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Sample records for enhances proteasome activity

  1. 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

  2. Formation of proteasome-PA700 complexes directly correlates with activation of peptidase activity.

    PubMed

    Adams, G M; Crotchett, B; Slaughter, C A; DeMartino, G N; Gogol, E P

    1998-09-15

    The proteolytic activity of the eukaryotic 20S proteasome is stimulated by a multisubunit activator, PA700, which forms both 1:1 and 2:1 complexes with the proteasome. Formation of the complexes is enhanced by an additional protein assembly called modulator, which also stimulates the enzymatic activity of the proteasome only in the presence of PA700. Here we show that the binding of PA700 to the proteasome is cooperative, as is the activation of the proteasome's intrinsic peptidase activity. Modulator increases the extent of complex formation and peptidase activation, while preserving the cooperative kinetics. Furthermore, the increase in activity is not linear with the number of PA700 assemblies bound to the proteasome, but rather with the number of proteasome-PA700 complexes, regardless of the PA700:proteasome stoichiometry. Hence the stimulation of peptidase activity is fully (or almost fully) effected by the binding of a single PA700 to the 20S proteasome. The stimulation of peptidase by modulator is explained entirely by the increased number of proteasome-PA700 complexes formed in its presence, rather than by any substantial direct stimulation of catalysis. These observations are consistent with a model in which PA700, either alone or assisted by modulator, promotes conformational changes in the proteasome that activate the catalytic sites and/or facilitate access of peptide substrates to these sites. PMID:9737872

  3. 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.

  4. 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.

  5. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    DOE PAGES

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; et al

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

  6. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    SciTech Connect

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.

  7. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    PubMed Central

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-01-01

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens. PMID:25831519

  8. 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.

  9. 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.

  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. 20S proteasome activation promotes life span extension and resistance to proteotoxicity in Caenorhabditis elegans.

    PubMed

    Chondrogianni, Niki; Georgila, Konstantina; Kourtis, Nikos; Tavernarakis, Nektarios; Gonos, Efstathios S

    2015-02-01

    Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to stress. We also provide evidence that the observed life span extension is dependent on the transcriptional activity of Dauer formation abnormal/Forkhead box class O (DAF-16/FOXO), skinhead-1 (SKN-1), and heat shock factor-1 (HSF-1) factors through regulation of downstream longevity genes. We further show that the reported beneficial effects are not ubiquitous but they are dependent on the genetic context. Finally, we provide evidence that proteasome core activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases, such as Alzheimer's disease (AD) or Huntington's disease (HD). In summary, this is the first report demonstrating that 20S core proteasome up-regulation in terms of both content and activity is feasible in a multicellular eukaryotic organism and that in turn this modulation promotes extension of organismal health span and life span. PMID:25395451

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. Determination of Protein Carbonylation and Proteasome Activity in Seeds.

    PubMed

    Xia, Qiong; El-Maarouf-Bouteau, Hayat; Bailly, Christophe; Meimoun, Patrice

    2016-01-01

    Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements. PMID:27424756

  18. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation.

    PubMed

    Choi, Won Hoon; de Poot, Stefanie A H; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-01-01

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼ 200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells. PMID:26957043

  19. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation

    PubMed Central

    Choi, Won Hoon; de Poot, Stefanie A. H.; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-01-01

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells. PMID:26957043

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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

  7. Structural Models for Interactions between the 20S Proteasome and Its PAN/19S Activators

    SciTech Connect

    Stadtmueller, B.; Ferrell, K; Whitby, F; Heroux, A; Robinson, H; Myszka, D; Hill, C

    2009-01-01

    Proteasome activity is regulated by sequestration of its proteolytic centers in a barrel-shaped structure that limits substrate access. Substrates enter the proteasome by means of activator complexes that bind to the end rings of proteasome alpha subunits and induce opening of an axial entrance/exit pore. The PA26 activator binds in a pocket on the proteasome surface using main chain contacts of its C-terminal residues and uses an internal activation loop to trigger gate opening by repositioning the proteasome Pro-17 reverse turn. Subunits of the unrelated PAN/19S activators bind with their C termini in the same pockets but can induce proteasome gate opening entirely from interactions of their C-terminal peptides, which are reported to cause gate opening by inducing a rocking motion of proteasome alpha subunits rather than by directly contacting the Pro-17 turn. Here we report crystal structures and binding studies of proteasome complexes with PA26 constructs that display modified C-terminal residues, including those corresponding to PAN. These findings suggest that PA26 and PAN/19S C-terminal residues bind superimposably and that both classes of activator induce gate opening by using direct contacts to residues of the proteasome Pro-17 reverse turn. In the case of the PAN and 19S activators, a penultimate tyrosine/phenylalanine residue contacts the proteasome Gly-19 carbonyl oxygen to stabilize the open conformation.

  8. Dyclonine and alverine citrate enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells

    PubMed Central

    JU, DONGHONG; WANG, XIAOGANG; XIE, YOUMING

    2014-01-01

    Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors. PMID:19148544

  9. Dyclonine and alverine citrate enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells.

    PubMed

    Ju, Donghong; Wang, Xiaogang; Xie, Youming

    2009-02-01

    Proteasome is an important target in cancer therapy. To enhance the efficacy of proteasome inhibitors is a challenging task due to the paucity of understanding the functional interactions between proteasome and other cellular pathways in mammalian cells. Taking advantage of the knowledge gained from Saccharomyces cerevisiae, we show that dyclonine and alverine citrate, the major components of two over-the-counter medicines, can substantially enhance the cytotoxic effects of proteasome inhibitor MG132 on breast cancer cells. This study also highlights an important yeast genetic approach to identification of potential therapeutics that can be used for combination therapy with proteasome inhibitors. PMID:19148544

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  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. 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

  17. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

    PubMed Central

    Lier, Johanna Maria; Burmühl, Stephan; Struchtrup, Andreas; Deutschmann, Kathleen; Vetter, Maik; Leu, Tristan; Reeg, Sandra; Grune, Tilman; Rüther, Ulrich

    2015-01-01

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. PMID:26150391

  18. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

    DOE PAGES

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B.; Darwin, K. Heran; Li, Huilin

    2016-03-21

    Here, the human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, themore » truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.« less

  19. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis.

    PubMed

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B; Darwin, K Heran; Li, Huilin

    2016-04-01

    The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria. PMID:27001842

  20. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

    PubMed Central

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B.; Darwin, K. Heran; Li, Huilin

    2016-01-01

    The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria. PMID:27001842

  1. 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.

  2. Ubiquitin, Proteasomes and Proteolytic Mechanisms Activated by Kidney Disease

    PubMed Central

    Rajan, Vik; Mitch, William E.

    2008-01-01

    Summary The ubiquitin-proteasome system (UPS) includes 3 enzymes that conjugate ubiquitin to intracellular proteins that are then recognized and degraded in the proteasome. The process participates in the regulation of cell metabolism. In the kidney, the UPS regulates the turnover of transporters and signaling proteins and its activity is down regulated in acidosis-induced proximal tubular cell hypertrophy. In chronic kidney disease (CKD), muscle wasting occurs because complications of CKD including acidosis, insulin resistance, inflammation, and increased angiotensin II levels stimulate the UPS to degrade muscle proteins. This response also includes caspase-3 and calpains which act to cleave muscle proteins to provide substrates for the UPS. For example, caspase-3 degrades actomyosin, leaving a 14kD fragment of actin in muscle. The 14 kD actin fragment is increased in muscle of patient with kidney disease, burn injury and surgery. In addition, acidosis, insulin resistance, inflammation and angiotensin II stimulate glucocorticoid production. Glucocorticoids are also required for the muscle wasting that occurs in CKD. Thus, the UPS is involved in regulating kidney function and participates in highly organized responses that degrade muscle protein in response to loss of kidney function. PMID:18723090

  3. Ubiquitin enzymes, ubiquitin and proteasome activity in blood mononuclear cells of MCI, Alzheimer and Parkinson patients.

    PubMed

    Ullrich, C; Mlekusch, R; Kuschnig, A; Marksteiner, J; Humpel, C

    2010-09-01

    Alzheimer's disease (AD) is a severe chronic neurodegenerative disease. During aging and neurodegeneration, misfolded proteins accumulate and activate the ubiquitin-proteasome system. The aim of the present study is to explore whether ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin or proteasome activity are affected in peripheral blood mononuclear cells (PBMC) of AD, mild cognitive impairment (MCI) and Parkinson's disease (PD) patients compared to healthy subjects. PBMCs were isolated from EDTA blood samples and extracts were analyzed by Western Blot. Proteasome activity was measured with fluorogenic substrates. When compared to healthy subjects, the concentration of enzyme E1 was increased in PBMCs of AD patients, whereas the concentration of the enzyme E2 was decreased in these same patients. Ubiquitin levels and proteasome activity were unchanged in AD patients. No changes in enzyme expression or proteasome activity was observed in MCI patients compared to healthy and AD subjects. In PD patients E2 levels and proteasomal activity were significantly reduced, while ubiquitin and E1 levels were unchanged. The present investigation demonstrates the differences in enzyme and proteasome activity patterns of AD and PD patients. These results suggest that different mechanisms are involved in regulating the ubiquitin-proteasomal system in different neurodegenerative diseases.

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity

    PubMed Central

    Haller, Martina; Hock, Andreas K; Giampazolias, Evangelos; Oberst, Andrew; Green, Douglas R; Debnath, Jayanta; Ryan, Kevin M; Vousden, Karen H; Tait, Stephen W G

    2015-01-01

    During macroautophagy, conjugation of ATG12 to ATG5 is essential for LC3 lipidation and autophagosome formation. Additionally, ATG12 has ATG5-independent functions in diverse processes including mitochondrial fusion and mitochondrial-dependent apoptosis. In this study, we investigated the regulation of free ATG12. In stark contrast to the stable ATG12–ATG5 conjugate, we find that free ATG12 is highly unstable and rapidly degraded in a proteasome-dependent manner. Surprisingly, ATG12, itself a ubiquitin-like protein, is directly ubiquitinated and this promotes its proteasomal degradation. As a functional consequence of its turnover, accumulation of free ATG12 contributes to proteasome inhibitor-mediated apoptosis, a finding that may be clinically important given the use of proteasome inhibitors as anticancer agents. Collectively, our results reveal a novel interconnection between autophagy, proteasome activity, and cell death mediated by the ubiquitin-like properties of ATG12. PMID:25629932

  14. 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

  15. 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

  16. 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

  17. 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.

  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. Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S

    2011-02-25

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-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 transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.

  20. Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity*

    PubMed Central

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S.

    2011-01-01

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-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 transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting. PMID:21135093

  1. A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex)

    NASA Technical Reports Server (NTRS)

    Weitman, D.; Etlinger, J. D.

    1992-01-01

    Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.

  2. Why does threonine, and not serine, function as the active site nucleophile in proteasomes?

    PubMed

    Kisselev, A F; Songyang, Z; Goldberg, A L

    2000-05-19

    Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine. PMID:10809725

  3. 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

  4. 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

  5. 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

  6. Proteasome Inhibitor YSY01A Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant Human Ovarian Cancer Cells

    PubMed Central

    Huang, Wei; Zhou, Quan; Yuan, Xia; Ge, Ze-mei; Ran, Fu-xiang; Yang, Hua-yu; Qiang, Guang-liang; Li, Run-tao; Cui, Jing-rong

    2016-01-01

    Cisplatin is one of the most common drugs used for treatment of solid tumors such as ovarian cancer. Unfortunately, the development of resistance against this cytotoxic agent limits its clinical use. Here we report that YSY01A, a novel proteasome inhibitor, is capable of suppressing survival of cisplatin-resistant ovarian cancer cells by inducing apoptosis. And YSY01A treatment enhances the cytotoxicity of cisplatin in drug-resistant ovarian cancer cells. Specifically, YSY01A abrogates regulatory proteins important for cell proliferation and anti-apoptosis including NF-κB p65 and STAT3, resulting in down-regulation of Bcl-2. A dramatic increase in cisplatin uptake was also observed by inductively coupled plasma-mass spectrometry following exposure to YSY01A. Taken together, YSY01A serves as a potential candidate for further development as anticancer therapeutics targeting the proteasome. PMID:27326257

  7. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal. PMID:27176742

  8. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.

    PubMed

    Rut, Wioletta; Drag, Marcin

    2016-09-01

    The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.

  9. Proteasome activators, PA28γ and PA200, play indispensable roles in male fertility

    PubMed Central

    Huang, Lin; Haratake, Kousuke; Miyahara, Hatsumi; Chiba, Tomoki

    2016-01-01

    Protein degradation mediated by the proteasome is important for the protein homeostasis. Various proteasome activators, such as PA28 and PA200, regulate the proteasome function. Here we show double knockout (dKO) mice of Psme3 and Psme4 (genes for PA28γ and PA200), but not each single knockout mice, are completely infertile in male. The dKO sperms exhibited remarkable defects in motility, although most of them showed normal appearance in morphology. The proteasome activity of the mutant sperms decreased notably, and the sperms were strongly positive with ubiquitin staining. Quantitative analyses of proteins expressed in dKO sperms revealed up-regulation of several proteins involved in oxidative stress response. Furthermore, increased 8-OHdG staining was observed in dKO sperms head, suggesting defective response to oxidative damage. This report verified PA28γ and PA200 play indispensable roles in male fertility, and provides a novel insight into the role of proteasome activators in antioxidant response. PMID:27003159

  10. Effect of age on proteasomal activity of T cells and macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    T cell function is impaired with aging. Proteasome activity in T cells is important for T cell activation and its activity in macrophages is required for processing antigens in order to be presented via class I major histocompatibility complex to CD8+ T cells. Since studies have demonstrated that pr...

