Notch-ICD is prevented, and Notch signaling is sustained. PMID:24022895

  4. Ni(II), Cu(II), and Zn(II) Diethyldithiocarbamate Complexes Show Various Activities Against the Proteasome in Breast Cancer Cells

    PubMed Central

    Cvek, Boris; Milacic, Vesna; Taraba, Jan; Dou, Q. Ping

    2008-01-01

    A series of three complexes with diethyldithiocarbamate ligand and three different metals (Ni, Cu, Zn) was prepared, confirmed by X-ray crystallography, and tested in human breast cancer MDA-MB-231 cells. Zinc and copper complexes, but not nickel complex, were found to be more active against cellular 26S proteasome than against purified 20S proteasome core particle. One of the possible explanations is inhibition of JAMM domain in the 19S proteasome lid. PMID:18816109

  5. 3,4,5-tricaffeoylquinic acid attenuates proteasome inhibition-mediated programmed cell death in differentiated PC12 cells.

    PubMed

    Nam, Yoon Jeong; Lee, Da Hee; Kim, Yun Jeong; Shin, Yong Kyoo; Sohn, Dong Suep; Lee, Min Sung; Lee, Chung Soo

    2014-08-01

    The dysfunction of the proteasome system is suggested to be implicated in neuronal degeneration. Caffeoylquinic acid derivatives have demonstrated anti-oxidant and anti-inflammatory effects. However, the effect of 3,4,5-tricaffeoylquinic acid on the neuronal cell death induced by proteasome inhibition has not been studied. Therefore, in the respect of cell death process, we assessed the effect of 3,4,5-tricaffeoylquinic acid on the proteasome inhibition-induced programmed cell death using differentiated PC12 cells. The proteasome inhibitors MG132 and MG115 induced a decrease in Bid, Bcl-2, and survivin protein levels, an increase in Bax, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9 and -3), and an increase in the tumor suppressor p53 levels. Treatment with 3,4,5-tricaffeoylquinic acid attenuated the proteasome inhibitor-induced changes in the programmed cell death-related protein levels, formation of reactive oxygen species, GSH depletion and cell death. The results show that 3,4,5-tricaffeoylquinic acid may attenuate the proteasome inhibitor-induced programmed cell death in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The preventive effect of 3,4,5-tricaffeoylquinic acid appears to be attributed to its inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

  6. Fundamental reaction pathway and free energy profile for proteasome inhibition by syringolin A (SylA)

    PubMed Central

    Wei, Donghui; Tang, Mingsheng; Zhan, Chang-Guo

    2015-01-01

    In this study, molecular dynamics (MD) simulations and first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations have been performed to uncover the fundamental reaction pathway of proteasome with a representative inhibitor syringolin A (SylA). The calculated results reveal that the reaction process consists of three steps. The first step is a proton transfer process, activating Thr1-Oγ directly by Thr1-Nz to form a zwitterionic intermediate. The next step is nucleophilic attack on the olefin carbon of SylA by the negatively charged Thr1-Oγ atom. The last step is a proton transfer from Thr1-Nz to another olefin carbon of SylA to complete the inhibition reaction process. The calculated free energy profile demonstrates that the second step should be the rate-determining step and has the highest free energy barrier of 24.6 kcal/mol, which is reasonably close to the activation free energy (∼22.4 – 23.0 kcal/mol) derived from available experimental kinetic data. In addition, our computational results indicate that no water molecule can assist the rate-determining step, since the second step is not involved a proton transfer process. The obtained mechanistic insights should be valuable for understanding the inhibition process of proteasome by SylA and structurally related inhibitors at molecular level, and thus provide a solid mechanistic base and valuable clues for future rational design of novel, more potent inhibitors of proteasome. PMID:26018983

  7. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity☆

    PubMed Central

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q. Ping

    2013-01-01

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 13.8 μM, which was less potent than copper(II) chloride (IC50 5.3 μM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells. PMID:18501397

  8. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity

    SciTech Connect

    Milacic, Vesna; Chen Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q. Ping

    2008-08-15

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC{sub 50} value of 13.8 {mu}M, which was less potent than copper(II) chloride (IC{sub 50} 5.3 {mu}M). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells.

  9. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity.

    PubMed

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q Ping

    2008-08-15

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells. PMID:18501397

  10. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs.

  11. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs. PMID:23053530

  12. Troxerutin inhibits 2,2',4,4'-tetrabromodiphenyl ether (BDE-47)-induced hepatocyte apoptosis by restoring proteasome function.

    PubMed

    Zhang, Zi-Feng; Shan, Qun; Zhuang, Juan; Zhang, Yan-Qiu; Wang, Xin; Fan, Shao-Hua; Lu, Jun; Wu, Dong-Mei; Hu, Bin; Zheng, Yuan-Lin

    2015-03-18

    Proteasome dysfunction has been associated with the pathogeneses of a variety of diseases and with the neurotoxicities of environmental chemicals; however, whether proteasome dysfunction plays a role in the cellular toxicity of polybrominated diphenyl ethers (PBDEs) has not been investigated to date. Emerging evidence suggests that antioxidants exhibit evident beneficial effects on the cellular toxicity associated with PBDEs. In the present study, we investigated whether troxerutin attenuates BDE-47-induced hepatocyte apoptosis by restoring proteasome function and explored the mechanisms underlying this effect. Our results revealed that proteasome dysfunction was involved in the BDE-47-induced hepatocyte apoptosis in the mouse liver. Furthermore, our results revealed that troxerutin effectively inhibited hepatocyte apoptosis by restoring oxidative stress-mediated proteasome dysfunction in BDE-47-treated mice. Consequently, troxerutin markedly suppressed endoplasmic reticulum (ER) stress in the livers of the BDE-47-treated mice. The inhibitory effects of troxerutin on ER stress and apoptotic pathways were markedly blunted by treatment with epoxomicin (a selective inhibitor of proteasome). Ultimately, troxerutin dramatically blocked TRAF2-ASK1-JNK signaling and CHOP-mediated apoptosis signaling in the BDE-47-treated mouse livers. This study provides novel mechanistic insights into the toxicity of BDE-47 and indicates that troxerutin might be a candidate for the prevention of and therapy for BDE-47-induced hepatotoxicity. PMID:25639565

  13. Troxerutin inhibits 2,2',4,4'-tetrabromodiphenyl ether (BDE-47)-induced hepatocyte apoptosis by restoring proteasome function.

    PubMed

    Zhang, Zi-Feng; Shan, Qun; Zhuang, Juan; Zhang, Yan-Qiu; Wang, Xin; Fan, Shao-Hua; Lu, Jun; Wu, Dong-Mei; Hu, Bin; Zheng, Yuan-Lin

    2015-03-18

    Proteasome dysfunction has been associated with the pathogeneses of a variety of diseases and with the neurotoxicities of environmental chemicals; however, whether proteasome dysfunction plays a role in the cellular toxicity of polybrominated diphenyl ethers (PBDEs) has not been investigated to date. Emerging evidence suggests that antioxidants exhibit evident beneficial effects on the cellular toxicity associated with PBDEs. In the present study, we investigated whether troxerutin attenuates BDE-47-induced hepatocyte apoptosis by restoring proteasome function and explored the mechanisms underlying this effect. Our results revealed that proteasome dysfunction was involved in the BDE-47-induced hepatocyte apoptosis in the mouse liver. Furthermore, our results revealed that troxerutin effectively inhibited hepatocyte apoptosis by restoring oxidative stress-mediated proteasome dysfunction in BDE-47-treated mice. Consequently, troxerutin markedly suppressed endoplasmic reticulum (ER) stress in the livers of the BDE-47-treated mice. The inhibitory effects of troxerutin on ER stress and apoptotic pathways were markedly blunted by treatment with epoxomicin (a selective inhibitor of proteasome). Ultimately, troxerutin dramatically blocked TRAF2-ASK1-JNK signaling and CHOP-mediated apoptosis signaling in the BDE-47-treated mouse livers. This study provides novel mechanistic insights into the toxicity of BDE-47 and indicates that troxerutin might be a candidate for the prevention of and therapy for BDE-47-induced hepatotoxicity.

  14. Evidence for anti-apoptotic roles of proteasome activator 28γ via inhibiting caspase activity.

    PubMed

    Moncsek, Anja; Gruner, Melanie; Meyer, Hannes; Lehmann, Andrea; Kloetzel, Peter-Michael; Stohwasser, Ralf

    2015-09-01

    Proteasome activator PA28γ (REGγ, Ki antigen) has recently been demonstrated to display anti-apoptotic properties via enhancing Mdm2-p53 interaction, thereby facilitating ubiquitination and down-regulation of the tumor suppressor p53. In this study we demonstrate a correlation between cellular PA28γ levels and the sensitivity of cells towards apoptosis in different cellular contexts thereby confirming a role of proteasome activator PA28γ as an anti-apoptotic regulator. We investigated the anti-apoptotic role of PA28γ upon UV-C stimulation in B8 mouse fibroblasts stably overexpressing the PA28γ-encoding PSME3 gene and upon butyrate-induced apoptosis in human HT29 adenocarcinoma cells with silenced PSME3 gene. Interestingly, our results demonstrate that PA28γ has a strong influence on different apoptotic hallmarks, especially p53 phosphorylation and caspase activation. In detail, PA28γ and effector caspases mutually restrict each other. PA28γ is a caspase substrate, if PA28γ levels are low. In contrast, PA28γ overexpression reduces caspase activities, including the caspase-dependent processing of PA28γ. Furthermore, overexpression of PA28γ resulted in a nuclear accumulation of transcriptional active p53. In summary, our findings indicate that even in a p53-dominated cellular context, pro-apoptotic signaling might be overcome by PA28γ-mediated caspase inhibition. PMID:26201457

  15. Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2.

    PubMed

    Thacker, Gatha; Kumar, Yogesh; Khan, Mohd Parvez; Shukla, Nidhi; Kapoor, Isha; Kanaujiya, Jitendra Kumar; Lochab, Savita; Ahmed, Shakil; Sanyal, Sabyasachi; Chattopadhyay, Naibedya; Trivedi, Arun Kumar

    2016-04-01

    Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene. PMID:26778333

  16. Paeoniflorin inhibits human glioma cells via STAT3 degradation by the ubiquitin–proteasome pathway

    PubMed Central

    Nie, Xiao-hu; Ou-yang, Jia; Xing, Ying; Li, Dan-yan; Dong, Xing-yu; Liu, Ru-en; Xu, Ru-xiang

    2015-01-01

    We investigated the underlying mechanism for the potent proapoptotic effect of paeoniflorin (PF) on human glioma cells in vitro, focusing on signal transducer and activator of transcription 3 (STAT3) signaling. Significant time- and dose-dependent apoptosis and inhibition of proliferation were observed in PF-treated U87 and U251 glioma cells. Expression of STAT3, its active form phosphorylated STAT3 (p-STAT3), and several downstream molecules, including HIAP, Bcl-2, cyclin D1, and Survivin, were significantly downregulated upon PF treatment. Overexpression of STAT3 induced resistance to PF, suggesting that STAT3 was a critical target of PF. Interestingly, rapid downregulation of STAT3 was consistent with its accelerated degradation, but not with its dephosphorylation or transcriptional modulation. Using specific inhibitors, we demonstrated that the prodegradation effect of PF on STAT3 was mainly through the ubiquitin–proteasome pathway rather than via lysosomal degradation. These findings indicated that PF-induced growth suppression and apoptosis in human glioma cells through the proteasome-dependent degradation of STAT3. PMID:26508835

  17. mTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy.

    PubMed

    Zhao, Jinghui; Zhai, Bo; Gygi, Steven P; Goldberg, Alfred Lewis

    2015-12-29

    Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440-443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth. PMID:26669439

  18. Enhanced rate of degradation of basic proteins by 26S immunoproteasomes.

    PubMed

    Raule, Mary; Cerruti, Fulvia; Cascio, Paolo

    2014-09-01

    Immunoproteasomes are alternative forms of proteasomes specialized in the generation of MHC class I antigenic peptides and important for efficient cytokine production. We have identified a new biochemical property of 26S immunoproteasomes, namely the ability to hydrolyze basic proteins at greatly increased rates compared to constitutive proteasomes. This enhanced degradative capacity is specific for basic polypeptides, since substrates with a lower content in lysine and arginine residues are hydrolyzed at comparable rates by constitutive and immunoproteasomes. Crucially, selective inhibition of the immunoproteasome tryptic subunit β2i strongly reduces degradation of basic proteins. Therefore, our data demonstrate the rate limiting function of the proteasomal trypsin-like activity in controlling turnover rates of basic protein substrates and suggest new biological roles for immunoproteasomes in maintaining cellular homeostasis by rapidly removing a potentially harmful excess of free histones that can build up under different pathophysiological conditions.

  19. Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

    PubMed

    Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, M L B; Mishra, Durga Prasad

    2015-12-22

    Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer.

  20. Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-Gingerol in cervical cancer cells

    PubMed Central

    Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, Madan L.B.; Mishra, Durga Prasad

    2015-01-01

    Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer. PMID:26621832

  1. Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

    PubMed

    Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, M L B; Mishra, Durga Prasad

    2015-12-22

    Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer. PMID:26621832

  2. Gambogic acid induces apoptosis in diffuse large B-cell lymphoma cells via inducing proteasome inhibition

    PubMed Central

    Shi, Xianping; Lan, Xiaoying; Chen, Xin; Zhao, Chong; Li, Xiaofen; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Zang, Dan; Liao, Yuning; Zhang, Peiquan; Wang, Xuejun; Liu, Jinbao

    2015-01-01

    Resistance to chemotherapy is a great challenge to improving the survival of patients with diffuse large B-cell lymphoma (DLBCL), especially those with activated B-cell-like DLBCL (ABC-DLBCL). Therefore it is urgent to search for novel agents for the treatment of DLBCL. Gambogic acid (GA), a small molecule derived from Chinese herb gamboges, has been approved for Phase II clinical trial for cancer therapy by Chinese FDA. In the present study, we investigated the effect of GA on cell survival and apoptosis in DLBCL cells including both GCB- and ABC-DLBCL cells. We found that GA induced growth inhibition and apoptosis of both GCB- and ABC-DLBCL cells in vitro and in vivo, which is associated with proteasome malfunction. These findings provide significant pre-clinical evidence for potential usage of GA in DLBCL therapy particularly in ABC-DLBCL treatment. PMID:25853502

  3. Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation

    PubMed Central

    Lobbestael, E.; Civiero, L.; De Wit, T.; Taymans, J.-M.; Greggio, E.; Baekelandt, V.

    2016-01-01

    Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson’s disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies. PMID:27658356

  4. Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

    PubMed Central

    Tsvetkov, Peter; Mendillo, Marc L; Zhao, Jinghui; Carette, Jan E; Merrill, Parker H; Cikes, Domagoj; Varadarajan, Malini; van Diemen, Ferdy R; Penninger, Josef M; Goldberg, Alfred L; Brummelkamp, Thijn R; Santagata, Sandro; Lindquist, Susan

    2015-01-01

    Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans. DOI: http://dx.doi.org/10.7554/eLife.08467.001 PMID:26327695

  5. Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability.

    PubMed

    Lecomte, S; Desmots, F; Le Masson, F; Le Goff, P; Michel, D; Christians, E S; Le Dréan, Y

    2010-07-22

    A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

  6. Genome wide transcriptional profiling in breast cancer cells reveals distinct changes in hormone receptor target genes and chromatin modifying enzymes after proteasome inhibition

    PubMed Central

    Kinyamu, H. Karimi; Collins, Jennifer B.; Grissom, Sherry F.; Hebbar, Pratibha B.; Archer, Trevor K.

    2010-01-01

    Steroid hormone receptors, like glucocorticoid (GR) and estrogen receptors (ER), are master regulators of genes that control many biological processes implicated in health and disease. Gene expression is dependent on receptor levels which are tightly regulated by the ubiquitin-proteasome system. Previous studies have shown that proteasome inhibition increases GR, but decreases ER-mediated gene expression. At the gene expression level this divergent role of the proteasome in receptor-dependent transcriptional regulation is not well understood. We have used a genomic approach to examine the impact of proteasome activity on GR and ER-mediated gene expression in MCF-7 breast cancer cells treated with dexamethasone (DEX) or 17β-estradiol (E2), the proteasome inhibitor MG132 (MG) or MG132 and either hormone (MD or ME2) for 24h. Transcript profiling reveals that inhibiting proteasome activity modulates gene expression by GR and ER in a similar manner in that several GR and ER target genes are up-regulated and down-regulated after proteasome inhibition. In addition, proteasome inhibition modulates receptor-dependent genes involved in the etiology of a number of human pathological states, including multiple myeloma, leukemia, breast/prostate cancer, HIV/AIDS and neurodegenerative disorders. Importantly, our analysis reveals that a number of transcripts encoding histone and DNA modifying enzymes, prominently histone/DNA methyltransferases and demethylases, are altered after proteasome inhibition. As proteasome inhibitors are currently in clinical trials as therapy for multiple myeloma, HIV/AIDs and leukemia, the possibility that some of the target molecules are hormone regulated and by chromatin modifying enzymes is intriguing in this era of epigenetic therapy. PMID:18381591

  7. Bortezomib Amplifies Effect on Intracellular Proteasomes by Changing Proteasome Structure.

    PubMed

    Pitcher, David S; de Mattos-Shipley, Kate; Tzortzis, Konstantinos; Auner, Holger W; Karadimitris, Anastasios; Kleijnen, Maurits F

    2015-07-01

    The proteasome inhibitor Bortezomib is used to treat multiple myeloma (MM). Bortezomib inhibits protein degradation by inactivating proteasomes' active-sites. MM cells are exquisitely sensitive to Bortezomib - exhibiting a low-nanomolar IC(50) - suggesting that minimal inhibition of degradation suffices to kill MM cells. Instead, we report, a low Bortezomib concentration, contrary to expectation, achieves severe inhibition of proteasome activity in MM cells: the degree of inhibition exceeds what one would expect from the small proportion of active-sites that Bortezomib inhibits. Our data indicate that Bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity. Comparing MM cells to other, Bortezomib-resistant, cancer cells shows that the degree of proteasome inhibition is the greatest in MM cells and only there leads to proteasome stress, providing an explanation for why Bortezomib is effective against MM but not other cancers.

  8. Genetically induced moderate inhibition of 20S proteasomes in cardiomyocytes facilitates heart failure in mice during systolic overload

    PubMed Central

    Ranek, Mark J.; Zheng, Hanqiao; Huang, Wei; Kumarapeli, Asangi R.; Li, Jie; Liu, Jinbao; Wang, Xuejun

    2015-01-01

    The in vivo function status of the ubiquitin-proteasome system (UPS) in pressure overloaded hearts remains undefined. Cardiotoxicity was observed during proteasome inhibitor chemotherapy, especially in those with preexisting cardiovascular conditions; however, proteasome inhibition (PsmI) was also suggested by some experimental studies as a potential therapeutic strategy to curtail cardiac hypertrophy. Here we used genetic approaches to probe cardiac UPS performance and determine the impact of cardiomyocyte-restricted PsmI (CR-PsmI) on cardiac responses to systolic overload. Transgenic mice expressing an inverse reporter of the UPS (GFPdgn) were subject to transverse aortic constriction (TAC) to probe myocardial UPS performance during systolic overload. Mice with or without moderate CR-PsmI were subject to TAC and temporally characterized for cardiac responses to moderate and severe systolic overload. After moderate TAC (pressure gradient: ~40mmHg), cardiac UPS function was upregulated during the first two weeks but turned to functional insufficiency between 6 and 12 weeks as evidenced by the dynamic changes in GFPdgn protein levels, proteasome peptidase activities, and total ubiquitin conjugates. Severe TAC (pressure gradients >60mmHg) led to UPS functional insufficiency within a week. Moderate TAC elicited comparable hypertrophic responses between mice with and without genetic CR-PsmI but caused cardiac malfunction in CR-PsmI mice significantly earlier than those without CR-PsmI. In mice subject to severe TAC, CR-PsmI inhibited cardiac hypertrophy but led to rapidly progressed heart failure and premature death, associated with a pronounced increase in cardiomyocyte death. It is concluded that cardiac UPS function is dynamically altered, with the initial brief upregulation of proteasome function being adaptive; and CR-PsmI facilitates cardiac malfunction during systolic overload. PMID:26116868

  9. Genetically induced moderate inhibition of 20S proteasomes in cardiomyocytes facilitates heart failure in mice during systolic overload.

    PubMed

    Ranek, Mark J; Zheng, Hanqiao; Huang, Wei; Kumarapeli, Asangi R; Li, Jie; Liu, Jinbao; Wang, Xuejun

    2015-08-01

    The in vivo function status of the ubiquitin-proteasome system (UPS) in pressure overloaded hearts remains undefined. Cardiotoxicity was observed during proteasome inhibitor chemotherapy, especially in those with preexisting cardiovascular conditions; however, proteasome inhibition (PsmI) was also suggested by some experimental studies as a potential therapeutic strategy to curtail cardiac hypertrophy. Here we used genetic approaches to probe cardiac UPS performance and determine the impact of cardiomyocyte-restricted PsmI (CR-PsmI) on cardiac responses to systolic overload. Transgenic mice expressing an inverse reporter of the UPS (GFPdgn) were subject to transverse aortic constriction (TAC) to probe myocardial UPS performance during systolic overload. Mice with or without moderate CR-PsmI were subject to TAC and temporally characterized for cardiac responses to moderate and severe systolic overload. After moderate TAC (pressure gradient: ~40mmHg), cardiac UPS function was upregulated during the first two weeks but turned to functional insufficiency between 6 and 12weeks as evidenced by the dynamic changes in GFPdgn protein levels, proteasome peptidase activities, and total ubiquitin conjugates. Severe TAC (pressure gradients >60mmHg) led to UPS functional insufficiency within a week. Moderate TAC elicited comparable hypertrophic responses between mice with and without genetic CR-PsmI but caused cardiac malfunction in CR-PsmI mice significantly earlier than those without CR-PsmI. In mice subject to severe TAC, CR-PsmI inhibited cardiac hypertrophy but led to rapidly progressed heart failure and premature death, associated with a pronounced increase in cardiomyocyte death. It is concluded that cardiac UPS function is dynamically altered, with the initial brief upregulation of proteasome function being adaptive; and CR-PsmI facilitates cardiac malfunction during systolic overload.

  10. Novel 8-hydroxylquinoline analogs induce copper-dependent proteasome inhibition and cell death in human breast cancer cells.

    PubMed

    Milacic, Vesna; Jiao, Peifu; Zhang, Bin; Yan, Bing; Dou, Q Ping

    2009-12-01

    An elevated level of copper (Cu), which is necessary for the growth and metastasis of tumor cells, has been found in many types of cancer, including breast, prostate, lung and brain. Although its molecular basis is unclear, this tumor-specific Cu elevation has been proposed to be a novel target for developing selective anti-cancer therapies. We previously reported that 8-hydroxylquinoline (8-OHQ) is able to form a Cu complex that inhibits the proteasome and induces apoptosis in cultured cancer cells. Toward the goal of discovering novel 8-OHQ analogs as potential anti-copper and anti-cancer drugs, in the current study we synthesized several 8-OHQ analogs and their copper complexes and evaluated their biological activities in human breast cancer cells. We report that when substitutions are made on the hydroxyl group of 8-OHQ, their copper mixtures have profound effects on the proteasome-inhibitory and apoptosis-inducing abilities in breast cancer MDA-MB-231 cells. In addition, the proteasome-inhibitory and apoptosis-inducing activities of 8-OHQ analog-copper mixtures are determined by both the polarity and position of the substituents. Finally, a synthetic complex of 8-OHQ analog-copper was able to inhibit the proteasome activity, induce cell death and suppress the growth selectively in breast cancer MDA-MB-231 cells, but not in normal immortalized human breast MCF-10A cells. Our results support the concept that human cancer cells and tissues, which contain an elevated copper level and are highly dependent on proteasome activity for their survival, should be sensitive to treatment with anti-copper drugs such as the novel 8-OHQ analogs described here.

  11. Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation

    SciTech Connect

    Honma, Yuichi; Harada, Masaru

    2013-08-15

    Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. -- Graphical abstract: Display Omitted -- Highlights: •We examined the cytotoxic mechanisms of sorafenib in hepatoma cells. •Sorafenib induces cell death via apoptotic and necrotic fashion. •Sorafenib inhibits protein ubiquitination and unfolded protein response. •Autophagy induced by sorafenib may affect its cytotoxicity. •Sorafenib inhibits keratin phosphorylation and cytoplasmic inclusion formation.

  12. Consequences of individual N-glycan deletions and of proteasomal inhibition on secretion of active BACE.

    PubMed

    Vanoni, Omar; Paganetti, Paolo; Molinari, Maurizio

    2008-10-01

    BACE is an aspartic protease involved in the production of a toxic peptide accumulating in the brain of Alzheimer's disease patients. After attainment of the native structure in the endoplasmic reticulum (ER), BACE is released into the secretory pathway. To better understand the mechanisms regulating protein biogenesis in the mammalian ER, we determined the fate of five variants of soluble BACE with 4, 3, 2, 1, or 0 N-linked glycans. The number of N-glycans displayed on BACE correlated directly with folding and secretion rates and with the yield of active BACE harvested from the cell culture media. Addition of a single N-glycan was sufficient to recruit the calnexin chaperone system and/or for oligosaccharide de-glucosylation by the ER-resident alpha-glucosidase II. Addition of 1-4 N-glycans progressively enhanced the dissociation rate from BiP and reduced the propensity of newly synthesized BACE to enter aberrant soluble and insoluble aggregates. Finally, inhibition of the proteasome increased the yield of active BACE. This shows that active protein normally targeted for destruction can be diverted for secretion, as if for BACE the quality control system would be acting too stringently in the ER lumen, thus causing loss of functional polypeptides. PMID:18632981

  13. Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis

    PubMed Central

    Ito, Wulf D.; Lund, Natalie; Zhang, Ziyang; Buck, Friedrich; Lellek, Heinrich; Horst, Andrea; Machens, Hans-Günther; Schunkert, Heribert; Schaper, Wolfgang; Meinertz, Thomas

    2015-01-01

    Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo. PMID:26146628

  14. Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study.

    PubMed

    Santoro, Anna Maria; Monaco, Irene; Attanasio, Francesco; Lanza, Valeria; Pappalardo, Giuseppe; Tomasello, Marianna Flora; Cunsolo, Alessandra; Rizzarelli, Enrico; De Luigi, Ada; Salmona, Mario; Milardi, Danilo

    2016-01-01

    Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components. PMID:27633879

  15. Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study

    PubMed Central

    Santoro, Anna Maria; Monaco, Irene; Attanasio, Francesco; Lanza, Valeria; Pappalardo, Giuseppe; Tomasello, Marianna Flora; Cunsolo, Alessandra; Rizzarelli, Enrico; De Luigi, Ada; Salmona, Mario; Milardi, Danilo

    2016-01-01

    Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components. PMID:27633879

  16. Inhibition of all-trans-retinoic acid-induced proteasome activation potentiates the differentiating effect of retinoid in acute myeloid leukemia cells.

    PubMed

    Fang, Yanfen; Zhou, Xinglu; Lin, Meihua; Ying, Meidan; Luo, Peihua; Zhu, Difeng; Lou, Jianshu; Yang, Bo; He, Qiaojun

    2011-01-01

    All-trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation-based therapy in leukemia; however, the mechanisms of ATRA's biological effects remain largely unknown. Here we first reported that ATRA-induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin-protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA-induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA-elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRA's effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin-proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA-dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA-elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy.

  17. The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes.

    PubMed

    Żabka, Aneta; Winnicki, Konrad; Polit, Justyna Teresa; Maszewski, Janusz

    2015-11-01

    DNA topoisomerase II (Topo II), a highly specialized nuclear enzyme, resolves various entanglement problems concerning DNA that arise during chromatin remodeling, transcription, S-phase replication, meiotic recombination, chromosome condensation and segregation during mitosis. The genotoxic effects of two Topo II inhibitors known as potent anti-cancer drugs, etoposide (ETO) and ellipticine (EPC), were assayed in root apical meristem cells of Allium cepa. Despite various types of molecular interactions between these drugs and DNA-Topo II complexes at the chromatin level, which have a profound negative impact on the genome integrity (production of double-strand breaks, chromosomal bridges and constrictions, lagging fragments of chromosomes and their uneven segregation to daughter cell nuclei), most of the elicited changes were apparently similar, regarding both their intensity and time characteristics. No essential changes between ETO- and EPC-treated onion roots were noticed in the frequency of G1-, S-, G2-and M-phase cells, nuclear morphology, chromosome structures, tubulin-microtubule systems, extended distribution of mitosis-specific phosphorylation sites of histone H3, and the induction of apoptosis-like programmed cell death (AL-PCD). However, the important difference between the effects induced by the ETO and EPC concerns their catalytic activities in the presence of MG132 (proteasome inhibitor engaged in Topo II-mediated formation of cleavage complexes) and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. This result implies that proteasome-dependent mechanisms may contribute to the course of physiological effects generated by DNA lesions under conditions that affect the ability of plant cells to resolve topological problems that associated with the nuclear metabolic activities. PMID:26233708

  18. Association of metals and proteasome activity in erythrocytes of prostate cancer patients and controls.

    PubMed

    Neslund-Dudas, Christine; Mitra, Bharati; Kandegedara, Ashoka; Chen, Di; Schmitt, Sara; Shen, Min; Cui, Qiuzhi; Rybicki, Benjamin A; Dou, Q Ping

    2012-10-01

    Information is lacking on the effects toxic environmental metals may have on the 26S proteasome. The proteasome is a primary vehicle for selective degradation of damaged proteins in a cell and due to its role in cell proliferation, inhibition of the proteasome has become a target for cancer therapy. Metals are essential to the proteasome's normal function and have been used within proteasome-inhibiting complexes for cancer therapy. This study evaluated the association of erythrocyte metal levels and proteasome chymotrypsin-like (CT-like) activity in age- and race-matched prostate cancer cases (n=61) and controls (n=61). Erythrocyte metals were measured by inductively coupled plasma mass spectrometry (ICP-MS). CT-like activity was measured by proteasome activity assay using a fluorogenic peptide substrate. Among cases, significant correlations between individual toxic metals were observed (r(arsenic-cadmium)=0.49, p<0.001; r(arsenic-lead)=0.26, p=0.04, r(cadmium-lead) 0.53, p<0.001), but there were no significant associations between metals and CT-like activity. In contrast, within controls there were no significant associations between metals, however, copper and lead levels were significantly associated with CT-like activity. The associations between copper and lead and proteasome activity (r(copper-CT-like)=-0.28, p=0.002 ; r(lead-CT-like)=0.23, p=0.011) remained significant in multivariable models that included all of the metals. These findings suggest that biologically essential metals and toxic metals may affect proteasome activity in healthy controls and, further, show that prostate cancer cases and controls differ in associations between metals and proteasome activity in erythrocytes. More research on toxic metals and the proteasome in prostate cancer is warranted.

  19. Inhibition of Proteasome Activity by Low-dose Bortezomib Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm in Apo E−/− Mice

    PubMed Central

    Ren, Hualiang; Li, Fangda; Tian, Cui; Nie, Hao; Wang, Lei; Li, Hui-Hua; Zheng, Yuehong

    2015-01-01

    Abdominal aortic aneurysm (AAA) is a leading cause of sudden death in aged people. Activation of ubiquitin proteasome system (UPS) plays a critical role in the protein quality control and various diseases. However, the functional role of UPS in AAA formation remains unclear. In this study, we found that the proteasome activities and subunit expressions in AAA tissues from human and angiotensin II (Ang II)-infused apolipoprotein E knockout (Apo E−/−) mice were significantly increased. To investigate the effect of proteasome activation on the AAA formation, Apo E−/− mice were cotreated with bortezomib (BTZ) (a proteasome inhibitor, 50 μg/kg, 2 times per week) and Ang II (1000 ng/kg/min) up to 28 days. Ang II infusion significantly increased the incidence and severity of AAA in Apo E−/− mice, whereas BTZ treatment markedly inhibited proteasome activities and prevented AAA formation. Furthermore, BTZ treatment significantly reduced the inflammation, inhibited the metal matrix metalloprotease activity, and reversed the phenotypic SMC modulation in AAA tissue. In conclusion, these results provide a new evidence that proteasome activation plays a critical role in AAA formation through multiple mechanisms, and suggest that BTZ might be a novel therapeutic target for treatment of AAA formation. PMID:26508670

  20. Inhibition of human preadipocyte proteasomal activity by HIV protease inhibitors or specific inhibitor lactacystin leads to a defect in adipogenesis, which involves matrix metalloproteinase-9.

    PubMed

    De Barros, Sandra; Zakaroff-Girard, Alexia; Lafontan, Max; Galitzky, Jean; Bourlier, Virginie

    2007-01-01

    In a previous publication, we reported that human immunodeficiency virus (HIV) protease inhibitors (PIs) inhibited the differentiation of human preadipocytes in primary culture, reducing the expression and secretion of matrix metalloproteinase 9 (MMP-9). The present work was performed to clarify this mechanism. Interestingly, HIV-PIs have been reported to be inhibitors of the proteasome complex, which is known to regulate nuclear factor (NF)-kappaB activation and transcription of its target genes, among them MMP-9. We thus investigated the potential involvement of the proteasome in the antiadipogenic effects of HIV-PIs. The effect of four HIV-PIs was tested on preadipocyte proteasomal activity, and chronic treatment with the specific proteasome inhibitor lactacystin was performed to evaluate alterations of adipogenesis and MMP-9 expression/secretion. Finally, modifications of the NF-kappaB pathway induced by either HIV-PIs or lactacystin were studied. We demonstrated that preadipocyte proteasomal activity was decreased by several HIV-PIs and that chronic treatment with lactacystin mimicked the effects of HIV-PIs by reducing adipogenesis and MMP-9 expression/secretion. Furthermore, we observed an intracellular accumulation of the NF-kappaB inhibitor, IkappaBbeta, with chronic treatment with HIV-PIs or lactacystin as well as a decrease in MMP-9 expression induced by acute tumor necrosis factor-alpha stimulation. These results indicate that inhibition of the proteasome by specific (lactacystin) or nonspecific (HIV-PIs) inhibitors leads to a reduction of human adipogenesis, and they therefore implicate deregulation of the NF-kappaB pathway and the related decrease of the key adipogenic factor, MMP-9. This study adds significantly to recent reports that have linked HIV-PI-related lipodystrophic syndrome with altered proteasome function, endoplasmic reticulum stress, and metabolic disorders.

  1. Inhibition of TRIP1/S8/hSug1, a component of the human 19S proteasome, enhances mitotic apoptosis induced by spindle poisons.

    PubMed

    Yamada, Hiroshi Y; Gorbsky, Gary J

    2006-01-01

    Mitotic spindle poisons (e.g., Taxol and vinblastine), used as chemotherapy drugs, inhibit mitotic spindle function, activate the mitotic spindle checkpoint, arrest cells in mitosis, and then cause cell death by mechanisms that are poorly understood. By expression cloning, we identified a truncated version of human TRIP1 (also known as S8, hSug1), an AAA (ATPases associated with diverse cellular activities) family ATPase subunit of the 19S proteasome regulatory complex, as an enhancer of spindle poison-mediated apoptosis. Stable expression of the truncated TRIP1/S8/hSug1 in HeLa cells [OP-TRIP1(88-406)] resulted in a decrease of measurable cellular proteasome activity, indicating that OP-TRIP1(88-406) had a dominant-negative effect on proteasome function. OP-TRIP1(88-406) revealed an increased apoptotic response after treatment with spindle poisons or with proteasome inhibitors. The increased apoptosis coincided with a significant decrease in expression of BubR1, a kinase required for activation and maintenance of the mitotic spindle checkpoint in response to treatment with spindle poisons. Small interfering RNA (siRNA)-mediated knockdown of TRIP1/S8/hSug1 resulted in a reduction of general proteasome activity and an increase in mitotic index. The siRNA treatment also caused increased cell death after spindle poison treatment. These results indicate that inhibition of TRIP1/S8/hSug1 function by expression of a truncated version of the protein or by siRNA-mediated suppression enhances cell death in response to spindle poison treatment. Current proteasome inhibitor drugs in trial as anticancer agents target elements of the 20S catalytic subcomplex. Our results suggest that targeting the ATPase subunits in 19S regulatory complex in the proteasome may enhance the antitumor effects of spindle poisons.

  2. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

    PubMed

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion.

  3. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death

    PubMed Central

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A.; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion. PMID:27529620

  4. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

    PubMed

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion. PMID:27529620

  5. A novel dithiocarbamate analogue with potentially decreased ALDH inhibition has copper-dependent proteasome-inhibitory and apoptosis-inducing activity in human breast cancer cells

    PubMed Central

    Wang, Fei; Zhai, Shumei; Liu, Xiaojun; Li, Liwen; Wu, Shirley; Dou, Q. Ping; Yan, Bing

    2013-01-01

    Dithiocarbamates are a class of sulfur-based metal-chelating compounds with various applications in medicine. We reported previously that certain members of dithiocarbamates, such as diethyldithiocarbamate, disulfiram (DSF) and pyrrolidine dithiocarbamate (PDTC), were able to bind with tumor cellular copper to inhibit tumor growth through the inhibition of proteasome activity and induction of cancer cell apoptosis. Since the DSF is an irreversible inhibitor of aldehyde dehydrogenase (ALDH), its ALDH-inhibitory activity might potentially affect its usefulness as an anti-cancer drug. For the purpose of selecting potent anti-cancer compounds that are not ALDH inhibitors and mapping out preliminary structure–activity relationship trends for these novel compounds, we synthesized a series of PDTC analogues and chose three novel compounds to study their ALDH-inhibitory activity, proteasome-inhibitory activity as well as the cancer cell apoptosis-inducing activity. The results showed that compared to DSF, compound 9 has less ALDH inhibition activity, and the in vitro results also proved the positive effects of 9-Cu in proteasome inhibition and apoptosis induction in breast cancer cells, suggesting that 9 as a lead compound could be developed into a novel proteasome inhibitor anti-cancer drug. PMID:21035945

  6. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  7. The proteasome.

    PubMed

    Dalton, William S

    2004-12-01

    The proteasome is an abundant multicatalytic enzyme complex present in the cytoplasm and nucleus of all eukaryotic cells. The primary function of the proteasome is to degrade proteins. While it was once thought to act primarily as a cellular "garbage disposal" that removed damaged or misfolded proteins from cells, the proteasome is now known to also remove various short-lived proteins that regulate the cell cycle, cell growth, and differentiation. By regulating the turnover of these proteins via timely degradation and recycling, the proteasome plays a critical role in the maintenance of cellular homeostasis. Substrates of the proteasome include cell-cycle regulators, signaling molecules, tumor suppressors, transcription factors, and antiapoptotic proteins; over 80% of all cellular proteins are recycled through the proteasome. This article discusses the structure and function of the proteasome, and its role in malignant cells and as a therapeutic target.

  8. Proteasome structure, function, and lessons learned from beta-lactone inhibitors.

    PubMed

    Groll, Michael; Potts, Barbara C

    2011-12-01

    The 26S proteasome is the enzymatic core engine of the ubiquitin and proteasome dependent proteolytic system (UPS), the major eukaryotic pathway for regulated protein degradation. The UPS plays a pivotal role in cellular protein turnover, protein quality control, antigen processing, signal transduction, cell cycle regulation, cell differentiation and apoptosis, inspiring in-depth studies of proteasome structure and function and the search for selective inhibitors. Structural studies revealed that the 26S proteasome comprises up to two 19S regulatory caps flanking a cylindrical 20S core particle, which houses the proteolytic subunits and is present in all kingdoms of life. This review highlights current understanding of 20S architecture, maturation and assembly, the mechanism for selective degradation of protein substrates targeted for destruction, and relationships to other proteases. This knowledge base has benefited from structurally diverse proteasome inhibitors discovered from unique sources, including terrestrial and marine actinomycetes that produce the β-lactone-γ- lactam superfamily of inhibitors, including omuralide, salinosporamide A (marizomib; NPI-0052) and the cinnabaramides. These "minimalist inhibitors" utilize dense functionality to maximum efficiency for potent and selective proteasome inhibition and have advanced from biochemical tools to potential agrochemicals and anticancer agents. In this review, lessons learned from the β-lactone-γ-lactam superfamily are presented, with an emphasis on their unique binding mechanisms elucidated through structural biology in concert with medicinal chemistry. Distinctions between slowly reversible and irreversible inhibitors are discussed, together with the relationship of irreversible binding at the molecular level to prolonged duration proteasome inhibition in tumor cells, and in vitro and in vivo efficacy.

  9. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    SciTech Connect

    Li Lihua; Yang Huanjie; Chen Di; Cui, Cindy; Ping Dou, Q.

    2008-06-01

    The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC{sub 50} value of 32 {mu}mol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

  10. Altered ubiquitin-proteasome signaling in right ventricular hypertrophy and failure.

    PubMed

    Rajagopalan, Viswanathan; Zhao, Mingming; Reddy, Sushma; Fajardo, Giovanni; Wang, Xuejun; Dewey, Shannamar; Gomes, Aldrin V; Bernstein, Daniel

    2013-08-15

    Alterations in the ubiquitin-proteasome system (UPS) have been described in left ventricular hypertrophy and failure, although results have been inconsistent. The role of the UPS in right ventricular (RV) hypertrophy (RVH) and RV failure (RVF) is unknown. Given the greater percent increase in RV mass associated with RV afterload stress, as present in many congenital heart lesions, we hypothesized that alterations in the UPS could play an important role in RVH/RVF. UPS expression and activity were measured in the RV from mice with RVH/RVF secondary to pulmonary artery constriction (PAC). Epoxomicin and MG132 were used to inhibit the proteasome, and overexpression of the 11S PA28α subunit was used to activate the proteasome. PAC mice developed RVH (109.3% increase in RV weight to body weight), RV dilation with septal shift, RV dysfunction, and clinical RVF. Proteasomal function (26S β₅ chymotrypsin-like activity) was decreased 26% (P < 0.05). Protein expression of 19S subunit Rpt5 (P < 0.05), UCHL1 deubiquitinase (P < 0.0001), and Smurf1 E3 ubiquitin ligase (P < 0.01) were increased, as were polyubiquitinated proteins (P < 0.05) and free-ubiquitins (P = 0.05). Pro-apoptotic Bax was increased (P < 0.0001), whereas anti-apoptotic Bcl-2 decreased (P < 0.05), resulting in a sixfold increase in the Bax/Bcl-2 ratio. Proteasomal inhibition did not accelerate RVF. However, proteasome enhancement by cardiac-specific proteasome overexpression partially improved survival. Proteasome activity is decreased in RVH/RVF, associated with upregulation of key UPS regulators and pro-apoptotic signaling. Enhancement of proteasome function partially attenuates RVF, suggesting that UPS dysfunction contributes to RVF.

  11. The recognition of ubiquitinated proteins by the proteasome.

    PubMed

    Grice, Guinevere L; Nathan, James A

    2016-09-01

    The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome. PMID:27137187

  12. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  13. Regulation of the proteasome by ATP: implications for ischemic myocardial injury and donor heart preservation.

    PubMed

    Majetschak, Matthias

    2013-08-01

    Several lines of evidence suggest that proteasomes are involved in multiple aspects of myocardial physiology and pathology, including myocardial ischemia-reperfusion injury. It is well established that the 26S proteasome is an ATP-dependent enzyme and that ischemic heart disease is associated with changes in the ATP content of the cardiomyocyte. A functional link between the 26S proteasome, myocardial ATP concentrations, and ischemic cardiac injury, however, has been suggested only recently. This review discusses the currently available data on the pathophysiological role of the cardiac proteasome during ischemia and reperfusion in the context of the cellular ATP content. Depletion of the myocardial ATP content during ischemia appears to activate the 26S proteasome via direct regulatory effects of ATP on 26S proteasome stability and activity. This implies pathological degradation of target proteins by the proteasome and could provide a pathophysiological basis for beneficial effects of proteasome inhibitors in various models of myocardial ischemia. In contrast to that in the ischemic heart, reduced and impaired proteasome activity is detectable in the postischemic heart. The paradoxical findings that proteasome inhibitors showed beneficial effects when administered during reperfusion in some studies could be explained by their anti-inflammatory and immune suppressive actions, leading to reduction of leukocyte-mediated myocardial reperfusion injury. The direct regulatory effects of ATP on the 26S proteasome have implications for the understanding of the contribution of the 26S proteasome to the pathophysiology of the ischemic heart and its possible role as a therapeutic target.

  14. Proteasome Regulator Marizomib (NPI-0052) Exhibits Prolonged Inhibition, Attenuated Efflux, and Greater Cytotoxicity than Its Reversible Analogs

    PubMed Central

    Obaidat, Amanda; Weiss, Jeffrey; Wahlgren, Brett; Manam, Rama R.; Macherla, Venkat R.; McArthur, Katherine; Chao, Ta-Hsiang; Palladino, Michael A.; Lloyd, G. Kenneth; Potts, Barbara C.; Enna, Salvatore J.; Neuteboom, Saskia T. C.

    2011-01-01

    The present study was undertaken to compare the cellular transport characteristics of [3H]NPI-0052 (1R,4R,5S)-4-(2-chloroethyl)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (marizomib; salinosporamide A) and [3H]NPI-0047 (1R,4R, 5S)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-4-ethyl-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione in RPMI 8226 multiple myeloma and PC-3 prostate adenocarcinoma cells to determine whether these properties explain differences in the cytotoxic potencies of these chemical analogs. The results indicate that marizomib, which possesses a chemical-leaving group, is more cytotoxic to both cell lines and inhibits proteasome activity more completely at lower concentrations than NPI-0047, a nonleaving-group analog. Moreover, it was found that both compounds accumulate in these cells by simple diffusion and the same carrier-mediated transport system. Although the rate of uptake is similar, the cellular efflux, which does not seem to be mediated by a major ATP-binding cassette (ABC)-efflux transporter, is more rapid for NPI-0047 than for marizomib. Experiments revealed that the irreversible binding of marizomib to the proteasome is responsible for its slower efflux, longer duration of action, and greater cytotoxicity compared with NPI-0047. The discovery that major ABC transporters of the multidrug resistance-associated protein family do not seem to be involved in the accumulation or removal of these agents suggests they may not be affected by multidrug resistance mechanisms during prolonged administration. PMID:21303921

  15. Inhibition of Stat5a/b enhances proteasomal degradation of androgen receptor liganded by antiandrogens in prostate cancer

    PubMed Central

    Hoang, David T.; Gu, Lei; Liao, Zhiyong; Talati, Pooja G.; Shen, Feng; Koptyra, Mateusz; Tan, Shyh-Han; Ellsworth, Elyse; Gupta, Shilpa; Montie, Heather; Dagvadorj, Ayush; Savolainen, Saija; Leiby, Benjamin; Mirtti, Tuomas; Merry, Diane E.; Nevalainen, Marja T.

    2015-01-01

    Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, Bicalutamide, Flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the Prostate Specific Antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b. PMID:25552366

  16. Breast cancer cell line MCF7 escapes from G1/S arrest induced by proteasome inhibition through a GSK-3β dependent mechanism

    PubMed Central

    Gavilán, Elena; Giráldez, Servando; Sánchez-Aguayo, Inmaculada; Romero, Francisco; Ruano, Diego; Daza, Paula

    2015-01-01

    Targeting the ubiquitin proteasome pathway has emerged as a rational approach in the treatment of human cancers. Autophagy has been described as a cytoprotective mechanism to increase tumor cell survival under stress conditions. Here, we have focused on the role of proteasome inhibition in cell cycle progression and the role of autophagy in the proliferation recovery. The study was performed in the breast cancer cell line MCF7 compared to the normal mammary cell line MCF10A. We found that the proteasome inhibitor MG132 induced G1/S arrest in MCF10A, but G2/M arrest in MCF7 cells. The effect of MG132 on MCF7 was reproduced on MCF10A cells in the presence of the glycogen synthase kinase 3β (GSK-3β) inhibitor VII. Similarly, MCF7 cells overexpressing constitutively active GSK-3β behaved like MCF10A cells. On the other hand, MCF10A cells remained arrested after MG132 removal while MCF7 recovered the proliferative capacity. Importantly, this recovery was abolished in the presence of the autophagy inhibitor 3-methyladenine (3-MA). Thus, our results support the relevance of GSK-3β and autophagy as two targets for controlling cell cycle progression and proliferative capacity in MCF7, highlighting the co-treatment of breast cancer cells with 3-MA to synergize the effect of the proteasome inhibition. PMID:25941117

  17. Proteasome inhibition blocks ligand-induced dynamic processing and internalization of epidermal growth factor receptor via altered receptor ubiquitination and phosphorylation.

    PubMed

    Kesarwala, Aparna H; Samrakandi, Mustapha M; Piwnica-Worms, David

    2009-02-01

    Epidermal growth factor (EGF) receptor (EGFR), a member of the EGF superfamily of receptor tyrosine kinases, is a critical regulator of cell growth and an important target for single agent and combination anticancer therapeutics. To further investigate the dynamics of ligand-induced EGFR processing and regulation noninvasively, we developed a chimeric EGFR-firefly luciferase (FLuc) fusion reporter to directly monitor processing of EGFR in real-time. In a stable HeLa cell line expressing the reporter at physiologically relevant levels, bioluminescence imaging continuously monitored reporter dynamics, correlating with the ligand-induced response of endogenous EGFR as determined by Western blot, subcellular localization of an EGFR-green fluorescent protein (GFP) fusion protein, and validated pharmacologic responses. The signaling competency of the reporter was confirmed by gene rescue experiments in EGFR-null cells. Bioluminescence analysis further showed that proteasome inhibition with bortezomib or MG132 attenuated overall ligand-induced degradation of EGFR. In cells expressing EGFR-GFP, pretreatment with proteasome inhibitors trapped essentially all of the receptor at the cell membrane both before and after ligand-induced activation with EGF. Furthermore, proteasome inhibition enhanced receptor ubiquitination in both the basal and ligand-activated states as well as delayed the processing of ligand-activated phosphorylation of the receptor, kinetically correlating with attenuated receptor degradation. These observations point to a potential mechanism for the synergistic therapeutic effects of combination EGFR- and proteasome-targeted therapies.

  18. BRICHOS binds to a designed amyloid-forming β-protein and reduces proteasomal inhibition and aggresome formation.

    PubMed

    Dolfe, Lisa; Winblad, Bengt; Johansson, Jan; Presto, Jenny

    2016-01-15

    The BRICHOS domain is associated with proliferative, degenerative and amyloid diseases, and it has been shown to inhibit fibril formation and toxicity of the Alzheimer's disease-associated amyloid β-peptide. ProSP-C (prosurfactant protein C) BRICHOS binds to stretches of hydrophobic amino acid residues, which are unfolded or in β-strand conformation, suggesting that it may have broad anti-amyloid activity. We have studied the effect of the proSP-C BRICHOS domain on the designed amyloidogenic β-sheet proteins β17 and β23. β17 expressed in the secretory pathway of HEK (human embryonic kidney)-293 cells forms intracellular inclusions, whereas β23 is rapidly degraded. Co-expression of BRICHOS leads to a reduction in β17 inclusion size and increased levels of soluble β17 and β23. Furthermore, BRICHOS interacts with the β-proteins intracellularly, reduces their ubiquitination and decreases aggresome formation and proteasomal inhibition. Collectively, these data suggest that BRICHOS is capable of delaying the aggregation process and toxicity of amyloidogenic proteins in a generic manner. PMID:26578816

  19. MiR-29b replacement inhibits proteasomes and disrupts aggresome+autophagosome formation to enhance the antimyeloma benefit of bortezomib

    PubMed Central

    Jagannathan, S; Vad, N; Vallabhapurapu, S; Vallabhapurapu, S; Anderson, K C; Driscoll, J J

    2015-01-01

    Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Although proteasome inhibitors (PIs) have transformed management of multiple myeloma (MM), drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism. Genome-wide profiling identified microRNAs (miRs) differentially expressed in bortezomib-resistant myeloma cells compared with drug-naive cells. The effect of individual miRs on proteasomal degradation of short-lived fluorescent reporter proteins was then determined in live cells. MiR-29b was significantly reduced in bortezomib-resistant cells as well as in cells resistant to second-generation PIs carfilzomib and ixazomib. Luciferase reporter assays demonstrated that miR-29b targeted PSME4 that encodes the proteasome activator PA200. Synthetically engineered miR-29b replacements impaired the growth of myeloma cells, patient tumor cells and xenotransplants. MiR-29b replacements also decreased PA200 association with proteasomes, reduced the proteasome's peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through ubiquitin-independent mechanisms. Immunofluorescence studies revealed that miR-29b replacements enhanced the bortezomib-induced accumulation of ubiquitinated proteins but did not reveal aggresome or autophagosome formation. Taken together, our study identifies miR-29b replacements as the first-in-class miR-based PIs that also disrupt the autophagy pathway and highlight their potential to synergistically enhance the antimyeloma effect of bortezomib. PMID:25234165

  20. Protease inhibition by Heterodera glycines cyst content: evidence for effects on the Meloidogyne incognita proteasome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteases from Heterodera glycines and Meloidogyne incognita juveniles were inhibited by heat-stable content of H. glycines female cysts (HglCE), and by the plant polyphenol epigallocatechin gallate (EGCG). General protease activities detected using the nematode peptide KSAYMRFa were inhibited by EG...

  1. Direct cellular delivery of human proteasomes to delay tau aggregation.

    PubMed

    Han, Dong Hoon; Na, Hee-Kyung; Choi, Won Hoon; Lee, Jung Hoon; Kim, Yun Kyung; Won, Cheolhee; Lee, Seung-Han; Kim, Kwang Pyo; Kuret, Jeff; Min, Dal-Hee; Lee, Min Jae

    2014-01-01

    The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins. PMID:25476420

  2. Proteasome Inhibition Enhances the Induction and Impairs the Maintenance of Late-Phase Long-Term Potentiation

    ERIC Educational Resources Information Center

    Dong, Chenghai; Upadhya, Sudarshan C.; Ding, Lan; Smith, Thuy K.; Hegde, Ashok N.

    2008-01-01

    Protein degradation by the ubiquitin-proteasome pathway plays important roles in synaptic plasticity, but the molecular mechanisms by which proteolysis regulates synaptic strength are not well understood. We investigated the role of the proteasome in hippocampal late-phase long-term potentiation (L-LTP), a model for enduring synaptic plasticity.…

  3. Postnatal Proteasome Inhibition Induces Neurodegeneration and Cognitive Deficiencies in Adult Mice: A New Model of Neurodevelopment Syndrome

    PubMed Central

    Romero-Granados, Rocío; Fontán-Lozano, Ángela; Aguilar-Montilla, Francisco Javier; Carrión, Ángel Manuel

    2011-01-01

    Defects in the ubiquitin-proteasome system have been related to aging and the development of neurodegenerative disease, although the effects of deficient proteasome activity during early postnatal development are poorly understood. Accordingly, we have assessed how proteasome dysfunction during early postnatal development, induced by administering proteasome inhibitors daily during the first 10 days of life, affects the behaviour of adult mice. We found that this regime of exposure to the proteasome inhibitors MG132 or lactacystin did not produce significant behavioural or morphological changes in the first 15 days of life. However, towards the end of the treatment with proteasome inhibitors, there was a loss of mitochondrial markers and activity, and an increase in DNA oxidation. On reaching adulthood, the memory of mice that were injected with proteasome inhibitors postnatally was impaired in hippocampal and amygdala-dependent tasks, and they suffered motor dysfunction and imbalance. These behavioural deficiencies were correlated with neuronal loss in the hippocampus, amygdala and brainstem, and with diminished adult neurogenesis. Accordingly, impairing proteasome activity at early postnatal ages appears to cause morphological and behavioural alterations in adult mice that resemble those associated with certain neurodegenerative diseases and/or syndromes of mental retardation. PMID:22174927

  4. Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2

    PubMed Central

    Arlow, Tim; Scott, Kristan; Wagenseller, Aubrey; Gammie, Alison

    2013-01-01

    MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies. PMID:23248292

  5. Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

    PubMed Central

    Metz, Philippe; Chiramel, Abhilash; Chatel-Chaix, Laurent; Alvisi, Gualtiero; Bankhead, Peter; Mora-Rodríguez, Rodrigo; Long, Gang; Hamacher-Brady, Anne

    2015-01-01

    ABSTRACT Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCE Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the

  6. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    PubMed Central

    Li, Lihua; Yang, Huanjie; Chen, Di; Cui, Cindy; Dou, Q. Ping

    2013-01-01

    The ubiquitinproteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF–Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF–Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF–Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd. PMID:18304598

  7. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

    PubMed Central

    Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

  8. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    PubMed

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

  9. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    PubMed

    Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

  10. Simultaneous inhibition of the ubiquitin-proteasome system and autophagy enhances apoptosis induced by ER stress aggravators in human pancreatic cancer cells.

    PubMed

    Li, Xu; Zhu, Feng; Jiang, Jianxin; Sun, Chengyi; Zhong, Qing; Shen, Ming; Wang, Xin; Tian, Rui; Shi, Chengjian; Xu, Meng; Peng, Feng; Guo, Xingjun; Hu, Jun; Ye, Dawei; Wang, Min; Qin, Renyi

    2016-09-01

    In contrast to normal tissue, cancer cells display profound alterations in protein synthesis and degradation. Therefore, proteins that regulate endoplasmic reticulum (ER) homeostasis are being increasingly recognized as potential therapeutic targets. The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. However, interactions between autophagy, the proteasome, and ER stress pathways in cancer remain largely undefined. This study demonstrated that withaferin-A (WA), the biologically active withanolide extracted from Withania somnifera, significantly increased autophagosomes, but blocked the degradation of autophagic cargo by inhibiting SNARE-mediated fusion of autophagosomes and lysosomes in human pancreatic cancer (PC) cells. WA specifically induced proteasome inhibition and promoted the accumulation of ubiquitinated proteins, which resulted in ER stress-mediated apoptosis. Meanwhile, the impaired autophagy at early stage induced by WA was likely activated in response to ER stress. Importantly, combining WA with a series of ER stress aggravators enhanced apoptosis synergistically. WA was well tolerated in mice, and displayed synergism with ER stress aggravators to inhibit tumor growth in PC xenografts. Taken together, these findings indicate that simultaneous suppression of 2 key intracellular protein degradation systems rendered PC cells vulnerable to ER stress, which may represent an avenue for new therapeutic combinations for this disease. PMID:27308733

  11. Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

    PubMed

    Kwak, Juri; Shim, Joo Hee; Tiwari, Indira; Jang, Kyung Lib

    2016-09-28

    The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV.

  12. Tumor Cellular Proteasome Inhibition and Growth Suppression by 8-Hydroxyquinoline and Clioquinol Requires Their Capabilities to Bind Copper and Transport Copper into Cells

    PubMed Central

    Zhai, Shumei; Yang, Lei; Cui, Qiuzhi Cindy; Sun, Ying; Dou, Q. Ping; Yan, Bing

    2009-01-01

    We have previously reported that when mixed with copper, 8-hydroxyquinoline (8-OHQ) and its analog clioquinol (CQ) inhibited the proteasomal activity and proliferation in cultured human cancer cells. CQ treatment of high copper-containing human tumor xenografts also caused cancer suppression, associated with proteasome inhibition in vivo. However, the nature of copper dependence of these events has not been elucidated experimentally. In the current study, by using chemical probe molecules that mimic structures of 8-OHQ and CQ, but have no copper binding capability, we dissected the complex cellular processes elicited by 8-OHQ-Cu or CQ-Cu mixture and revealed that copper-binding to 8-OHQ or CQ is required for transportation of copper complex into human breast cancer cells and the consequent proteasome-inhibitory, growth-suppressive and apoptosis-inducing activities. In contrast, the non-copper-binding analogs of 8-OHQ or CQ blocked the very first step – copper binding in this chain of events mediated by 8-OHQ-Cu or CQ-Cu. PMID:19809836

  13. Nigral proteasome inhibition in mice leads to motor and non-motor deficits and increased expression of Ser129 phosphorylated α-synuclein

    PubMed Central

    Bentea, Eduard; Van der Perren, Anke; Van Liefferinge, Joeri; El Arfani, Anissa; Albertini, Giulia; Demuyser, Thomas; Merckx, Ellen; Michotte, Yvette; Smolders, Ilse; Baekelandt, Veerle; Massie, Ann

    2015-01-01

    Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 μg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets. PMID:25873870

  14. How the ubiquitin proteasome system regulates the regulators of transcription.

    PubMed

    Ee, Gary; Lehming, Norbert

    2012-01-01

    The ubiquitin proteasome system plays an important role in transcription. Monoubiquitination of activators is believed to aid their function, while the 26S proteasomal degradation of repressors is believed to restrict their function. What remains controversial is the question of whether the degradation of activators aids or restricts their function.

  15. Ubiquitin-proteasome pathway and cellular responses to oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

  16. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

    PubMed

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

  17. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution

    PubMed Central

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism. PMID:26186340

  18. Human cytomegalovirus inhibits apoptosis by proteasome-mediated degradation of Bax at endoplasmic reticulum-mitochondrion contacts.