  11. 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.

  12. 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

  13. 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

  14. Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1

    PubMed Central

    Lehrbach, Nicolas J; Ruvkun, Gary

    2016-01-01

    Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies. DOI: http://dx.doi.org/10.7554/eLife.17721.001 PMID:27528192

  15. Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1.

    PubMed

    Lehrbach, Nicolas J; Ruvkun, Gary

    2016-01-01

    Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies. PMID:27528192

  16. Denervation-Induced Activation of the Ubiquitin-Proteasome System Reduces Skeletal Muscle Quantity Not Quality.

    PubMed

    Baumann, Cory W; Liu, Haiming M; Thompson, LaDora V

    2016-01-01

    It is well known that the ubiquitin-proteasome system is activated in response to skeletal muscle wasting and functions to degrade contractile proteins. The loss of these proteins inevitably reduces skeletal muscle size (i.e., quantity). However, it is currently unknown whether activation of this pathway also affects function by impairing the muscle's intrinsic ability to produce force (i.e., quality). Therefore, the purpose of this study was twofold, (1) document how the ubiquitin-proteasome system responds to denervation and (2) identify the physiological consequences of these changes. To induce soleus muscle atrophy, C57BL6 mice underwent tibial nerve transection of the left hindlimb for 7 or 14 days (n = 6-8 per group). At these time points, content of several proteins within the ubiquitin-proteasome system were determined via Western blot, while ex vivo whole muscle contractility was specifically analyzed at day 14. Denervation temporarily increased several key proteins within the ubiquitin-proteasome system, including the E3 ligase MuRF1 and the proteasome subunits 19S, α7 and β5. These changes were accompanied by reductions in absolute peak force and power, which were offset when expressed relative to physiological cross-sectional area. Contrary to peak force, absolute and relative forces at submaximal stimulation frequencies were significantly greater following 14 days of denervation. Taken together, these data represent two keys findings. First, activation of the ubiquitin-proteasome system is associated with reductions in skeletal muscle quantity rather than quality. Second, shortly after denervation, it appears the muscle remodels to compensate for the loss of neural activity via changes in Ca2+ handling. PMID:27513942

  17. Denervation-Induced Activation of the Ubiquitin-Proteasome System Reduces Skeletal Muscle Quantity Not Quality

    PubMed Central

    Liu, Haiming M.; Thompson, LaDora V.

    2016-01-01

    It is well known that the ubiquitin-proteasome system is activated in response to skeletal muscle wasting and functions to degrade contractile proteins. The loss of these proteins inevitably reduces skeletal muscle size (i.e., quantity). However, it is currently unknown whether activation of this pathway also affects function by impairing the muscle’s intrinsic ability to produce force (i.e., quality). Therefore, the purpose of this study was twofold, (1) document how the ubiquitin-proteasome system responds to denervation and (2) identify the physiological consequences of these changes. To induce soleus muscle atrophy, C57BL6 mice underwent tibial nerve transection of the left hindlimb for 7 or 14 days (n = 6–8 per group). At these time points, content of several proteins within the ubiquitin-proteasome system were determined via Western blot, while ex vivo whole muscle contractility was specifically analyzed at day 14. Denervation temporarily increased several key proteins within the ubiquitin-proteasome system, including the E3 ligase MuRF1 and the proteasome subunits 19S, α7 and β5. These changes were accompanied by reductions in absolute peak force and power, which were offset when expressed relative to physiological cross-sectional area. Contrary to peak force, absolute and relative forces at submaximal stimulation frequencies were significantly greater following 14 days of denervation. Taken together, these data represent two keys findings. First, activation of the ubiquitin-proteasome system is associated with reductions in skeletal muscle quantity rather than quality. Second, shortly after denervation, it appears the muscle remodels to compensate for the loss of neural activity via changes in Ca2+ handling. PMID:27513942

  18. 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

  19. 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

  20. 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

  1. 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.

  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. 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

  4. The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection.

    PubMed

    Dieudé, Mélanie; Bell, Christina; Turgeon, Julie; Beillevaire, Deborah; Pomerleau, Luc; Yang, Bing; Hamelin, Katia; Qi, Shijie; Pallet, Nicolas; Béland, Chanel; Dhahri, Wahiba; Cailhier, Jean-François; Rousseau, Matthieu; Duchez, Anne-Claire; Lévesque, Tania; Lau, Arthur; Rondeau, Christiane; Gingras, Diane; Muruve, Danie; Rivard, Alain; Cardinal, Héloise; Perreault, Claude; Desjardins, Michel; Boilard, Éric; Thibault, Pierre; Hébert, Marie-Josée

    2015-12-16

    Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. PMID:26676607

  5. 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.

  6. Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway

    PubMed Central

    Chinchankar, Meghna N.; Ferguson, Annabel A.; Ghazi, Arjumand; Fisher, Alfred L.

    2016-01-01

    The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant provides a unique

  7. Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway.

    PubMed

    Keith, Scott A; Maddux, Sarah K; Zhong, Yayu; Chinchankar, Meghna N; Ferguson, Annabel A; Ghazi, Arjumand; Fisher, Alfred L

    2016-02-01

    The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant provides a unique

  8. 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.

  9. 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

  10. 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.

  11. 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

  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. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome

    PubMed Central

    Huber, Eva M.; Heinemeyer, Wolfgang; Li, Xia; Arendt, Cassandra S.; Hochstrasser, Mark; Groll, Michael

    2016-01-01

    Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease. PMID:26964885

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. Diaphragm Muscle Fiber Weakness and Ubiquitin–Proteasome Activation in Critically Ill Patients

    PubMed Central

    Hooijman, Pleuni E.; Beishuizen, Albertus; Witt, Christian C.; de Waard, Monique C.; Girbes, Armand R. J.; Spoelstra-de Man, Angelique M. E.; Niessen, Hans W. M.; Manders, Emmy; van Hees, Hieronymus W. H.; van den Brom, Charissa E.; Silderhuis, Vera; Lawlor, Michael W.; Labeit, Siegfried; Stienen, Ger J. M.; Hartemink, Koen J.; Paul, Marinus A.; Heunks, Leo M. A.

    2015-01-01

    Rationale: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency, and increases morbidity and duration of hospital stay. To date, the nature of diaphragm weakness and its underlying pathophysiologic mechanisms are poorly understood. Objectives: We hypothesized that diaphragm muscle fibers of mechanically ventilated critically ill patients display atrophy and contractile weakness, and that the ubiquitin–proteasome pathway is activated in the diaphragm. Methods: We obtained diaphragm muscle biopsies from 22 critically ill patients who received mechanical ventilation before surgery and compared these with biopsies obtained from patients during thoracic surgery for resection of a suspected early lung malignancy (control subjects). In a proof-of-concept study in a muscle-specific ring finger protein-1 (MuRF-1) knockout mouse model, we evaluated the role of the ubiquitin–proteasome pathway in the development of contractile weakness during mechanical ventilation. Measurements and Main Results: Both slow- and fast-twitch diaphragm muscle fibers of critically ill patients had approximately 25% smaller cross-sectional area, and had contractile force reduced by half or more. Markers of the ubiquitin–proteasome pathway were significantly up-regulated in the diaphragm of critically ill patients. Finally, MuRF-1 knockout mice were protected against the development of diaphragm contractile weakness during mechanical ventilation. Conclusions: These findings show that diaphragm muscle fibers of critically ill patients display atrophy and severe contractile weakness, and in the diaphragm of critically ill patients the ubiquitin–proteasome pathway is activated. This study provides rationale for the development of treatment strategies that target the contractility of diaphragm fibers to facilitate weaning. PMID:25760684

  20. 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

  1. 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.

  2. Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

    PubMed Central

    Fang, Nancy N.; Zhu, Mang; Rose, Amalia; Wu, Kuen-Phon; Mayor, Thibault

    2016-01-01

    Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions. PMID:27698423

  3. DNA damage-induced activation of CUL4B targets HUWE1 for proteasomal degradation.

    PubMed

    Yi, Juan; Lu, Guang; Li, Li; Wang, Xiaozhen; Cao, Li; Lin, Ming; Zhang, Sha; Shao, Genze

    2015-05-19

    The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in integrating/coordinating diverse cellular processes such as DNA damage repair and apoptosis. A previous study has shown that HUWE1 is required for the early step of DNA damage-induced apoptosis, by targeting MCL-1 for proteasomal degradation. However, HUWE1 is subsequently inactivated, promoting cell survival and the subsequent DNA damage repair process. The mechanism underlying its regulation during this process remains largely undefined. Here, we show that the Cullin4B-RING E3 ligase (CRL4B) is required for proteasomal degradation of HUWE1 in response to DNA damage. CUL4B is activated in a NEDD8-dependent manner, and ubiquitinates HUWE1 in vitro and in vivo. The depletion of CUL4B stabilizes HUWE1, which in turn accelerates the degradation of MCL-1, leading to increased induction of apoptosis. Accordingly, cells deficient in CUL4B showed increased sensitivity to DNA damage reagents. More importantly, upon CUL4B depletion, these phenotypes can be rescued through simultaneous depletion of HUWE1, consistent with the role of CUL4B in regulating HUWE1. Collectively, these results identify CRL4B as an essential E3 ligase in targeting the proteasomal degradation of HUWE1 in response to DNA damage, and provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase.

  4. Reactive center loop moiety is essential for the maspin activity on cellular invasion and ubiquitin-proteasome level.

    PubMed

    Khanaree, Chakkrit; Chairatvit, Kongthawat; Roytrakul, Sittiruk; Wongnoppavich, Ariyaphong

    2013-01-01

    Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin's action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain. PMID:23924927

  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. 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

  7. The proteasome is responsible for caspase-3-like activity during xylem development.

    PubMed

    Han, Jia-Jia; Lin, Wei; Oda, Yoshihisa; Cui, Ke-Ming; Fukuda, Hiroo; He, Xin-Qiang

    2012-10-01

    Xylem development is a process of xylem cell terminal differentiation that includes initial cell division, cell expansion, secondary cell wall formation and programmed cell death (PCD). PCD in plants and apoptosis in animals share many common characteristics. Caspase-3, which displays Asp-Glu-Val-Asp (DEVD) specificity, is a crucial executioner during animal cells apoptosis. Although a gene orthologous to caspase-3 is absent in plants, caspase-3-like activity is involved in many cases of PCD and developmental processes. However, there is no direct evidence that caspase-3-like activity exists in xylem cell death. In this study, we showed that caspase-3-like activity is present and is associated with secondary xylem development in Populus tomentosa. The protease responsible for the caspase-3-like activity was purified from poplar secondary xylem using hydrophobic interaction chromatography (HIC), Q anion exchange chromatography and gel filtration chromatography. After identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS), it was revealed that the 20S proteasome (20SP) was responsible for the caspase-3-like activity in secondary xylem development. In poplar 20SP, there are seven α subunits encoded by 12 genes and seven β subunits encoded by 12 genes. Pharmacological assays showed that Ac-DEVD-CHO, a caspase-3 inhibitor, suppressed xylem differentiation in the veins of Arabidopsis cotyledons. Furthermore, clasto-lactacystin β-lactone, a proteasome inhibitor, inhibited PCD of tracheary element in a VND6-induced Arabidopsis xylogenic culture. In conclusion, the 20S proteasome is responsible for caspase-3-like activity and is involved in xylem development.

  8. Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system

    PubMed Central

    Elharar, Yifat; Roth, Ziv; Hecht, Nir; Rotkopf, Ron; Khalaila, Isam; Gur, Eyal

    2016-01-01

    The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for high-molecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions. PMID:26951665

  9. Chemical analysis of Greek pollen - Antioxidant, antimicrobial and proteasome activation properties

    PubMed Central

    2011-01-01

    Background Pollen is a bee-product known for its medical properties from ancient times. In our days is increasingly used as health food supplement and especially as a tonic primarily with appeal to the elderly to ameliorate the effects of ageing. In order to evaluate the chemical composition and the biological activity of Greek pollen which has never been studied before, one sample with identified botanical origin from sixteen different common plant taxa of Greece has been evaluated. Results Three different extracts of the studied sample of Greek pollen, have been tested, in whether could induce proteasome activities in human fibroblasts. The water extract was found to induce a highly proteasome activity, showing interesting antioxidant properties. Due to this activity the aqueous extract was further subjected to chemical analysis and seven flavonoids have been isolated and identified by modern spectral means. From the methanolic extract, sugars, lipid acids, phenolic acids and their esters have been also identified, which mainly participate to the biosynthetic pathway of pollen phenolics. The total phenolics were estimated with the Folin-Ciocalteau reagent and the total antioxidant activity was determined by the DPPH method while the extracts and the isolated compounds were also tested for their antimicrobial activity by the dilution technique. Conclusions The Greek pollen is rich in flavonoids and phenolic acids which indicate the observed free radical scavenging activity, the effects of pollen on human fibroblasts and the interesting antimicrobial profile. PMID:21699688

  10. 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

  11. 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.

  12. 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.

  13. Control of Death-associated Protein Kinase (DAPK) Activity by Phosphorylation and Proteasomal Degradation*

    PubMed Central

    Jin, Yijun; Blue, Emily K.; Gallagher, Patricia J.

    2010-01-01

    Activation of death-associated protein kinase (DAPK) occurs via dephosphorylation of Ser-308 and subsequent association of calcium/calmodulin. In this study, we confirmed the existence of the alternatively spliced human DAPK-β, and we examined the levels of DAPK autophosphorylation and DAPK catalytic activity in response to tumor necrosis factor or ceramide. It was found that DAPK is rapidly dephosphorylated in response to tumor necrosis factor or ceramide and then subsequently degraded via proteasome activity. Dephosphorylation and activation of DAPK are shown to temporally precede its subsequent degradation. This results in an initial increase in kinase activity followed by a decrease in DAPK expression and activity. The decline in DAPK expression is paralleled with increased caspase activity and cell apoptosis. These results suggest that the apoptosis regulatory activities mediated by DAPK are controlled both by phosphorylation status and protein stability. PMID:17056602

  14. Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation.