    PubMed

    Zhang, Aiping; Hildreth, Richard L; Colberg-Poley, Anamaris M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes the UL37 exon 1 protein (pUL37x1), which is the potent viral mitochondrion-localized inhibitor of apoptosis (vMIA), to increase survival of infected cells. HCMV vMIA traffics from the endoplasmic reticulum (ER) to ER subdomains, which are physically linked to mitochondria known as mitochondrion-associated membranes (MAM), and to mitochondria. The antiapoptotic function of vMIA is thought to primarily result from its ability to inhibit Bax-mediated permeabilization of the outer mitochondrial membrane (OMM). Here, we establish that vMIA retargets Bax to the MAM as well as to the OMM from immediate early through late times of infection. However, MAM localization of Bax results in its increased ubiquitination and proteasome-mediated degradation. Surprisingly, HCMV infection does not increase OMM-associated degradation (OMMAD) of Bax, even though the ER and mitochondria are physically connected at the MAM. It was recently found that lipid rafts at the plasma membrane can connect extrinsic and intrinsic apoptotic pathways and can serve as sites of apoptosome assembly. In transfected permissive human fibroblasts, vMIA mediates, through its cholesterol affinity, association of Bax and apoptosome components with MAM lipid rafts. While Bax association with MAM lipid rafts was detected in HCMV-infected cells, association of apoptosome components was not. These results establish that Bax recruitment to the MAM and its MAM-associated degradation (MAMAD) are a newly described antiapoptotic mechanism used by HCMV infection to increase cell survival for its growth.

  19. Synthetic and structural studies on syringolin A and B reveal critical determinants of selectivity and potency of proteasome inhibition

    PubMed Central

    Clerc, Jérôme; Groll, Michael; Illich, Damir J.; Bachmann, André S.; Huber, Robert; Schellenberg, Barbara; Dudler, Robert; Kaiser, Markus

    2009-01-01

    Syrbactins, a family of natural products belonging either to the syringolin or glidobactin class, are highly potent proteasome inhibitors. Although sharing similar structural features, they differ in their macrocyclic lactam core structure and exocyclic side chain. These structural variations critically influence inhibitory potency and proteasome subsite selectivity. Here, we describe the total synthesis of syringolin A and B, which together with enzyme kinetic and structural studies, allowed us to elucidate the structural determinants underlying the proteasomal subsite selectivity and binding affinity of syrbactins. These findings were used successfully in the rational design and synthesis of a syringolin A-based lipophilic derivative, which proved to be the most potent syrbactin-based proteasome inhibitor described so far. With a Ki′ of 8.65 ± 1.13 nM for the chymotryptic activity, this syringolin A derivative displays a 100-fold higher potency than the parent compound syringolin A. In light of the medicinal relevance of proteasome inhibitors as anticancer compounds, the present findings may assist in the rational design and development of syrbactin-based chemotherapeutics. PMID:19359491

  20. Targeting Tumor Proteasome with Traditional Chinese Medicine

    PubMed Central

    Yang, Huanjie; Liu, Jinbao; Dou, Q. Ping

    2012-01-01

    The proteasome is a multicatalytic protease complex whose activity is required for the growth of normal or tumor cells. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that the proteasome could be a target of chemotherapy. Studies suggest that traditional Chinese medicine (TCM) is an effective approach for cancer treatment. Here we reviewed several TCMs for their potential in treatment of cancer. This short review focuses mainly on the TCMs that potentially target the tumor cellular proteasome and NF-κB pathway whose activation is dependent on the proteasome activity. PMID:20156140

  1. Ubiquitin-proteasome pathway and cellular responses to oxidative stress

    PubMed Central

    Taylor, Allen

    2011-01-01

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in non-canonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. While many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin conjugation enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin conjugation enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells appear to be an indicator of mild oxidative stress. PMID:21530648

  2. Proteasome Activators

    PubMed Central

    Stadtmueller, Beth M.; Hill, Christopher P.

    2011-01-01

    Summary Proteasomes degrade a multitude of protein substrates in the cytosol and nucleus, and thereby are essential for many aspects of cellular function. Because the proteolytic sites are sequestered in a closed barrel-shaped structure, activators are required to facilitate substrate access. Structural and biochemical studies of two activator families, 11S and Blm10, have provided insights to proteasome activation mechanisms, although the biological functions of these factors remain obscure. Recent advances have improved our understanding of the third activator family, including the 19S activator, which targets polyubiquitylated proteins for degradation. PMID:21211719

  3. A Bowman–Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition

    PubMed Central

    Mehdad, A; Brumana, G; Souza, AA; Barbosa, JARG; Ventura, MM; de Freitas, SM

    2016-01-01

    Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman–Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment. PMID:27551492

  4. A Bowman-Birk inhibitor induces apoptosis in human breast adenocarcinoma through mitochondrial impairment and oxidative damage following proteasome 20S inhibition.

    PubMed

    Mehdad, A; Brumana, G; Souza, A A; Barbosa, Jarg; Ventura, M M; de Freitas, S M

    2016-01-01

    Proteasome inhibitors are emerging as a new class of chemopreventive agents and have gained huge importance as potential pharmacological tools in breast cancer treatment. Improved understanding of the role played by proteases and their specific inhibitors in humans offers novel and challenging opportunities for preventive and therapeutic intervention. In this study, we demonstrated that the Bowman-Birk protease inhibitor from Vigna unguiculata seeds, named black-eyed pea trypsin/chymotrypsin Inhibitor (BTCI), potently suppresses human breast adenocarcinoma cell viability by inhibiting the activity of proteasome 20S. BTCI induced a negative growth effect against a panel of breast cancer cells, with a concomitant cytostatic effect at the G2/M phase of the cell cycle and an increase in apoptosis, as observed by an augmented number of cells at the sub-G1 phase and annexin V-fluorescin isothiocyanate (FITC)/propidium iodide (PI) staining. In contrast, BTCI exhibited no cytotoxic effect on normal mammary epithelial cells. Moreover, the increased levels of intracellular reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in cells treated with BTCI indicated mitochondrial damage as a crucial cellular event responsible for the apoptotic process. The higher activity of caspase in tumoral cells treated with BTCI in comparison with untreated cells suggests that BTCI induces apoptosis in a caspase-dependent manner. BTCI affected NF-kB target gene expression in both non invasive and invasive breast cancer cell lines, with the effect highly pronounced in the invasive cells. An increased expression of interleukin-8 (IL-8) in both cell lines was also observed. Taken together, these results suggest that BTCI promotes apoptosis through ROS-induced mitochondrial damage following proteasome inhibition. These findings highlight the pharmacological potential and benefit of BTCI in breast cancer treatment. PMID:27551492

  5. Hinokitiol inhibits vasculogenic mimicry activity of breast cancer stem/progenitor cells through proteasome-mediated degradation of epidermal growth factor receptor

    PubMed Central

    TU, DOM-GENE; YU, YUN; LEE, CHE-HSIN; KUO, YU-LIANG; LU, YIN-CHE; TU, CHI-WEN; CHANG, WEN-WEI

    2016-01-01

    Hinokitiol, alternatively known as β-thujaplicin, is a tropolone-associated natural compound with antimicrobial, anti-inflammatory and antitumor activity. Breast cancer stem/progenitor cells (BCSCs) are a subpopulation of breast cancer cells associated with tumor initiation, chemoresistance and metastatic behavior, and may be enriched by mammosphere cultivation. Previous studies have demonstrated that BCSCs exhibit vasculogenic mimicry (VM) activity via the epidermal growth factor receptor (EGFR) signaling pathway. The present study investigated the anti-VM activity of hinokitiol in BCSCs. At a concentration below the half maximal inhibitory concentration, hinokitiol inhibited VM formation of mammosphere cells derived from two human breast cancer cell lines. Hinokitiol was additionally indicated to downregulate EGFR protein expression in mammosphere-forming BCSCs without affecting the expression of messenger RNA. The protein stability of EGFR in BCSCs was also decreased by hinokitiol. The EGFR protein expression and VM formation capability of hinokitiol-treated BCSCs were restored by co-treatment with MG132, a proteasome inhibitor. In conclusion, the present study indicated that hinokitiol may inhibit the VM activity of BCSCs through stimulating proteasome-mediated EGFR degradation. Hinokitiol may act as an anti-VM agent, and may be useful for the development of novel breast cancer therapeutic agents. PMID:27073579

  6. Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues

    PubMed Central

    Svozil, Julia; Gruissem, Wilhelm; Baerenfaller, Katja

    2015-01-01

    Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions. PMID:26074939

  7. PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

    PubMed Central

    Errafiy, Rajaa; Aguado, Carmen; Ghislat, Ghita; Esteve, Juan M.; Gil, Anabel; Loutfi, Mohammed; Knecht, Erwin

    2013-01-01

    Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation. PMID:24349488

  8. TSGΔ154-1054 splice variant increases TSG101 oncogenicity by inhibiting its E3-ligase-mediated proteasomal degradation

    PubMed Central

    Weng, Pei-Lun; Yeh, Te-Huei

    2016-01-01

    Tumor susceptibility gene 101 (TSG101) elicits an array of cellular functions, including promoting cytokinesis, cell cycle progression and proliferation, as well as facilitating endosomal trafficking and viral budding. TSG101 protein is highly and aberrantly expressed in various human cancers. Specifically, a TSG101 splicing variant missing nucleotides 154 to 1054 (TSGΔ154-1054), which is linked to progressive tumor-stage and metastasis, has puzzled investigators for more than a decade. TSG101-associated E3 ligase (Tal)- and MDM2-mediated proteasomal degradation are the two major routes for posttranslational regulation of the total amount of TSG101. We reveal that overabundance of TSG101 results from TSGΔ154-1054 stabilizing the TSG101 protein by competitively binding to Tal, but not MDM2, thereby perturbing the Tal interaction with TSG101 and impeding subsequent polyubiquitination and proteasomal degradation of TSG101. TSGΔ154-1054 therefore specifically enhances TSG101-stimulated cell proliferation, clonogenicity, and tumor growth in nude mice. This finding shows the functional significance of TSGΔ154-1054 in preventing the ubiquitin-proteasome proteolysis of TSG101, which increases tumor malignancy and hints at its potential as a therapeutic target in cancer treatment. PMID:26811492

  9. Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

    PubMed

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-06-20

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

  10. Novel sphingosine-containing analogues selectively inhibit sphingosine kinase (SK) isozymes, induce SK1 proteasomal degradation and reduce DNA synthesis in human pulmonary arterial smooth muscle cells

    PubMed Central

    Byun, Hoe-Sup; Pyne, Susan; MacRitchie, Neil; Pyne, Nigel J.

    2013-01-01

    Sphingosine 1-phosphate (S1P) is involved in hyper-proliferative diseases such as cancer and pulmonary arterial hypertension. We have synthesized inhibitors that are selective for the two isoforms of sphingosine kinase (SK1 and SK2) that catalyze the synthesis of S1P. A thiourea adduct of sphinganine (F02) is selective for SK2 whereas the 1-deoxysphinganines 55-21 and 77-7 are selective for SK1. (2S,3R)-1-Deoxysphinganine (55-21) induced the proteasomal degradation of SK1 in human pulmonary arterial smooth muscle cells and inhibited DNA synthesis, while the more potent SK1 inhibitors PF-543 and VPC96091 failed to inhibit DNA synthesis. These findings indicate that moderate potency inhibitors such as 55-21 are likely to have utility in unraveling the functions of SK1 in inflammatory and hyperproliferative disorders. PMID:24396570

  11. [Proteasome inhibitors in cancer therapy].

    PubMed

    Romaniuk, Wioletta; Ołdziej, Agnieszka Ewa; Zińczuk, Justyna; Kłoczko, Janusz

    2015-01-01

    Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052). PMID:27259216

  12. E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models

    PubMed Central

    Prideaux, Matt; Allen, Steve; Buttle, David J.; Pitsillides, Andrew A.; Farquharson, Colin

    2015-01-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast–osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO‐A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post‐translational degradation. We found that exogenous treatment of MLO‐A5 and osteocytic IDG‐SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome‐selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin‐A) produced similar dose‐dependent increases in E11 protein levels in MLO‐A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO‐A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome‐mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. J. Cell

  13. MCPIP1 contributes to the toxicity of proteasome inhibitor MG-132 in HeLa cells by the inhibition of NF-κB.

    PubMed

    Skalniak, Lukasz; Dziendziel, Monika; Jura, Jolanta

    2014-10-01

    Recently, we have shown that the treatment of cells with proteasome inhibitor MG-132 results in the induction of expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1). MCPIP1 is a ribonuclease, responsible for the degradation of transcripts encoding certain pro-inflammatory cytokines. The protein is also known as an inhibitor of NF-κB transcription factor. Thanks to its molecular properties, MCPIP1 is considered as a regulator of inflammation, differentiation, and survival. Using siRNA technology, we show here that MCPIP1 expression contributes to the toxic properties of MG-132 in HeLa cells. The inhibition of proteasome by MG-132 and epoxomicin markedly increased MCPIP1 expression. While MG-132 induces HeLa cell death, down-regulation of MCPIP1 expression by siRNA partially protects HeLa cells from MG-132 toxicity and restores Nuclear factor-κB (NF-κB) activity, inhibited by MG-132 treatment. Inversely, overexpression of MCPIP1 decreased constitutive activity of NF-κB and limited the survival of HeLa cells, as we have shown in the previous study. Interestingly, although MG-132 decreased the expression of IκBα and increased p65 phosphorylation, the inhibition of constitutive NF-κB activity was observed in MG-132-treated cells. Since the elevated constitutive activity of NF-κB is one of the mechanisms providing increased survival of cancer cells, including HeLa cells, we propose that death-promoting properties of MCPIP1 in MG-132-treated HeLa cells may, at least partially, derive from the negative effect on the constitutive NF-κB activity.

  14. An evolutionarily conserved pathway controls proteasome homeostasis.

    PubMed

    Rousseau, Adrien; Bertolotti, Anne

    2016-08-11

    The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation. PMID:27462806

  15. Proteasome Inhibition Increases Recruitment of IκB Kinase β (IKKβ), S536P-p65, and Transcription Factor EGR1 to Interleukin-8 (IL-8) Promoter, Resulting in Increased IL-8 Production in Ovarian Cancer Cells*

    PubMed Central

    Singha, Bipradeb; Gatla, Himavanth Reddy; Manna, Subrata; Chang, Tzu-Pei; Sanacora, Shannon; Poltoratsky, Vladimir; Vancura, Ales; Vancurova, Ivana

    2014-01-01

    Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase β (IKKβ) and an increased recruitment of the nuclear IKKβ, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKβ and with p65, with preferential binding to S536P-p65. Both IKKβ activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKβ and EGR-1 as potential new targets in ovarian cancer combination therapies. PMID:24337575

  16. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji Saeki, Yasushi

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

  17. Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy.

    PubMed

    Sakata, Eri; Bohn, Stefan; Mihalache, Oana; Kiss, Petra; Beck, Florian; Nagy, Istvan; Nickell, Stephan; Tanaka, Keiji; Saeki, Yasushi; Förster, Friedrich; Baumeister, Wolfgang

    2012-01-31

    Two canonical subunits of the 26S proteasome, Rpn10 and Rpn13, function as ubiquitin (Ub) receptors. The mutual arrangement of these subunits--and all other non-ATPase subunits--in the regulatory particle is unknown. Using electron cryomicroscopy, we calculated difference maps between wild-type 26S proteasome from Saccharomyces cerevisiae and deletion mutants (rpn10Δ, rpn13Δ, and rpn10Δrpn13Δ). These maps allowed us to localize the two Ub receptors unambiguously. Rpn10 and Rpn13 mapped to the apical part of the 26S proteasome, above the N-terminal coiled coils of the AAA-ATPase heterodimers Rpt4/Rpt5 and Rpt1/Rpt2, respectively. On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome.

  18. Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy

    PubMed Central

    Sakata, Eri; Bohn, Stefan; Mihalache, Oana; Kiss, Petra; Beck, Florian; Nagy, Istvan; Nickell, Stephan; Tanaka, Keiji; Saeki, Yasushi; Förster, Friedrich; Baumeister, Wolfgang

    2012-01-01

    Two canonical subunits of the 26S proteasome, Rpn10 and Rpn13, function as ubiquitin (Ub) receptors. The mutual arrangement of these subunits—and all other non-ATPase subunits—in the regulatory particle is unknown. Using electron cryomicroscopy, we calculated difference maps between wild-type 26S proteasome from Saccharomyces cerevisiae and deletion mutants (rpn10Δ, rpn13Δ, and rpn10Δrpn13Δ). These maps allowed us to localize the two Ub receptors unambiguously. Rpn10 and Rpn13 mapped to the apical part of the 26S proteasome, above the N-terminal coiled coils of the AAA-ATPase heterodimers Rpt4/Rpt5 and Rpt1/Rpt2, respectively. On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome. PMID:22215586

  19. Proteasome as a Molecular Target of Microcystin-LR

    PubMed Central

    Zhu, Zhu; Zhang, Li; Shi, Guoqing

    2015-01-01

    Proteasome degrades proteins in eukaryotic cells. As such, the proteasome is crucial in cell cycle and function. This study proved that microcystin-LR (MC-LR), which is a toxic by-product of algal bloom, can target cellular proteasome and selectively inhibit proteasome trypsin-like (TL) activity. MC-LR at 1 nM can inhibit up to 54% of the purified 20S proteasome TL activity and 43% of the proteasome TL activity in the liver of the cyprinid rare minnow (Gobiocypris rarus). Protein degradation was retarded in GFP-CL1-transfected PC-3 cells because MC-LR inhibited the proteasome TL activity. Docking studies indicated that MC-LR blocked the active site of the proteasome β2 subunit; thus, the proteasome TL activity was inhibited. In conclusion, MC-LR can target proteasome, selectively inhibit proteasome TL activity, and retard protein degradation. This study may be used as a reference of future research on the toxic mechanism of MC-LR. PMID:26090622

  20. Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization.

    PubMed

    Song, Won-Hee; Yi, Young-Joo; Sutovsky, Miriam; Meyers, Stuart; Sutovsky, Peter

    2016-09-01

    Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules. PMID:27551072

  1. Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

    PubMed Central

    Jazirehi, Ali R.; Economou, James S.

    2012-01-01

    Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

  2. Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3{beta}

    SciTech Connect

    Choi, Cheol-Hee; Lee, Byung-Hoon; Ahn, Sang-Gun; Oh, Seon-Hee

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer MG132 induces the phosphorylation of GSK3{beta}{sup Ser9} and, to a lesser extent, of GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer MG132 induces dephosphorylation of p70S6K{sup Thr389} and phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 dephosphorylates GSK3{beta}{sup Ser9} and phosphorylates GSK3{beta}{sup Thr390}. Black-Right-Pointing-Pointer Inactivation of p38 phosphorylates p70S6K{sup Thr389} and increases the phosphorylation of p70S6K{sup Thr421/Ser424}. Black-Right-Pointing-Pointer Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3{beta} (GSK3{beta}) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3{beta} at Ser{sup 9} and, to a lesser extent, Thr{sup 390}, the dephosphorylation of p70S6K at Thr{sup 389}, and the phosphorylation of p70S6K at Thr{sup 421} and Ser{sup 424}. The specific p38 inhibitor SB203080 reduced the p-GSK3{beta}{sup Ser9} and autophagy through the phosphorylation of p70S6K{sup Thr389}; however, it augmented the levels of p-ERK, p-GSK3{beta}{sup Thr390}, and p-70S6K{sup Thr421/Ser424} induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our

  3. Suppression of BRCA1 sensitizes cells to proteasome inhibitors

    PubMed Central

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  4. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

    The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome. PMID:20008553

  5. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S [beta]5-subunit

    SciTech Connect

    Blackburn, Christopher; Gigstad, Kenneth M.; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J.; Barrett, Cynthia; Liu, Jane X.; Soucy, Teresa A.; Sappal, Darshan S.; Bump, Nancy; Olhava, Edward J.; Fleming, Paul; Dick, Lawrence R.; Tsu, Christopher; Sintchak, Michael D.; Blank, Jonathan L.

    2012-04-30

    The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the {beta}5 (chymotrypsin-like) site over the {beta}1 (caspase-like) and {beta}2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC{sub 50} values for the human 20S {beta}5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NF{Kappa}B (nuclear factor {Kappa}B) in response to TNF-{alpha} (tumor necrosis factor-{alpha}) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the {beta}5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the {beta}5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

  6. Gambogic Acid Induces Apoptosis in Imatinib-Resistant Chronic Myeloid Leukemia Cells via Inducing Proteasome Inhibition and Caspase-Dependent Bcr-Abl Downregulation

    PubMed Central

    Shi, Xianping; Chen, Xin; Li, Xiaofen; Lan, Xiaoying; Zhao, Chong; Liu, Shouting; Huang, Hongbiao; Liu, Ningning; Liao, Siyan; Song, Wenbin; Zhou, Ping; Wang, Shunqing; Xu, Li; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    Purpose Chronic myelogenous leukemia (CML) is characterized by the constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl-T315I is the predominant mutation that causes resistance to imatinib, cytotoxic drugs, and the second-generation tyrosine kinase inhibitors. The emergence of imatinib resistance in patients with CML leads to searching for novel approaches to the treatment of CML. Gambogic acid, a small molecule derived from Chinese herb gamboges, has been approved for phase II clinical trial for cancer therapy by the Chinese Food and Drug Administration (FDA). In this study, we investigated the effect of gambogic acid on cell survival or apoptosis in CML cells bearing Bcr-Abl-T315I or wild-type Bcr-Abl. Experimental Design CML cell lines (KBM5, KBM5-T315I, and K562), primary cells from patients with CML with clinical resistance to imatinib, and normal monocytes from healthy volunteers were treated with gambogic acid, imatinib, or their combination, followed by measuring the effects on cell growth, apoptosis, and signal pathways. The in vivo antitumor activity of gambogic acid and its combination with imatinib was also assessed with nude xenografts. Results Gambogic acid induced apoptosis and cell proliferation inhibition in CML cells and inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Our data suggest that GA-induced proteasome inhibition is required for caspase activation in both imatinib-resistant and -sensitive CML cells, and caspase activation is required for gambogic acid–induced Bcr-Abl downregulation and apoptotic cell death. Conclusions These findings suggest an alternative strategy to overcome imatinib resistance by enhancing Bcr-Abl downregulation with the medicinal compound gambogic acid, which may have great clinical significance in imatinib-resistant cancer therapy. PMID:24334603

  7. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

    PubMed

    Otero, Maria G; Alloatti, Matías; Cromberg, Lucas E; Almenar-Queralt, Angels; Encalada, Sandra E; Pozo Devoto, Victorio M; Bruno, Luciana; Goldstein, Lawrence S B; Falzone, Tomás L

    2014-04-01

    Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

  8. Inhibition of nuclear factor-{kappa}B and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma

    SciTech Connect

    Van Waes, Carter . E-mail: vanwaesc@nidcd.nih.gov; Chang, Angela A.; Lebowitz, Peter F.; Druzgal, Colleen H.; Chen, Zhong; Elsayed, Yusri A.; Sunwoo, John B.; Rudy, Susan; Morris, John C.; Mitchell, James B.; Camphausen, Kevin; Gius, David; Adams, Julian; Sausville, Edward A.; Conley, Barbara A.

    2005-12-01

    Purpose: To examine the effects the proteasome inhibitor bortezomib (VELCADE) on transcription factor nuclear factor-{kappa}B (NF-{kappa}B) and target genes and the feasibility of combination therapy with reirradiation in patients with recurrent head-and-neck squamous cell carcinoma (HNSCC). Methods and Materials: The tolerability and response to bortezomib 0.6 mg/m{sup 2} and 0.9 mg/m{sup 2} given twice weekly concurrent with daily reirradiation to 50-70 Gy was explored. Blood proteasome inhibition and NF-{kappa}B-modulated cytokines and factors were measured. Proteasome inhibition, nuclear localization of NF-{kappa}B phospho-p65, apoptosis, and expression of NF-{kappa}B-modulated mRNAs were compared in serial biopsies from accessible tumors. Results: The maximally tolerated dose was exceeded, and study was limited to 7 and 2 patients, respectively, given bortezomib 0.6 mg/m{sup 2} and 0.9 mg/m{sup 2}/dose with reirradiation. Grade 3 hypotension and hyponatremia were dose limiting. Mucositis was Grade 3 or less and was delayed. The mean blood proteasome inhibition at 1, 24, and 48 h after 0.6 mg/m{sup 2} was 32%, 16%, and 7% and after 0.9 mg/m{sup 2} was 56%, 26%, and 14%, respectively. Differences in proteasome and NF-{kappa}B activity, apoptosis, and expression of NF-{kappa}B-modulated cell cycle, apoptosis, and angiogenesis factor mRNAs were detected in 2 patients with minor tumor reductions and in serum NF-{kappa}B-modulated cytokines in 1 patient with a major tumor reduction. Conclusions: In combination with reirradiation, the maximally tolerated dose of bortezomib was exceeded at a dose of 0.6 mg/m{sup 2} and the threshold of proteasome inhibition. Although this regimen with reirradiation is not feasible, bortezomib induced detectable differences in NF-{kappa}B localization, apoptosis, and NF-{kappa}B-modulated genes and cytokines in tumor and serum in association with tumor reduction, indicating that other schedules of bortezomib combined with primary

  9. Evolution of proteasome regulators in eukaryotes.

    PubMed

    Fort, Philippe; Kajava, Andrey V; Delsuc, Fredéric; Coux, Olivier

    2015-05-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles.

  10. Evolution of Proteasome Regulators in Eukaryotes

    PubMed Central

    Fort, Philippe; Kajava, Andrey V.; Delsuc, Fredéric; Coux, Olivier

    2015-01-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles. PMID:25943340

  11. TRIM13 (RFP2) downregulation decreases tumour cell growth in multiple myeloma through inhibition of NF Kappa B pathway and proteasome activity

    PubMed Central

    Gatt, Moshe E; Takada, Kohichi; Mani, Mala; Lerner, Mikael; Pick, Marjorie; Hideshima, Teru; Carrasco, Daniel E.; Protopopov, Alexei; Ivanova, Elena; Sangfelt, Olle; Grandér, Dan; Barlogie, Bart; Shaughnessy, John D.; Anderson, Kenneth C.; Carrasco, Daniel R.

    2013-01-01

    Multiple myeloma (MM) is an incurable neoplasm caused by proliferation of malignant plasma cells in the bone marrow (BM). MM is characterized frequently by a complete or partial deletion of chromosome 13q14, seen in more than 50% of patients at diagnosis. Within this deleted region the tripartite motif containing 13 (TRIM13, also termed RFP2) gene product has been proposed to be a tumour suppressor gene (TSG). Here, we show that low expression levels of TRIM13 in MM are associated with chromosome 13q deletion and poor clinical outcome. We present a functional analysis of TRIM13 using a loss-of-function approach, and demonstrate that TRIM13 downregulation decreases tumour cell survival as well as cell cycle progression and proliferation of MM cells. In addition, we provide evidence for the involvement of TRIM13 downregulation in inhibiting the NF kappa B pathway and the activity of the 20S proteasome. Although this data does not support a role of TRIM13 as a TSG, it substantiates important roles of TRIM13 in MM tumour survival and proliferation, underscoring its potential role as a novel target for therapeutic intervention. PMID:23647456

  12. Hydrated and anhydrous forms of copper(II) complex of 3-methylpicolinic acid, and spectroscopic studies of their ROS-inducing and proteasome inhibition

    NASA Astrophysics Data System (ADS)

    Lai, Jing Wei; Chan, Cheang Wei; Ng, Chew Hee; Ooi, Ing Hong; Tan, Kong Wai; Maah, Mohd Jamil; Ng, Seik Weng

    2016-02-01

    The hydrated and anhydrous forms of the copper(II) complex of 3-methylpicolinic acid, monomeric [Cu(3Me-pic)2(H2O)]·H2O 1 and polymeric [Cu(3Me-pic)2]n2, were synthesized and characterized by FTIR, UV-visible spectroscopy, conductivity measurement, magnetic susceptibility determination, electron paramagnetic resonance (EPR) and light scattering. Crystal structure analysis of 2 shows that it has a doubly-bridged polymeric structure, involving diagonally stacked Cu(3Me-pic)2 units which are linked via carbonyl oxygen atoms of the 3Me-pic moieties. Analysis of EPR spectra at 133 K and 293 K suggests isotropic intermolecular spin interaction only in 2. Complex 2 dissolved in DMF and DMSO solvents to yield nano-size particles. Solution studies show aqueous solutions of 1 and 2 contain the same neutral Cu(3Me-pic)2 species, which generates less hydroxyl radicals from the reaction with hydrogen peroxide than their precursor CuCl2. The Cu(3Me-pic)2 species exhibit more selective inhibition of the β2 site of the 20S proteasome, among the three proteolytic sites.

  13. Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation.

    PubMed

    Hsu, Hsien-Yeh; Lin, Tung-Yi; Wu, Yu-Chung; Tsao, Shu-Ming; Hwang, Pai-An; Shih, Yu-Wei; Hsu, Jason

    2014-09-15

    Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.

  14. Mechanisms involved in 3',5'-cyclic adenosine monophosphate-mediated inhibition of the ubiquitin-proteasome system in skeletal muscle.

    PubMed

    Gonçalves, Dawit A P; Lira, Eduardo C; Baviera, Amanda M; Cao, Peirang; Zanon, Neusa M; Arany, Zoltan; Bedard, Nathalie; Tanksale, Preeti; Wing, Simon S; Lecker, Stewart H; Kettelhut, Isis C; Navegantes, Luiz C C

    2009-12-01

    Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

  15. Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase: Signal transduction and synaptic transmission.

    PubMed

    Rezvani, Khosrow; Baalman, Kelli; Teng, Yanfen; Mee, Maureen P; Dawson, Simon P; Wang, Hongmin; De Biasi, Mariella; Mayer, R John

    2012-07-01

    The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.

  16. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  17. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  18. Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

    PubMed Central

    Cacan, Ercan; Spring, Alexander M.; Kumari, Anita; Greer, Susanna F.; Garnett-Benson, Charlie

    2015-01-01

    Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity. PMID:26703577

  19. The novel β2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

    PubMed

    Kraus, Marianne; Bader, Juergen; Geurink, Paul P; Weyburne, Emily S; Mirabella, Anne C; Silzle, Tobias; Shabaneh, Tamer B; van der Linden, Wouter A; de Bruin, Gerjan; Haile, Sarah R; van Rooden, Eva; Appenzeller, Christina; Li, Nan; Kisselev, Alexei F; Overkleeft, Herman; Driessen, Christoph

    2015-10-01

    Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.