    PubMed

    Martín-Garrido, A; Boyano-Adánez, M C; Alique, M; Calleros, L; Serrano, I; Griera, M; Rodríguez-Puyol, D; Griendling, K K; Rodríguez-Puyol, M

    2009-11-15

    Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.

  15. 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.

  16. Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form

    SciTech Connect

    Sugiyama, Masaaki; Sahashi, Hiroki; Kurimoto, Eiji; Takata, Shin-ichi; Yagi, Hirokazu; Kanai, Keita; Sakata, Eri; Minami, Yasufumi; Tanaka, Keiji; Kato, Koichi

    2013-03-01

    Highlights: ► Homologous α and β subunits are alternatively arranged in the PA28 heptameric ring. ► The flexible loops of the three α subunits surround the site of substrate entry. ► The loops serve as gatekeepers that selectively hinder passage of longer peptides. - Abstract: A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and β subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four β subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.

  17. 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

  18. 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

  19. Rescue of Murine F508del CFTR Activity in Native Intestine by Low Temperature and Proteasome Inhibitors

    PubMed Central

    Wilke, Martina; Bot, Alice; Jorna, Huub; Scholte, Bob J.; de Jonge, Hugo R.

    2012-01-01

    Most patients with Cystic Fibrosis (CF) carry at least one allele with the F508del mutation, resulting in a CFTR chloride channel protein with a processing, gating and stability defect, but with substantial residual activity when correctly sorted to the apical membranes of epithelial cells. New therapies are therefore aimed at improving the folding and trafficking of F508del CFTR, (CFTR correctors) or at enhancing the open probability of the CFTR chloride channel (CFTR potentiators). Preventing premature breakdown of F508del CFTR is an alternative or additional strategy, which is investigated in this study. We established an ex vivo assay for murine F508del CFTR rescue in native intestinal epithelium that can be used as a pre-clinical test for candidate therapeutics. Overnight incubation of muscle stripped ileum in modified William's E medium at low temperature (26°C), and 4 h or 6 h incubation at 37°C with different proteasome inhibitors (PI: ALLN, MG-132, epoxomicin, PS341/bortezomib) resulted in fifty to hundred percent respectively of the wild type CFTR mediated chloride secretion (forskolin induced short-circuit current). The functional rescue was accompanied by enhanced expression of the murine F508del CFTR protein at the apical surface of intestinal crypts and a gain in the amount of complex-glycosylated CFTR (band C) up to 20% of WT levels. Sustained rescue in the presence of brefeldin A shows the involvement of a post-Golgi compartment in murine F508del CFTR degradation, as was shown earlier for its human counterpart. Our data show that proteasome inhibitors are promising candidate compounds for improving rescue of human F508del CFTR function, in combination with available correctors and potentiators. PMID:23284872

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. Pathogenesis of human mitochondrial diseases is modulated by reduced activity of the ubiquitin/proteasome system.

    PubMed

    Segref, Alexandra; Kevei, Éva; Pokrzywa, Wojciech; Schmeisser, Kathrin; Mansfeld, Johannes; Livnat-Levanon, Nurit; Ensenauer, Regina; Glickman, Michael H; Ristow, Michael; Hoppe, Thorsten

    2014-04-01

    Mitochondria maintain cellular homeostasis by coordinating ATP synthesis with metabolic activity, redox signaling, and apoptosis. Excessive levels of mitochondria-derived reactive oxygen species (ROS) promote mitochondrial dysfunction, triggering numerous metabolic disorders. However, the molecular basis for the harmful effects of excessive ROS formation is largely unknown. Here, we identify a link between mitochondrial stress and ubiquitin-dependent proteolysis, which supports cellular surveillance both in Caenorhabditis elegans and humans. Worms defective in respiration with elevated ROS levels are limited in turnover of a GFP-based substrate protein, demonstrating that mitochondrial stress affects the ubiquitin/proteasome system (UPS). Intriguingly, we observed similar proteolytic defects for disease-causing IVD and COX1 mutations associated with mitochondrial failure in humans. Together, these results identify a conserved link between mitochondrial metabolism and ubiquitin-dependent proteostasis. Reduced UPS activity during pathological conditions might potentiate disease progression and thus provides a valuable target for therapeutic intervention. PMID:24703696

  5. 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.

  6. Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

    PubMed Central

    Doi, Kenichiro; Sharma, Arun K.; Wang, Hong-Gang; Amin, Shantu

    2013-01-01

    Background and purpose Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells. Experimental approach For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis. Key results The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC50 of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids. Conclusions and implications Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors. PMID:24223823

  7. alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress.

    PubMed

    Sharma, Nijee; Brandis, Katrina A; Herrera, Sara K; Johnson, Brandon E; Vaidya, Tulaza; Shrestha, Ruja; Debburman, Shubhik K

    2006-01-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.

  8. 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

  9. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    PubMed

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Long-term memory consolidation depends on proteasome activity in the crab Chasmagnathus.

    PubMed

    Merlo, E; Romano, A

    2007-06-15

    Long-term memory formation depends on protein and mRNA synthesis that subserves synaptic reorganization. The removal of pre-existing inhibitory proteins by the ubiquitin-proteasome system (UPS) is proposed as a crucial step to support these modifications. The activation of the constitutive transcription factor nuclear factor kappaB (NF-kappaB) depends on the degradation of the inhibitor of NF-kappaB (IkappaB) by the UPS. Here we study the effect of a UPS inhibitor, MG132, on long-term memory consolidation and NF-kappaB activation in the learning paradigm of the crab Chasmagnathus, a model in which this transcription factor plays a key role. Here we found that administration of MG132 interferes with long-term memory but not with short-term memory, and no facilitatory effects were found. Then we studied the effect of the UPS inhibitor on NF-kappaB pathway, finding that MG132 blocks the activation of NF-kappaB induced by training. These results suggest that the UPS is necessary for long-term memory consolidation, allowing for the activation of NF-kappaB as one of the target molecular pathways.

  16. 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

  17. Intracellular colocalization of HAP1/STBs with steroid hormone receptors and its enhancement by a proteasome inhibitor

    SciTech Connect

    Fujinaga, Ryutaro; Takeshita, Yukio; Yoshioka, Kazuhiro; Nakamura, Hiroyuki; Shinoda, Shuhei; Islam, Md. Nabiul; Jahan, Mir Rubayet; Yanai, Akie; Kokubu, Keiji; Shinoda, Koh

    2011-07-15

    The stigmoid body (STB) is a cytoplasmic inclusion containing huntingtin-associated protein 1 (HAP1), and HAP1/STB formation is induced by transfection of the HAP1 gene into cultured cells. In the present study, we examined the intracellular colocalization of HAP1/STBs with steroid hormone receptors (SHRs), including the androgen receptor (AR), estrogen receptor, glucocorticoid receptor (GR), and mineralocorticoid receptor, in COS-7 cells cotransfected with HAP1 and each receptor. We found that C-terminal ligand-binding domains of all SHRs had potential for colocalization with HAP1/STBs, whereas only AR and GR were clearly colocalized with HAP1/STBs when each full-length SHR was coexpressed with HAP1. In addition, it appeared that HAP1/STBs did not disrupt GR and AR functions because the receptors on HAP1/STBs maintained nuclear translocation activity in response to their specific ligands. When the cells were treated with a proteasome inhibitor, GR and AR localized outside HAP1/STBs translocated into the nucleus, whereas the receptors colocalized with HAP1/STBs persisted in their colocalization even after treatment with their ligands. Therefore, HAP1/STBs may be involved in cytoplasmic modifications of the nuclear translocation of GR and AR in a ubiquitin-proteasome system.

  18. 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

  19. 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

  20. Biochemical and toxicological evaluation of nano-heparins in cell functional properties, proteasome activation and expression of key matrix molecules.

    PubMed

    Piperigkou, Zoi; Karamanou, Konstantina; Afratis, Nikolaos A; Bouris, Panagiotis; Gialeli, Chrysostomi; Belmiro, Celso L R; Pavão, Mauro S G; Vynios, Dimitrios H; Tsatsakis, Aristidis M

    2016-01-01

    The glycosaminoglycan heparin and its derivatives act strongly on blood coagulation, controlling the activity of serine protease inhibitors in plasma. Nonetheless, there is accumulating evidence highlighting different anticancer activities of these molecules in numerous types of cancer. Nano-heparins may have great biological significance since they can inhibit cell proliferation and invasion as well as inhibiting proteasome activation. Moreover, they can cause alterations in the expression of major modulators of the tumor microenvironment, regulating cancer cell behavior. In the present study, we evaluated the effects of two nano-heparin formulations: one isolated from porcine intestine and the other from the sea squirt Styela plicata, on a breast cancer cell model. We determined whether these nano-heparins are able to affect cell proliferation, apoptosis and invasion, as well as proteasome activity and the expression of extracellular matrix molecules. Specifically, we observed that nano-Styela compared to nano-Mammalian analogue has higher inhibitory role on cell proliferation, invasion and proteasome activity. Moreover, nano-Styela regulates cell apoptosis, expression of inflammatory molecules, such as IL-6 and IL-8 and reduces the expression levels of extracellular matrix macromolecules, such as the proteolytic enzymes MT1-MMP, uPA and the cell surface proteoglycans syndecan-1 and -2, but not on syndecan-4. The observations reported in the present article indicate that nano-heparins and especially ascidian heparin are effective agents for heparin-induced effects in critical cancer cell functions, providing an important possibility in pharmacological targeting.

  1. 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

  2. Sepiapterin attenuates 1-methyl-4-phenylpyridinium-induced apoptosis in neuroblastoma cells transfected with neuronal NOS: role of tetrahydrobiopterin, nitric oxide, and proteasome activation.

    PubMed

    Shang, Tiesong; Kotamraju, Srigiridhar; Zhao, Hongtao; Kalivendi, Shasi V; Hillard, Cecilia J; Kalyanaraman, B

    2005-10-15

    In this study, we investigated the molecular mechanism of toxicity of 1-methyl-4-phenylpyridinium (MPP+), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes parkinsonism in experimental animals and humans. Using wild-type and human neuronal nitric oxide synthase (nNOS) stably transfected neuroblastoma cells (SH-SY5Y), we showed that nNOS overexpression in SH-SY5Y cells greatly enhanced proteasome activity and mitigated MPP+-induced apoptosis. During MPP+-induced oxidative stress, intracellular BH4 levels decreased, resulting in nNOS "uncoupling" (i.e., switching from nitric oxide to superoxide generation). Increasing the intracellular BH4 levels by sepiapterin supplementation restored the nNOS activity, inhibited superoxide formation, increased proteasome activity, decreased protein ubiquitination, and attenuated apoptosis in MPP+-treated cells. Implications of BH4 depletion in dopaminergic cells and sepiapterin supplementation to augment the striatal nNOS activity in the pathogenesis mechanism and treatment of Parkinson disease are discussed. PMID:16198233

  3. Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics*

    PubMed Central

    Fabre, Bertrand; Lambour, Thomas; Delobel, Julien; Amalric, François; Monsarrat, Bernard; Burlet-Schiltz, Odile; Bousquet-Dubouch, Marie-Pierre

    2013-01-01

    Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins—which modulate proteasome activity, stability, localization, or substrate uptake—rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells. PMID:23242550

  4. Tight Junction Protein 1 Modulates Proteasome Capacity and Proteasome Inhibitor Sensitivity in Multiple Myeloma via EGFR/JAK1/STAT3 Signaling.

    PubMed

    Zhang, Xing-Ding; Baladandayuthapani, Veerabhadran; Lin, Heather; Mulligan, George; Li, Bin; Esseltine, Dixie-Lee W; Qi, Lin; Xu, Jianliang; Hunziker, Walter; Barlogie, Bart; Usmani, Saad Z; Zhang, Qing; Crowley, John; Hoering, Antje; Shah, Jatin J; Weber, Donna M; Manasanch, Elisabet E; Thomas, Sheeba K; Li, Bing-Zong; Wang, Hui-Han; Zhang, Jiexin; Kuiatse, Isere; Tang, Jin-Le; Wang, Hua; He, Jin; Yang, Jing; Milan, Enrico; Cenci, Simone; Ma, Wen-Cai; Wang, Zhi-Qiang; Davis, Richard Eric; Yang, Lin; Orlowski, Robert Z

    2016-05-01

    Proteasome inhibitors have revolutionized outcomes in multiple myeloma, but they are used empirically, and primary and secondary resistance are emerging problems. We have identified TJP1 as a determinant of plasma cell proteasome inhibitor susceptibility. TJP1 suppressed expression of the catalytically active immunoproteasome subunits LMP7 and LMP2, decreased proteasome activity, and enhanced proteasome inhibitor sensitivity in vitro and in vivo. This occurred through TJP1-mediated suppression of EGFR/JAK1/STAT3 signaling, which modulated LMP7 and LMP2 levels. In the clinic, high TJP1 expression in patient myeloma cells was associated with a significantly higher likelihood of responding to bortezomib and a longer response duration, supporting the use of TJP1 as a biomarker to identify patients most likely to benefit from proteasome inhibitors. PMID:27132469

  5. 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

  6. 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

  7. Interactive effects of CO₂ and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

    PubMed

    Götze, Sandra; Matoo, Omera B; Beniash, Elia; Saborowski, Reinhard; Sokolova, Inna M

    2014-04-01

    Increased anthropogenic emission of CO2 changes the carbonate chemistry and decreases the pH of the ocean. This can affect the speciation and the bioavailability of metals in polluted habitats such as estuaries. However, the effects of acidification on metal accumulation and stress response in estuarine organisms including bivalves are poorly understood. We studied the interactive effects of CO2 and two common metal pollutants, copper (Cu) and cadmium (Cd), on metal accumulation, intracellular ATP/ubiquitin-dependent protein degradation, stress response and energy metabolism in two common estuarine bivalves-Crassostrea virginica (eastern oyster) and Mercenaria mercenaria (hard shell clam). Bivalves were exposed for 4-5 weeks to clean seawater (control) and to either 50 μg L(-1) Cu or 50 μg L(-1) Cd at one of three partial pressures of CO2 ( [Formula: see text] ∼ 395, ∼ 800 and ∼ 1500 μatm) representative of the present-day conditions and projections of the Intergovernmental Panel for Climate Change (IPCC) for the years 2100 and 2250, respectively. Clams accumulated lower metal burdens than oysters, and elevated [Formula: see text] enhanced the Cd and Cu accumulation in mantle tissues in both species. Higher Cd and Cu burdens were associated with elevated mRNA expression of metal binding proteins metallothionein and ferritin. In the absence of added metals, proteasome activities of clams and oysters were robust to elevated [Formula: see text] , but [Formula: see text] modulated the proteasome response to metals. Cd exposure stimulated the chymotrypsin-like activity of the oyster proteasome at all CO2 levels. In contrast, trypsin- and caspase-like activities of the oyster proteasome were slightly inhibited by Cd exposure in normocapnia but this inhibition was reversed at elevated [Formula: see text] . Cu exposure inhibited the chymotrypsin-like activity of the oyster proteasome regardless of the exposure [Formula: see text] . The effects of metal exposure on

  8. 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

  9. LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites

    PubMed Central

    Reis, Julia; Guan, Xiu Qin; Kisselev, Alexei F.; Papasian, Christopher J.; Qureshi, Asaf A.; Morrison, David C.; Van Way, Charles W.; Vogel, Stefanie N.