  20. Proteasome Inhibitors Block Development of Plasmodium spp.

    PubMed Central

    Gantt, Soren M.; Myung, Joon Mo; Briones, Marcelo R. S.; Li, Wei Dong; Corey, E. J.; Omura, Satoshi; Nussenzweig, Victor; Sinnis, Photini

    1998-01-01

    Proteasomes degrade most of the proteins inside eukaryotic cells, including transcription factors and regulators of cell cycle progression. Here we show that nanomolar concentrations of lactacystin, a specific irreversible inhibitor of the 20S proteasome, inhibit development of the exoerythrocytic and erythrocytic stages of the malaria parasite. Although lactacystin-treated Plasmodium berghei sporozoites are still invasive, their development into exoerythrocytic forms (EEF) is inhibited in vitro and in vivo. Erythrocytic schizogony of P. falciparum in vitro is also profoundly inhibited when drug treatment of the synchronized parasites is prior, but not subsequent, to the initiation of DNA synthesis, suggesting that the inhibitory effect of lactacystin is cell cycle specific. Lactacystin reduces P. berghei parasitemia in rats, but the therapeutic index is very low. Along with other studies showing that lactacystin inhibits stage-specific transformation in Trypanosoma and Entamoeba spp., these findings highlight the potential of proteasome inhibitors as drugs for the treatment of diseases caused by protozoan parasites. PMID:9756786

  1. Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

    PubMed Central

    Delley, Cyrille L.; Laederach, Juerg; Ziemski, Michal; Bolten, Marcel; Boehringer, Daniel; Weber-Ban, Eilika

    2014-01-01

    The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins. PMID:25469515

  2. Phosphorylation of the C-terminal tail of proteasome subunit α7 is required for binding of the proteasome quality control factor Ecm29

    PubMed Central

    Wani, Prashant S.; Suppahia, Anjana; Capalla, Xavier; Ondracek, Alex; Roelofs, Jeroen

    2016-01-01

    The proteasome degrades many short-lived proteins that are labeled with an ubiquitin chain. The identification of phosphorylation sites on the proteasome subunits suggests that degradation of these substrates can also be regulated at the proteasome. In yeast and humans, the unstructured C-terminal region of α7 contains an acidic patch with serine residues that are phosphorylated. Although these were identified more than a decade ago, the molecular implications of α7 phosphorylation have remained unknown. Here, we showed that yeast Ecm29, a protein involved in proteasome quality control, requires the phosphorylated tail of α7 for its association with proteasomes. This is the first example of proteasome phosphorylation dependent binding of a proteasome regulatory factor. Ecm29 is known to inhibit proteasomes and is often found enriched on mutant proteasomes. We showed that the ability of Ecm29 to bind to mutant proteasomes requires the α7 tail binding site, besides a previously characterized Rpt5 binding site. The need for these two binding sites, which are on different proteasome subcomplexes, explains the specificity of Ecm29 for proteasome holoenzymes. We propose that alterations in the relative position of these two sites in different conformations of the proteasome provides Ecm29 the ability to preferentially bind specific proteasome conformations. PMID:27302526

  3. Genetics of proteasome diseases.

    PubMed

    Gomes, Aldrin V

    2013-01-01

    The proteasome is a large, multiple subunit complex that is capable of degrading most intracellular proteins. Polymorphisms in proteasome subunits are associated with cardiovascular diseases, diabetes, neurological diseases, and cancer. One polymorphism in the proteasome gene PSMA6 (-8C/G) is associated with three different diseases: type 2 diabetes, myocardial infarction, and coronary artery disease. One type of proteasome, the immunoproteasome, which contains inducible catalytic subunits, is adapted to generate peptides for antigen presentation. It has recently been shown that mutations and polymorphisms in the immunoproteasome catalytic subunit PSMB8 are associated with several inflammatory and autoinflammatory diseases including Nakajo-Nishimura syndrome, CANDLE syndrome, and intestinal M. tuberculosis infection. This comprehensive review describes the disease-related polymorphisms in proteasome genes associated with human diseases and the physiological modulation of proteasome function by these polymorphisms. Given the large number of subunits and the central importance of the proteasome in human physiology as well as the fast pace of detection of proteasome polymorphisms associated with human diseases, it is likely that other polymorphisms in proteasome genes associated with diseases will be detected in the near future. While disease-associated polymorphisms are now readily discovered, the challenge will be to use this genetic information for clinical benefit. PMID:24490108

  4. Genetics of Proteasome Diseases

    PubMed Central

    Gomes, Aldrin V.

    2013-01-01

    The proteasome is a large, multiple subunit complex that is capable of degrading most intracellular proteins. Polymorphisms in proteasome subunits are associated with cardiovascular diseases, diabetes, neurological diseases, and cancer. One polymorphism in the proteasome gene PSMA6 (−8C/G) is associated with three different diseases: type 2 diabetes, myocardial infarction, and coronary artery disease. One type of proteasome, the immunoproteasome, which contains inducible catalytic subunits, is adapted to generate peptides for antigen presentation. It has recently been shown that mutations and polymorphisms in the immunoproteasome catalytic subunit PSMB8 are associated with several inflammatory and autoinflammatory diseases including Nakajo-Nishimura syndrome, CANDLE syndrome, and intestinal M. tuberculosis infection. This comprehensive review describes the disease-related polymorphisms in proteasome genes associated with human diseases and the physiological modulation of proteasome function by these polymorphisms. Given the large number of subunits and the central importance of the proteasome in human physiology as well as the fast pace of detection of proteasome polymorphisms associated with human diseases, it is likely that other polymorphisms in proteasome genes associated with diseases will be detected in the near future. While disease-associated polymorphisms are now readily discovered, the challenge will be to use this genetic information for clinical benefit. PMID:24490108

  5. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

    PubMed

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  6. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases

    PubMed Central

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  7. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling

    PubMed Central

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  8. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    PubMed

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  9. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    SciTech Connect

    Singh, Chingakham R.; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q.; Geanes, Eric S.; Battaile, Kevin P.; Roelofs, Jeroen

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  10. Proteasome inhibitors induce auditory hair cell death through peroxisome dysfunction.

    PubMed

    Lee, Joon No; Kim, Seul-Gi; Lim, Jae-Young; Kim, Se-Jin; Choe, Seong-Kyu; Park, Raekil

    2015-01-01

    Even though bortezomib, a proteasome inhibitor, is a powerful chemotherapeutic agent used to treat multiple myeloma (MM) and other lymphoma cells, recent clinical reports suggest that the proteasome inhibitor therapy may be associated with severe bilateral hearing loss. We herein investigated the adverse effect of proteasome inhibitor on auditory hair cells. Treatment of a proteasome inhibitor destroys stereocilia bundles of hair cells resulting in the disarray of stereocilia in the organ of Corti explants. Since proteasome activity may be potentially important for biogenesis and function of the peroxisome, we tested whether proteasome activity is necessary for maintaining functional peroxisomes. Our results showed that treatment of a proteasome inhibitor significantly decreases both the number of peroxisomes and expression of peroxisomal proteins such as PMP70 and Catalase. In addition, we also found that proteasome inhibitor impairs the import pathway of PTS1-peroxisome matrix proteins. Taken together, our findings support recent clinical reports of hearing loss associated with proteasome inhibition. Mechanistically, peroxisome dysfunction may contribute to hair cell damage and hearing loss in response to the treatment of a proteasome inhibitor.

  11. Structural Basis for the Assembly and Gate Closure Mechanisms of the Mycobacterium tuberculosis 20S Proteasome

    SciTech Connect

    Lin, D.; Li, H; Wang, T; Pan, H; Lin, G; Li, H

    2010-01-01

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  12. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

    SciTech Connect

    Li, D.; Li, H.; Li, H.; Wang, T.; Pan, H.; Lin, G.

    2010-06-16

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  13. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    SciTech Connect

    Cho, Eun-Ah; Juhnn, Yong-Sung

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  14. In AβPP-overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AβPP751 and GSK3β activation; effects mitigated by lithium and apparently relevant to sporadic inclusion-body myositis

    PubMed Central

    Terracciano, Chiara; Nogalska, Anna; Engel, W. King; Askanas, Valerie

    2009-01-01

    Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-β-precursor protein 751 (AβPP751), amyloid-β (Aβ), phosphorylated tau (p-tau), and other “Alzheimer-characteristic” proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer disease (AD) patients and AD transgenic mouse models, phosphorylation of neuronal AβPP695 (p-AβPP) on Threonine668 (T668) (equivalent to T724 of AβPP751) is considered detrimental because it increases generation of cytotoxic Aβ and induces tau phosphorylation. Activated glycogen synthase kinase3β (GSK3β) is involved in phosphorylation of both AβPP and tau. Lithium, an inhibitor of GSK3β, was reported to reduce levels of both the total AβPP and p-AβPP in AD animal models. In relation to s-IBM, we now show for the first time that: 1. In AβPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: a) proteasome inhibition significantly increases GSK3β activity and AβPP phosphorylation; b) treatment with lithium decreases i) phosphorylated-AβPP; ii) total amount of AβPP, iii) Aβ oligomers, and iv) GSK3β activity; and c) lithium improves proteasome function. 2. In biopsied s-IBM muscle fibers, GSK3β is significantly activated and AβPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3β inhibitors, might benefit s-IBM patients. PMID:19878439

  15. Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease.

    PubMed

    Wang, Dong-Tao; Yang, Ya-Jun; Huang, Ren-Hua; Zhang, Zhi-Hua; Lin, Xin

    2015-01-01

    Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems. PMID:26448817

  16. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Nawabi, Atta; Komatsu, Masaaki; Huang, Heqing; Ding, Wen-Xing

    2016-01-01

    Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation. PMID:26769846

  17. Proteasome proteolysis supports stimulated platelet function and thrombosis

    PubMed Central

    Gupta, Nilaksh; Li, Wei; Willard, Belinda; Silverstein, Roy L.; McIntyre, Thomas M.

    2014-01-01

    Objective Proteasome inhibitors are in use to treat hematologic cancers, but also reduce thrombosis. Whether the proteasome participates in platelet activation or function is opaque since little is known of the proteasome in these terminally differentiated cells. Approach and Results Platelets displayed all three primary proteasome protease activities, which MG132 and bortezomib (Velcade®) inhibited. Proteasome substrates are marked by ubiquitin, and platelets contained a functional ubiquitination system that modified the proteome by mono- and poly-ubiquitination. Systemic MG132 strongly suppressed formation of occlusive, platelet-rich thrombi in FeCl3-damaged carotid arteries. Transfusion of platelets treated ex vivo with MG132 and washed prior to transfusion into thrombocytopenic mice also reduced carotid artery thrombosis. Proteasome inhibition reduced platelet aggregation by low thrombin concentrations and ristocetin-stimulated agglutination through the GPIb-IX-V complex. This receptor was not appropriately internalized after proteasome inhibition in stimulated platelets, and spreading and clot retraction after MG132 exposure also were decreased. The effects of proteasome inhibitors were not confined to a single receptor as MG132 suppressed thrombin-, ADP-, and LPS-stimulated microparticle shedding. Proteasome inhibition increased ubiquitin decoration of cytoplasmic proteins, including the cytoskeletal proteins Filamin A and Talin-1. Mass spectrometry revealed a single MG132-sensitive tryptic cleavage after R1745 in an extended Filamin A loop, which would separate its actin-binding domain from its carboxy terminal GPIbα binding domain. Conclusions Platelets contain a ubiquitin/proteasome system that marks cytoskeletal proteins for proteolytic modification to promote productive platelet-platelet and platelet-wall interactions. PMID:24177323

  18. Proteasome inhibitors suppress the protein expression of mutant p53.

    PubMed

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53.

  19. Proteasome inhibitors suppress the protein expression of mutant p53

    PubMed Central

    Halasi, Marianna; Pandit, Bulbul; Gartel, Andrei L

    2014-01-01

    Tumor suppressor p53 is one of the most frequently mutated genes in cancer, with almost 50% of all types of cancer expressing a mutant form of p53. p53 transactivates the expression of its primary negative regulator, HDM2. HDM2 is a ubiquitin ligase, which initiates the proteasomal degradation of p53 following ubiquitination. Proteasome inhibitors, by targeting the ubiquitin proteasome pathway inhibit the degradation of the majority of cellular proteins including wild-type p53. In contrast, in this study we found that the protein expression of mutant p53 was suppressed following treatment with established or novel proteasome inhibitors. Furthermore, for the first time we demonstrated that Arsenic trioxide, which was previously shown to suppress mutant p53 protein level, exhibits proteasome inhibitory activity. Proteasome inhibitor-mediated suppression of mutant p53 was partially rescued by the knockdown of HDM2, suggesting that the stabilization of HDM2 by proteasome inhibitors might be responsible for mutant p53 suppression to some extent. This study suggests that suppression of mutant p53 is a general property of proteasome inhibitors and it provides additional rationale to use proteasome inhibitors for the treatment of tumors with mutant p53. PMID:25485499

  20. The proteasome assembly line

    PubMed Central

    Madura, Kiran

    2013-01-01

    The assembly of the proteasome — the cellular machine that eliminates unwanted proteins — is a carefully choreographed affair, involving a complex sequence of steps overseen by dedicated protein chaperones. PMID:19516331

  1. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells

    PubMed Central

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M.; Pyne, Nigel J.; Pyne, Susan

    2016-01-01

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest. PMID:26934645

  2. Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

    PubMed

    McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

    2016-03-29

    Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

  3. Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis[C][W][OA

    PubMed Central

    Liu, Xiaomin; Qin, Tao; Ma, Qianqian; Sun, Jingbo; Liu, Ziqiang; Yuan, Ming; Mao, Tonglin

    2013-01-01

    Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation. PMID:23653471

  4. Proteomic profiling of expression of proteasomal subunits from livers of mice treated with diethylnitrosamine.

    PubMed

    Yuan, Fuqiang; Lu, Jia; You, Pan; Yang, Zengming; Yang, Pengyuan; Ma, Qiling; Tao, Tao

    2013-01-01

    The liver plays a central role in transforming and clearing chemicals and is susceptible to the toxicity from these agents. Diethylnitrosamine is metabolized primarily in the liver by cytochrome P-450 and can cause DNA damage. The 26S proteasome is a large proteolytic complex that degrades ubiquitinated proteins, and regulates many physiological processes. We used proteomics-based approaches to examine expressional differences of liver proteasomal subunits from diethylnitrosamine-treated mice. The expression of most proteasomal subunits was observed to be upregulated in the analysis of 2DE and MALDI-TOF MS/MS. Some of these differentially expressed proteasomal subunits were further confirmed by Western blot, RT-PCR, and immunohistochemistry. Our results provided useful information on the relationship between the proteasomal complex and related diseases.

  5. N-terminal residues regulate proteasomal degradation of AANAT.

    PubMed

    Huang, Zheping; Liu, Tiecheng; Borjigin, Jimo

    2010-04-01

    Serotonin N-acetyltransferase (AANAT) catalyzes the conversion of serotonin to N-acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT activity and melatonin release and examined the amino acid residues in AANAT that contribute to the proteasome degradation. We have shown that inhibition of proteasome activities in vivo in the intact pineal gland fails to prevent the light-induced suppression of melatonin secretion. Furthermore, in cell lines stably expressing AANAT, inhibition of proteasomal proteolysis, which resulted in a large accumulation of AANAT protein, similarly failed to increase AANAT enzyme activity proportional to the amount of proteins accumulated. Site-directed mutagenesis analysis of AANAT revealed that the AANAT degradation is independent of lysine and the two surface cysteine residues. Deletion analysis of N-terminus identified the second amino acid leucine (L2) as the key residue that contributes to the proteasomal proteolysis of AANAT protein. These results suggest that rat AANAT protein is degraded via the N-end rule pathway of proteasomal proteolysis and the leucine at the N-terminus appears to be the key residue recognized by N-end rule pathway.

  6. Small-Molecule RA-9 Inhibits Proteasome-Associated DUBs and Ovarian Cancer in Vitro and in Vivo Via Exacerbating Unfolded Protein Responses

    PubMed Central

    Coughlin, Kathleen; Anchoori, Ravi; Iizuka, Yoshie; Meints, Joyce; MacNeill, Lauren; Vogel, Rachel Isaksson; Orlowski, Robert Z.; Lee, Michael K.; Roden, Richard BS; Bazzaro, Martina

    2014-01-01

    Purpose Ovarian cancer is the deadliest of the gynecological malignancies. Carcinogenic progression is accompanied by up-regulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUBs) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer. Experimental Design The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action was evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intra-peritoneal ES-2 xenograft model of human ovarian cancer. Results Here we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis, in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an Unfolded Protein Response (UPR) as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival and was well tolerated by the host. Conclusions Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic. PMID:24727327

  7. ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction.

    PubMed

    Choi, Chang-Yong; Oh, Hae-Na; Lee, Suk Jun; Chun, Taehoon

    2015-11-01

    Defining how each ORF of porcine circovirus type 2 (PCV2) manipulates the host immune system may be helpful to understand the disease progression of post-weaning multisystemic wasting syndrome. In this study, we demonstrated a direct interaction between the PCV2 ORF2 and complement component 1, q subcomponent binding protein (C1QBP) within the cytoplasm of host macrophages. The physical interaction between PCV2 ORF2 and C1QBP inhibited ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of C1QBP by the interaction with PCV2 ORF2 further enhanced the phagocytic activity of porcine macrophages through the phosphoinositol 3-kinase signalling pathway. This may explain the molecular basis of how PCV2 ORF2 enhances the phagocytic activity of host macrophages. PMID:26361775

  8. ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction.

    PubMed

    Choi, Chang-Yong; Oh, Hae-Na; Lee, Suk Jun; Chun, Taehoon

    2015-11-01

    Defining how each ORF of porcine circovirus type 2 (PCV2) manipulates the host immune system may be helpful to understand the disease progression of post-weaning multisystemic wasting syndrome. In this study, we demonstrated a direct interaction between the PCV2 ORF2 and complement component 1, q subcomponent binding protein (C1QBP) within the cytoplasm of host macrophages. The physical interaction between PCV2 ORF2 and C1QBP inhibited ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of C1QBP by the interaction with PCV2 ORF2 further enhanced the phagocytic activity of porcine macrophages through the phosphoinositol 3-kinase signalling pathway. This may explain the molecular basis of how PCV2 ORF2 enhances the phagocytic activity of host macrophages.

  9. The proteasome stress regulon is controlled by a pair of NAC transcription factors in arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteotoxic stress is mitigated by a variety of mechanisms, including activation of the unfolded protein response and co-ordinated increases in protein chaperones and activities that direct proteolysis such as the 26S proteasome. Using RNA-seq analyses combined with either chemical inhibitors or mut...

  10. The proteasome stress regulon is controlled by a pair of NAC transcription factors in arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteotoxic stress is mitigated by a variety of mechanisms, including activation of the unfolded protein response and coordinated increases in protein chaperones and activities that direct proteolysis such as the 26S proteasome. Using RNA-seq analyses combined with either chemical inhibitors or mut...

  11. Development of novel proteasome inhibitors based on phthalazinone scaffold.

    PubMed

    Yang, Lingfei; Wang, Wei; Sun, Qi; Xu, Fengrong; Niu, Yan; Wang, Chao; Liang, Lei; Xu, Ping

    2016-06-15

    In this study we designed a series of proteasome inhibitors using pyridazinone as initial scaffold, and extended the structure with rational design by computer aided drug design (CADD). Two different synthetic routes were explored and the biological evaluation of the phthalazinone derivatives was investigated. Most importantly, electron positive triphenylphosphine group was first introduced in the structure of proteasome inhibitors and potent inhibition was achieved. As 6c was the most potent inhibitor of proteasome, we examined the structure-activity relationship (SAR) of 6c analogs. PMID:27158142

  12. Fellutamide B is a Potent Inhibitor of the Mycobacterium tuberculosis Proteasome

    SciTech Connect

    Lin, G.; Li, D; Chidawanyika, T; Nathan, C; Li, H

    2010-01-01

    Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K{sub i} = 6.8 nM, whereas it inhibits the human proteasome {beta}5 active site following a two-step mechanism with K{sub i} = 11.5 nM and K*{sub i} = 0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.

  13. Proteasome Activation is a Mechanism for Pyrazolone Small Molecules Displaying Therapeutic Potential in Amyotrophic Lateral Sclerosis

    PubMed Central

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1G93A cells. PC12-SOD1G93A cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1G93A cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS. PMID:25001311

  14. Base-CP proteasome can serve as a platform for stepwise lid formation.

    PubMed

    Yu, Zanlin; Livnat-Levanon, Nurit; Kleifeld, Oded; Mansour, Wissam; Nakasone, Mark A; Castaneda, Carlos A; Dixon, Emma K; Fushman, David; Reis, Noa; Pick, Elah; Glickman, Michael H

    2015-01-27

    26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11-m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11-Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.

  15. Calcium-dependent proteasome activation is required for axonal neurofilament degradation.

    PubMed

    Park, Joo Youn; Jang, So Young; Shin, Yoon Kyung; Suh, Duk Joon; Park, Hwan Tae

    2013-12-25

    Even though many studies have identified roles of proteasomes in axonal degeneration, the molecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were significantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration.

  16. Overview of Proteasome Inhibitor-Based Anti-cancer Therapies: Perspective on Bortezomib and Second Generation Proteasome Inhibitors versus Future Generation Inhibitors of Ubiquitin-Proteasome System

    PubMed Central

    Dou, Q. Ping; Zonder, Jeffrey A.

    2014-01-01

    Over the past ten years, proteasome inhibition has emerged as an effective therapeutic strategy for treating multiple myeloma (MM) and some lymphomas. In 2003, Bortezomib (BTZ) became the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA). BTZ-based therapies have become a staple for the treatment of MM at all stages of the disease. The survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has several limitations. Not all patients respond to BTZ-based therapies and relapse occurs in many patients who initially responded. Solid tumors, in particular, are often resistant to BTZ. Furthermore, BTZ can induce dose-limiting peripheral neuropathy (PN). The second generation proteasome inhibitor Carfizomib (CFZ; U.S. FDA approved in August 2012) induces responses in a minority of MM patients relapsed from or refractory to BTZ. There is less PN compared to BTZ. Four other second-generation proteasome inhibitors (Ixazomib, Delanzomib, Oprozomib and Marizomib) with different pharmacologic properties and broader anticancer activities, have also shown some clinical activity in bortezomib-resistant cancers. While the mechanism of resistance to bortezomib in human cancers still remains to be fully understood, targeting the immunoproteasome, ubiquitin E3 ligases, the 19S proteasome and deubiquitinases in pre-clinical studies represents possible directions for future generation inhibitors of ubiquitin-proteasome system in the treatment of MM and other cancers. PMID:25092212

  17. Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

    PubMed Central

    Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

    2010-01-01

    Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. PMID:20478262

  18. The ubiquitin-proteasome system regulates plant hormone signaling

    PubMed Central

    Santner, Aaron; Estelle, Mark

    2011-01-01

    SUMMARY Plants utilize the ubiquitin-proteasome system (UPS) to modulate nearly every aspect of growth and development. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target protein in the 26S proteasome. During the last 10 years it has become clear that the UPS plays a prominent regulatory role in hormone biology. E3 ubiquitin ligases in particular actively participate in hormone perception, de-repression of hormone signaling pathways, degradation of hormone specific transcription factors, and regulation of hormone biosynthesis. It is certain that additional functions will be discovered as more of the nearly 1200 potential E3s in plants are elucidated. PMID:20409276

  19. Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.

    PubMed

    Deroo, Bonnie J; Archer, Trevor K

    2002-06-01

    Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

  20. Nuclear Import of Yeast Proteasomes

    PubMed Central

    Burcoglu, Julianne; Zhao, Liang; Enenkel, Cordula

    2015-01-01

    Proteasomes are highly conserved protease complexes responsible for the degradation of aberrant and short-lived proteins. In highly proliferating yeast and mammalian cells, proteasomes are predominantly nuclear. During quiescence and cell cycle arrest, proteasomes accumulate in granules in close proximity to the nuclear envelope/ER. With prolonged quiescence in yeast, these proteasome granules pinch off as membraneless organelles, and migrate as stable entities through the cytoplasm. Upon exit from quiescence, the proteasome granules clear and the proteasomes are rapidly transported into the nucleus, a process reflecting the dynamic nature of these multisubunit complexes. Due to the scarcity of studies on the nuclear transport of mammalian proteasomes, we summarised the current knowledge on the nuclear import of yeast proteasomes. This pathway uses canonical nuclear localisation signals within proteasomal subunits and Srp1/Kap95, and the canonical import receptor, named importin/karyopherin αβ. Blm10, a conserved 240 kDa protein, which is structurally related to Kap95, provides an alternative import pathway. Two models exist upon which either inactive precursor complexes or active holo-enzymes serve as the import cargo. Here, we reconcile both models and suggest that the import of inactive precursor complexes predominates in dividing cells, while the import of mature enzymes mainly occurs upon exit from quiescence. PMID:26262643

  1. The proteasome: a proteolytic nanomachine of cell regulation and waste disposal.

    PubMed

    Wolf, Dieter H; Hilt, Wolfgang

    2004-11-29

    The final destination of the majority of proteins that have to be selectively degraded in eukaryotic cells is the proteasome, a highly sophisticated nanomachine essential for life. 26S proteasomes select target proteins via their modification with polyubiquitin chains or, in rare cases, by the recognition of specific motifs. They are made up of different subcomplexes, a 20S core proteasome harboring the proteolytic active sites hidden within its barrel-like structure and two 19S caps that execute regulatory functions. Similar complexes equipped with PA28 regulators instead of 19S caps are a variation of this theme specialized for the production of antigenic peptides required in immune response. Structure analysis as well as extensive biochemical and genetic studies of the 26S proteasome and the ubiquitin system led to a basic model of substrate recognition and degradation. Recent work raised new concepts. Additional factors involved in substrate acquisition and delivery to the proteasome have been discovered. Moreover, first insights in the tasks of individual subunits or subcomplexes of the 19S caps in substrate recognition and binding as well as release and recycling of polyubiquitin tags have been obtained. PMID:15571806

  2. TM-233, a novel analog of 1′-acetoxychavicol acetate, induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities

    PubMed Central

    Sagawa, Morihiko; Tabayashi, Takayuki; Kimura, Yuta; Tomikawa, Tatsuki; Nemoto-Anan, Tomoe; Watanabe, Reiko; Tokuhira, Michihide; Ri, Masaki; Hashimoto, Yuichi; Iida, Shinsuke; Kizaki, Masahiro

    2015-01-01

    Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well-tolerated agents for myeloma, we have previously reported that 1′-acetoxychavicol acetate (ACA), a natural condiment from South-East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF-κB-related functions. Searching for more potent NF-κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM-233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC50 than ACA. Treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not Bcl-2 and Bcl-xL proteins. In addition, TM-233 rapidly decreased the nuclear expression of NF-κB and also decreased the accumulation of cytosolic NF-κB. We also examined the effects of TM-233 on bortezomib-resistant myeloma cells that we recently established, KMS-11/BTZ and OPM-2/BTZ. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant myeloma cells through inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM-233 might be a more potent NF-κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells. PMID:25613668

  3. TM-233, a novel analog of 1'-acetoxychavicol acetate, induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities.

    PubMed

    Sagawa, Morihiko; Tabayashi, Takayuki; Kimura, Yuta; Tomikawa, Tatsuki; Nemoto-Anan, Tomoe; Watanabe, Reiko; Tokuhira, Michihide; Ri, Masaki; Hashimoto, Yuichi; Iida, Shinsuke; Kizaki, Masahiro

    2015-04-01

    Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well-tolerated agents for myeloma, we have previously reported that 1'-acetoxychavicol acetate (ACA), a natural condiment from South-East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF-κB-related functions. Searching for more potent NF-κB inhibitors, we developed several ACA analogs based on quantitative structure-activity relationship analysis. TM-233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC50 than ACA. Treatment with TM-233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti-apoptotic Mcl-1 protein, but not Bcl-2 and Bcl-xL proteins. In addition, TM-233 rapidly decreased the nuclear expression of NF-κB and also decreased the accumulation of cytosolic NF-κB. We also examined the effects of TM-233 on bortezomib-resistant myeloma cells that we recently established, KMS-11/BTZ and OPM-2/BTZ. TM-233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS-11/BTZ and OPM-2/BTZ cells. Interestingly, the combination of TM-233 and bortezomib significantly induced cell death in these bortezomib-resistant myeloma cells through inhibition of NF-κB activity. These results indicate that TM-233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM-233 might be a more potent NF-κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.

  4. The Ubiquitin-Proteasome Pathway and Proteasome Inhibitors

    PubMed Central

    Myung, Jayhyuk; Kim, Kyung Bo

    2008-01-01

    The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases. PMID:11410931

  5. Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

    PubMed Central

    Singh Gautam, Amit Kumar; Balakrishnan, Satish; Venkatraman, Prasanna

    2012-01-01

    Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases

  6. Quiescent fibroblasts are protected from proteasome inhibition–mediated toxicity

    PubMed Central

    Legesse-Miller, Aster; Raitman, Irene; Haley, Erin M.; Liao, Albert; Sun, Lova L.; Wang, David J.; Krishnan, Nithya; Lemons, Johanna M. S.; Suh, Eric J.; Johnson, Elizabeth L.; Lund, Benjamin A.; Coller, Hilary A.