    2011-01-01

    Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin–proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators. PMID:21455682

  10. 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.…

  11. Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro.

    PubMed

    Phukan, Geetika; Shin, Tae Hwan; Shim, Jeom Soon; Paik, Man Jeong; Lee, Jin-Kyu; Choi, Sangdun; Kim, Yong Man; Kang, Seong Ho; Kim, Hyung Sik; Kang, Yup; Lee, Soo Hwan; Mouradian, M Maral; Lee, Gwang

    2016-07-05

    The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α-synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles.

  12. Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro

    PubMed Central

    Phukan, Geetika; Shin, Tae Hwan; Shim, Jeom Soon; Paik, Man Jeong; Lee, Jin-Kyu; Choi, Sangdun; Kim, Yong Man; Kang, Seong Ho; Kim, Hyung Sik; Kang, Yup; Lee, Soo Hwan; Mouradian, M. Maral; Lee, Gwang

    2016-01-01

    The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α–synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles. PMID:27378605

  13. 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

  14. Regulation of HTLV-1 tax stability, cellular trafficking and NF-κB activation by the ubiquitin-proteasome pathway.

    PubMed

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-10-23

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis.

  15. Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome.

    PubMed

    Tomaić, V; Banks, L

    2015-01-01

    Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology. PMID:25633294

  16. Negatively charged metal oxide nanoparticles interact with the 20S proteasome and differentially modulate its biologic functional effects.

    PubMed

    Falaschetti, Christine A; Paunesku, Tatjana; Kurepa, Jasmina; Nanavati, Dhaval; Chou, Stanley S; De, Mrinmoy; Song, MinHa; Jang, Jung-tak; Wu, Aiguo; Dravid, Vinayak P; Cheon, Jinwoo; Smalle, Jan; Woloschak, Gayle E

    2013-09-24

    The multicatalytic ubiquitin-proteasome system (UPS) carries out proteolysis in a highly orchestrated way and regulates a large number of cellular processes. Deregulation of the UPS in many disorders has been documented. In some cases, such as carcinogenesis, elevated proteasome activity has been implicated in disease development, while the etiology of other diseases, such as neurodegeneration, includes decreased UPS activity. Therefore, agents that alter proteasome activity could suppress as well as enhance a multitude of diseases. Metal oxide nanoparticles, often developed as diagnostic tools, have not previously been tested as modulators of proteasome activity. Here, several types of metal oxide nanoparticles were found to adsorb to the proteasome and show variable preferential binding for particular proteasome subunits with several peptide binding "hotspots" possible. These interactions depend on the size, charge, and concentration of the nanoparticles and affect proteasome activity in a time-dependent manner. Should metal oxide nanoparticles increase proteasome activity in cells, as they do in vitro, unintended effects related to changes in proteasome function can be expected.

  17. Negatively Charged Metal Oxide Nanoparticles Interact with the 20S Proteasome and Differentially Modulate Its Biologic Functional Effects

    PubMed Central

    Falaschetti, Christine A.; Paunesku, Tatjana; Kurepa, Jasmina; Nanavati, Dhaval; Chou, Stanley S.; De, Mrinmoy; Song, MinHa; Jang, Jung-tak; Wu, Aiguo; Dravid, Vinayak P.; Cheon, Jinwoo; Smalle, Jan; Woloschak, Gayle E.

    2013-01-01

    The multicatalytic ubiquitin-proteasome system (UPS) carries out proteolysis in a highly orchestrated way and regulates a large number of cellular processes. Deregulation of the UPS in many disorders has been documented. In some cases, e.g. carcinogenesis, elevated proteasome activity has been implicated in disease development, while the etiology of other diseases, e.g. neurodegeneration, includes decreased UPS activity. Therefore, agents that alter proteasome activity could suppress as well as enhance a multitude of diseases. Metal oxide nanoparticles, often developed as diagnostic tools, have not previously been tested as modulators of proteasome activity. Here, several types of metal oxide nanoparticles were found to adsorb to the proteasome and show variable preferential binding for particular proteasome subunits with several peptide binding “hotspots” possible. These interactions depend on the size, charge, and concentration of the nanoparticles and affect proteasome activity in a time-dependent manner. Should metal oxide nanoparticles increase proteasome activity in cells, as they do in vitro, unintended effects related to changes in proteasome function can be expected. PMID:23930940

  18. ADD66, a Gene Involved in the Endoplasmic Reticulum-associated Degradation of α-1-Antitrypsin-Z in Yeast, Facilitates Proteasome Activity and Assembly

    PubMed Central

    Scott, Craig M.; Kruse, Kristina B.; Schmidt, Béla Z.; Perlmutter, David H.; McCracken, Ardythe A.

    2007-01-01

    Antitrypsin deficiency is a primary cause of juvenile liver disease, and it arises from expression of the “Z” variant of the α-1 protease inhibitor (A1Pi). Whereas A1Pi is secreted from the liver, A1PiZ is retrotranslocated from the endoplasmic reticulum (ER) and degraded by the proteasome, an event that may offset liver damage. To better define the mechanism of A1PiZ degradation, a yeast expression system was developed previously, and a gene, ADD66, was identified that facilitates A1PiZ turnover. We report here that ADD66 encodes an ∼30-kDa soluble, cytosolic protein and that the chymotrypsin-like activity of the proteasome is reduced in add66Δ mutants. This reduction in activity may arise from the accumulation of 20S proteasome assembly intermediates or from qualitative differences in assembled proteasomes. Add66p also seems to be a proteasome substrate. Consistent with its role in ER-associated degradation (ERAD), synthetic interactions are observed between the genes encoding Add66p and Ire1p, a transducer of the unfolded protein response, and yeast deleted for both ADD66 and/or IRE1 accumulate polyubiquitinated proteins. These data identify Add66p as a proteasome assembly chaperone (PAC), and they provide the first link between PAC activity and ERAD. PMID:17634286

  19. 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.

  20. Fatty acids regulate CREBh via transcriptional mechanisms that are dependent on proteasome activity and insulin

    PubMed Central

    Gentile, CL; Wang, D; Pfaffenbach, KT; Cox, R; Wei, Y; Pagliassotti, MJ

    2011-01-01

    Excess fatty acids are closely associated with metabolic dysfunction. The deleterious effects of fatty acids relate, in part, to their ability to up-regulate proinflammatory cytokines and propagate a state of systemic inflammation. CREBh is a recently identified transcription factor that appears to be required for hepatic synthesis of C-reactive protein (CRP). Recent data suggest that fatty acids can up-regulate CREBh, thus establishing a potential molecular link between fatty acids and inflammation. The aim of the current study was to examine the nature and mechanisms of fatty acid-mediated regulation of CREBh. H4IIE liver cells were incubated in the absence or presence of varying concentrations (50–500 μM) of albumin-bound, long-chain saturated (palmitate, stearate) or unsaturated (oleate, linoleate) fatty acids (1–16 hours). All fatty acids significantly increased CREBh gene expression via transcriptional mechanisms, at concentrations as low as 50 μM. Palmitate- or oleate-mediated upregulation of CREBh was not inhibited by triacsin C, an inhibitor of long-chain fatty acyl CoA synthetase, or by the PPARα antagonist, MK886. Inhibition of proteasome activity with MG132 or lactacystin, or inclusion of insulin reduced palmitate- and oleate-mediated increases in CREBh mRNA. Finally, we examined fatty acid regulation of CREBh in vivo. Male Wistar rats were exposed to a 4-hour pancreatic clamp combined with infusion of saline or a mixed lipid emulsion. CREBh mRNA and protein were significantly increased in rats exposed to the lipid infusion compared to the saline group. Collectively, these results may have important implications for metabolic diseases characterized by excess fatty acids, insulin resistance and inflammation. PMID:20607591

  1. Proteasomes, Sir2, and Hxk2 form an interconnected aging network that impinges on the AMPK/Snf1-regulated transcriptional repressor Mig1.

    PubMed

    Yao, Yanhua; Tsuchiyama, Scott; Yang, Ciyu; Bulteau, Anne Laure; He, Chong; Robison, Brett; Tsuchiya, Mitsuhiro; Miller, Delana; Briones, Valeria; Tar, Krisztina; Potrero, Anahi; Friguet, Bertrand; Kennedy, Brian K; Schmidt, Marion

    2015-01-01

    Elevated proteasome activity extends lifespan in model organisms such as yeast, worms and flies. This pro-longevity effect might be mediated by improved protein homeostasis, as this protease is an integral module of the protein homeostasis network. Proteasomes also regulate cellular processes through temporal and spatial degradation of signaling pathway components. Here we demonstrate that the regulatory function of the proteasome plays an essential role in aging cells and that the beneficial impact of elevated proteasome capacity on lifespan partially originates from deregulation of the AMPK signaling pathway. Proteasome-mediated lifespan extension activity was carbon-source dependent and cells with enhancement proteasome function exhibited increased respiratory activity and oxidative stress response. These findings suggested that the pro-aging impact of proteasome upregulation might be related to changes in the metabolic state through a premature induction of respiration. Deletion of yeast AMPK, SNF1, or its activator SNF4 abrogated proteasome-mediated lifespan extension, supporting this hypothesis as the AMPK pathway regulates metabolism. We found that the premature induction of respiration in cells with increased proteasome activity originates from enhanced turnover of Mig1, an AMPK/Snf1 regulated transcriptional repressor that prevents the induction of genes required for respiration. Increasing proteasome activity also resulted in partial relocation of Mig1 from the nucleus to the mitochondria. Collectively, the results argue for a model in which elevated proteasome activity leads to the uncoupling of Snf1-mediated Mig1 regulation, resulting in a premature activation of respiration and thus the induction of a mitohormetic response, beneficial to lifespan. In addition, we observed incorrect Mig1 localization in two other long-lived yeast aging models: cells that overexpress SIR2 or deleted for the Mig1-regulator HXK2. Finally, compromised proteasome function

  2. Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants.

    PubMed

    Djebali, Wahbi; Gallusci, Philippe; Polge, Cécile; Boulila, Latifa; Galtier, Nathalie; Raymond, Philippe; Chaibi, Wided; Brouquisse, Renaud

    2008-02-01

    The effects of cadmium (Cd) on cellular proteolytic responses were investigated in the roots and leaves of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 3 and 10 days in the presence of 0.3-300 microM Cd and compared to control plants grown in the absence of Cd. Roots of Cd treated plants accumulated four to fivefold Cd as much as mature leaves. Although 10 days of culture at high Cd concentrations inhibited plant growth, tomato plants recovered and were still able to grow again after Cd removal. Tomato roots and leaves are not modified in their proteolytic response with low Cd concentrations (< or =3 microM) in the incubation medium. At higher Cd concentration, protein oxidation state and protease activities are modified in roots and leaves although in different ways. The soluble protein content of leaves decreased and protein carbonylation level increased indicative of an oxidative stress. Conversely, protein content of roots increased from 30 to 50%, but the amount of oxidized proteins decreased by two to threefold. Proteolysis responded earlier in leaves than in root to Cd stress. Additionally, whereas cysteine- and metallo-endopeptidase activities, as well as proteasome chymotrypsin activity and subunit expression level, increased in roots and leaves, serine-endopeptidase activities increased only in leaves. This contrasted response between roots and leaves may reflect differences in Cd compartmentation and/or complexation, antioxidant responses and metabolic sensitivity to Cd between plant tissues. The up-regulation of the 20S proteasome gene expression and proteolytic activity argues in favor of the involvement of the 20S proteasome in the degradation of oxidized proteins in plants.

  3. N-Acetylcysteine in Combination with IGF-1 Enhances Neuroprotection against Proteasome Dysfunction-Induced Neurotoxicity in SH-SY5Y Cells

    PubMed Central

    Anand, Pinki; Kuang, Anxiu; Akhtar, Feroz; Scofield, Virginia L.