    2012-01-01

    Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition–mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition–induced cytotoxicity. PMID:22875985

  7. Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

    PubMed Central

    Wang, Feng; Deng, Xing Wang

    2011-01-01

    The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth. PMID:21788985

  8. The Role of the Proteasome in Heart Disease

    PubMed Central

    Li, Yi-Fan; Wang, Xuejun

    2010-01-01

    Intensive investigations into the pathophysiological significance of the proteasome in the heart did not start until the beginning of the past decade but exciting progresses have been made and are summarized here as two fronts. First, strong evidence continues to emerge to support a novel hypothesis that proteasome functional insufficiency represents a common pathological phenomenon in a large subset of heart disease, compromises protein quality control in heart muscle cells, and thereby acts as a major pathogenic factor promoting the progression of the subset of heart disease to congestive heart failure. This front is represented by the studies on the ubiquitin-proteasome system (UPS) in cardiac proteinopathy, which have taken advantage of a transgenic mouse model expressing a fluorescence reporter for UPS proteolytic function. Second, pharmacological inhibition of the proteasome has been explored experimentally as a potential therapeutic strategy to intervene some forms of heart disease, such as pressure overload cardiac hypertrophy, viral myocarditis, and myocardial ischemic injury. Not only between the two fronts but also within each one, a multitude of inconsistency and controversy remain to be explained and clarified. At present, the controversy perhaps reflects the sophistication of cardiac proteasomes in terms of the composition, assembly, and regulation, as well as the intricacy and diversity of heart disease in terms of its etiology and pathogenesis. A definitive role of altered proteasome function in the development of various forms of heart disease remains to be established. PMID:20840877

  9. Structure and function based design of Plasmodium-selective proteasome inhibitors

    PubMed Central

    Li, Hao; O'Donoghue, Anthony J.; van der Linden, Wouter A.; Xie, Stanley C.; Yoo, Euna; Foe, Ian T.; Tilley, Leann; Craik, Charles S.; da Fonseca, Paula C. A.; Bogyo, Matthew

    2016-01-01

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the

  10. Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

    PubMed

    Li, Hao; O'Donoghue, Anthony J; van der Linden, Wouter A; Xie, Stanley C; Yoo, Euna; Foe, Ian T; Tilley, Leann; Craik, Charles S; da Fonseca, Paula C A; Bogyo, Matthew

    2016-02-11

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a

  11. Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

    PubMed

    Li, Hao; O'Donoghue, Anthony J; van der Linden, Wouter A; Xie, Stanley C; Yoo, Euna; Foe, Ian T; Tilley, Leann; Craik, Charles S; da Fonseca, Paula C A; Bogyo, Matthew

    2016-02-11

    The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a

  12. A role for the proteasome in the turnover of Sup35p and in [PSI(+) ] prion propagation.

    PubMed

    Kabani, Mehdi; Redeker, Virginie; Melki, Ronald

    2014-05-01

    Yeast prions are superb models for understanding the mechanisms of self-perpetuating protein aggregates formation. [PSI(+) ] stands among the most documented yeast prions and results from self-assembly of the translation termination factor Sup35p into protein fibrils. A plethora of cellular factors were shown to affect [PSI(+) ] formation and propagation. Clearance of Sup35p prion particles is however poorly understood and documented. Here, we investigated the role of the proteasome in the degradation of Sup35p and in [PSI(+) ] prion propagation. We found that cells lacking the RPN4 gene, which have reduced intracellular proteasome pools, accumulated Sup35p and have defects in [PSI(+) ] formation and propagation. Sup35p is degraded in vitro by the 26S and 20S proteasomes in a ubiquitin-independent manner, generating an array of amyloidogenic peptides derived from its prion-domain. We also demonstrate the formation of a proteasome-resistant fragment spanning residues 83-685 which is devoid of the prion-domain that is essential for [PSI(+) ] propagation. Most important was the finding that the 26S and 20S proteasomes degrade Sup35p fibrils in vitro and abolish their infectivity. Our results point to an overlooked role of the proteasome in clearing toxic protein aggregates, and have important implications for a better understanding of the life cycle of infectious protein assemblies.

  13. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells.

    PubMed

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-01

    Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-κB inhibitory molecule (IκBα) and suppressed the transcriptional activity of NF-κB in PEL cells. The NF-κB specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-κB signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-κB signaling is upregulated by proteasome-dependent degradation of IκBα. The suppression of NF-κB signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

  14. Molecular Architecture and Assembly of the Eukaryotic Proteasome

    PubMed Central

    Tomko, Robert J.; Hochstrasser, Mark

    2013-01-01

    The eukaryotic ubiquitin-proteasome system is responsible for most cellular quality-control and regulatory protein degradation. Its substrates, which are usually modified by polymers of ubiquitin, are ultimately degraded by the 26S proteasome. This 2.6 MDa protein complex is separated into a barrel-shaped proteolytic 20S core particle (CP) of 28 subunits capped on one or both ends by a 19S regulatory particle (RP) comprising at least 19 subunits. The RP coordinates substrate recognition, removal of substrate polyubiquitin chains, and substrate unfolding and translocation into the CP for degradation. While many atomic structures of the CP have been determined, the RP has resisted high-resolution analysis. Recently, however, a combination of cryo-electron microscopy (cryo-EM), biochemical analysis, and crystal structure determination of several RP subunits has yielded a near-atomic resolution view of much of the complex. Major new insights into chaperone-assisted proteasome assembly have also recently been made. Here we review these novel findings. PMID:23495936

  15. The proteasome is an integral part of solar ultraviolet a radiation-induced gene expression.

    PubMed

    Catalgol, Betul; Ziaja, Isabella; Breusing, Nicolle; Jung, Tobias; Höhn, Annika; Alpertunga, Buket; Schroeder, Peter; Chondrogianni, Niki; Gonos, Efstathios S; Petropoulos, Isabelle; Friguet, Bertrand; Klotz, Lars-Oliver; Krutmann, Jean; Grune, Tilman

    2009-10-30

    Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response.

  16. The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction

    PubMed Central

    Koizumi, Shun; Irie, Taro; Hirayama, Shoshiro; Sakurai, Yasuyuki; Yashiroda, Hideki; Naguro, Isao; Ichijo, Hidenori; Hamazaki, Jun; Murata, Shigeo

    2016-01-01

    In response to proteasome dysfunction, mammalian cells upregulate proteasome gene expression by activating Nrf1. Nrf1 is an endoplasmic reticulum-resident transcription factor that is continually retrotranslocated and degraded by the proteasome. Upon proteasome inhibition, Nrf1 escapes degradation and is cleaved to become active. However, the processing enzyme for Nrf1 remains obscure. Here we show that the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is required to cleave and activate Nrf1. Deletion of DDI2 reduced the cleaved form of Nrf1 and increased the full-length cytosolic form of Nrf1, resulting in poor upregulation of proteasomes in response to proteasome inhibition. These defects were restored by adding back wild-type DDI2 but not protease-defective DDI2. Our results provide a clue for blocking compensatory proteasome synthesis to improve cancer therapies targeting proteasomes. DOI: http://dx.doi.org/10.7554/eLife.18357.001 PMID:27528193

  17. From Bortezomib to other Inhibitors of the Proteasome and Beyond

    PubMed Central

    Buac, Daniela; Shen, Min; Schmitt, Sara; Kona, Fathima Rani; Deshmukh, Rahul; Zhang, Zhen; Neslund-Dudas, Christine; Mitra, Bharati; Dou, Q. Ping

    2013-01-01

    The cancer drug discovery field has placed much emphasis on the identification of novel and cancer-specific molecular targets. A rich source of such targets for the design of novel anti-tumor agents is the ubiqutin-proteasome system (UP-S), a tightly regulated, highly specific pathway responsible for the vast majority of protein turnover within the cell. Because of its critical role in almost all cell processes that ensure normal cellular function, its inhibition at one point in time was deemed non-specific and therefore not worth further investigation as a molecular drug target. However, today the proteasome is one of the most promising anti-cancer drug targets of the century. The discovery that tumor cells are in fact more sensitive to proteasome inhibitors than normal cells indeed paved the way for the design of its inhibitors. Such efforts have led to bortezomib, the first FDA approved proteasome inhibitor now used as a frontline treatment for newly diagnosed multiple myeloma (MM), relapsed/refractory MM and mantle cell lymphoma. Though successful in improving clinical outcomes for patients with hematological malignancies, relapse often occurs in those who initially responded to bortezomib. Therefore, the acquisition of bortezomib resistance is a major issue with its therapy. Furthermore, some neuro-toxicities have been associated with bortezomib treatment and its efficacy in solid tumors is lacking. These observations have encouraged researchers to pursue the next generation of proteasome inhibitors, which would ideally overcome bortezomib resistance, have reduced toxicities and a broader range of anti-cancer activity. This review summarizes the success and limitations of bortezomib, and describes recent advances in the field, including, and most notably, the most recent FDA approval of carfilzomib in July, 2012, a second generation proteasome inhibitor. Other proteasome inhibitors currently in clinical trials and those that are currently experimental grade

  18. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain.

    PubMed

    Singh, Chingakham R; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q; Geanes, Eric S; Battaile, Kevin P; Roelofs, Jeroen

    2014-04-01

    The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  19. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    PubMed Central

    Singh, Chingakham R.; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q.; Geanes, Eric S.; Battaile, Kevin P.; Roelofs, Jeroen

    2014-01-01

    The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly. PMID:24699731

  20. Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base.

    PubMed

    Funakoshi, Minoru; Tomko, Robert J; Kobayashi, Hideki; Hochstrasser, Mark

    2009-05-29

    The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here, we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.

  1. Induction of Caspase-3-like activity in Rice following release of cytochrome-f from the chloroplast and subsequent interaction with the Ubiquitin-Proteasome System

    PubMed Central

    Wang, Hongjuan; Zhu, Xiaonan; Li, Huan; Cui, Jing; Liu, Cheng; Chen, Xi; Zhang, Wei

    2014-01-01

    It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD. PMID:25103621

  2. Potential Roles for Ubiquitin and the Proteasome during Ribosome Biogenesis‡

    PubMed Central

    Stavreva, Diana A.; Kawasaki, Miyuki; Dundr, Miroslav; Koberna, Karel; Müller, Waltraud G.; Tsujimura-Takahashi, Teruko; Komatsu, Wataru; Hayano, Toshiya; Isobe, Toshiaki; Raska, Ivan; Misteli, Tom; Takahashi, Nobuhiro; McNally, James G.

    2006-01-01

    We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome. PMID:16782897

  3. Proteasome Inhibitors in the Treatment of Multiple Myeloma

    PubMed Central

    Shah, Jatin J.; Orlowski, Robert Z.

    2016-01-01

    Targeting intracellular protein turnover by inhibiting the ubiquitin-proteasome pathway as a strategy for cancer therapy is a new addition to our chemotherapeutic armamentarium, and has seen its greatest successes against multiple myeloma. The first-in-class proteasome inhibitor bortezomib was initially approved for treatment of patients in the relapsed/refractory setting as a single agent, and was recently shown to induce even greater benefits as part of rationally-designed combinations that overcome chemoresistance. Modulation of proteasome function is also a rational approach to achieve chemosensitization to other anti-myeloma agents, and bortezomib has now been incorporated into the front-line setting. Bortezomib-based induction regimens are able to achieve higher overall response rates and response qualities than was the case with prior standards of care, and unlike these older approaches, maintain efficacy in patients with clinically- and molecularly-defined high-risk disease. Second-generation proteasome inhibitors with novel properties, such as NPI-0052 and carfilzomib, are entering the clinical arena, and showing evidence of anti-myeloma activity. In this spotlight review, we provide an overview of the current state of the art use of bortezomib and other proteasome inhibitors against multiple myeloma, and highlight areas for future study that will further optimize our ability to benefit patients with this disease. PMID:19741722

  4. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

    PubMed

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F S; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  5. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form

    PubMed Central

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F. S.; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  6. Proteasome inhibitors induce p53-independent apoptosis in human cancer cells.

    PubMed

    Pandit, Bulbul; Gartel, Andrei L

    2011-01-01

    Proteasome inhibitors are used against human cancer, but their mechanisms of action are not entirely understood. For example, the role of the tumor suppressor p53 is controversial. We reevaluated the role of p53 in proteasome inhibitor-induced apoptosis by using isogenic human cancer cell lines with different p53 status. We found that well-known proteasome inhibitors such as MG132 and bortezomib, as well as the recently discovered proteasome inhibitor thiostrepton, induced p53-independent apoptosis in human cancer cell lines that correlated with p53-independent induction of proapoptotic Noxa but not Puma protein. In addition, these drugs inhibited growth of several cancer cell lines independently of p53 status. Notably, thiostrepton induced more potent apoptosis in HepG2 cells with p53 knockdown than in parental cells with wild-type p53. Our data confirm that proteasome inhibitors generally induce p53-independent apoptosis in human cancer cells.

  7. Functions of the Proteasome on Chromatin

    PubMed Central

    McCann, Tyler S.; Tansey, William P.

    2014-01-01

    The proteasome is a large self-compartmentalized protease complex that recognizes, unfolds, and destroys ubiquitylated substrates. Proteasome activities are required for a host of cellular functions, and it has become clear in recent years that one set of critical actions of the proteasome occur on chromatin. In this review, we discuss some of the ways in which proteasomes directly regulate the structure and function of chromatin and chromatin regulatory proteins, and how this influences gene transcription. We discuss lingering controversies in the field, the relative importance of proteolytic versus non-proteolytic proteasome activities in this process, and highlight areas that require further investigation. Our intention is to show that proteasomes are involved in major steps controlling the expression of the genetic information, that proteasomes use both proteolytic mechanisms and ATP-dependent protein remodeling to accomplish this task, and that much is yet to be learned about the full spectrum of ways that proteasomes influence the genome. PMID:25422899

  8. Changes in the Expression and the Enzymic Properties of the 20S Proteasome in Sugar-Starved Maize Roots. Evidence for an in Vivo Oxidation of the Proteasome1

    PubMed Central

    Basset , Gilles; Raymond, Philippe; Malek, Lada; Brouquisse, Renaud

    2002-01-01

    The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of α3 and β6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations. PMID:11891269

  9. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    SciTech Connect

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

  10. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.

    PubMed

    Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L; Remmers, Elaine F; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina I; Rother, Kristina; Hildebrand, Peter W; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-11-01

    Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

  11. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

    PubMed Central

    Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L.; Remmers, Elaine F.; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J.; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D.; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L.; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina; Hildebrand, Peter W.; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-01-01

    Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

  12. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    SciTech Connect

    Zangar, Richard C. ); Kocarek, Thomas A.; Shen, Shang; Bollinger, Nikki ); Dahn, Michael S.; Lee, Donna W.

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3 A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  13. Suppression of cytochrome P450 3A protein levels by proteasome inhibitors.

    PubMed

    Zangar, Richard C; Kocarek, Thomas A; Shen, Shang; Bollinger, Nikki; Dahn, Michael S; Lee, Donna W

    2003-06-01

    We have previously reported that CYP3A cross-links with polyubiquitinated proteins in microsomes from nicardipine-treated rats in a process that is distinct from classical polyubiquitination. To further examine the role of the proteasome in CYP3A degradation, we investigated the effects of proteasome inhibitors lactacystin, MG132, proteasome inhibitor 1, and hemin in primary cultures of rat and human hepatocytes. With the exception of hemin, these agents increased the total pool of ubiquitinated proteins in microsomes isolated from rat hepatocytes, indicating that lactacystin, MG132, and proteasome inhibitor 1 effectively inhibited the proteasome in these cells. All four agents caused a reduction in the amount of the major approximately 55-kDa CYP3A band, opposite to what would be expected if the ubiquitin-proteasome pathway degraded CYP3A. Only hemin treatment caused an increase in high molecular mass (HMM) CYP3A bands. Because hemin treatment did not alter levels of ubiquitin in CYP3A immunoprecipitates, the HMM CYP3A bands formed in response to hemin treatment clearly were not due to proteasome inhibition. Rather, because hemin treatment also caused an increase in HMM CYP3A in the detergent-insoluble fraction of the 10,000g pellet, the HMM CYP3A seems to represent a large protein complex that is unlikely to primarily represent ubiquitination.

  14. Biochemical characterization and role of the proteasome in the oxidative stress response of adult Schistosoma mansoni worms.

    PubMed

    de Paula, Renato Graciano; Ornelas, Alice Maria de Magalhães; Morais, Enyara Rezende; Borges, William de Castro; Natale, Massimo; Magalhães, Lizandra Guidi; Rodrigues, Vanderlei

    2014-08-01

    The trematode Schistosoma mansoni, an important parasite of humans, is the principle agent of the disease schistosomiasis. In the human host, one of the most important stress factors of this parasite is the oxidative stress generated by both the metabolism of the worm and the immune system of the host. The proteasomal system is responsible for protein homeostasis during oxidative stress. The 26S proteasome is a multicatalytic protease formed by two compartments, a 20S core and regulatory particle 19S, and controls the degradation of intracellular proteins, hence regulating many cellular processes. In the present report, we describe the biochemical characterization and role of the 20S proteasome in the response of adult S. mansoni worms exposed to hydrogen peroxide. Characterization of the response to the oxidative stress included the evaluation of viability, egg production, mortality, tegument integrity, and both expression and activity of proteasome. We observed decreases in viability, egg production as well as 100% mortality at the higher concentrations of hydrogen peroxide tested. The main changes observed in the tegument of adult worms were peeling as well as the appearance of bubbles and a decrease of spines on the tubercles. Furthermore, there were increases in 26S activity to the same extent as 20S proteasome activity, although there was increase of 20S proteasome content, suggesting that degradation of protein oxidized in adult worms is due to the 20S proteasome. It was demonstrated that adult S. mansoni worms are sensitive to oxidative stress, and that a variety of processes in this parasite are altered under this condition. The work contributes to a better understanding of the mechanisms employed by S. mansoni to survive under oxidative stress. PMID:24870249

  15. Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development.

    PubMed

    Götze, Sandra; Saborowski, Reinhard

    2011-10-01

    Decapod crustaceans grow discontinuously and gain size through complex molt processes. The molt comprises the loss of the old cuticle and, moreover, substantial reduction and re-organization of muscles and connective tissues. In adult lobsters, the muscle tissue of the massive claws undergoes significant atrophy of 40-75% before ecdysis. The degradation of this tissue is facilitated by calcium-dependent proteases and by the proteasome, an intra-cellular proteolytic multi-enzyme complex. In contrast to the adults, the involvement of the proteasome during the larval development is yet not validated. Therefore, we developed micro-methods to measure the 20S and the 26S proteasomal activities within mg- and sub-mg-quantities of the larval claw tissue of the European lobster, Homarus gammarus. Within the three larval stages (Z1-3) we distinguished between sub-stages of freshly molted/hatched (post-molt), inter-molt, and ready to molt (pre-molt) larvae. Juveniles were analyzed in the post-molt and in the inter-molt stage. The trypsin-like, the chymotrypsin-like, and the peptidyl-glutamyl peptide hydrolase activity (PGPH) of the 20S proteasome increased distinctly from freshly hatched larvae to pre-molt Z1. During the Z2 stage, the activities were highest in the post-molt animals, decreased in the inter-molt animals and increased again in the pre-molt animals. A similar but less distinct trend was evident in the Z3 stages. In the juveniles, the proteasomal activities decreased toward the lowest values. A similar pattern was present for the chymotrypsin-like activity of the 26S proteasome. The results show that the proteasome plays a significant role during the larval development of lobsters. This is not only reflected by the elevated activities, but also by the continuous change of the trypsin/chymotrypsin-ratio which may indicate a shift in the subunit composition of the proteasome and, thus, a biochemical adjustment to better cope with elevated protein turnover rates

  16. Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway

    PubMed Central

    Xu, Bing-Can; Long, Hui-Bao; Luo, Ke-Qin

    2016-01-01

    Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice. PMID:27435826

  17. Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei Wu Yachun; Yu Le; Li Zhijie; Sung, Joseph Jao Yiu; Cho, C.H.

    2008-09-19

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G{sub 2}/M cell cycle arrest which was associated with the formation of LC3{sup +} autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3{sup +} autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.

  18. Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Jao Yiu; Yu Le; Cho, C.H.

    2008-06-27

    Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21{sup Waf1/Cip1} mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21{sup Waf1/Cip1} induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

  19. Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis.

    PubMed

    Zheng, Jianzheng; Bizzozero, Oscar A

    2011-04-01

    Carbonylated (oxidized) proteins are known to accumulate in the cerebral white matter (WM) and gray matter (GM) of patients with multiple sclerosis (MS). Although oxidative stress is necessary for carbonyl generation, it is the failure of the degradation systems that ultimately leads to the build-up of carbonylated proteins within tissues. In this study, we measured the activity of the 20S proteasome and other proteolytic systems in the cerebral WM and GM of 13 MS patients and 13 controls. We report that the activities of the three peptidases of the 20S proteasome (i.e. chymotrypsin-like, caspase-like and trypsin-like) in both MS-WM and MS-GM are greatly reduced. Interestingly, neither the amount of proteasome nor the levels of the catalytic subunits (β1, β2, and β5) are diminished in this disease. Proteins containing Lys-48 poly-ubiquitin also accumulate in MS tissues, indicating failure of the 26S proteasome as well. Levels of the regulatory caps 11S α and 19S are also lower in MS than in controls, suggesting that the activity of the more complex proteasomes may be reduced further. Finally, the activities of other proteases that might also remove oxidized proteins (calpain, cathepsin B, mitochondrial LonP) are not lessened in MS. Together, these studies suggest that direct inactivation of proteolytic centers in the 20S particle and/or the presence of specific inhibitors is the underlying cause of proteasomal dysfunction in MS.

  20. Proteasome Modulates Positive and Negative Translational Regulators in Long-Term Synaptic Plasticity

    PubMed Central

    Dong, Chenghai; Bach, Svitlana V.; Haynes, Kathryn A.

    2014-01-01

    Proteolysis by the ubiquitin-proteasome pathway appears to have a complex role in synaptic plasticity, but its various functions remain to be elucidated. Using late phase long-term potentiation (L-LTP) in the hippocampus of the mouse as a model for long-term synaptic plasticity, we previously showed that inhibition of the proteasome enhances induction but blocks maintenance of L-LTP. In this study, we investigated the possible mechanisms by which proteasome inhibition has opposite effects on L-LTP induction and maintenance. Our results show that inhibiting phosphatidyl inositol-3 kinase or blocking the interaction between eukaryotic initiation factors 4E (eIF4E) and 4G (eIF4G) reduces the enhancement of L-LTP induction brought about by proteasome inhibition suggesting interplay between proteolysis and the signaling pathway mediated by mammalian target of rapamycin (mTOR). Also, proteasome inhibition leads to accumulation of translational activators in the mTOR pathway such as eIF4E and eukaryotic elongation factor 1A (eEF1A) early during L-LTP causing increased induction. Furthermore, inhibition of the proteasome causes a buildup of translational repressors, such as polyadenylate-binding protein interacting protein 2 (Paip2) and eukaryotic initiation factor 4E-binding protein 2 (4E-BP2), during late stages of L-LTP contributing to the blockade of L-LTP maintenance. Thus, the proteasome plays a critical role in regulating protein synthesis during L-LTP by tightly controlling translation. Our results provide novel mechanistic insights into the interplay between protein degradation and protein synthesis in long-term synaptic plasticity. PMID:24573276

  1. ATP binds to proteasomal ATPases in pairs with distinct functional effects implying an ordered reaction cycle

    PubMed Central

    Smith, David M.; Fraga, Hugo; Reis, Christian; Kafri, Galit; Goldberg, Alfred L.

    2011-01-01

    In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits, and in archaea by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules, or two ATPγS plus two ADP molecules it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate-opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and “wobble” on top of the heptameric 20S proteasome. PMID:21335235

  2. Subnormothermic Perfusion in the Isolated Rat Liver Preserves the Antioxidant Glutathione and Enhances the Function of the Ubiquitin Proteasome System

    PubMed Central

    Alva, Norma; Sanchez-Nuño, Sergio; Dewey, Shannamar; Gomes, Aldrin V.

    2016-01-01

    The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated rat livers after 15 min perfusion at temperatures ranging from normothermia (37°C) to subnormothermia (26°C and 22°C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22°C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22°C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like (β5) proteasome activity was significantly increased in the 26°C (46%) and 22°C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus. PMID:27800122

  3. Identification of proteasome subunit beta type 6 (PSMB6) associated with deltamethrin resistance in mosquitoes by proteomic and bioassay analyses.

    PubMed

    Sun, Linchun; Ye, Yuting; Sun, Haibo; Yu, Jing; Zhang, Li; Sun, Yan; Zhang, Donghui; Ma, Lei; Shen, Bo; Zhu, Changliang

    2013-01-01

    Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.

  4. Proteasome activity is required for the stage-specific transformation of a protozoan parasite

    PubMed Central

    1996-01-01

    A prominent feature of the life cycle of intracellular parasites is the profound morphological changes they undergo during development in the vertebrate and invertebrate hosts. In eukaryotic cells, most cytoplasmic proteins are degraded in proteasomes. Here, we show that the transformation in axenic medium of trypomastigotes of Trypanosoma cruzi into amastigote-like organisms, and the intracellular development of the parasite from amastigotes into trypomastigotes, are prevented by lactacystin, or by a peptide aldehyde that inhibits proteasome function. Clasto-lactacystin, an inactive analogue of lactacystin, and cell-permeant peptide aldehyde inhibitors of T. cruzi cysteine proteinases have no effect. We have also identified the 20S proteasomes from T. cruzi as a target of lactacystin in vivo. Our results document the essential role of proteasomes in the stage-specific transformation of a protozoan. PMID:8920878

  5. Secondary Metabolites Produced by an Endophytic Fungus Pestalotiopsis sydowiana and Their 20S Proteasome Inhibitory Activities.

    PubMed

    Xia, Xuekui; Kim, Soonok; Liu, Changheng; Shim, Sang Hee

    2016-01-01

    Fungal endophytes have attracted attention due to their functional diversity. Secondary metabolites produced by Pestalotiopsis sydowiana from a halophyte, Phragmites communis Trinus, were investigated. Eleven compounds, including four penicillide derivatives (1-4) and seven α-pyrone analogues (5-10) were isolated from cultures of P. sydowiana. The compounds were identified based on spectroscopic data. The inhibitory activities against the 20S proteasome were evaluated. Compounds 1-3, 5, and 9-10 showed modest proteasome inhibition activities, while compound 8 showed strong activity with an IC50 of 1.2 ± 0.3 μM. This is the first study on the secondary metabolites produced by P. sydowiana and their proteasome inhibitory activities. The endophytic fungus P. sydowiana might be a good resource for proteasome inhibitors. PMID:27447600

  6. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    PubMed Central

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    2009-01-01

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic α- and β-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy in coping with crisis of misfolded proteins. PMID:19682429

  7. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    SciTech Connect

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    2009-10-16

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic {alpha}- and {beta}-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.

  8. Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast

    SciTech Connect

    Mannen, Taro; Andoh, Tomoko; Tani, Tokio

    2008-01-25

    Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1{sup +} gene ({delta}dss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A){sup +} RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in {delta}dss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1{sup +} gene, which encodes a component of the 26S proteasome, as a suppressor for the ts{sup -} phenotype of {delta}dss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.

  9. ahg12 is a dominant proteasome mutant that affects multiple regulatory systems for germination of Arabidopsis.

    PubMed

    Hayashi, Shimpei; Hirayama, Takashi

    2016-01-01

    The ubiquitin-proteasome system is fundamentally involved in myriad biological phenomena of eukaryotes. In plants, this regulated protein degradation system has a pivotal role in the cellular response mechanisms for both internal and external stimuli, such as plant hormones and environmental stresses. Information about substrate selection by the ubiquitination machinery has accumulated, but there is very little information about selectivity for substrates at the proteasome. Here, we report characterization of a novel abscisic acid (ABA)-hypersensitive mutant named ABA hypersensitive germination12 (ahg12) in Arabidopsis. The ahg12 mutant showed a unique pleiotropic phenotype, including hypersensitivity to ABA and ethylene, and hyposensitivity to light. Map-based cloning identified the ahg12 mutation to cause an amino acid conversion in the L23 loop of RPT5a, which is predicted to form the pore structure of the 19S RP complex of the proteasome. Transient expression assays demonstrated that some plant-specific signaling components accumulated at higher levels in the ahg12 mutant. These results suggest that the ahg12 mutation led to changes in the substrate preference of the 26S proteasome. The discovery of the ahg12 mutation thus will contribute to elucidate the characteristics of the regulated protein degradation system. PMID:27139926

  10. ahg12 is a dominant proteasome mutant that affects multiple regulatory systems for germination of Arabidopsis

    PubMed Central

    Hayashi, Shimpei; Hirayama, Takashi

    2016-01-01

    The ubiquitin-proteasome system is fundamentally involved in myriad biological phenomena of eukaryotes. In plants, this regulated protein degradation system has a pivotal role in the cellular response mechanisms for both internal and external stimuli, such as plant hormones and environmental stresses. Information about substrate selection by the ubiquitination machinery has accumulated, but there is very little information about selectivity for substrates at the proteasome. Here, we report characterization of a novel abscisic acid (ABA)-hypersensitive mutant named ABA hypersensitive germination12 (ahg12) in Arabidopsis. The ahg12 mutant showed a unique pleiotropic phenotype, including hypersensitivity to ABA and ethylene, and hyposensitivity to light. Map-based cloning identified the ahg12 mutation to cause an amino acid conversion in the L23 loop of RPT5a, which is predicted to form the pore structure of the 19S RP complex of the proteasome. Transient expression assays demonstrated that some plant-specific signaling components accumulated at higher levels in the ahg12 mutant. These results suggest that the ahg12 mutation led to changes in the substrate preference of the 26S proteasome. The discovery of the ahg12 mutation thus will contribute to elucidate the characteristics of the regulated protein degradation system. PMID:27139926

  11. The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

    PubMed Central

    Pathare, Ganesh Ramnath; Nagy, István; Bohn, Stefan; Unverdorben, Pia; Hubert, Agnes; Körner, Roman; Nickell, Stephan; Lasker, Keren; Sali, Andrej; Tamura, Tomohiro; Nishioka, Taiki; Förster, Friedrich; Baumeister, Wolfgang; Bracher, Andreas

    2012-01-01

    Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP. PMID:22187461

  12. The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together.

    PubMed

    Pathare, Ganesh Ramnath; Nagy, István; Bohn, Stefan; Unverdorben, Pia; Hubert, Agnes; Körner, Roman; Nickell, Stephan; Lasker, Keren; Sali, Andrej; Tamura, Tomohiro; Nishioka, Taiki; Förster, Friedrich; Baumeister, Wolfgang; Bracher, Andreas

    2012-01-01

    Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP.

  13. Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

    PubMed Central

    Kushwaha, Deepa S.; Hsieh, Grace; Merzon, Dmitry; Rameseder, Jonathan; Chen, Clark C.; D’Andrea, Alan D.; Kozono, David

    2013-01-01

    Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes. PMID:24040035

  14. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

    SciTech Connect

    Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-09-04

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of {sup 35}S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

  15. Proteasome inhibitors induce apoptosis of prostate cancer cells by inducing nuclear translocation of IkappaBalpha.

    PubMed

    Vu, Hai-Yen; Juvekar, Ashish; Ghosh, Chandra; Ramaswami, Sitharam; Le, Dung Hong; Vancurova, Ivana

    2008-07-15

    Proteasome inhibitors are known to suppress the proteasome-mediated degradation of IkappaBalpha in stimulated cells. This results in the cytoplasmic retention of NFkappaB and its reduced nuclear transcriptional activity. In this study, we show that in the metastatic prostate cancer cells, the proteasome inhibitors exhibit a novel, previously unrecognized effect: they increase the cellular levels of IkappaBalpha, which then translocates to the nucleus, associates with the nuclear p65 NFkappaB, thus inhibiting the constitutive NFkappaB DNA binding activity and inducing apoptosis. The proteasome inhibition-induced nuclear translocation of IkappaBalpha is dependent on de novo protein synthesis, occurs also in other cell types, and does not require IkappaBalpha phosphorylation on Ser-32. Since NFkappaB activity is constitutively increased in many human cancers as well as in inflammatory disorders, the proteasome inhibition-induced nuclear translocation of IkappaBalpha could thus provide a new therapeutic strategy aimed at the specific inhibition of NFkappaB activity by the nuclear IkappaBalpha.