    2016-01-01

    Ubiquitin proteasome system (UPS) dysfunction has been implicated in the development of many neuronal disorders, including Parkinson's disease (PD). Previous studies focused on individual neuroprotective agents and their respective abilities to prevent neurotoxicity following a variety of toxic insults. However, the effects of the antioxidant N-acetylcysteine (NAC) on proteasome impairment-induced apoptosis have not been well characterized in human neuronal cells. The aim of this study was to determine whether cotreatment of NAC and insulin-like growth factor-1 (IGF-1) efficiently protected against proteasome inhibitor-induced cytotoxicity in SH-SY5Y cells. Our results demonstrate that the proteasome inhibitor, MG132, initiates poly(ADP-ribose) polymerase (PARP) cleavage, caspase 3 activation, and nuclear condensation and fragmentation. In addition, MG132 treatment leads to endoplasmic reticulum (ER) stress and autophagy-mediated cell death. All of these events can be attenuated without obvious reduction of MG132 induced protein ubiquitination by first treating the cells with NAC and IGF-1 separately or simultaneously prior to exposure to MG132. Moreover, our data demonstrated that the combination of the two proved to be significantly more effective for neuronal protection. Therefore, we conclude that the simultaneous use of growth/neurotrophic factors and a free radical scavenger may increase overall protection against UPS dysfunction-mediated cytotoxicity and neurodegeneration. PMID:27774335

  4. Accumulation of wildtype and ALS-linked mutated VAPB impairs activity of the proteasome.

    PubMed

    Moumen, Anice; Virard, Isabelle; Raoul, Cédric

    2011-01-01

    Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER) quality control and the ubiquitin proteasome system (UPS) have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ER-resident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB) leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAPB aggregates. Our results suggest that ER stress and corruption of the proteasome function might contribute to the aberrant protein homeostasis associated with hVAPB.

  5. Molecular and functional characterization of a putative PA28γ proteasome activator orthologue in Schistosoma mansoni

    PubMed Central

    Soares, Cláudia Sossai; Morais, Enyara Rezende; Magalhães, Lizandra G.; Machado, Carla Botelho; Moreira, Érika Bueno de Carvalho; Teixeira, Felipe Roberti; Rodrigues, Vanderlei; Yoshino, Timothy P.

    2013-01-01

    PA28γ is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28γ of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28γ, this transcript has a 753 bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28γ with multiple PA28γ orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28γ from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28γ to its sister group that contains well-characterized PA28γ sequences from Drosophila spp., as well as sharing the same branch with PA28γ from S. japonicum. Gene expression profiling of SmPA28γ using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28γ antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28γ from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28γ transcript and protein in these stages may be correlated with an important role of the PA28γ in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28γ double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28γ dsRNA treatment resulted in gene and protein knockdown of ~60% and ~80%, respectively, which were correlated with a significant decrease in larval length

  6. 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.

  7. Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system

    PubMed Central

    Xu, Daichao; Shan, Bing; Lee, Byung-Hoon; Zhu, Kezhou; Zhang, Tao; Sun, Huawang; Liu, Min; Shi, Linyu; Liang, Wei; Qian, Lihui; Xiao, Juan; Wang, Lili; Pan, Lifeng; Finley, Daniel; Yuan, Junying

    2015-01-01

    Regulation of ubiquitin-proteasome system (UPS), which controls the turnover of short-lived proteins in eukaryotic cells, is critical in maintaining cellular proteostasis. Here we show that USP14, a major deubiquitinating enzyme that regulates the UPS, is a substrate of Akt, a serine/threonine-specific protein kinase critical in mediating intracellular signaling transducer for growth factors. We report that Akt-mediated phosphorylation of USP14 at Ser432, which normally blocks its catalytic site in the inactive conformation, activates its deubiquitinating activity in vitro and in cells. We also demonstrate that phosphorylation of USP14 is critical for Akt to regulate proteasome activity and consequently global protein degradation. Since Akt can be activated by a wide range of growth factors and is under negative control by phosphoinosotide phosphatase PTEN, we suggest that regulation of UPS by Akt-mediated phosphorylation of USP14 may provide a common mechanism for growth factors to control global proteostasis and for promoting tumorigenesis in PTEN-negative cancer cells. DOI: http://dx.doi.org/10.7554/eLife.10510.001 PMID:26523394

  8. 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

  9. Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

    NASA Technical Reports Server (NTRS)

    Baracos, V. E.; DeVivo, C.; Hoyle, D. H.; Goldberg, A. L.

    1995-01-01

    Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

  10. The role of hypercholesterolemic diet and vitamin E on Nrf2 pathway, endoplasmic reticulum stress and proteasome activity.

    PubMed

    Bozaykut, Perinur; Sozen, Erdi; Yazgan, Burak; Karademir, Betul; Kartal-Ozer, Nesrin

    2014-10-01

    Hypercholesterolemia is the major risk factor for the development of atherosclerosis and vitamin E is suggested to have a preventive role in this process (1), although the mechanism of action still remains unclear.The ubiquitin-proteasome system (UPS) may in?uence atherosclerosis by affecting disease-relevant cellular processes such as apoptosis, proliferation, and differentiation, or by affecting cellular stress responses and/or adaptive phenomena, such as ER stress, in?ammation, and redox homeostasis (2). NF-E2-related factor 2 (Nrf2) is a transcription factor that controls the expression of phase II detoxi?cation and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits (3). In the present study, we investigated the role of Nrf2 pathway on oxidative and ER stress conditions induced by cholesterol diet and the effects of vitamin E on related signaling pathways in in vivo model of atherosclerosis. All experimental procedures were approved by the Marmara University Ethics Committee. Twenty-one male albino rabbits (23 months old) were assigned randomly to four groups fed for 8 weeks: (i) vitamin E deficient diet, (ii) vitamin E deficient diet containing 2% cholesterol, and (iii) vitamin E deficient diet containing 2% cholesterol with daily intramuscular injections of vitamin E (50mg/kg), (iv) vitamin E deficient diet with daily intramuscular injections of vitamin E (50mg/kg). In order to elucidate in vivo role of oxidative stress and ER stress in cardiovascular system of hypercholesterolemic rabbits, we investigated serum levels of cholesterol, MDA and vitamin E and Nrf2, GST-1, GRP78, GRP94, PERK, IRE1 protein levels and the proteasomal activity in aortic tissues will be discussed. PMID:26461313

  11. Chromium-Insulin Reduces Insulin Clearance and Enhances Insulin Signaling by Suppressing Hepatic Insulin-Degrading Enzyme and Proteasome Protein Expression in KKAy Mice.

    PubMed

    Wang, Zhong Q; Yu, Yongmei; Zhang, Xian H; Komorowski, James

    2014-01-01

    JDS-chromium-insulin (CRI)-003 is a novel form of insulin that has been directly conjugated with chromium (Cr) instead of zinc. Our hypothesis was that CRI enhances insulin's effects by altering insulin-degrading enzyme (IDE) and proteasome enzymes. To test this hypothesis, we measured hepatic IDE content and proteasome parameters in a diabetic animal model. Male KKAy mice were randomly divided into three groups (n = 8/group); Sham (saline), human regular insulin (Reg-In), and chromium conjugated human insulin (CRI), respectively. Interventions were initiated at doses of 2 U insulin/kg body weight daily for 8-weeks. Plasma glucose and insulin were measured. Hepatic IDE, proteasome, and insulin signaling proteins were determined by western blotting. Insulin tolerance tests at week 7 showed that both insulin treatments significantly reduced glucose concentrations and increased insulin levels compared with the Sham group, CRI significantly reduced glucose at 4 and 6 h relative to Reg-In (P < 0.05), suggesting the effects of CRI on reducing glucose last longer than Reg-In. CRI treatment significantly increased hepatic IRS-1 and Akt1 and reduced IDE, 20S as well as 19S protein abundance (P < 0.01, P < 0.05, and P < 0.001, respectively), but Reg-In only significantly increased Akt1 (P < 0.05). Similar results were also observed in Reg-In- and CRI-treated HepG2 cells. This study, for the first time, demonstrates that CRI reduces plasma insulin clearance by inhibition of hepatic IDE protein expression and enhances insulin signaling as well as prevents degradation of IRS-1 and IRS-2 by suppressing ubiquitin-proteasome pathway in diabetic mice.

  12. [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

  13. MAP17 (PDZKIP1) Expression Determines Sensitivity to the Proteasomal Inhibitor Bortezomib by Preventing Cytoprotective Autophagy and NFκB Activation in Breast Cancer.

    PubMed

    Muñoz-Galván, Sandra; Gutierrez, Gabriel; Perez, Marco; Carnero, Amancio

    2015-06-01

    MAP17 is a small nonglycosylated membrane protein that is overexpressed in a high percentage of carcinomas. High levels of MAP17 enhance the tumorigenic properties of tumor cells by increasing oxidative stress, which is dependent on Na(+)-coupled cotransport. Here, we show that MAP17 is associated with proteins involved in protein degradation and that proteasome inhibition induces autophagy. To analyze whether MAP17 could also alter this process, we used the proteasome inhibitor bortezomib (Velcade, PS-341), which is approved for the treatment of multiple myeloma and mantle cell lymphoma, although it has a high rate of resistance emergence and poor efficacy in solid tumors. We provide evidence that bortezomib induces a cytoprotective effect by activating autophagy and NFκB nuclear translocation, responses that are repressed in the presence of high levels of MAP17 both in vitro and in vivo. Indeed, patients with multiple myeloma treated with bortezomib showed higher response rates and a longer time to progression associated with increased levels of MAP17 expression. The MAP17-induced sensitivity to bortezomib is dependent on the oxidative status of the cells and the activity of Na(+)-coupled transporters because treatment with antioxidants or the inhibitor furosemide restores the cytoprotective activity induced by bortezomib. Therefore, bortezomib induces a prosurvival response through cytoprotective autophagy and NFκB nuclear translocation, which is repressed by high levels of MAP17. We propose that the levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematologic malignancies and in other tissues that are not commonly responsive to the drug.

  14. Proteasomal degradation of retinoid X receptor α reprograms transcriptional activity of PPARγ in obese mice and humans

    PubMed Central

    Lefebvre, Bruno; Benomar, Yacir; Guédin, Aurore; Langlois, Audrey; Hennuyer, Nathalie; Dumont, Julie; Bouchaert, Emmanuel; Dacquet, Catherine; Pénicaud, Luc; Casteilla, Louis; Pattou, Francois; Ktorza, Alain; Staels, Bart; Lefebvre, Philippe

    2010-01-01

    Obese patients have chronic, low-grade inflammation that predisposes to type 2 diabetes and results, in part, from dysregulated visceral white adipose tissue (WAT) functions. The specific signaling pathways underlying WAT dysregulation, however, remain unclear. Here we report that the PPARγ signaling pathway operates differently in the visceral WAT of lean and obese mice. PPARγ in visceral, but not subcutaneous, WAT from obese mice displayed increased sensitivity to activation by its agonist rosiglitazone. This increased sensitivity correlated with increased expression of the gene encoding the ubiquitin hydrolase/ligase ubiquitin carboxyterminal esterase L1 (UCH-L1) and with increased degradation of the PPARγ heterodimerization partner retinoid X receptor α (RXRα), but not RXRβ, in visceral WAT from obese humans and mice. Interestingly, increased UCH-L1 expression and RXRα proteasomal degradation was induced in vitro by conditions mimicking hypoxia, a condition that occurs in obese visceral WAT. Finally, PPARγ-RXRβ heterodimers, but not PPARγ-RXRα complexes, were able to efficiently dismiss the transcriptional corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) upon agonist binding. Increasing the RXRα/RXRβ ratio resulted in increased PPARγ responsiveness following agonist stimulation. Thus, the selective proteasomal degradation of RXRα initiated by UCH-L1 upregulation modulates the relative affinity of PPARγ heterodimers for SMRT and their responsiveness to PPARγ agonists, ultimately activating the PPARγ-controlled gene network in visceral WAT of obese animals and humans. PMID:20364085

  15. 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

  16. 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.

  17. 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.

  18. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    PubMed

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms.

  19. 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

  20. Loss of a 20S Proteasome Activator in Saccharomyces cerevisiae Downregulates Genes Important for Genomic Integrity, Increases DNA Damage, and Selectively Sensitizes Cells to Agents With Diverse Mechanisms of Action

    PubMed Central

    Doherty, Kevin M.; Pride, Leah D.; Lukose, James; Snydsman, Brian E.; Charles, Ronald; Pramanik, Ajay; Muller, Eric G.; Botstein, David; Moore, Carol Wood

    2012-01-01

    Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae Blm10 and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking Blm10 and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. Blm10 loss or truncation of the Ubp3/Blm3 deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that Blm10 and Ubp3/Blm3 function to stabilize the genome and protect against cell death. Diploids lacking Blm10 also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged Blm10 localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of Blm10 did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated Blm10 can interact with the proteasome apart from this region. Without its carboxyl-terminus, Blm10(−339aa) localized to nuclei in untreated, nonproliferating (G0) cells, but not during G1 S, G2, and M. The results indicate Blm10 functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting Blm10/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious. PMID:22908043

  1. Novel green synthesis of gold nanoparticles using Citrullus lanatus rind and investigation of proteasome inhibitory activity, antibacterial, and antioxidant potential.