  16. Proteasome-mediated effects on amyloid precursor protein processing at the γ-secretase site

    PubMed Central

    2004-01-01

    Aβ (β-amyloid) peptides are found aggregated in the cortical amyloid plaques associated with Alzheimer's disease neuropathology. Inhibition of the proteasome alters the amount of Aβ produced from APP (amyloid precursor protein) by various cell lines in vitro. Proteasome activity is altered during aging, a major risk factor for Alzheimer's disease. In the present study, a human neuroblastoma cell line expressing the C-terminal 100 residues of APP (SH-SY5Y-SPA4CT) was used to determine the effect of proteasome inhibition, by lactacystin and Bz-LLL-COCHO (benzoyl-Leu-Leu-Leu-glyoxal), on APP processing at the γ-secretase site. Proteasome inhibition caused a significant increase in Aβ peptide levels in medium conditioned by SH-SY5Y-SPA4CT cells, and was also associated with increased cell death. APP is a substrate of the apoptosis-associated caspase 3 protease, and we therefore investigated whether the increased Aβ levels could reflect caspase activation. We report that caspase activation was not required for proteasome-inhibitor-mediated effects on APP (SPA4CT) processing. Cleavage of Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin), a caspase substrate, was reduced following exposure of SH-SY5Y-SPA4CT cells to lactacystin, and co-treatment of cells with lactacystin and a caspase inhibitor [Z-DEVD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone)] resulted in higher Aβ levels in medium, augmenting those seen with lactacystin alone. This study indicated that proteasome inhibition could increase APP processing specifically at the γ-secretase site, and increase release of Aβ, in the absence of caspase activation. This indicates that the decline in proteasome function associated with aging would contribute to increased Aβ levels. PMID:15473868

  17. Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis

    PubMed Central

    Semren, Nora; Habel-Ungewitter, Nunja C.; Fernandez, Isis E.; Königshoff, Melanie; Eickelberg, Oliver; Stöger, Tobias; Meiners, Silke

    2015-01-01

    Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution. PMID:26340365

  18. FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing.

    PubMed

    Wang, Z; Dove, P; Wang, X; Shamas-Din, A; Li, Z; Nachman, A; Oh, Y J; Hurren, R; Ruschak, A; Climie, S; Press, B; Griffin, C; Undzys, E; Aman, A; Al-awar, R; Kay, L E; O'Neill, D; Trudel, S; Slassi, M; Schimmer, A D

    2015-01-01

    Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.

  19. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    SciTech Connect

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors

  20. Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

    PubMed Central

    Pedersen, Nina Marie; Thorvaldsen, Tor Espen; Schultz, Sebastian Wolfgang; Wenzel, Eva Maria; Stenmark, Harald

    2016-01-01

    In canonical Wnt signaling, the protein levels of the key signaling mediator β-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of β-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of β-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi. PMID:27482906

  1. Identification of a new series of amides as non-covalent proteasome inhibitors.

    PubMed

    Scarbaci, Kety; Troiano, Valeria; Micale, Nicola; Ettari, Roberta; Tamborini, Lucia; Di Giovanni, Carmen; Cerchia, Carmen; Grasso, Silvana; Novellino, Ettore; Schirmeister, Tanja; Lavecchia, Antonio; Zappalà, Maria

    2014-04-01

    Proteasome inhibition has emerged as an important therapeutic strategy for the treatment of multiple myeloma (MM) and some forms of lymphoma, with potential application in other types of cancers. 20S proteasome consists of three different catalytic activities known as chymotrypsin-like (ChT-L), trypsin-like (T-L), and, post-glutamyl peptide hydrolyzing (PGPH) or caspase-like (C-L), which are located respectively on the β5, β2, and β1 subunits of each heptameric β rings. Currently a wide number of covalent proteasome inhibitors are reported in literature; however, the less widely investigated non-covalent inhibitors might be a promising alternative to employ in therapy, because of the lack of all drawbacks and side-effects related to irreversible inhibition. In the present work we identified a series of amides, two of which (1b and 1f) are good candidates to non-covalent inhibition of the chymotrypsin-like activity of the β5 proteasome subunit. The non-covalent binding mode was corroborated by docking simulations of the most active inhibitors 1b, 1f and 2h into the yeast 20S proteasome crystal structure. PMID:24561716

  2. The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals.

    PubMed

    Götze, Sandra; Bose, Aneesh; Sokolova, Inna M; Abele, Doris; Saborowski, Reinhard

    2014-05-01

    The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg(2+), Zn(2+), Cu(2+), and Cd(2+) on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg(2+) and Cu(2+) while Zn(2+) had only a moderate effect and Cd(2+) caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus. PMID:24721378

  3. The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals.

    PubMed

    Götze, Sandra; Bose, Aneesh; Sokolova, Inna M; Abele, Doris; Saborowski, Reinhard

    2014-05-01

    The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg(2+), Zn(2+), Cu(2+), and Cd(2+) on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg(2+) and Cu(2+) while Zn(2+) had only a moderate effect and Cd(2+) caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus.

  4. Molecular mechanisms of proteasome plasticity in aging

    PubMed Central

    Rodriguez, Karl; Gaczynska, Maria; Osmulski, Pawel A.

    2010-01-01

    The ubiquitin-proteasome pathway plays a crucial role in regulation of intracellular protein turnover. Proteasome, the central protease of the pathway, encompasses multisubunit assemblies sharing a common catalytic core supplemented by regulatory modules and localizing to different subcellular compartments. To better comprehend age-related functions of the proteasome we surveyed content, composition and catalytic properties of the enzyme in cytosolic, microsomal and nuclear fractions. obtained from mouse livers subjected to organismal aging. We found that during aging subunit composition and subcellular distribution of proteasomes changed without substantial alterations in the total level of core complexes. We observed that the general decline in proteasomes functions was limited to nuclear and cytosolic compartments. Surprisingly, the observed changes in activity and specificity were linked to the amount of the activator module and distinct composition of the catalytic subunits. In contrast, activity, specificity and composition of the microsomal-associated proteasomes remained mostly unaffected by aging; however their relative contribution to the total activity was substantially elevated. Unexpectedly, the nuclear proteasomes were affected most profoundly by aging possibly triggering significant changes in cellular signaling and transcription. Collectively, the data indicate an age-related refocusing of proteasome from the compartment specific functions towards general protein maintenance. PMID:20080121

  5. Nrf1 can be processed and activated in a proteasome-independent manner.

    PubMed

    Vangala, Janakiram R; Sotzny, Franziska; Krüger, Elke; Deshaies, Raymond J; Radhakrishnan, Senthil K

    2016-09-26

    In response to proteasome inhibition, the transcription factor Nrf1 facilitates de novo synthesis of proteasomes by inducing proteasome subunit (PSM) genes [1,2]. Previously, we showed that activation of the p120 form of Nrf1, a membrane-bound protein in the endoplasmic reticulum (ER) with the bulk of its polypeptide in the lumen, involves its retrotranslocation into the cytosol in a manner that depends on the AAA-ATPase p97/VCP [3]. This is followed by proteolytic processing and mobilization of the transcriptionally active p110 form of Nrf1 to the nucleus. A subsequent study suggested that site-specific proteolytic processing of Nrf1 by the proteasome yields an active 75 kDa fragment [4]. We show here that under conditions where all three active sites of the proteasome are completely blocked, p120 Nrf1 can still be proteolytically cleaved to the p110 form, which is translocated to the nucleus to activate transcription of PSM genes. Thus, our results indicate that a proteasome-independent pathway can promote the release of active p110 Nrf1 from the ER membrane. PMID:27676297

  6. NAC1 regulates the recruitment of the proteasome complex into dendritic spines.

    PubMed

    Shen, Haowei; Korutla, Laxminarayana; Champtiaux, Nicholas; Toda, Shigenobu; LaLumiere, Ryan; Vallone, Joseph; Klugmann, Matthias; Blendy, Julie A; Mackler, Scott A; Kalivas, Peter W

    2007-08-15

    Coordinated proteolysis of synaptic proteins is required for synaptic plasticity, but a mechanism for recruiting the ubiquitin-proteasome system (UPS) into dendritic spines is not known. NAC1 is a cocaine-regulated transcriptional protein that was found to complex with proteins in the UPS, including cullins and Mov34. NAC1 and the proteasome were cotranslocated from the nucleus into dendritic spines in cortical neurons in response to proteasome inhibition or disinhibiting synaptic activity with bicuculline. Bicuculline also produced a progressive accumulation of the proteasome and NAC1 in the postsynaptic density. Recruitment of the proteasome into dendrites and postsynaptic density by bicuculline was prevented in neurons from mice harboring an NAC1 gene deletion or in neurons transfected with mutated NAC1 lacking the proteasome binding domain. These experiments show that NAC1 modulates the translocation of the UPS from the nucleus into dendritic spines, thereby suggesting a potential missing link in the recruitment of necessary proteolysis machinery for synaptic remodeling.

  7. Nrf1 can be processed and activated in a proteasome-independent manner.

    PubMed

    Vangala, Janakiram R; Sotzny, Franziska; Krüger, Elke; Deshaies, Raymond J; Radhakrishnan, Senthil K

    2016-09-26

    In response to proteasome inhibition, the transcription factor Nrf1 facilitates de novo synthesis of proteasomes by inducing proteasome subunit (PSM) genes [1,2]. Previously, we showed that activation of the p120 form of Nrf1, a membrane-bound protein in the endoplasmic reticulum (ER) with the bulk of its polypeptide in the lumen, involves its retrotranslocation into the cytosol in a manner that depends on the AAA-ATPase p97/VCP [3]. This is followed by proteolytic processing and mobilization of the transcriptionally active p110 form of Nrf1 to the nucleus. A subsequent study suggested that site-specific proteolytic processing of Nrf1 by the proteasome yields an active 75 kDa fragment [4]. We show here that under conditions where all three active sites of the proteasome are completely blocked, p120 Nrf1 can still be proteolytically cleaved to the p110 form, which is translocated to the nucleus to activate transcription of PSM genes. Thus, our results indicate that a proteasome-independent pathway can promote the release of active p110 Nrf1 from the ER membrane.

  8. Dithiocarbamate-based coordination compounds as potent proteasome inhibitors in human cancer cells.

    PubMed

    Buac, Daniela; Schmitt, Sara; Ventro, George; Kona, Fathima Rani; Dou, Q Ping

    2012-10-01

    Dithiocarbamates are a class of metal-chelating compounds with various applications in medicine. They have been used for the treatment of bacterial and fungal infections, possible treatment of AIDS, and most recently cancer. Their anti-tumor effects can in part be attributed to their ability to complex tumor cellular copper, leading to binding to and inhibition of the proteasome and in turn initiating tumor cell-specific apoptosis. Current chemotherapeutic agents are highly toxic and therefore their efficacy in the eradication of tumors is greatly limited. As a result many scientists have joined the quest for novel targeted therapies in hopes of reducing toxicity while maximizing potency and proteasome inhibition has become an attractive therapy in this regard. Here we discuss the origins, mechanism, and evolution of dithiocarbamates as potent proteasome inhibitors and therefore anti-cancer agents. PMID:22931591

  9. Dithiocarbamate-Based Coordination Compounds as Potent Proteasome Inhibitors in Human Cancer Cells

    PubMed Central

    Buac, Daniela; Schmitt, Sara; Ventro, George; Kona, Fathima Rani; Dou, Q. Ping

    2013-01-01

    Dithiocarbamates are a class of metal-chelating compounds with various applications in medicine. They have been used for the treatment of bacterial and fungal infections, possible treatment of AIDS, and most recently cancer. Their anti-tumor effects can in part be attributed to their ability to complex tumor cellular copper, leading to binding to and inhibition of the proteasome and in turn initiating tumor cell-specific apoptosis. Current chemotherapeutic agents are highly toxic and therefore their efficacy in the eradication of tumors is greatly limited. As a result many scientists have joined the quest for novel targeted therapies in hopes of reducing toxicity while maximizing potency and proteasome inhibition has become an attractive therapy in this regard. Here we discuss the origins, mechanism, and evolution of dithiocarbamates as potent proteasome inhibitors and therefore anti-cancer agents. PMID:22931591

  10. Insights into the relationship between the proteasome and autophagy in human and yeast cells.

    PubMed

    Athané, Axel; Buisson, Anthony; Challier, Marion; Beaumatin, Florian; Manon, Stéphen; Bhatia-Kiššová, Ingrid; Camougrand, Nadine

    2015-07-01

    In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions. PMID:25882491

  11. Proteasome regulates turnover of toxic human amylin in pancreatic cells

    PubMed Central

    Singh, Sanghamitra; Trikha, Saurabh; Sarkar, Anjali; Jeremic, Aleksandar M.

    2016-01-01

    Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin’s clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma β-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, keeping the net cytosolic amylin content low. An increase in hA accumulation in the nucleus of pancreatic cells correlated with its cytotoxicity, suggesting that its excessive accumulation in the nucleus is detrimental. hA interacted with 20S core and 19S lid subunits of the β-cell proteasomal complex, as suggested by immunoprecipitation and confocal microscopy studies, which subsequently resulted in a decrease in the proteasome’s proteolytic activity in these cells. In vitro binding and activity assays confirmed an intrinsic and potent ability of amylin to interact with the 20S core complex thereby modulating its proteolytic activity. Interestingly, less toxic and aggregation incapable rat amylin (rA) showed a comparable inhibitory effect on proteasome activity and protein ubiquitination, decoupling amylin aggregation/toxicity and amylin-induced protein stress. In agreement with these studies, inhibition of proteasomal proteolytic activity significantly increased intracellular amylin content and toxicity. Taken together, our results suggest a pivotal role of proteasomes in amylin’s turnover and detoxification in pancreatic cells. PMID:27340132

  12. Effect of ajoene, a natural antitumor small molecule, on human 20S proteasome activity in vitro and in human leukemic HL60 cells.

    PubMed

    Xu, Bo; Monsarrat, Bernard; Gairin, Jean Edouard; Girbal-Neuhauser, Elisabeth

    2004-04-01

    The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

  13. Cyclic AMP signaling reduces sirtuin 6 expression in non-small cell lung cancer cells by promoting ubiquitin-proteasomal degradation via inhibition of the Raf-MEK-ERK (Raf/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase) pathway.

    PubMed

    Kim, Eui-Jun; Juhnn, Yong-Sung

    2015-04-10

    The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gαs plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.

  14. Native structure of rat liver immune proteasomes.

    PubMed

    Stepanova, A A; Lyupina, Yu V; Sharova, N P; Erokhov, P A

    2016-05-01

    Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of "rotated" ubiquitinated proteins. PMID:27417720

  15. Ubiquitination and proteasome degradation of the E6 proteins of human papillomavirus types 11 and 18.

    PubMed

    Stewart, Deborah; Kazemi, Shirin; Li, Suiyang; Massimi, Paola; Banks, Lawrence; Koromilas, Antonis E; Matlashewski, Greg

    2004-06-01

    Human papillomaviruses (HPVs) are aetiological agents for genital warts and cervical cancer, the different pathologies of which are dependent on the type of HPV infection. Oncogenic HPV types associated with cancer are carcinogens by virtue of their oncogene products, which target key regulators of cell proliferation and apoptosis. The viral E6 protein from oncogenic HPV types plays a central role in carcinogenesis by exploiting the cellular proteasome degradation pathway in order to mediate the degradation of cellular proteins, most notably the prototype tumour suppressor protein p53. Much less is known about the cellular targets of E6 from the non-oncogenic HPV types associated with genital warts. It is also unclear what factors influence the level and stability of the viral E6 proteins in cells. This report demonstrates that both oncogenic and non-oncogenic HPV E6 proteins (from types 18 and 11, respectively) are ubiquitinated and targeted for degradation by the 26S proteasome. E6 domains required for the induction of p53 or DLG degradation, or E6AP binding, are not involved in proteasome-mediated degradation of HPV-18 E6. These results provide insight into the cellular modulation of E6 protein levels from both high-risk and low-risk HPV types. PMID:15166424

  16. Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation

    PubMed Central

    Elharar, Yifat; Roth, Ziv; Hermelin, Inna; Moon, Alexandra; Peretz, Gabriella; Shenkerman, Yael; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2014-01-01

    Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S-proteasome, bacterial prokaryotic ubiquitin-like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup-proteasome system (PPS) is important for virulence, yet its physiological role in non-pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto-regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation. PMID:24986881

  17. Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

    PubMed

    Dos Santos, Maísa Pavani; Batistela, Emanuele; Pereira, Mayara Peron; Paula-Gomes, Silvia; Zanon, Neusa Maria; Kettelhut, Isis do Carmo; Karatzaferi, Christina; Andrade, Claudia Marlise Balbinotti; de França, Suélem Aparecida; Baviera, Amanda Martins; Kawashita, Nair Honda

    2016-08-01

    Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis.

  18. Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein

    PubMed Central

    Rothbarth, Karsten; Stammer, Hermann; Werner, Dieter

    2002-01-01

    The C1D gene is expressed in a broad spectrum of mammalian cells and tissues but its product induces apoptotic cell death when exceeding a critical level. Critical levels are achieved in a fraction of cells by transient transfection with EGFP-tagged C1D expression constructs. However, transfected cells expressing sub-critical levels of C1D(EGFP) escape apoptotic cell death by activation of a proteasome-mediated rescue mechanism. Inhibition of the proteasome-dependent degradation of the C1D(EGFP) protein results in a parallel increase of the intracellular C1D level and in the fraction of apoptotic cells. PMID:12379155

  19. Serendipity in discovery of proteasome inhibitors.

    PubMed

    Dunn, Derek; Iqbal, Mohamed; Husten, Jean; Ator, Mark A; Chatterjee, Sankar

    2012-05-15

    Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported. PMID:22503349

  20. Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28

    PubMed Central

    Hernebring, Malin; Fredriksson, Åsa; Liljevald, Maria; Cvijovic, Marija; Norrman, Karin; Wiseman, John; Semb, Henrik; Nyström, Thomas

    2013-01-01

    In embryonic stem cells, removal of oxidatively damaged proteins is triggered upon the first signs of cell fate specification but the underlying mechanism is not known. Here, we report that this phase of differentiation encompasses an unexpected induction of genes encoding the proteasome activator PA28αβ (11S), subunits of the immunoproteasome (20Si), and the 20Si regulator TNFα. This induction is accompanied by assembly of mature PA28-20S(i) proteasomes and elevated proteasome activity. Inhibiting accumulation of PA28α using miRNA counteracted the removal of damaged proteins demonstrating that PA28αβ has a hitherto unidentified role required for resetting the levels of protein damage at the transition from self-renewal to cell differentiation. PMID:23459332

  1. Deimination of the myelin basic protein decelerates its proteasome-mediated metabolism.

    PubMed

    Kuzina, E S; Kudriaeva, A A; Glagoleva, I S; Knorre, V D; Gabibov, A G; Belogurov, A A

    2016-07-01

    Deimination of myelin basic protein (MBP) by peptidylarginine deiminase (PAD) prevents its binding to the proteasome and decelerates its degradation by the proteasome in mammalian cells. Potential anticancer drug tetrazole analogue of chloramidine 2, at concentrations greater than 1 µM inhibits the enzymatic activity of PAD in vitro. The observed acceleration of proteasome hydrolysis of MBP to antigenic peptides in the presence of PAD inhibitor may increase the efficiency of lesion of the central nervous system by cytotoxic lymphocytes in multiple sclerosis. We therefore suggest that clinical trials and the introduction of PAD inhibitors in clinical practice for the treatment of malignant neoplasms should be performed only after a careful analysis of their potential effect on the induction of autoimmune neurodegeneration processes. PMID:27599511

  2. The ubiquitin–proteasome pathway protects Chlamydomonas reinhardtii against selenite toxicity, but is impaired as reactive oxygen species accumulate

    PubMed Central

    Vallentine, Patrick; Hung, Chiu-Yueh; Xie, Jiahua; Van Hoewyk, Doug

    2014-01-01

    The ubiquitin–proteasome pathway (UPP) coordinates a myriad of physiological processes in higher plants, including abiotic stress responses, but it is less well characterized in algal species. In this study, the green alga Chlamydomonas reinhardtii was used to gain insights into the role of the UPP during moderate and severe selenite stress at three different time points. The data indicate that activity of the UPP in response to selenium (Se) stress was both time and dose dependent. Moderate selenite stress increased proteasome activity, protein ubiquitination and the proteasomal removal of malformed selenoproteins. However, severe Se stress caused by prolonged selenite treatment or high selenite concentration decreased proteasome activity, inhibited protein ubiquitination and prevented the proteasomal removal of selenoproteins. The UPP impairment during severe Se stress was associated with the observed accumulation of reactive oxygen species (ROS), including mitochondrial superoxide. Additionally, proteasomal inhibition decreased the concentration of chlorophyll in cultures challenged with Se. Therefore, although the UPP protects Chlamydomonas against Se stress, severe oxidative stress induced by selenite toxicity likely hinders the UPP's capacity to mediate a stress response. The possibility that stress tolerance in plants is dependent upon optimal UPP activity and maintenance is discussed. PMID:25301821

  3. Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana.

    PubMed

    Kim, Moonil; Yang, Kyoung-Sil; Kim, Yu-Kyung; Paek, Kyung-Hee; Pai, Hyun-Sook

    2003-02-28

    The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus. PMID:12661772

  4. Breast cancer cells: Modulation by melatonin and the ubiquitin-proteasome system--a review.

    PubMed

    Vriend, Jerry; Reiter, Russel J

    2015-12-01

    Melatonin inhibits human breast cancer cells stimulated with estrogen. This antiproliferative action depends on the presence of the estrogen receptor alpha (ERα) in the human MCF-7 cell line and is strictly dose-dependent. Since researchers concerned with melatonin and breast cancer have not considered the relevance of the ubiquitin-proteasome system to this research in this review we do so. The fact that the first breast cancer susceptibility gene to be identified, Brca1, functions as a ubiquitin ligase indicates that the ubiquitin-proteasome system has a role in regulating susceptibility to breast cancer. While mutations of this gene increase the incidence of breast cancer, the wild type gene suppresses estrogen-dependent transcriptional events relying on the estrogen receptor ERα. Three other ubiquitin ligases, SCF(Skp2), E6AP and APC, interact directly with ERα at the ERE and AP-1 promoters of ERα target genes. Melatonin, like proteasome inhibitors, decreases estrogen-induced gene transcription. Indeed, it has been reported that melatonin specifically inhibits estrogen-induced transcription mediated by ERα at the ERE and AP1 gene promoters. Herein, we present a model in which the inhibitory action of melatonin on MCF-7 cells is mediated, directly or indirectly, by the ubiquitin-proteasome system. In this model ERα, apoptotic proteins, and cell cycle proteins, all influenced by melatonin, are substrates of key ubiquitin ligases including SCF(Skp2), E6AP, and SCF(B-TrCP). Since dysfunction of the ubiquitin-proteasome system is a risk factor for breast cancer, this model provides a context in which to test the clinical potential, and limitations, of melatonin and proteasome inhibitors. PMID:26363225

  5. The 19S proteasome activator promotes human cytomegalovirus immediate early gene expression through proteolytic and nonproteolytic mechanisms.

    PubMed

    Winkler, Laura L; Kalejta, Robert F

    2014-10-01

    Proteasomes are large, multisubunit complexes that support normal cellular activities by executing the bulk of protein turnover. During infection, many viruses have been shown to promote viral replication by using proteasomes to degrade cellular factors that restrict viral replication. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomal degradation of Daxx, a cellular transcriptional repressor that can silence viral immediate early (IE) gene expression. We previously showed that this degradation requires both the proteasome catalytic 20S core particle (CP) and the 19S regulatory particle (RP). The 19S RP associates with the 20S CP to facilitate protein degradation but also plays a 20S CP-independent role promoting transcription. Here, we present a nonproteolytic role of the 19S RP in HCMV IE gene expression. We demonstrate that 19S RP subunits are recruited to the major immediate early promoter (MIEP) that directs IE transcription. Depletion of 19S RP subunits generated a defect in RNA polymerase II elongation through the MIE locus during HCMV infection. Our results reveal that HCMV commandeers proteasome components for both proteolytic and nonproteolytic roles to promote HCMV lytic infection. Importance: Proteasome inhibitors decrease or eliminate 20S CP activity and are garnering increasing interest as chemotherapeutics. However, an increasing body of evidence implicates 19S RP subunits in important proteolytic-independent roles during transcription. Thus, pharmacological inhibition of the 20S CP as a means to modulate proteasome function toward therapeutic effect is an incomplete capitalization on the potential of this approach. Here, we provide an additional example of nonproteolytic 19S RP function in promoting HCMV transcription. These data provide a novel system with which to study the roles of different proteasome components during transcription, a rationale for previously described shifts in 19S RP subunit localization during

  6. Proteasome activity influences UV-mediated subnuclear localization changes of NPM.

    PubMed

    Moore, Henna M; Bai, Baoyan; Matilainen, Olli; Colis, Laureen; Peltonen, Karita; Laiho, Marikki

    2013-01-01

    UV damage activates cellular stress signaling pathways, causes DNA helix distortions and inhibits transcription by RNA polymerases I and II. In particular, the nucleolus, which is the site of RNA polymerase I transcription and ribosome biogenesis, disintegrates following UV damage. The disintegration is characterized by reorganization of the subnucleolar structures and change of localization of many nucleolar proteins. Here we have queried the basis of localization change of nucleophosmin (NPM), a nucleolar granular component protein, which is increasingly detected in the nucleoplasm following UV radiation. Using photobleaching experiments of NPM-fluorescent fusion protein in live human cells we show that NPM mobility increases after UV damage. However, we show that the increase in NPM nucleoplasmic abundance after UV is independent of UV-activated cellular stress and DNA damage signaling pathways. Unexpectedly, we find that proteasome activity affects NPM redistribution. NPM nucleolar expression was maintained when the UV-treated cells were exposed to proteasome inhibitors or when the expression of proteasome subunits was inhibited using RNAi. However, there was no evidence of increased NPM turnover in the UV damaged cells, or that ubiquitin or ubiquitin recycling affected NPM localization. These findings suggest that proteasome activity couples to nucleolar protein localizations in UV damage stress.

  7. The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

    PubMed Central

    Pinto, Maria J.; Alves, Pedro L.; Martins, Luís; Pedro, Joana R.; Ryu, Hyun R.; Jeon, Noo Li; Taylor, Anne M.

    2016-01-01

    Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin–proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses. PMID:27022091

  8. Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16

    PubMed Central

    Bigler, Laurent; Dudler, Robert

    2014-01-01

    Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts. PMID:24727275

  9. Identification of proteasome subunit beta type 2 associated with deltamethrin detoxification in Drosophila Kc cells by cDNA microarray analysis and bioassay analyses.

    PubMed

    Hu, Junli; Jiao, Dongxu; Xu, Qin; Ying, Xiaoli; Liu, Wei; Chi, Qingping; Ye, Yuting; Li, Xueyu; Cheng, Luogen

    2016-05-10

    Insecticide deltamethrin resistance has presented a difficult obstacle for pest control and the resistance development is complex and associated with many genes. To better understand the possible molecular mechanisms involved in DM stress, in this study, cDNA microarray analysis was employed. 448 differentially expressed genes with at least a 2-fold expression difference were identified in Drosophila cells after DM exposure. Moreover, some genes were confirmed with qPCR, which yielded results consistent with the microarray analysis. Three members of the ubiquitin-proteasome system were significantly elevated in DM-stressed cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM detoxification. The proteasome beta2 subunit (Prosbeta2) is a member of 20S proteasome subunit family, which forms the proteolytic core of 26S proteasome. Whether Prosbeta2 participates in DM detoxification requires further study. RNAi and heterologous expression were conducted to investigate the contribution of Prosbeta2 in DM detoxification. The results revealed Prosbeta2 knockdown significantly reduce the level of DM detoxification in RNAi-treated cells after 48 h. Overexpression of Prosbeta2 increased cellular viability. These detoxification results represent the first evidence that Prosbeta2 plays a role in the detoxification of DM, which may provide new idea and target for studying the molecular mechanisms of insect resistance.

  10. Controlled access of p53 to the nucleus regulates its proteasomal degradation by MDM2.

    PubMed

    Davis, James R; Mossalam, Mohanad; Lim, Carol S

    2013-04-01

    The tumor suppressor p53 can be sent to the proteasome for degradation by placing its nucleo-cytoplasmic shuttling under ligand control. Endogenous p53 is ubiquitinated by MDM2 in the nucleus, and controlling the access of p53 to the nuclear compartment regulates its ubiquitination and proteasomal degradation. This was accomplished by the use of a protein switch that places nuclear translocation under the control of externally applied dexamethasone. Fluorescence microscopy revealed that sending protein switch p53 (PS-p53) to the nucleus produces a distinct punctate distribution in both the cytoplasm and nucleus. The nuclear role in accessing the proteasome was investigated by inhibiting classical nuclear export with leptomycin B. Trapping PS-p53 in the nucleus only allows this punctate staining in that compartment, suggesting that PS-p53 must translocate first to the nuclear compartment for cytoplasmic punctate staining to occur. The role of MDM2 binding was explored by inhibiting MDM2/p53 binding with nutlin-3. Inhibition of this interaction blocked both nuclear export and cytoplasmic and nuclear punctate staining, providing evidence that any change in localization after nuclear translocation is due to MDM2 binding. Further, blocking the proteolytic activity of the proteasome maintained the nuclear localization of the construct. Truncations of p53 were made to determine smaller constructs still capable of interacting with MDM2, and their subcellular localization and degradation potential was observed. PS-p53 and a smaller construct containing the two MDM2 binding regions of p53 (Box I + V) were indeed degraded by the proteasome as measured by loss of enhanced green fluorescent protein that was also fused to the construct. The influence of these constructs on p53 gene transactivation function was assessed and revealed that PS-p53 decreased gene transactivation, while PS-p53(Box I + V) did not significantly change baseline gene transactivation.

  11. Synergy between Proteasome Inhibitors and Imatinib Mesylate in Chronic Myeloid Leukemia

    PubMed Central

    Liu, Da-Peng; Liu, Ying; Feng, Ting-Ting; Meng, Fan-Yi; Liu, Xiao-Li; Jiang, Qian-Li; Chen, Xiao-Qin; Liu, Jing-Lei; Liu, Ping; Chen, Zhu; Chen, Sai-Juan; Zhou, Guang-Biao

    2009-01-01

    Background Resistance developed by leukemic cells, unsatisfactory efficacy on patients with chronic myeloid leukemia (CML) at accelerated and blastic phases, and potential cardiotoxity, have been limitations for imatinib mesylate (IM) in treating CML. Whether low dose IM in combination with agents of distinct but related mechanisms could be one of the strategies to overcome these concerns warrants careful investigation. Methods and Findings We tested the therapeutic efficacies as well as adverse effects of low dose IM in combination with proteasome inhibitor, Bortezomib (BOR) or proteasome inhibitor I (PSI), in two CML murine models, and investigated possible mechanisms of action on CML cells. Our results demonstrated that low dose IM in combination with BOR exerted satisfactory efficacy in prolongation of life span and inhibition of tumor growth in mice, and did not cause cardiotoxicity or body weight loss. Consistently, BOR and PSI enhanced IM-induced inhibition of long-term clonogenic activity and short-term cell growth of CML stem/progenitor cells, and potentiated IM-caused inhibition of proliferation and induction of apoptosis of BCR-ABL+ cells. IM/BOR and IM/PSI inhibited Bcl-2, increased cytoplasmic cytochrome C, and activated caspases. While exerting suppressive effects on BCR-ABL, E2F1, and β-catenin, IM/BOR and IM/PSI inhibited proteasomal degradation of protein phosphatase 2A (PP2A), leading to a re-activation of this important negative regulator of BCR-ABL. In addition, both combination therapties inhibited Bruton's tyrosine kinase via suppression of NFκB. Conclusion These data suggest that combined use of tyrosine kinase inhibitor and proteasome inhibitor might be helpful for optimizing CML treatment. PMID:19606213

  12. Trial Watch: Proteasomal inhibitors for anticancer therapy

    PubMed Central

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called “ubiquitin-proteasome system” (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  13. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  14. Proteasome inhibitors in multiple myeloma: 10 years later

    PubMed Central

    Richardson, Paul G.; Cavo, Michele; Orlowski, Robert Z.; San Miguel, Jesús F.; Palumbo, Antonio; Harousseau, Jean-Luc

    2012-01-01

    Proteasome inhibition has emerged as an important therapeutic strategy in multiple myeloma (MM). Since the publication of the first phase 1 trials of bortezomib 10 years ago, this first-in-class proteasome inhibitor (PI) has contributed substantially to the observed improvement in survival in MM patients over the past decade. Although first approved as a single agent in the relapsed setting, bortezomib is now predominantly used in combination regimens. Furthermore, the standard twice-weekly schedule may be replaced by weekly infusion, especially when bortezomib is used as part of combination regimens in frontline therapy. Indeed, bortezomib is an established component of induction therapy for patients eligible or ineligible for autologous stem cell transplantation. Bortezomib has also been incorporated into conditioning regimens before autologous stem cell transplantation, as well as into post-ASCT consolidation therapy, and in the maintenance setting. In addition, a new route of bortezomib administration, subcutaneous infusion, has recently been approved. Recently, several new agents have been introduced into the clinic, including carfilzomib, marizomib, and MLN9708, and trials investigating these “second-generation” PIs in patients with relapsed/refractory MMs have demonstrated positive results. This review provides an overview of the role of PIs in the treatment of MM, focusing on developments over the past decade. PMID:22645181

  15. Proteasome Inhibitors Evoke Latent Tumor Suppression Programs in Pro-B MLL Leukemias Through MLL-AF4

    PubMed Central

    Liu, Han; Westergard, Todd D.; Cashen, Amanda; Piwnica-Worms, David R.; Kunkle, Lori; Vij, Ravi; Pham, Can G.; DiPersio, John; Cheng, Emily H.; Hsieh, James J.

    2014-01-01

    SUMMARY Chromosomal translocations disrupting MLL generate MLL-fusion proteins that induce aggressive leukemias. Unexpectedly, MLL-fusion proteins are rarely observed at high levels, suggesting excessive MLL-fusions may be incompatible with a malignant phenotype. Here, we used clinical proteasome inhibitors, bortezomib and carfilzomib, to reduce the turnover of endogenous MLL-fusions and discovered that accumulated MLL-fusions induce latent, context-dependent tumor suppression programs. Specifically, in MLL pro-B lymphoid, but not myeloid, leukemias, proteasome inhibition triggers apoptosis and cell cycle arrest involving activation cleavage of BID by Caspase-8 and upregulation of p27, respectively. Furthermore, proteasome inhibition conferred preliminary benefit to MLL-AF4 leukemia patients. Hence, feasible strategies to treat cancer-type and oncogene specific cancers can be improvised through harnessing inherent tumor suppression properties of individual oncogenic fusions. PMID:24735925

  16. Intracellular Dynamics of the Ubiquitin-Proteasome-System.

    PubMed

    Chowdhury, Maisha; Enenkel, Cordula

    2015-01-01

    The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body's cells. PMID:26339477

  17. The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT61[OPEN

    PubMed Central

    2015-01-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. PMID:25739698

  18. Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity and Induces Tumor Cell Death.

    PubMed

    Bachmann, André S; Opoku-Ansah, John; Ibarra-Rivera, Tannya R; Yco, Lisette P; Ambadi, Sudhakar; Roberts, Christopher C; Chang, Chia-En A; Pirrung, Michael C

    2016-04-15

    Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (β1, β2, and β5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.

  19. Proteasome activity and proteasome subunit transcripts in human spermatozoa separated by a discontinuous Percoll gradient.

    PubMed

    Rosales, O; Opazo, C; Diaz, E S; Villegas, J V; Sanchez, R; Morales, P

    2011-04-01

    Human semen is composed of a heterogeneous population of spermatozoa with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. Using a discontinuous Percoll gradient, we separated three subsets of spermatozoa (65/45%, 90/65% and 90% fractions) from normozoospermic semen samples from healthy donors and proceeded to characterise their morphology, viability, motility and proteasome activity. In addition, the presence of proteasome subunit transcripts was investigated using reverse transcription-polymerase chain reaction (RT-PCR). The results obtained showed significant differences in sperm motility, viability and morphology between the cells collected from each of the fractions. In particular, normal sperm morphology was 4.5 times higher in the 90% pellet in comparison with the 65/45% interface. In addition, there were significant differences in proteasomal activity between spermatozoa recovered from the 90% pellet and spermatozoa recovered from the 65/45% interface. Finally, there was a positive correlation between sperm proteasomal enzymatic activity and sperm motility and normal morphology after separation by a discontinuous Percoll gradient. The results of the RT-PCR revealed the presence of transcripts for the proteasome subunits β1, β2 and β5 in the human spermatozoa analysed. In conclusion, poor quality spermatozoa isolated from a Percoll gradient display an intrinsic proteasome activity deficiency, which may be associated with their low fertilising potential.

  20. Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

    PubMed

    Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L; Hink, Mark A; Bates, Gillian P; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

    2014-01-01

    Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin-proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington's disease, and support the reported absence of proteasome impairment in mouse models of Huntington's disease.

  1. Commentary on "Proteasome Inhibitors: A Novel Class of Potent and Effective Antitumor Agents".

    PubMed

    Tew, Kenneth D

    2016-09-01

    The relatively recent clinical success of bortezomib, particularly in multiple myeloma, has established the validity of the proteasome as a viable target for anticancer drug development. This highly cited 1999 Cancer Research article from Adams and colleagues was published during the period when this drug was transitioning from preclinical studies to phase I clinical trial status. Their results detail structure-activity analyses using a series of boronic acid proteasome inhibitors and correlate cytotoxicity with inhibition of proteasome activity. In and of itself, the recognition that interference with proteasome functions represented a novel therapeutic approach likely underlies the popularity of this article. In addition, the provision of in vitro (at that time using the NCI 60 cell line panel) and in vivo antitumor activity, toxicology, and mouse pharmacokinetic and pharmacodynamic data provided a solid basis for establishing the future credentials for bortezomib to gain initial FDA approval in 2003. Cancer Res; 76(17); 4916-7. ©2016 AACRSee related article by Adams et al., Cancer Res 1999;59:2615-22Visit the Cancer Research 75(th) Anniversary timeline. PMID:27587650

  2. Defective regulation of the ubiquitin/proteasome system in the hypothalamus of obese male mice.

    PubMed

    Ignacio-Souza, Leticia M; Bombassaro, Bruna; Pascoal, Livia B; Portovedo, Mariana A; Razolli, Daniela S; Coope, Andressa; Victorio, Sheila C; de Moura, Rodrigo F; Nascimento, Lucas F; Arruda, Ana P; Anhe, Gabriel F; Milanski, Marciane; Velloso, Licio A

    2014-08-01

    In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity.

  3. The Ubiquitin–Proteasome System Regulates the Stability of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Rezvani, Khosrow; Teng, Yanfen

    2010-01-01

    Ubiquitination is a key event for protein degradation by the proteasome system, membrane protein internalization, and protein trafficking among cellular compartments. Few data are available on the role of the ubiquitin–proteasome system (UPS) in the trafficking of neuronal nicotinic acetylcholine receptors (nAChRs). Experiments conducted in neuron-like differentiated rat pheochromocytoma cells (PC12 cells) show that the α3, β2, and β4 nAChR subunits are ubiquitinated and that their ubiquitination is necessary for degradation. A 24-h treatment with the proteasome inhibitor PS-341 increased the total levels of α3 and the two β subunits in both whole cell lysates and fractions enriched for the ER/Golgi compartment. nAChR subunit upregulation was also detected in plasma membrane-enriched fractions. Inhibition of the lysosomal degradation machinery by E-64 had a significantly smaller effect on nAChR turnover. The present data, together with previous results showing that the α7 nAChR subunit is a target of the UPS, point to a prominent role of the proteasome in nAChR trafficking. PMID:19693707

  4. Commentary on "Proteasome Inhibitors: A Novel Class of Potent and Effective Antitumor Agents".

    PubMed

    Tew, Kenneth D

    2016-09-01

    The relatively recent clinical success of bortezomib, particularly in multiple myeloma, has established the validity of the proteasome as a viable target for anticancer drug development. This highly cited 1999 Cancer Research article from Adams and colleagues was published during the period when this drug was transitioning from preclinical studies to phase I clinical trial status. Their results detail structure-activity analyses using a series of boronic acid proteasome inhibitors and correlate cytotoxicity with inhibition of proteasome activity. In and of itself, the recognition that interference with proteasome functions represented a novel therapeutic approach likely underlies the popularity of this article. In addition, the provision of in vitro (at that time using the NCI 60 cell line panel) and in vivo antitumor activity, toxicology, and mouse pharmacokinetic and pharmacodynamic data provided a solid basis for establishing the future credentials for bortezomib to gain initial FDA approval in 2003. Cancer Res; 76(17); 4916-7. ©2016 AACRSee related article by Adams et al., Cancer Res 1999;59:2615-22Visit the Cancer Research 75(th) Anniversary timeline.

  5. Assaying Proteasomal Degradation in a Cell-free System in Plants

    PubMed Central

    García-Cano, Elena; Zaltsman, Adi; Citovsky, Vitaly

    2014-01-01

    The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions. The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions. PMID:24747194

  6. Differential regulation of the REGγ–proteasome pathway by p53/TGF-β signalling and mutant p53 in cancer cells

    PubMed Central

    Ali, Amjad; Wang, Zhuo; Fu, Junjiang; Ji, Lei; Liu, Jiang; Li, Lei; Wang, Hui; Chen, Jiwu; Caulin, Carlos; Myers, Jeffrey N.; Zhang, Pei; Xiao, Jianru; Zhang, Bianhong; Li, Xiaotao

    2013-01-01

    Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-β signalling inhibits the REGγ–20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ–20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-β signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ–20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-β signalling and the REGγ–20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop. PMID:24157709

  7. The regulation of glucose on milk fat synthesis is mediated by the ubiquitin-proteasome system in bovine mammary epithelial cells.

    PubMed

    Liu, Lily; Jiang, Li; Ding, Xiang-dong; Liu, Jian-feng; Zhang, Qin

    2015-09-11

    Glucose as one of the nutrition factors plays a vital role in the regulation of milk fat synthesis. Ubiquitin-proteasome system (UPS) is a vital proteolytic pathway in all eukaryotic cells through timely marking, recognizing and degrading the poly-ubiquitinated protein substrates. Previous studies indicated that UPS plays a considerable role in controlling the triglyceride (TG) synthesis. Therefore, the aim of this study is to confirm the link between high-glucose and UPS and its regulation mechanism on milk fat synthesis in BMEC (bovine mammary epithelial cells). We incubated BMEC with normal (17.5 mm/L) and high-glucose (25 mm/L) with and without proteasome inhibitor epoxomicin and found that, compared with the control (normal glucose and without proteasome inhibitor), both high-glucose concentration and proteasome inhibitor epoxomicin could increase the accumulation of TG and poly-ubiquitinated proteins, and reduce significantly three proteasome activities (chymotrypsin-like, caspase-like, and trypsin-like). In addition, high-glucose concentration combined with proteasome inhibitor further enhanced the increase of the poly-ubiquitinated protein level and the decrease of proteasome activities. Our results suggest that the regulation of high-glucose on milk fat synthesis is mediated by UPS in BMEC, and high-glucose exposure could lead to a hypersensitization of BMEC to UPS inhibition which in turn results in increased milk fat synthesis.

  8. Design Principles Involving Protein Disorder Facilitate Specific Substrate Selection and Degradation by the Ubiquitin-Proteasome System.

    PubMed

    Guharoy, Mainak; Bhowmick, Pallab; Tompa, Peter

    2016-03-25

    The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss post-translational modifications within disordered regions that regulate E3 ligase localization, conformation, and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons, and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core.

  9. Pupylation-dependent and -independent proteasomal degradation in mycobacteria.

    PubMed

    Imkamp, Frank; Ziemski, Michal; Weber-Ban, Eilika

    2015-08-01

    Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway. PMID:26352358

  10. Activity-based imaging probes of the proteasome.

    PubMed

    Carmony, Kimberly Cornish; Kim, Kyung Bo

    2013-09-01

    Over the years, the proteasome has been extensively investigated due to its crucial roles in many important signaling pathways and its implications in diseases. Two proteasome inhibitors--bortezomib and carfilzomib--have received FDA approval for the treatment of multiple myeloma, thereby validating the proteasome as a chemotherapeutic target. As a result, further research efforts have been focused on dissecting the complex biology of the proteasome to gain the insight required for developing next-generation proteasome inhibitors. It is clear that chemical probes have made significant contributions to these efforts, mostly by functioning as inhibitors that selectively block the catalytic activity of proteasomes. Analogues of these inhibitors are now providing additional tools for visualization of catalytically active proteasome subunits, several of which allow real-time monitoring of proteasome activity in living cells as well as in in vivo settings. These imaging probes will provide powerful tools for assessing the efficacy of proteasome inhibitors in clinical settings. In this review, we will focus on the recent efforts towards developing imaging probes of proteasomes, including the latest developments in immunoproteasome-selective imaging probes. PMID:23700161

  11. Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells

    PubMed Central

    Daniel, Kenyon G; Chen, Di; Orlu, Shirley; Cui, Qiuzhi Cindy; Miller, Fred R; Dou, Q Ping

    2005-01-01

    Introduction A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison. Methods Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage. Results When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations

  12. Proteasomal Degradation of γ-Aminobutyric AcidB Receptors Is Mediated by the Interaction of the GABAB2 C Terminus with the Proteasomal ATPase Rtp6 and Regulated by Neuronal Activity*

    PubMed Central

    Zemoura, Khaled; Benke, Dietmar

    2014-01-01

    Regulation of cell surface expression of neurotransmitter receptors is crucial for determining synaptic strength and plasticity, but the underlying mechanisms are not well understood. We previously showed that proteasomal degradation of GABAB receptors via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery determines the number of cell surface GABAB receptors and thereby GABAB receptor-mediated neuronal inhibition. Here, we show that proteasomal degradation of GABAB receptors requires the interaction of the GABAB2 C terminus with the proteasomal AAA-ATPase Rpt6. A mutant of Rpt6 lacking ATPase activity prevented degradation of GABAB receptors but not the removal of Lys48-linked ubiquitin from GABAB2. Blocking ERAD activity diminished the interaction of Rtp6 with GABAB receptors resulting in increased total as well as cell surface expression of GABAB receptors. Modulating neuronal activity affected proteasomal activity and correspondingly the interaction level of Rpt6 with GABAB2. This resulted in altered cell surface expression of the receptors. Thus, neuronal activity-dependent proteasomal degradation of GABAB receptors by the ERAD machinery is a potent mechanism regulating the number of GABAB receptors available for signaling and is expected to contribute to homeostatic neuronal plasticity. PMID:24482233

  13. Marizomib, a Proteasome Inhibitor for All Seasons: Preclinical Profile and a Framework for Clinical Trials

    PubMed Central

    Potts, B.C.; Albitar, M.X.; Anderson, K.C.; Baritaki, S.; Berkers, C.; Bonavida, B.; Chandra, J.; Chauhan, D.; Cusack, J.C.; Fenical, W.; Ghobrial, I.M.; Groll, M.; Jensen, P.R.; Lam, K.S.; Lloyd, G.K.; McBride, W.; McConkey, D.J.; Miller, C.P.; Neuteboom, S.T.C.; Oki, Y.; Ovaa, H.; Pajonk, F.; Richardson, P.G.; Roccaro, A.M.; Sloss, C.M.; Spear, M.A.; Valashi, E.; Younes, A.; Palladino, M.A.

    2013-01-01

    The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade®) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique β-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, che-motherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom’s macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid®), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside. PMID:21247382

  14. Cystic Fibrosis Transmembrane Conductance Regulator Controls Lung Proteasomal Degradation and Nuclear Factor-κB Activity in Conditions of Oxidative Stress

    PubMed Central

    Boncoeur, Emilie; Roque, Telma; Bonvin, Elise; Saint-Criq, Vinciane; Bonora, Monique; Clement, Annick; Tabary, Olivier; Henrion-Caude, Alexandra; Jacquot, Jacky

    2008-01-01

    Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-κB/IκB-α signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr−/−) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr+/+) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-κB inhibitor IκB-α. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-κB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl− channel by CFTRinh-172 in the normal bronchial immortalized cell line 16HBE14o− increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-κB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl− channel activity is crucial for regulation of lung proteasomal degradation and NF-κB activity in conditions of oxidative stress. PMID:18372427

  15. Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia

    PubMed Central

    Taylor, Cormac T.; Furuta, Glenn T.; Synnestvedt, Kristin; Colgan, Sean P.

    2000-01-01

    Hypoxia activates a number of gene products through degradation of the transcriptional coactivator cAMP response element binding protein (CREB). Other transcriptional regulators (e.g., β-catenin and NF-κB) are controlled through phosphorylation-targeted proteasomal degradation, and thus, we hypothesized a similar degradative pathway for CREB. Differential display analysis of mRNA derived from hypoxic epithelia revealed a specific and time-dependent repression of protein phosphatase 1 (PP1), a serine phosphatase important in CREB dephosphorylation. Subsequent studies identified a previously unappreciated proteasomal-targeting motif within the primary structure of CREB (DSVTDS), which functions as a substrate for PP1. Ambient hypoxia resulted in temporally sequential CREB serine phosphorylation, ubiquitination, and degradation (in vitro and in vivo). HIV-tat peptide-facilitated loading of intact epithelia with phosphopeptides corresponding to this proteasome targeting motif resulted in inhibition of CREB ubiquitination. Further studies revealed that PP1 inhibitors mimicked hypoxia-induced gene expression, whereas proteasome inhibitors reversed the hypoxic phenotype. Thus, hypoxia establishes conditions that target CREB to proteasomal degradation. These studies may provide unique insight into a general mechanism of transcriptional regulation by hypoxia. PMID:11035795

  16. Evaluation of copper-dependent proteasome-inhibitory and apoptosis-inducing activities of novel pyrrolidine dithiocarbamate analogues.

    PubMed

    Yu, Zhiyong; Wang, Fei; Milacic, Vesna; Li, Xiaofeng; Cui, Qiuzhi Cindy; Zhang, Bin; Yan, Bing; Dou, Q Ping

    2007-12-01

    Apoptosis has a central role in the pathogenesis of many human diseases, one of which is cancer. One of the most important strategies to regulate apoptosis is via the ubiquitin-proteasome pathway. It has been shown that inhibition of proteasomal chymotrypsin-like activity is a strong apoptosis-inducing stimulus and that actively proliferating cancer cells are more sensitive to proteasome inhibitors than normal or untransformed cells. Dithioscarbamates are a class of metal-chelating compounds with various applications in medicine. We reported previously that certain members of dithiocarbamates, such as pyrrolidine dithiocarbamate (PDTC), diethyldithiocarbamate and disulfiram, are able to bind with tumor cellular copper, forming an active complex with proteasome-inhibitory, apoptosis-inducing and anti-cancer activities. In the current study, we synthesized eight PDTC analogues with substitutions made to the pyrrolidine ring and studied their structure-activity relationships. We found that substitution of the pyrrolidine ring with piperidine had almost no effect on their proteasome-inhibitory and anti-proliferative potencies in human breast cancer cells. However, after the pyrrolidine ring was substituted with morpholine, the activity of the mixtures slightly decreased but was completely lost when piperazine with the attached ethyl group was used for the substitution. This structure-activity relationship was confirmed by the results generated with the corresponding copper complexes. Our data further support the novel concept of using accumulated copper in human cancer cells as a selective approach for chemotherapy. PMID:17982703

  17. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    PubMed Central

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  18. Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome.

    PubMed

    Reichard, Eden L; Chirico, Giavanna G; Dewey, William J; Nassif, Nicholas D; Bard, Katelyn E; Millas, Nickolas E; Kraut, Daniel A

    2016-08-26

    In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We find that the architecture of polyubiquitin chains attached to a substrate affects the ability of the proteasome to unfold and degrade the substrate, with K48- or mixed-linkage chains leading to greater processivity than K63-linked chains. Ubiquitin-independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin-dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. Our work supports a model in which a polyubiquitin chain associated with a substrate switches the proteasome into an activated state that persists throughout the degradation process. PMID:27405762

  19. A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster

    PubMed Central

    Pickering, Andrew M.; Staab, Trisha A.; Tower, John; Sieburth, Derek; Davies, Kelvin J. A.

    2013-01-01

    SUMMARY In mammalian cells, hydrogen peroxide (H2O2)-induced adaptation to oxidative stress is strongly dependent on an Nrf2 transcription factor-mediated increase in the 20S proteasome. Here, we report that both Caenorhabditis elegans nematode worms and Drosophila melanogaster fruit flies are also capable of adapting to oxidative stress with H2O2 pre-treatment. As in mammalian cells, this adaptive response in worms and flies involves an increase in proteolytic activity and increased expression of the 20S proteasome, but not of the 26S proteasome. We also found that the increase in 20S proteasome expression in both worms and flies, as in mammalian cells, is important for the adaptive response, and that it is mediated by the SKN-1 and CNC-C orthologs of the mammalian Nrf2 transcription factor, respectively. These studies demonstrate that stress mechanisms operative in cell culture also apply in disparate intact organisms across a wide biological diversity. PMID:23038734

  20. Association of plasminogen activator inhibitor type 2 (PAI-2) with proteasome within endothelial cells activated with inflammatory stimuli.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2011-12-16

    Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.

  1. Association of Plasminogen Activator Inhibitor Type 2 (PAI-2) with Proteasome within Endothelial Cells Activated with Inflammatory Stimuli*

    PubMed Central

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Cierniewski, Czeslaw S.

    2011-01-01

    Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling. PMID:21976669

  2. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde.

    PubMed

    Ortega-Atienza, Sara; Green, Samantha E; Zhitkovich, Anatoly

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA-protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions.

  3. Targeting the 19S proteasomal subunit, Rpt4, for the treatment of colon cancer.

    PubMed

    Boland, Karen; Flanagan, Lorna; McCawley, Niamh; Pabari, Ritesh; Kay, Elaine W; McNamara, Deborah A; Murray, Frank; Byrne, Annette T; Ramtoola, Zebunnissa; Concannon, Caoimhín G; Prehn, Jochen H M

    2016-06-01

    Deregulation of the ubiquitin-proteasome pathway has been frequently observed in a number of malignancies. Using quantitative Western blotting of normal and matched tumour tissue, we here identified a significant increase in the 19S proteasome subunit Rpt4 in response to chemoradiation in locally advanced rectal cancer patients with unfavourable outcome. We therefore explored the potential of Rpt4 reduction as a therapeutic strategy in colorectal cancer (CRC). Utilizing siRNA to down regulate Rpt4 expression, we show that silencing of Rpt4 reduced proteasomal activity and induced endoplasmic reticulum stress. Gene silencing of Rpt4 also inhibited cell proliferation, reduced clonogenic survival and induced apoptosis in HCT-116 colon cancer cells. We next developed a cell penetrating peptide-based nanoparticle delivery system to achieve in vivo gene silencing of Rpt4. Administration of Rpt4 siRNA nanoparticles reduced tumour growth and improved survival in a HCT-116 colon cancer xenograft tumour model in vivo. Collectively, our data suggest that inhibition of Rpt4 represents a novel strategy for the treatment of CRC. PMID:26997367

  4. Replication of the rotavirus genome requires an active ubiquitin-proteasome system.

    PubMed

    López, Tomás; Silva-Ayala, Daniela; López, Susana; Arias, Carlos F

    2011-11-01

    Here we show that the ubiquitin-proteasome system is required for the efficient replication of rotavirus RRV in MA104 cells. The proteasome inhibitor MG132 decreased the yield of infectious virus under conditions where it severely reduces the synthesis of not only viral but also cellular proteins. Addition of nonessential amino acids to the cell medium restored both viral protein synthesis and cellular protein synthesis, but the production of progeny viruses was still inhibited. In medium supplemented with nonessential amino acids, we showed that MG132 does not affect rotavirus entry but inhibits the replication of the viral genome. It was also shown that it prevents the efficient incorporation into viroplasms of viral polymerase VP1 and the capsid proteins VP2 and VP6, which could explain the inhibitory effect of MG132 on genome replication and infectious virus yield. We also showed that ubiquitination is relevant for rotavirus replication since the yield of rotavirus progeny in cells carrying a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme was reduced at the restrictive temperature. In addition, overexpression of ubiquitin in MG132-treated MA104 cells partially reversed the effect of the inhibitor on virus yield. Altogether, these data suggest that the ubiquitin-proteasome (UP) system has a very complex interaction with the rotavirus life cycle, with both the ubiquitination and proteolytic activities of the system being relevant for virus replication.

  5. Proteasomes and protein conjugation across domains of life

    PubMed Central

    Maupin-Furlow, Julie

    2012-01-01

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes. PMID:22183254

  6. Isolation and purification of proteasomes from primary cells.

    PubMed

    Steers, Nicholas J; Peachman, Kristina K; Alving, Carl R; Rao, Mangala

    2014-11-03

    Proteasomes play an important role in cell homeostasis and in orchestrating the immune response by systematically degrading foreign proteins and misfolded or damaged host cell proteins. We describe a protocol to purify functionally active proteasomes from human CD4(+) T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three-step process involving ion-exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described.

  7. Regulation of Cardiac Proteasomes by Ubiquitination, Sumoylation, and Beyond

    PubMed Central

    Cui, Ziyou; Scruggs, Sarah B.; Gilda, Jennifer E.; Ping, Peipei; Gomes, Aldrin V.

    2013-01-01

    The ubiquitin-proteasome system (UPS) is the major intracellular degradation system, and its proper function is critical to the health and function of cardiac cells. Alterations in cardiac proteasomes have been linked to several pathological phenotypes, including cardiomyopathies, ischemia-reperfusion injury, heart failure, and hypertrophy. Defects in proteasome-dependent cellular protein homeostasis can be causal for the initiation and progression of certain cardiovascular diseases. Emerging evidence suggests that the UPS can specifically target proteins that govern pathological signaling pathways for degradation, thus altering downstream effectors and disease outcomes. Alterations in UPS-substrate interactions in disease occur, in part, due to direct modifications of 19S, 11S or 20S proteasome subunits. Post-translational modifications (PTMs) are one facet of this proteasomal regulation, with over 400 known phosphorylation sites, over 500 ubiquitination sites and 83 internal lysine acetylation sites, as well as multiple sites for caspase cleavage, glycosylation (such as O-GlcNAc modification), methylation, nitrosylation, oxidation, and sumoylation. Changes in cardiac proteasome PTMs, which occur in ischemia and cardiomyopathies, are associated with changes in proteasome activity and proteasome assembly; however several features of this regulation remain to be explored. In this review, we focus on how some of the less common PTMs affect proteasome function and alter cellular protein homeostasis. PMID:24140722

  8. Small Molecule Control of Intracellular Protein Levels Through Modulation of the Ubiquitin Proteasome System

    PubMed Central

    Buckley, Dennis L.

    2015-01-01

    Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be easily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activities can be targeted instead, increasing the number of druggable targets. While targeting the proteasome itself can lead to a global increase in protein levels, targeting other components of the UPS (e.g., the hundreds of E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets such as enzymes and receptors. PMID:24459094

  9. A Conserved 20S Proteasome Assembly Factor Requires a C-terminal HbYX Motif for Proteasomal Precursor Binding

    PubMed Central

    Kusmierczyk, Andrew R.; Kunjappu, Mary J.; Kim, Roger Y.; Hochstrasser, Mark

    2011-01-01

    Dedicated chaperones facilitate eukaryotic proteasome assembly, yet how they function remains largely unknown. Here we demonstrate that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal HbYX (hydrophobic-tyrosine-X) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX-dependent fashion using the same proteasomal α-ring surface pockets bound by activators. Remarkably, PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data suggest an allosteric mechanism in which proteasome active-site maturation determines assembly chaperone binding, potentially shielding assembly intermediates or misassembled complexes from non-productive associations until assembly is complete. PMID:21499243

  10. Phylogenetic relationships within Cornus (Cornaceae) based on 26S rDNA sequences.

    PubMed

    Fan, C

    2001-06-01

    Phylogenetic relationships within the dogwood genus Cornus have been highly controversial due to the great morphological heterogeneity. Earlier phylogenetic analyses of Cornus using chloroplast DNA (cpDNA) data (including rbcL and matK sequences, as well as restriction sites) and morphological characters suggested incongruent relationships within the genus. The present study generated sequence data from the nuclear gene 26S rDNA for Cornus to test the phylogenetic hypotheses based on cpDNA and morphological data. The 26S rDNA sequence data obtained represent 16 species, 13 from Cornus and three from outgroups, having an aligned length of 3380 bp. Both parsimony and maximum likelihood analyses of these sequences were conducted. Trees resulting from these analyses suggest relationships among subgroups of Cornus consistent with those inferred from cpDNA data. That is, the dwarf dogwood (subg. Arctocrania) and the big-bracted dogwood (subg. Cynoxylon and subg. Syncarpea) clades are sisters, which are, in turn, sister to the cornelian cherries (subg. Cornus and subg. Afrocrania). This red-fruited clade is sister to the blue- or white-fruited dogwoods (subg. Mesomora, subg. Kraniopsis, and subg. Yinquania). Within the blue- or white-fruited clade, C. oblonga (subg. Yinquania) is sister to the remainder, and subg. Mesomora is sister to subg. Kraniopsis. These relationships were also suggested by the combined 26S rDNA and cpDNA data, but with higher bootstrap and Bremer support in the combined analysis. The 26S rDNA sequence data of Cornus consist of 12 expansion segments spanning 1034 bp. These expansion segments evolve approximately four times as fast as the conserved core regions. The study provides an example of phylogenetic utility of 26S rDNA sequences below the genus level. PMID:11410478

  11. Structure of a Proteasome Pba1-Pba2 Complex

    PubMed Central

    Stadtmueller, Beth M.; Kish-Trier, Erik; Ferrell, Katherine; Petersen, Charisse N.; Robinson, Howard; Myszka, David G.; Eckert, Debra M.; Formosa, Tim; Hill, Christopher P.