    PubMed

    Patra, Jayanta Kumar; Baek, Kwang-Hyun

    2015-01-01

    Biological synthesis of nanoparticles using nontoxic, eco-friendly approaches is gaining importance owing to their fascinating biocompatibility and environmentally benign nature. This study describes the green synthesis approach for synthesis of gold nanoparticles (ANPs) using aqueous extract of the rind of watermelon as a fruit waste and evaluate its biopotential in terms of proteasome inhibitory activity, antibacterial, and antioxidant potential. The synthesized ANPs were characterized using UV-vis spectroscopy, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The surface plasmon resonance spectra of ANPs were obtained at 560 nm. Scanning electron microscopy image revealed that particles had a spherical shape and have a size distribution of 20-140 nm, followed by the elemental analysis by energy-dispersive X-ray spectroscopy. X-ray diffraction analysis confirmed the crystallite nature of the ANPs and Fourier-transform infrared spectroscopy revealed the involvement of bioactive compounds from watermelon rind in the synthesis, capping, and stabilization of ANPs. ANPs exhibited potential antibacterial activity against five different foodborne pathogenic bacteria with diameter of inhibition zones ranged between 9.23 and 11.58 mm. They also displayed strong synergistic antibacterial activity together with kanamycin (11.93-21.08 mm inhibition zones) and rifampicin (10.32-24.84 mm inhibition zones). ANPs displayed strong antioxidant activity in terms of DPPH radical scavenging (24.69%), nitric oxide scavenging (25.62%), ABTS scavenging (29.42%), and reducing power. Significantly high proteasome inhibitory potential of the ANPs (28.16%) could be highly useful for cancer treatment and targeted cancer drug delivery. Overall, results highlight a potential low-cost green method of synthesizing ANPs from food waste materials. Significant biopotentials of synthesized ANPs could make it a potential

  2. 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

  3. Novel green synthesis of gold nanoparticles using Citrullus lanatus rind and investigation of proteasome inhibitory activity, antibacterial, and antioxidant potential.

    PubMed

    Patra, Jayanta Kumar; Baek, Kwang-Hyun

    2015-01-01

    Biological synthesis of nanoparticles using nontoxic, eco-friendly approaches is gaining importance owing to their fascinating biocompatibility and environmentally benign nature. This study describes the green synthesis approach for synthesis of gold nanoparticles (ANPs) using aqueous extract of the rind of watermelon as a fruit waste and evaluate its biopotential in terms of proteasome inhibitory activity, antibacterial, and antioxidant potential. The synthesized ANPs were characterized using UV-vis spectroscopy, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The surface plasmon resonance spectra of ANPs were obtained at 560 nm. Scanning electron microscopy image revealed that particles had a spherical shape and have a size distribution of 20-140 nm, followed by the elemental analysis by energy-dispersive X-ray spectroscopy. X-ray diffraction analysis confirmed the crystallite nature of the ANPs and Fourier-transform infrared spectroscopy revealed the involvement of bioactive compounds from watermelon rind in the synthesis, capping, and stabilization of ANPs. ANPs exhibited potential antibacterial activity against five different foodborne pathogenic bacteria with diameter of inhibition zones ranged between 9.23 and 11.58 mm. They also displayed strong synergistic antibacterial activity together with kanamycin (11.93-21.08 mm inhibition zones) and rifampicin (10.32-24.84 mm inhibition zones). ANPs displayed strong antioxidant activity in terms of DPPH radical scavenging (24.69%), nitric oxide scavenging (25.62%), ABTS scavenging (29.42%), and reducing power. Significantly high proteasome inhibitory potential of the ANPs (28.16%) could be highly useful for cancer treatment and targeted cancer drug delivery. Overall, results highlight a potential low-cost green method of synthesizing ANPs from food waste materials. Significant biopotentials of synthesized ANPs could make it a potential

  4. Novel green synthesis of gold nanoparticles using Citrullus lanatus rind and investigation of proteasome inhibitory activity, antibacterial, and antioxidant potential

    PubMed Central

    Patra, Jayanta Kumar; Baek, Kwang-Hyun

    2015-01-01

    Biological synthesis of nanoparticles using nontoxic, eco-friendly approaches is gaining importance owing to their fascinating biocompatibility and environmentally benign nature. This study describes the green synthesis approach for synthesis of gold nanoparticles (ANPs) using aqueous extract of the rind of watermelon as a fruit waste and evaluate its biopotential in terms of proteasome inhibitory activity, antibacterial, and antioxidant potential. The synthesized ANPs were characterized using UV–vis spectroscopy, scanning electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The surface plasmon resonance spectra of ANPs were obtained at 560 nm. Scanning electron microscopy image revealed that particles had a spherical shape and have a size distribution of 20–140 nm, followed by the elemental analysis by energy-dispersive X-ray spectroscopy. X-ray diffraction analysis confirmed the crystallite nature of the ANPs and Fourier-transform infrared spectroscopy revealed the involvement of bioactive compounds from watermelon rind in the synthesis, capping, and stabilization of ANPs. ANPs exhibited potential antibacterial activity against five different foodborne pathogenic bacteria with diameter of inhibition zones ranged between 9.23 and 11.58 mm. They also displayed strong synergistic antibacterial activity together with kanamycin (11.93–21.08 mm inhibition zones) and rifampicin (10.32–24.84 mm inhibition zones). ANPs displayed strong antioxidant activity in terms of DPPH radical scavenging (24.69%), nitric oxide scavenging (25.62%), ABTS scavenging (29.42%), and reducing power. Significantly high proteasome inhibitory potential of the ANPs (28.16%) could be highly useful for cancer treatment and targeted cancer drug delivery. Overall, results highlight a potential low-cost green method of synthesizing ANPs from food waste materials. Significant biopotentials of synthesized ANPs could make it a potential

  5. 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

  6. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    SciTech Connect

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  7. Changes in autophagy, proteasome activity and metabolism to determine a specific signature for acute and chronic senescent mesenchymal stromal cells

    PubMed Central

    Capasso, Stefania; Alessio, Nicola; Squillaro, Tiziana; Di Bernardo, Giovanni; Melone, Mariarosa A.; Cipollaro, Marilena; Peluso, Gianfranco; Galderisi, Umberto

    2015-01-01

    A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions. We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes. PMID:26540573

  8. Changes in autophagy, proteasome activity and metabolism to determine a specific signature for acute and chronic senescent mesenchymal stromal cells.

    PubMed

    Capasso, Stefania; Alessio, Nicola; Squillaro, Tiziana; Di Bernardo, Giovanni; Melone, Mariarosa A; Cipollaro, Marilena; Peluso, Gianfranco; Galderisi, Umberto

    2015-11-24

    A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions. We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes. PMID:26540573

  9. Profound Activity of the Anti-cancer Drug Bortezomib against Echinococcus multilocularis Metacestodes Identifies the Proteasome as a Novel Drug Target for Cestodes

    PubMed Central

    Stadelmann, Britta; Aeschbacher, Denise; Huber, Cristina; Spiliotis, Markus; Müller, Joachim; Hemphill, Andrew

    2014-01-01

    A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors. PMID:25474446

  10. A binuclear complex constituted by diethyldithiocarbamate and copper(I) functions as a proteasome activity inhibitor in pancreatic cancer cultures and xenografts

    SciTech Connect

    Han, Jinbin; 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. - Highlights: • A new structure of DDTC–Cu(I) was reported for the first time. • DDTC–Cu(I) dissolved directly in water was for in vitro and in vivo uses. • DDTC–Cu(I) demonstrated significant anticancer effect in vitro and in vivo. • DDTC–Cu(I) is capable of inhibiting proteasome activity in vitro and in vivo.

  11. Decreased proteasomal function accelerates cigarette smoke-induced pulmonary emphysema in mice.

    PubMed

    Yamada, Yosuke; Tomaru, Utano; Ishizu, Akihiro; Ito, Tomoki; Kiuchi, Takayuki; Ono, Ayako; Miyajima, Syota; Nagai, Katsura; Higashi, Tsunehito; Matsuno, Yoshihiro; Dosaka-Akita, Hirotoshi; Nishimura, Masaharu; Miwa, Soichi; Kasahara, Masanori

    2015-06-01

    Chronic obstructive pulmonary disease (COPD) is a disease common in elderly people, characterized by progressive destruction of lung parenchyma and chronic inflammation of the airways. The pathogenesis of COPD remains unclear, but recent studies suggest that oxidative stress-induced apoptosis in alveolar cells contributes to emphysematous lung destruction. The proteasome is a multicatalytic enzyme complex that plays a critical role in proteostasis by rapidly destroying misfolded and modified proteins generated by oxidative and other stresses. Proteasome activity decreases with aging in many organs including lungs, and an age-related decline in proteasomal function has been implicated in various age-related pathologies. However, the role of the proteasome system in the pathogenesis of COPD has not been investigated. Recently, we have established a transgenic (Tg) mouse model with decreased proteasomal chymotrypsin-like activity, showing age-related phenotypes. Using this model, we demonstrate here that decreased proteasomal function accelerates cigarette smoke (CS)-induced pulmonary emphysema. CS-exposed Tg mice showed remarkable airspace enlargement and increased foci of inflammation compared with wild-type controls. Importantly, apoptotic cells were found in the alveolar walls of the affected lungs. Impaired proteasomal activity also enhanced apoptosis in cigarette smoke extract (CSE)-exposed fibroblastic cells derived from mice and humans in vitro. Notably, aggresome formation and prominent nuclear translocation of apoptosis-inducing factor were observed in CSE-exposed fibroblastic cells isolated from Tg mice. Collective evidence suggests that CS exposure and impaired proteasomal activity coordinately enhance apoptotic cell death in the alveolar walls that may be involved in the development and progression of emphysema in susceptible individuals such as the elderly.

  12. 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

  13. Multiple Interactions between Peroxisome Proliferators-Activated Receptors and the Ubiquitin-Proteasome System and Implications for Cancer Pathogenesis

    PubMed Central

    Genini, Davide; Carbone, Giuseppina M.; Catapano, Carlo V.

    2008-01-01

    The peroxisome proliferator-activated receptors (PPAR) α, β/δ, and γ are ligand-activated nuclear receptors involved in a number of physiological processes, including lipid and glucose homeostasis, inflammation, cell growth, differentiation, and death. PPAR agonists are used in the treatment of human diseases, like type 2 diabetes and dyslipidemia, and PPARs appear as promising therapeutic targets in other conditions, including cancer. A better understanding of the functions and regulation of PPARs in normal and pathological processes is of primary importance to devise appropriate therapeutic strategies. The ubiquitin-proteasome system (UPS) plays an important role in controlling level and activity of many nuclear receptors and transcription factors. PPARs are subjected to UPS-dependent regulation. Interestingly, the three PPAR isotypes are differentially regulated by the UPS in response to ligand-dependent activation, a phenomenon that may be intrinsically connected to their distinct cellular functions and behaviors. In addition to their effects ongene expression, PPARs appear to affect protein levels and downstream pathways also by modulating the activity of the UPS in target-specific manners. Here we review the current knowledge of the interactions between the UPS and PPARs in light of the potential implications for their effects on cell fate and tumorigenesis. PMID:18551186

  14. Tea enhances insulin activity.

    PubMed

    Anderson, Richard A; Polansky, Marilyn M

    2002-11-20

    The most widely known health benefits of tea relate to the polyphenols as the principal active ingredients in protection against oxidative damage and in antibacterial, antiviral, anticarcinogenic, and antimutagenic activities, but polyphenols in tea may also increase insulin activity. The objective of this study was to determine the insulin-enhancing properties of tea and its components. Tea, as normally consumed, was shown to increase insulin activity >15-fold in vitro in an epididymal fat cell assay. Black, green, and oolong teas but not herbal teas, which are not teas in the traditional sense because they do not contain leaves of Camellia senensis, were all shown to increase insulin activity. High-performance liquid chromatography fractionation of tea extracts utilizing a Waters SymmetryPrep C18 column showed that the majority of the insulin-potentiating activity for green and oolong teas was due to epigallocatechin gallate. For black tea, the activity was present in several regions of the chromatogram corresponding to, in addition to epigallocatechin gallate, tannins, theaflavins, and other undefined compounds. Several known compounds found in tea were shown to enhance insulin with the greatest activity due to epigallocatechin gallate followed by epicatechin gallate, tannins, and theaflavins. Caffeine, catechin, and epicatechin displayed insignificant insulin-enhancing activities. Addition of lemon to the tea did not affect the insulin-potentiating activity. Addition of 5 g of 2% milk per cup decreased the insulin-potentiating activity one-third, and addition of 50 g of milk per cup decreased the insulin-potentiating activity approximately 90%. Nondairy creamers and soy milk also decreased the insulin-enhancing activity. These data demonstrate that tea contains in vitro insulin-enhancing activity and the predominant active ingredient is epigallocatechin gallate. PMID:12428980

  15. HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans

    PubMed Central

    Kinet, Maxime J; Malin, Jennifer A; Abraham, Mary C; Blum, Elyse S; Silverman, Melanie R; Lu, Yun; Shaham, Shai

    2016-01-01

    Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.12821.001 PMID:26952214

  16. 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

  17. 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

  18. The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways.

    PubMed

    Tisoncik, Jennifer R; Billharz, Rosalind; Burmakina, Svetlana; Belisle, Sarah E; Proll, Sean C; Korth, Marcus J; García-Sastre, Adolfo; Garcíia-Sastre, Adolfo; Katze, Michael G

    2011-09-01

    The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-κB signalling-pathway genes and IFN-α and -β target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein.