    2012-01-01

    The 20S proteasome is an essential, 28-subunit protease that sequesters proteolytic sites within a central chamber, thereby repressing substrate degradation until proteasome activators open the entrance/exit gate. Two established activators, Blm10 and PAN/19S, induce gate opening by binding to the pockets between proteasome α-subunits using C-terminal HbYX (hydrophobic-tyrosine-any residue) motifs. Equivalent HbYX motifs have been identified in Pba1 and Pba2, which function in proteasome assembly. Here, we demonstrate that Pba1-Pba2 proteins form a stable heterodimer that utilizes its HbYX motifs to bind mature 20S proteasomes in vitro and that the Pba1-Pba2 HbYX motifs are important for a physiological function of proteasomes, the maintenance of mitochondrial function. Other factors that contribute to proteasome assembly or function also act in the maintenance of mitochondrial function and display complex genetic interactions with one another, possibly revealing an unexpected pathway of mitochondrial regulation involving the Pba1-Pba2 proteasome interaction. Our determination of a proteasome Pba1-Pba2 crystal structure reveals a Pba1 HbYX interaction that is superimposable with those of known activators, a Pba2 HbYX interaction that is different from those reported previously, and a gate structure that is disrupted but not sufficiently open to allow entry of even small peptides. These findings extend understanding of proteasome interactions with HbYX motifs and suggest multiple roles for Pba1-Pba2 interactions throughout proteasome assembly and function. PMID:22930756

  12. Novel proteasome inhibitor ixazomib sensitizes neuroblastoma cells to doxorubicin treatment

    PubMed Central

    Li, Haoyu; Chen, Zhenghu; Hu, Ting; Wang, Long; Yu, Yang; Zhao, Yanling; Sun, Wenijing; Guan, Shan; Pang, Jonathan C.; Woodfield, Sarah E.; Liu, Qing; Yang, Jianhua

    2016-01-01

    Neuroblastoma (NB) is the most common extracranial malignant solid tumor seen in children and continues to lead to the death of many pediatric cancer patients. The poor outcome in high risk NB is largely attributed to the development of chemoresistant tumor cells. Doxorubicin (dox) has been widely employed as a potent anti-cancer agent in chemotherapeutic regimens; however, it also leads to chemoresistance in many cancer types including NB. Thus, developing novel small molecules that can overcome dox-induced chemoresistance is a promising strategy in cancer therapy. Here we show that the second generation proteasome inhibitor ixazomib (MLN9708) not only inhibits NB cell proliferation and induces apoptosis in vitro but also enhances dox-induced cytotoxicity in NB cells. Ixazomib inhibits dox-induced NF-κB activity and sensitizes NB cells to dox-induced apoptosis. More importantly, ixazomib demonstrated potent anti-tumor efficacy in vivo by enhancing dox-induced apoptosis in an orthotopic xenograft NB mouse model. Collectively, our study illustrates the anti-tumor efficacy of ixazomib in NB both alone and in combination with dox, suggesting that combination therapy including ixazomib with traditional therapeutic agents such as dox is a viable strategy that may achieve better outcomes for NB patients. PMID:27687684

  13. Ubiquitin proteasome system research in gastrointestinal cancer.

    PubMed

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-02-15

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design. PMID:26909134

  14. Ubiquitin proteasome system research in gastrointestinal cancer

    PubMed Central

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-01-01

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design. PMID:26909134

  15. Purification and characterization of Candida albicans 20S proteasome: identification of four proteasomal subunits.

    PubMed

    Fernández Murray, P; Biscoglio, M J; Passeron, S

    2000-03-15

    The 20S proteasome from yeast cells of Candida albicans was purified by successive chromatographic steps to apparent homogeneity, as judged by nondenaturing and denaturing polyacrylamide gel electrophoresis. Its molecular mass was estimated to be 640 kDa by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave at least 10 bands in the range 20-32 kDa. Two-dimensional electrophoresis revealed the presence of at least 14 polypeptides. By electron microscopy after negative staining, the proteasome preparation appeared as typical symmetrical barrel-shaped particles. The enzyme cleaved the peptidyl-arylamide bonds in the model synthetic substrates Cbz-G-G-L-p-nitroanilide, Cbz-G-G-R-beta-naphthylamide, and Cbz-L-L-E-beta-naphthylamide (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide-hydrolyzing activities). The differential sensitivity of these activities to aldehyde peptides and sodium dodecyl sulfate supported the multicatalytic nature of this enzyme. Three proteasomal subunits were identified as alpha6/Pre5, alpha3/Y13, and alpha5/Pup2 by internal sequencing of tryptic fragments. Their sequences perfectly matched the corresponding deduced amino acid sequences of the C. albicans genes. A fourth subunit was identified as alpha7/Prs1 by immunorecognition with a monoclonal antibody specific for C8, the human proteasome subunit homologue. Treatment of the intact isolated 20S proteasome with acid phosphatase and Western blot analysis of the separated components indicated that the alpha7/Prs1 subunit is obtained as a multiply phosphorylated protein.

  16. Lysine Ubiquitination and Acetylation of Human Cardiac 20S Proteasomes

    PubMed Central

    Lau, Edward; Choi, Howard JH; Ng, Dominic CM; Meyer, David; Fang, Caiyun; Li, Haomin; Wang, Ding; Zelaya, Ivette M; Yates, John R; Lam, Maggie PY

    2016-01-01

    Purpose Altered proteasome functions are associated with multiple cardiomyopathies. While the proteasome targets poly-ubiquitinated proteins for destruction, it itself is modifiable by ubiquitination. We aim to identify the exact ubiquitination sites on cardiac proteasomes and examine whether they are also subject to acetylations. Experimental design Assembled cardiac 20S proteasome complexes were purified from five human hearts with ischemic cardiomyopathy, then analyzed by high-resolution MS to identify ubiquitination and acetylation sites. We developed a library search strategy that may be used to complement database search in identifying PTM in different samples. Results We identified 63 ubiquitinated lysines from intact human cardiac 20S proteasomes. In parallel, 65 acetylated residues were also discovered, 39 of which shared with ubiquitination sites. Conclusion and clinical relevance This is the most comprehensive characterization of cardiac proteasome ubiquitination to-date. There are significant overlaps between the discovered ubiquitination and acetylation sites, permitting potential crosstalk in regulating proteasome functions. The information presented here will aid future therapeutic strategies aimed at regulating the functions of cardiac proteasomes. PMID:24957502

  17. L-Ornithine Schiff base-copper and -cadmium complexes as new proteasome inhibitors and apoptosis inducers in human cancer cells.

    PubMed

    Zhang, Zhongyu; Bi, Caifeng; Fan, Yuhua; Zhang, Nan; Deshmukh, Rahul; Yan, Xingchen; Lv, Xiuwen; Zhang, Pengfei; Zhang, Xia; Dou, Q Ping

    2015-01-01

    Ubiquitin-proteasome system (UPS) plays a crucial role in many cellular processes such as cell cycle, proliferation and apoptosis. Aberrant activation of UPS may result in cellular transformation or other altered pathological conditions. Previous studies have shown that metal-based complexes could inhibit proteasome activity and induce apoptosis in certain human cancer cells. In the current study, we report that the cadmium and copper complexes with heterocycle-ornithine Schiff base are potent inhibitors of proteasomal chymotrypsin-like (CT-like) activity, leading to induction of apoptosis in cancer cells. Two novel copper-containing complexes and two novel cadmium-containing complexes with different heterocycle-ornithine Schiff base structures as ligands were synthesized and characterized. We found that complexes Cu1, Cd1 and Cd2 show proteasome-inhibitory activities in human breast cancer MDA-MB-231 and human prostate cancer LNCaP cells, resulting in the accumulation of p27, a natural proteasome substrate and other ubiquitinated proteins, followed by the induction of apoptosis. Our results suggest that metal complexes with heterocycle-ornithine Schiff base have proteasome-inhibitory capabilities and have the potential to be developed into novel anticancer drugs.

  18. Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors

    PubMed Central

    Wójcik, Sławomir; Spodnik, Jan Henryk; Dziewiątkowski, Jerzy; Spodnik, Edyta; Moryś, Janusz

    2015-01-01

    The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)—known as proteasome inhibitors—have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132

  19. Morphological Changes within the Rat Lateral Ventricle after the Administration of Proteasome Inhibitors.

    PubMed

    Wójcik, Sławomir; Spodnik, Jan Henryk; Dziewiątkowski, Jerzy; Spodnik, Edyta; Moryś, Janusz

    2015-01-01

    The broad variety of substances that inhibit the action of the ubiquitin-proteasome system (UPS)-known as proteasome inhibitors-have been used extensively in previous studies, and they are currently frequently proposed as a novel form of cancer treatment and as a protective factor in intracerebral hemorrhage treatment. The experimental data on the safest route of proteasome inhibitor administration, their associated side effects, and the possible ways of minimizing these effects have recently become a very important topic. The aim of our present study was to determine the effects of administering of MG-132, lactacystin and epoxomicin, compounds belonging to three different classes of proteasome inhibitors, on the ependymal walls of the lateral ventricle. Observations were made 2 and 8 weeks after the intraventricular administration of the studied substances dissolved in dimethyl sulfoxide (DMSO) into the lateral ventricle of adult Wistar rats. Qualitative and quantitative analysis of brain sections stained with histochemical and inmmunofluorescence techniques showed that the administration of proteasome inhibitors caused a partial occlusion of the injected ventricle in all of the studied animals. The occlusion was due to ependymal cells damage and subsequent ependymal discontinuity, which caused direct contact between the striatum and the lateral nuclei of the septum, mononuclear cell infiltration and the formation of a glial scar between these structures (with the activation of astroglia, microglia and oligodendroglia). Morphologically, the ubiquitin-positive aggregates corresponded to aggresomes, indicating impaired activity of the UPS and the accumulation and aggregation of ubiquitinated proteins that coincided with the occurrence of glial scars. The most significant changes were observed in the wall covering the striatum in animals that were administered epoxomicin, and milder changes were observed in animals administered lactacystin and MG-132. Interestingly

  20. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    SciTech Connect

    Lu, Li; Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao; Li, Ang; Xiao, Zhi-Cheng

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  1. Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome.

    PubMed

    Sun, Lianhui; Fan, Guangjian; Shan, Peipei; Qiu, Xiaoying; Dong, Shuxian; Liao, Lujian; Yu, Chunlei; Wang, Tingting; Gu, Xiaoyang; Li, Qian; Song, Xiaoyu; Cao, Liu; Li, Xiaotao; Cui, Yongping; Zhang, Shengping; Wang, Chuangui

    2016-01-01

    Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits. PMID:27511885

  2. Next-generation proteasome inhibitor MLN9708 sensitizes breast cancer cells to doxorubicin-induced apoptosis

    PubMed Central

    Wang, Hao; Yu, Yang; Jiang, Zheng; Cao, Wen-Ming; Wang, Zhenyu; Dou, Jun; Zhao, Yanling; Cui, Yunfu; Zhang, Hong

    2016-01-01

    Doxorubicin (Dox), one of the most effective chemotherapy drug for cancer treatment, is limited by its severe side effects and chemoresistance. Dox induces DNA damage and leads to significant proteomic changes in the cancer cells, which makes the ubiquitin-proteasome system a potential target to enhance the efficacy of Dox therapy. The unsuccessful clinical trials of proteasome inhibitor PS-341 (bortezomib) in solid tumors led to the invention of MLN9708 (ixazomib), an orally bioavailable next-generation proteasome inhibitor with improved pharmacokinetic and pharmacodynamic features. In this preclinical study, we used eight human breast cancer cell lines, which represent the major molecular subtypes of breast cancer, to validate the cytotoxic effects of MLN9708, alone and in combination with Dox. We found that MLN9708 had cytotoxic effects, induced autophagy and MKP-1 expression, and enhanced Dox-induced apoptosis in these cell lines. MLN9708 also enhanced Dox-induced JNK and p38 phosphorylation and inhibited Dox-induced IκBα degradation. Our in vitro results suggest that MLN9708 has antitumor effects in breast cancer and can sensitize breast cancer cells to Dox treatment. This promising combination may be an effective and feasible therapeutic option for treating breast cancer and warrants clinical validation. PMID:27217076

  3. Reciprocal regulation of cilia and autophagy via the MTOR and proteasome pathways.

    PubMed

    Wang, Shixuan; Livingston, Man J; Su, Yunchao; Dong, Zheng

    2015-04-01

    Primary cilium is an organelle that plays significant roles in a number of cellular functions ranging from cell mechanosensation, proliferation, and differentiation to apoptosis. Autophagy is an evolutionarily conserved cellular function in biology and indispensable for cellular homeostasis. Both cilia and autophagy have been linked to different types of genetic and acquired human diseases. Their interaction has been suggested very recently, but the underlying mechanisms are still not fully understood. We examined autophagy in cells with suppressed cilia and measured cilium length in autophagy-activated or -suppressed cells. It was found that autophagy was repressed in cells with short cilia. Further investigation showed that MTOR activation was enhanced in cilia-suppressed cells and the MTOR inhibitor rapamycin could largely reverse autophagy suppression. In human kidney proximal tubular cells (HK2), autophagy induction was associated with cilium elongation. Conversely, autophagy inhibition by 3-methyladenine (3-MA) and chloroquine (CQ) as well as bafilomycin A1 (Baf) led to short cilia. Cilia were also shorter in cultured atg5-knockout (KO) cells and in atg7-KO kidney proximal tubular cells in mice. MG132, an inhibitor of the proteasome, could significantly restore cilium length in atg5-KO cells, being concomitant with the proteasome activity. Together, the results suggest that cilia and autophagy regulate reciprocally through the MTOR signaling pathway and ubiquitin-proteasome system.

  4. Copper-binding compounds as proteasome inhibitors and apoptosis inducers in human cancer.

    PubMed

    Daniel, Kenyon G; Chen, Di; Yan, Bing; Dou, Q Ping

    2007-01-01

    The trace element copper is vital to the healthy functioning of organisms. Copper is used in a multitude of cellular activities including respiration, angiogenesis, and immune responses. Recently, copper has become a focus in medical research ranging from Alzheimer's disease to cancer. Copper modulation has been suggested to be a potential modality for therapy in these diseases. Several copper-binding compounds have been found to spontaneously complex with copper and form active proteasome inhibitors and apoptosis inducers. This review examines compounds in the quinoline and dithiocarbamate families and from the National Cancer Institute (NCI) Diversity Set that bind with copper and act as anticancer agents. In each case, it is shown that these compounds can bind with copper, inhibit the proteasome activity, and induce apoptosis in cancer cells. These activities are absent when copper is not present. Compounds alone, clioquinol and pyrrolidinedithiocarbamate as examples, are shown to have no effects in normal breast cells. Current research suggests that a possible therapeutic modality for cancer may be developed using the difference of high copper load in tumors versus low copper load in normal cells. This strategy would convert tumor cellular copper into a potent, specific proteasome inhibitor and apoptosis inducer. Thus, this approach could pave the way for the development of nontoxic anticancer therapy.

  5. Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome

    PubMed Central

    Sun, Lianhui; Fan, Guangjian; Shan, Peipei; Qiu, Xiaoying; Dong, Shuxian; Liao, Lujian; Yu, Chunlei; Wang, Tingting; Gu, Xiaoyang; Li, Qian; Song, Xiaoyu; Cao, Liu; Li, Xiaotao; Cui, Yongping; Zhang, Shengping; Wang, Chuangui

    2016-01-01

    Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits. PMID:27511885

  6. Regulation of energy homeostasis by the ubiquitin-independent REGγ proteasome.

    PubMed

    Sun, Lianhui; Fan, Guangjian; Shan, Peipei; Qiu, Xiaoying; Dong, Shuxian; Liao, Lujian; Yu, Chunlei; Wang, Tingting; Gu, Xiaoyang; Li, Qian; Song, Xiaoyu; Cao, Liu; Li, Xiaotao; Cui, Yongping; Zhang, Shengping; Wang, Chuangui

    2016-08-11

    Maintenance of energy homeostasis is essential for cell survival. Here, we report that the ATP- and ubiquitin-independent REGγ-proteasome system plays a role in maintaining energy homeostasis and cell survival during energy starvation via repressing rDNA transcription, a major intracellular energy-consuming process. Mechanistically, REGγ-proteasome limits cellular rDNA transcription and energy consumption by targeting the rDNA transcription activator SirT7 for ubiquitin-independent degradation under normal conditions. Moreover, energy starvation induces an AMPK-directed SirT7 phosphorylation and subsequent REGγ-dependent SirT7 subcellular redistribution and degradation, thereby further reducing rDNA transcription to save energy to overcome cell death. Energy starvation is a promising strategy for cancer therapy. Our report also shows that REGγ knockdown markedly improves the anti-tumour activity of energy metabolism inhibitors in mice. Our results underscore a control mechanism for an ubiquitin-independent process in maintaining energy homeostasis and cell viability under starvation conditions, suggesting that REGγ-proteasome inhibition has a potential to provide tumour-starving benefits.

  7. Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity

    PubMed Central

    Díaz-Ruiz, Alberto; Guzmán-Ruiz, Rocío; Moreno, Natalia R.; García-Rios, Antonio; Delgado-Casado, Nieves; Membrives, Antonio; Túnez, Isaac; El Bekay, Rajaa; Fernández-Real, José M.; Tovar, Sulay; Diéguez, Carlos; Tinahones, Francisco J.; Vázquez-Martínez, Rafael; López-Miranda, José

    2015-01-01

    Abstract Aims: Obesity is characterized by a low-grade systemic inflammatory state and adipose tissue (AT) dysfunction, which predispose individuals to the development of insulin resistance (IR) and metabolic disease. However, a subset of obese individuals, referred to as metabolically healthy obese (MHO) individuals, are protected from obesity-associated metabolic abnormalities. Here, we aim at identifying molecular factors and pathways in adipocytes that are responsible for the progression from the insulin-sensitive to the insulin-resistant, metabolically unhealthy obese (MUHO) phenotype. Results: Proteomic analysis of paired samples of adipocytes from subcutaneous (SC) and omental (OM) human AT revealed that both types of cells are altered in the MUHO state. Specifically, the glutathione redox cycle and other antioxidant defense systems as well as the protein-folding machinery were dysregulated and endoplasmic reticulum stress was increased in adipocytes from IR subjects. Moreover, proteasome activity was also compromised in adipocytes of MUHO individuals, which was associated with enhanced accumulation of oxidized and ubiquitinated proteins in these cells. Proteasome activity was also impaired in adipocytes of diet-induced obese mice and in 3T3-L1 adipocytes exposed to palmitate. In line with these data, proteasome inhibition significantly impaired insulin signaling in 3T3-L1 adipocytes. Innovation: This study provides the first evidence of the occurrence of protein homeostasis deregulation in adipocytes in human obesity, which, together with oxidative damage, interferes with insulin signaling in these cells. Conclusion: Our results suggest that proteasomal dysfunction and impaired proteostasis in adipocytes, resulting from protein oxidation and/or misfolding, constitute major pathogenic mechanisms in the development of IR in obesity. Antioxid. Redox Signal. 23, 597–612. PMID:25714483

  8. Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F

    PubMed Central

    Bosen, Felicitas; Celli, Anna; Crumrine, Debra; vom Dorp, Katharina; Ebel, Philipp; Jastrow, Holger; Dörmann, Peter; Winterhager, Elke; Mauro, Theodora; Willecke, Klaus

    2016-01-01

    The keratitis–ichthyosis–deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome. PMID:26070424

  9. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  10. A bis-Benzylidine Piperidone Targeting Proteasome Ubiquitin Receptor RPN13/ADRM1 as a therapy for cancer

    PubMed Central

    Anchoori, Ravi K.; Karanam, Balasubramanyam; Peng, Shiwen; Wang, Joshua W.; Jiang, Rosie; Tanno, Toshihiko; Orlowski, Robert Z.; Matsui, William; Zhao, Ming; Rudek, Michelle A.; Hung, Chien-fu; Chen, Xiang; Walters, Kylie J.; Roden, Richard B.S.

    2013-01-01

    The bis-benzylidine piperidone RA190 covalently binds to cysteine 88 of ubiquitin receptor RPN13 in the 19S regulatory particle and inhibits proteasome function, triggering rapid accumulation of polyubiquitinated proteins. Multiple myeloma (MM) lines, even those resistant to bortezomib, were sensitive to RA190 via endoplasmic reticulum stress-related apoptosis. RA190 stabilized targets of human papillomavirus (HPV) E6 oncoprotein, and preferentially killed HPV-transformed cells. After oral (p.o.) or intraperitoneal (i.p.) dosing of mice, RA190 distributed to plasma and major organs excepting brain, and inhibited proteasome function in skin and muscle. RA190 administration profoundly reduced growth of multiple myeloma and ovarian cancer xenografts, and oral RA190 treatment retarded HPV16+ syngeneic mouse tumor growth, without impacting spontaneous HPV-specific CD8+ T cell responses, suggesting its therapeutic potential. PMID:24332045

  11. A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly

    PubMed Central

    Tomko, Robert J.; Taylor, David W.; Chen, Zhuo A.; Wang, Hong-Wei; Rappsilber, Juri; Hochstrasser, Mark

    2015-01-01

    Summary Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes. PMID:26451487

  12. Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

    PubMed

    Gillissen, B; Richter, A; Richter, A; Overkamp, T; Essmann, F; Hemmati, P G; Preissner, R; Belka, C; Daniel, P T

    2013-01-01

    TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

  13. The Xanthomonas campestris Type III Effector XopJ Targets the Host Cell Proteasome to Suppress Salicylic-Acid Mediated Plant Defence

    PubMed Central

    Börnke, Frederik

    2013-01-01

    The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed. PMID:23785289

  14. A binuclear complex constituted by diethyldithiocarbamate and copper(I) functions as a proteasome activity inhibitor in pancreatic cancer cultures and xenografts.

    PubMed

    Han, Jinbin; Liu, Luming; Yue, Xiaoqiang; Chang, Jinjia; Shi, Weidong; Hua, Yongqiang

    2013-12-15

    It is a therapeutic strategy for cancers including pancreatic to inhibit proteasome activity. Disulfiram (DSF) may bind copper (Cu) to form a DSF-Cu complex. DSF-Cu is capable of inducing apoptosis in cancer cells by inhibiting proteasome activity. DSF is rapidly converted to diethyldithiocarbamate (DDTC) within bodies. Copper(II) absorbed by bodies is reduced to copper(I) when it enters cells. We found that DDTC and copper(I) could form a binuclear complex which might be entitled DDTC-Cu(I), and it had been synthesized by us in the laboratory. This study is to investigate the anticancer potential of this complex on pancreatic cancer and the possible mechanism. Pancreatic cancer cell lines, SW1990, PANC-1 and BXPC-3 were used for in vitro assays. Female athymic nude mice grown SW1990 xenografts were used as animal models. Cell counting kit-8 (cck-8) assay and flow cytometry were used for analyzing apoptosis in cells. A 20S proteasome assay kit was used in proteasome activity analysis. Western blot (WB) and immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used in tumor sample analysis. The results suggest that DDTC-Cu(I) inhibit pancreatic cancer cell proliferation and proteasome activity in vitro and in vivo. Accumulation of ubiquitinated proteins, and increased p27 as well as decreased NF-κB expression were detected in tumor tissues of DDTC-Cu(I)-treated group. Our data indicates that DDTC-Cu(I) is an effective proteasome activity inhibitor with the potential to be explored as a drug for pancreatic cancer.

  15. Regulation of dimethyl-fumarate toxicity by proteasome inhibitors.

    PubMed

    Booth, Laurence; Cruickshanks, Nichola; Tavallai, Seyedmehrad; Roberts, Jane L; Peery, Matthew; Poklepovic, Andrew; Dent, Paul

    2014-01-01

    The present studies examined the biology of the multiple sclerosis drug dimethyl-fumarate (DMF) or its in vivo breakdown product and active metabolite mono-methyl-fumarate (MMF), alone or in combination with proteasome inhibitors, in primary human glioblastoma (GBM) cells. MMF enhanced velcade and carfilzomib toxicity in multiple primary GBM isolates. Similar data were obtained in breast and colon cancer cells. MMF reduced the invasiveness of GBM cells, and enhanced the toxicity of ionizing radiation and temozolomide. MMF killed freshly isolated activated microglia which was associated with reduced IL-6, TGFβ and TNFα production. The combination of MMF and the multiple sclerosis drug Gilenya further reduced both GBM and activated microglia viability and cytokine production. Over-expression of c-FLIP-s or BCL(-)XL protected GBM cells from MMF and velcade toxicity. MMF and velcade increased plasma membrane localization of CD95, and knock down of CD95 or FADD blocked the drug interaction. The drug combination inactivated AKT, ERK1/2 and mTOR. Molecular inhibition of AKT/ERK/mTOR signaling enhanced drug combination toxicity whereas molecular activation of these pathways suppressed killing. MMF and velcade increased the levels of autophagosomes and autolysosomes and knock down of ATG5 or Beclin1 protected cells. Inhibition of the eIF2α/ATF4 arm or the IRE1α/XBP1 arm of the ER stress response enhanced drug combination lethality. This was associated with greater production of reactive oxygen species and quenching of ROS suppressed cell killing. PMID:25482938

  16. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  17. Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation*

    PubMed Central

    Goitea, Victor E.; Hallak, Marta E.

    2015-01-01

    Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm. PMID:25969538

  18. Rational Design of Proteasome Inhibitors as Antimalarial Drugs.

    PubMed

    Le Chapelain, Camille; Groll, Michael

    2016-05-23

    One life, two strategies: Crucial structural differences between the human and the Plasmodium falciparum proteasomes were recently identified. A combination of cryo-EM and functional characterization enabled the design of a selective antimalarial proteasome inhibitor that shows low toxicity in the host. When used with artemisinin, this ligand offers a new approach for the efficient treatment of malaria at all stages of the parasite lifecycle.

  19. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    SciTech Connect

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  20. Proteasomes are tightly associated to myofibrils in mature skeletal muscle.

    PubMed

    Bassaglia, Yann; Cebrian, José; Covan, Silvia; Garcia, Monica; Foucrier, Jean

    2005-01-15

    Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of alpha1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils. PMID:15561103

  1. Reconfiguration of the proteasome during chaperone-mediated assembly

    PubMed Central

    Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A.; Lovell, Scott; Battaile, Kevin P.; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, Daniel

    2013-01-01

    The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme. PMID:23644457

  2. Proteasome dysfunction induces muscle growth defects and protein aggregation.

    PubMed

    Kitajima, Yasuo; Tashiro, Yoshitaka; Suzuki, Naoki; Warita, Hitoshi; Kato, Masaaki; Tateyama, Maki; Ando, Risa; Izumi, Rumiko; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Ito, Hidefumi; Urushitani, Makoto; Nagatomi, Ryoichi; Takahashi, Ryosuke; Aoki, Masashi

    2014-12-15

    The ubiquitin-proteasome and autophagy-lysosome pathways are the two major routes of protein and organelle clearance. The role of the proteasome pathway in mammalian muscle has not been examined in vivo. In this study, we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice. Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice. Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions.

  3. Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells.

    PubMed

    Lee, Sanghwan; Hur, Eu-gene; Ryoo, In-geun; Jung, Kyeong-Ah; Kwak, Jiyeon; Kwak, Mi-Kyoung

    2012-11-01

    The ubiquitin-proteasome system plays a central role in protein quality control through endoplasmic reticulum (ER)-associated degradation (ERAD) of unfolded and misfolded proteins. NF-E2-related factor 2 (Nrf2) is a transcription factor that controls the expression of an array of phase II detoxification and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits in several cell types. Here, we investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine insulinoma β-cell line, βTC-6. shRNA-mediated silencing of Nrf2 expression in βTC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ER stress marker Chop10, and inhibited tunicamycin-inducible expression of the proteasomal catalytic subunits Psmb5 and Psmb6. The effects of 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, on ER stress were also examined in βTC-6 cells. D3T pretreatment reduced tunicamycin cytotoxicity and attenuated the tunicamycin-inducible Chop10 and protein kinase RNA-activated-like ER kinase (Perk). The protective effect of D3T was shown to be associated with increased ERAD. D3T increased the expression of Psmb5 and Psmb6 and elevated chymotrypsin-like peptidase activity; proteasome inhibitor treatment blocked D3T effects on tunicamycin cytotoxicity and ER stress marker changes. Similarly, silencing of Nrf2 abolished the protective effect of D3T against ER stress. These results indicate that the Nrf2 pathway contributes to the ER stress response in pancreatic β-cells by enhancing proteasome-mediated ERAD.

  4. A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

    PubMed Central

    Milan, Enrico; Perini, Tommaso; Resnati, Massimo; Orfanelli, Ugo; Oliva, Laura; Raimondi, Andrea; Cascio, Paolo; Bachi, Angela; Marcatti, Magda; Ciceri, Fabio; Cenci, Simone

    2015-01-01

    Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. An attractive target to enhance sensitivity is (macro)autophagy, recently found essential to bone marrow plasma cells, the normal counterpart of MM. Here, integrating proteomics with hypothesis-driven strategies, we identified the autophagic cargo receptor and adapter protein, SQSTM1/p62 as an essential component of an autophagic reserve that not only synergizes with the proteasome to maintain proteostasis, but also mediates a plastic adaptive response to PIs, and faithfully reports on inherent PI sensitivity. Lentiviral engineering revealed that SQSTM1 is essential for MM cell survival and affords specific PI protection. Under basal conditions, SQSTM1-dependent autophagy alleviates the degradative burden on the proteasome by constitutively disposing of substantial amounts of ubiquitinated proteins. Indeed, its inhibition or stimulation greatly sensitized to, or protected from, PI-induced protein aggregation and cell death. Moreover, under proteasome stress, myeloma cells selectively enhanced SQSTM1 de novo expression and reset its vast endogenous interactome, diverting SQSTM1 from signaling partners to maximize its association with ubiquitinated proteins. Saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested SQSTM1-positive aggregates, specifically discriminated patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. These aggregates correlated with accumulation of the endoplasmic reticulum, which comparative proteomics identified as the main cell compartment targeted by autophagy in MM. Altogether, the data integrate autophagy into our previously established proteasome load-versus-capacity model, and reveal SQSTM1 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM. PMID:26043024

  5. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    PubMed Central

    Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

    2012-01-01

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in an