  19. The NS1 protein of influenza A virus suppresses interferon-regulated activation of antigen-presentation and immune-proteasome pathways

    PubMed Central

    Tisoncik, Jennifer R.; Billharz, Rosalind; Burmakina, Svetlana; Belisle, Sarah E.; Proll, Sean C.; Korth, Marcus J.; García-Sastre, Adolfo

    2011-01-01

    The NS1 protein of influenza virus counters host antiviral defences primarily by antagonizing the type I interferon (IFN) response. Both the N-terminal dsRNA-binding domain and the C-terminal effector domain are required for optimal suppression of host responses during infection. To better understand the regulatory role of the NS1 effector domain, we used an NS1-truncated mutant virus derived from human H1N1 influenza isolate A/Texas/36/91 (Tx/91) and assessed global transcriptional profiles from two independent human lung cell-culture models. Relative to the wild-type Tx/91-induced gene expression, the NS1 mutant virus induced enhanced expression of innate immune genes, specifically NF-κB signalling-pathway genes and IFN-α and -β target genes. We queried an experimentally derived IFN gene set to gauge the proportion of IFN-responsive genes that are suppressed specifically by NS1. We show that the C-terminally truncated NS1 mutant virus is less efficient at suppressing IFN-regulated gene expression associated with activation of antigen-presentation and immune-proteasome pathways. This is the first report integrating genomic analysis from two independent human culture systems, including primary lung cells, using genetically similar H1N1 influenza viruses that differ only in the length of the NS1 protein. PMID:21593271

  20. The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma.

    PubMed

    Li, Bingzong; Fu, Jinxiang; Chen, Ping; Ge, Xueping; Li, Yali; Kuiatse, Isere; Wang, Hua; Wang, Huihan; Zhang, Xingding; Orlowski, Robert Z

    2015-12-11

    Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor.

  1. Vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector VH1 from lateral bodies.

    PubMed

    Schmidt, Florian Ingo; Bleck, Christopher Karl Ernst; Reh, Lucia; Novy, Karel; Wollscheid, Bernd; Helenius, Ari; Stahlberg, Henning; Mercer, Jason

    2013-08-15

    Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-γ-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors.

  2. 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

  3. PARK2/Parkin-mediated mitochondrial clearance contributes to proteasome activation during slow-twitch muscle atrophy via NFE2L1 nuclear translocation

    PubMed Central

    Furuya, Norihiko; Ikeda, Shin-Ichi; Sato, Shigeto; Soma, Sanae; Ezaki, Junji; Trejo, Juan Alejandro Oliva; Takeda-Ezaki, Mitsue; Fujimura, Tsutomu; Arikawa-Hirasawa, Eri; Tada, Norihiro; Komatsu, Masaaki; Tanaka, Keiji; Kominami, Eiki; Hattori, Nobutaka; Ueno, Takashi

    2014-01-01

    Skeletal muscle atrophy is thought to result from hyperactivation of intracellular protein degradation pathways, including autophagy and the ubiquitin–proteasome system. However, the precise contributions of these pathways to muscle atrophy are unclear. Here, we show that an autophagy deficiency in denervated slow-twitch soleus muscles delayed skeletal muscle atrophy, reduced mitochondrial activity, and induced oxidative stress and accumulation of PARK2/Parkin, which participates in mitochondrial quality control (PARK2-mediated mitophagy), in mitochondria. Soleus muscles from denervated Park2 knockout mice also showed resistance to denervation, reduced mitochondrial activities, and increased oxidative stress. In both autophagy-deficient and Park2-deficient soleus muscles, denervation caused the accumulation of polyubiquitinated proteins. Denervation induced proteasomal activation via NFE2L1 nuclear translocation in control mice, whereas it had little effect in autophagy-deficient and Park2-deficient mice. These results suggest that PARK2-mediated mitophagy plays an essential role in the activation of proteasomes during denervation atrophy in slow-twitch muscles. PMID:24451648

  4. Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways

    PubMed Central

    Doyle, Alexander; Zhang, Guohua; Abdel Fattah, Elmoataz A.; Eissa, N. Tony; Li, Yi-Ping

    2011-01-01

    Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.—Doyle, A., Zhang, G., Abdel Fattah, E. A., Eissa, N. T., Li, Y.-P. Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways. PMID:20826541

  5. 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

  6. 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.

  7. 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.

  8. Identification of noncovalent proteasome inhibitors with high selectivity for chymotrypsin-like activity by a multistep structure-based virtual screening.

    PubMed

    Di Giovanni, Carmen; Ettari, Roberta; Sarno, Serena; Rotondo, Archimede; Bitto, Alessandra; Squadrito, Francesco; Altavilla, Domenica; Schirmeister, Tanja; Novellino, Ettore; Grasso, Silvana; Zappalà, Maria; Lavecchia, Antonio

    2016-10-01

    Noncovalent proteasome inhibitors introduce an alternative mechanism of inhibition to that of covalent inhibitors, e.g. carfilzomib, used in cancer therapy. A multistep hierarchical structure-based virtual screening (SBVS) of the 65,375 NCI lead-like compound library led to the identification of two compounds (9 and 28) which noncovalently inhibited the chymotrypsin-like (ChT-L) activity (Ki = 2.18 and 2.12 μM, respectively) with little or no effects on the other two major proteasome proteolytic activities, trypsin-like (T-L) and post-glutamyl peptide hydrolase (PGPH) activities. A subsequent hierarchical similarity search over the full NCI database with the most active tripeptide-based inhibitor 9 resulted in the discovery of the β5/β6-specific tripeptide derivative 38 that noncovalently binds the ChT-L site (Ki = 0.42 μM). The solution structure of 9 and 38 was solved by (1)H NMR spectroscopy and the binding mode of the inhibitors was elucidated by docking experiments using the yeast 20S proteasome. Compound 38 (IC50 = 26.7 μM) is slightly more potent than 9 (IC50 = 34.3 μM) at inhibiting survival of dexamethasone-resistant (MM.1R) human multiple myeloma cells. The identified ligand thus provides valuable insights for the future structure-based design of subtype-specific proteasome inhibitors. PMID:27318981

  9. Wnt pathway activation and ABCB1 expression account for attenuation of Proteasome inhibitor-mediated apoptosis in multidrug-resistant cancer cells

    PubMed Central

    Chong, Kowit Yu; Hsu, Chih-Jung; Hung, Tsai-Hsien; Hu, Han-Shu; Huang, Tsung-Teng; Wang, Tzu-Hao; Wang, Chihuei; Chen, Chuan-Mu; Choo, Kong Bung; Tseng, Ching-Ping

    2015-01-01

    Multiple drug resistance (MDR) is a major obstacle to attenuating the effectiveness of chemotherapy to many human malignancies. Proteasome inhibition induces apoptosis in a variety of cancer cells and is recognized as a novel anticancer therapy approach. Despite its success, some multiple myeloma patients are resistant or become refractory to ongoing treatment by bortezomib suggesting that chemoresistant cancer cells may have developed a novel mechanism directed against the proteasome inhibitor. The present study aimed to investigate potential mechanism(s) of attenuation in a MDR cell line, MES-SA/Dx5. We found that compared to the parental human uterus sarcoma cell line MES-SA cells, MES-SA/Dx5 cells highly expressed the ABCB1 was more resistant to MG132 and bortezomib, escaping the proteasome inhibitor-induced apoptosis pathway. The resistance was reversed by co-treatment of MG132 and the ABCB1 inhibitor verapamil. The data indicated that ABCB1 might play a role in the efflux of MG132 from the MES-SA/Dx5 cells to reduce MG132-induced apoptosis. Furthermore, the canonical Wnt pathway was found activated only in the MES-SA/Dx5 cells through active β-catenin and related transactivation activities. Western blot analysis demonstrated that Wnt-targeting genes, including c-Myc and cyclin D1, were upregulated and were relevant in inhibiting the expression of p21 in MES-SA/Dx5 cells. On the other hand, MES-SA cells expressed high levels of p21 and downregulated cyclin D1 and caused cell cycle arrest. Together, our study demonstrated the existence and participation of ABCB1 and the Wnt pathway in an MDR cell line that attenuated proteasome inhibitor-induced apoptosis. PMID:25590413

  10. 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

  11. Nuclear Protein Quality Is Regulated by the Ubiquitin-Proteasome System through the Activity of Ubc4 and San1 in Fission Yeast*

    PubMed Central

    Matsuo, Yuzy; Kishimoto, Hayafumi; Tanae, Katsuhiro; Kitamura, Kenji; Katayama, Satoshi; Kawamukai, Makoto

    2011-01-01

    Eukaryotic cells monitor and maintain protein quality through a set of protein quality control (PQC) systems whose role is to minimize the harmful effects of the accumulation of aberrant proteins. Although these PQC systems have been extensively studied in the cytoplasm, nuclear PQC systems are not well understood. The present work shows the existence of a nuclear PQC system mediated by the ubiquitin-proteasome system in the fission yeast Schizosaccharomyces pombe. Asf1-30, a mutant form of the histone chaperone Asf1, was used as a model substrate for the study of the nuclear PQC. A temperature-sensitive Asf1-30 protein localized to the nucleus was selectively degraded by the ubiquitin-proteasome system. The Asf1-30 mutant protein was highly ubiquitinated at higher temperatures, and it remained stable in an mts2-1 mutant, which lacks proteasome activity. The E2 enzyme Ubc4 was identified among 11 candidate proteins as the ubiquitin-conjugating enzyme in this system, and San1 was selected among 100 candidates as the ubiquitin ligase (E3) targeting Asf1-30 for degradation. San1, but not other nuclear E3s, showed specificity for the mutant nuclear Asf1-30, but did not show activity against wild-type Asf1. These data clearly showed that the aberrant nuclear protein was degraded by a defined set of E1-E2-E3 enzymes through the ubiquitin-proteasome system. The data also show, for the first time, the presence of a nuclear PQC system in fission yeast. PMID:21324894

  12. Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications

    PubMed Central

    Huang, Qian; Figueiredo-Pereira, Maria E.

    2010-01-01

    The ubiquitin/proteasome pathway is the major proteolytic quality control system in cells. In this review we discuss the impact of a deregulation of this pathway on neuronal function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in pathological inclusion bodies in all the major chronic neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as amyotrophic lateral sclerosis. We describe the intricate nature of the ubiquitin/proteasome pathway and discuss the paradox of protein aggregation, i.e. its potential toxic/protective effect in neurodegeneration. The relations between some of the dysfunctional components of the pathway and neurodegeneration are presented. We highlight possible ubiquitin/proteasome pathway-targeting therapeutic approaches, such as activating the proteasome, enhancing ubiquitination and promoting SUMOylation that might be important to slow/treat the progression of neurodegeneration. Finally, a model time line is presented for neurodegeneration starting at the initial injurious events up to protein aggregation and cell death, with potential time points for therapeutic intervention. PMID:20131003

  13. 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

  14. 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

  15. 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

  16. 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

  17. Proteasome-mediated turnover of the transcriptional activator FIT is required for plant iron-deficiency responses.

    PubMed

    Sivitz, Alicia; Grinvalds, Claudia; Barberon, Marie; Curie, Catherine; Vert, Grégory

    2011-06-01

    Plants display a number of responses to low iron availability in order to increase iron uptake from the soil. In the model plant Arabidopsis thaliana, the ferric-chelate reductase FRO2 and the ferrous iron transporter IRT1 control iron entry from the soil into the root epidermis. To maintain iron homeostasis, the expression of FRO2 and IRT1 is tightly controlled by iron deficiency at the transcriptional level. The basic helix-loop-helix (bHLH) transcription factor FIT represents the most upstream actor known in the iron-deficiency signaling pathway, and directly regulates the expression of the root iron uptake machinery genes FRO2 and IRT1. However, how FIT is controlled by iron and acts to activate transcription of its targets remains obscure. Here we show that FIT mRNA and endogenous FIT protein accumulate in Arabidopsis roots upon iron deficiency. However, using plants constitutively expressing FIT, we observed that FIT protein accumulation is reduced in iron-limited conditions. This post-transcriptional regulation of FIT is perfectly synchronized with the accumulation of endogenous FIT and IRT1 proteins, and therefore is part of the early responses to low iron. We demonstrated that such regulation affects FIT protein stability under iron deficiency as a result of 26S proteasome-dependent degradation. In addition, we showed that FIT post-translational regulation by iron is required for FRO2 and IRT1 gene expression. Taken together our results indicate that FIT transcriptional and post-translational regulations are integrated in plant roots to ensure that the positive regulator FIT accumulates as a short-lived protein following iron shortage, and to allow proper iron-deficiency responses.

  18. S-nitrosylation-dependent proteasomal degradation restrains Cdk5 activity to regulate hippocampal synaptic strength

    PubMed Central

    Zhang, Peng; Fu, Wing-Yu; Fu, Amy K. Y.; Ip, Nancy Y.

    2015-01-01

    Precise regulation of synaptic strength requires coordinated activity and functions of synaptic proteins, which is controlled by a variety of post-translational modification. Here we report that S-nitrosylation of p35, the activator of cyclin-dependent kinase 5 (Cdk5), by nitric oxide (NO) is important for the regulation of excitatory synaptic strength. While blockade of NO signalling results in structural and functional synaptic deficits as indicated by reduced mature dendritic spine density and surface expression of glutamate receptor subunits, phosphorylation of numerous synaptic substrates of Cdk5 and its activity are aberrantly upregulated following reduced NO production. The results show that the NO-induced reduction in Cdk5 activity is mediated by S-nitrosylation of p35, resulting in its ubiquitination and degradation by the E3 ligase PJA2. Silencing p35 protein in hippocampal neurons partially rescues the NO blockade-induced synaptic deficits. These findings collectively demonstrate that p35 S-nitrosylation by NO signalling is critical for regulating hippocampal synaptic strength. PMID:26503494

  19. 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

  20. 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

  1. 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

  2. Comparative study of proteasome inhibitory, synergistic antibacterial, synergistic anticandidal, and antioxidant activities of gold nanoparticles biosynthesized using fruit waste materials

    PubMed Central

    Patra, Jayanta Kumar; Baek, Kwang-Hyun

    2016-01-01

    The aim of this study was to compare the biological synthesis of gold nanoparticles (AuNPs) generated using the aqueous extracts of outer oriental melon peel (OMP) and peach. The synthesized OMP-AuNPs and peach extract (PE)-AuNPs were characterized by ultraviolet–visible spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, X-ray powder diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The surface plasmon resonance spectra were obtained at 545 nm and 540 nm for OMP-AuNPs and PE-AuNPs, respectively. The estimated absolute crystallite size of the synthesized AuNPs was calculated to be 78.11 nm for OMP-AuNPs and 39.90 nm for PE-AuNPs based on the Scherer equation of the X-ray powder diffraction peaks. Fourier transform infrared spectroscopy results revealed the involvement of bioactive compounds present in OMP and peach extracts in the synthesis and stabilization of synthesized AuNPs. Both the OMP-AuNPs and PE-AuNPs showed a strong antibacterial synergistic activity when combined with kanamycin (9.38–20.45 mm inhibition zones) and rifampicin (9.52–25.23 mm inhibition zones), and they also exerted a strong synergistic anticandidal activity (10.09–15.47 mm inhibition zones) when combined with amphotericin B against five pathogenic Candida species. Both the OMP-AuNPs and PE-AuNPs exhibited a strong antioxidant potential in terms of 1,1-diphenyl-2-picrylhydraxyl radical scavenging, nitric oxide scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging, and a reducing power, along with a strong proteasome inhibitory potential that could be useful in cancer drug delivery and cancer treatments. The PE-AuNPs showed comparatively higher activity than OMP-AuNPs, which could be attributed to the presence of rich bioactive compounds in the PE that acted as reducing and capping agents in the synthesis of PE-AuNPs. Overall, the results of the current investigation

  3. Comparative study of proteasome inhibitory, synergistic antibacterial, synergistic anticandidal, and antioxidant activities of gold nanoparticles biosynthesized using fruit waste materials

    PubMed Central

    Patra, Jayanta Kumar; Baek, Kwang-Hyun

    2016-01-01

    The aim of this study was to compare the biological synthesis of gold nanoparticles (AuNPs) generated using the aqueous extracts of outer oriental melon peel (OMP) and peach. The synthesized OMP-AuNPs and peach extract (PE)-AuNPs were characterized by ultraviolet–visible spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, X-ray powder diffraction, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The surface plasmon resonance spectra were obtained at 545 nm and 540 nm for OMP-AuNPs and PE-AuNPs, respectively. The estimated absolute crystallite size of the synthesized AuNPs was calculated to be 78.11 nm for OMP-AuNPs and 39.90 nm for PE-AuNPs based on the Scherer equation of the X-ray powder diffraction peaks. Fourier transform infrared spectroscopy results revealed the involvement of bioactive compounds present in OMP and peach extracts in the synthesis and stabilization of synthesized AuNPs. Both the OMP-AuNPs and PE-AuNPs showed a strong antibacterial synergistic activity when combined with kanamycin (9.38–20.45 mm inhibition zones) and rifampicin (9.52–25.23 mm inhibition zones), and they also exerted a strong synergistic anticandidal activity (10.09–15.47 mm inhibition zones) when combined with amphotericin B against five pathogenic Candida species. Both the OMP-AuNPs and PE-AuNPs exhibited a strong antioxidant potential in terms of 1,1-diphenyl-2-picrylhydraxyl radical scavenging, nitric oxide scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging, and a reducing power, along with a strong proteasome inhibitory potential that could be useful in cancer drug delivery and cancer treatments. The PE-AuNPs showed comparatively higher activity than OMP-AuNPs, which could be attributed to the presence of rich bioactive compounds in the PE that acted as reducing and capping agents in the synthesis of PE-AuNPs. Overall, the results of the current investigation

  4. 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.

  5. 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.

  6. Immunocytochemical and ultrastructural analyses of the function of the ubiquitin-proteasome system during spermiogenesis with the use of the inhibitors of proteasome proteolytic activity in the alga, Chara vulgaris.

    PubMed

    Wojtczak, Agnieszka; Kwiatkowska, Maria

    2008-04-01

    Spermiogenesis in Chara vulgaris and in animals share many common features, including exchange of nucleohistones into nucleoprotamines, remodeling and extreme condensation of chromatin, formation of flagellae and of microtubule manchette, and decrease in cytoplasm volume. In C. vulgaris, spermiogenesis is not preceded by meiosis since this alga is a haplobiont. In the present work we showed that in early spermiogenesis characterized by a significant metabolic activity of spermatids, the inhibitors of proteasomes did not visibly change their ultrastructure but significantly prolonged this process. At late stages of spermiogenesis, MG-132 and epoxomicin dramatically changed the structure of nuclei: regular fibrillar and lamellar structure of chromatin was disturbed and clusters of grains corresponding to aggresomes appeared, but the nucleus shape and cytoplasm structure were the same as in the controls. Immunocytochemical studies revealed that these inhibitors blocked disappearance of histones from nuclei while the structures corresponding to aggresomes were clusters of undegraded ubiquitinated histones, since they gave positive immunosignals indicating the presence of ubiquitin and histones.

  7. 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

  8. Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems

    SciTech Connect

    Fanzani, Alessandro Zanola, Alessandra; Rovetta, Francesca; Rossi, Stefania; Aleo, Maria Francesca

    2011-02-01

    Cisplatin (cisPt) is an antineoplastic drug which causes an array of adverse effects on different organs and tissues, including skeletal muscle. In this work we show that cisPt behaves as a potent trigger to activate protein hypercatabolism in skeletal C2C12 myotubes. Within 24 h of 50 {mu}M cisPt administration, C2C12 myotubes displayed unchanged cell viability but showed a subset of hallmark signs typically recognized during atrophy, including severe reduction in body size, repression of Akt phosphorylation, transcriptional up-regulation of atrophy-related genes, such as atrogin-1, gabarap, beclin-1 and bnip-3, and loss of myogenic markers. As a consequence, proteasomal activity and formation of autophagosomes were remarkably increased in cisPt-treated myotubes, but forced stimulation of Akt pathway, as obtained through insulin administration or delivery of a constitutively activated Akt form, was sufficient to counter the cisPt-induced protein breakdown, leading to rescue of atrophic size. Overall, these results indicate that cisPt induces atrophy of C2C12 myotubes via activation of proteasome and autophagy systems, suggesting that the Akt pathway represents one sensitive target of cisPt molecular action in skeletal muscle.

  9. Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin-proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet.

    PubMed

    Batistela, Emanuele; Pereira, Mayara Peron; Siqueira, Juliany Torres; Paula-Gomes, Silvia; Zanon, Neusa Maria; Oliveira, Eduardo Brandt; Navegantes, Luiz Carlos Carvalho; Kettelhut, Isis C; Andrade, Claudia Marlise Balbinotti; Kawashita, Nair Honda; Baviera, Amanda Martins

    2014-06-01

    The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin-proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  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. 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.

  17. 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

  18. Vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector VH1 from lateral bodies.

    PubMed

    Schmidt, Florian Ingo; Bleck, Christopher Karl Ernst; Reh, Lucia; Novy, Karel; Wollscheid, Bernd; Helenius, Ari; Stahlberg, Henning; Mercer, Jason

    2013-08-15

    Host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (LBs) into the cytosol. Uncoating of viral cores is poorly characterized, and the composition and function of LBs remains enigmatic. We found that cytosolic cores rapidly dissociated from LBs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. We identified the abundant phosphoprotein F17, the dual-specificity phosphatase VH1, and the oxidoreductase G4 as bona fide LB components. After reaching the cytosol, F17 was degraded in a proteasome-dependent manner. Proteasome activity, and presumably LB disassembly, was required for the immediate immunomodulatory activity of VH1: dephosphorylation of STAT1 to prevent interferon-γ-mediated antiviral responses. These results reveal a mechanism used by poxviruses to deliver viral enzymes to the host cell cytosol and are likely to facilitate the identification of additional LB-resident viral effectors. PMID:23891003

  19. Detection of O-propargyl-puromycin with SUMO and ubiquitin by click chemistry at PML-nuclear bodies during abortive proteasome activities.

    PubMed

    Uozumi, Naoki; Matsumoto, Hotaru; Saitoh, Hisato

    2016-05-27

    The amino-nucleoside antibiotic, puromycin, acts by covalently linking to elongating polypeptide chains on ribosomes to generate prematurely terminated immature polypeptides. The trafficking of puromycin-conjugated (puromycylated) immature polypeptides within cell has, however, remained elusive. In this study, using O-propargyl-puromycin (OP-Puro), the distribution of puromycylated polypeptides was assessed in HeLa cells by click chemistry. Under standard culture conditions, OP-Puro signals were detected in the cytoplasm and nucleus with the highest concentrations in the nucleolus. Intriguingly, when proteasome activities were aborted using MG132, OP-Puro signals began to accumulate at promyelocytic leukemia nuclear bodies (PML-NBs) in addition to the nucleolus. We also found promiscuous association of OP-Puro signals with SUMO-2/3 and ubiquitin at PML-NBs, but not at the nucleolus, during abortive proteasome activities. This study reveals a previously unknown distribution of OP-Puro that argues for a nuclear function in regulating immature protein homeostasis.

  20. 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

  1. 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

  2. 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.

  3. Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen Receptor α Turnover and Functional Activity via the SCFSkp2 Proteasomal Complex

    PubMed Central

    Bhatt, Shweta; Xiao, Zhen; Meng, Zhaojing

    2012-01-01

    The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCFSkp2 proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers. PMID:22431515

  4. 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.

  5. 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.

  6. High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis.

    PubMed

    Abrigo, Johanna; Rivera, Juan Carlos; Aravena, Javier; Cabrera, Daniel; Simon, Felipe; Ezquer, Fernando; Ezquer, Marcelo; Cabello-Verrugio, Claudio

    2016-01-01

    Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes. PMID:27579157

  7. High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis.

    PubMed

    Abrigo, Johanna; Rivera, Juan Carlos; Aravena, Javier; Cabrera, Daniel; Simon, Felipe; Ezquer, Fernando; Ezquer, Marcelo; Cabello-Verrugio, Claudio

    2016-01-01

    Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes.

  8. High Fat Diet-Induced Skeletal Muscle Wasting Is Decreased by Mesenchymal Stem Cells Administration: Implications on Oxidative Stress, Ubiquitin Proteasome Pathway Activation, and Myonuclear Apoptosis

    PubMed Central

    Aravena, Javier; Cabrera, Daniel; Simon, Felipe; Ezquer, Fernando

    2016-01-01

    Obesity can lead to skeletal muscle atrophy, a pathological condition characterized by the loss of strength and muscle mass. A feature of muscle atrophy is a decrease of myofibrillar proteins as a result of ubiquitin proteasome pathway overactivation, as evidenced by increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF-1. Additionally, other mechanisms are related to muscle wasting, including oxidative stress, myonuclear apoptosis, and autophagy. Stem cells are an emerging therapy in the treatment of chronic diseases such as high fat diet-induced obesity. Mesenchymal stem cells (MSCs) are a population of self-renewable and undifferentiated cells present in the bone marrow and other mesenchymal tissues of adult individuals. The present study is the first to analyze the effects of systemic MSC administration on high fat diet-induced skeletal muscle atrophy in the tibialis anterior of mice. Treatment with MSCs reduced losses of muscle strength and mass, decreases of fiber diameter and myosin heavy chain protein levels, and fiber type transitions. Underlying these antiatrophic effects, MSC administration also decreased ubiquitin proteasome pathway activation, oxidative stress, and myonuclear apoptosis. These results are the first to indicate that systemically administered MSCs could prevent muscle wasting associated with high fat diet-induced obesity and diabetes. PMID:27579157

  9. 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.

  10. 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

  11. 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

  12. Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin–Proteasome Pathway

    PubMed Central

    Mitula, Filip; Tajdel, Malgorzata; Cieśla, Agata; Kasprowicz-Maluśki, Anna; Kulik, Anna; Babula-Skowrońska, Danuta; Michalak, Michal; Dobrowolska, Grazyna; Sadowski, Jan; Ludwików, Agnieszka

    2015-01-01

    Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18. PMID:26443375

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. TGF-β activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

    PubMed

    Pavlides, Savvas C; Lecanda, Jon; Daubriac, Julien; Pandya, Unnati M; Gama, Patricia; Blank, Stephanie; Mittal, Khushbakhat; Shukla, Pratibha; Gold, Leslie I

    2016-01-01

    We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome.

    PubMed

    Ferreira, Joana S; Schmidt, Jeannette; Rio, Pedro; Águas, Rodolfo; Rooyakkers, Amanda; Li, Ka Wan; Smit, August B; Craig, Ann Marie; Carvalho, Ana Luisa

    2015-06-01

    NMDA receptors play a central role in shaping the strength of synaptic connections throughout development and in mediating synaptic plasticity mechanisms that underlie some forms of learning and memory formation in the CNS. In the hippocampus and the neocortex, GluN1 is combined primarily with GluN2A and GluN2B, which are differentially expressed during development and confer distinct molecular and physiological properties to NMDA receptors. The contribution of each subunit to the synaptic traffic of NMDA receptors and therefore to their role during development and in synaptic plasticity is still controversial. We report a critical role for the GluN2B subunit in regulating NMDA receptor synaptic targeting. In the absence of GluN2B, the synaptic levels of AMPA receptors are increased and accompanied by decreased constitutive endocytosis of GluA1-AMPA receptor. We used quantitative proteomic analysis to identify changes in the composition of postsynaptic densities from GluN2B(-/-) mouse primary neuronal cultures and found altered levels of several ubiquitin proteasome system components, in particular decreased levels of proteasome subunits. Enhancing the proteasome activity with a novel proteasome activator restored the synaptic levels of AMPA receptors in GluN2B(-/-) neurons and their endocytosis, revealing that GluN2B-mediated anchoring of the synaptic proteasome is responsible for fine tuning AMPA receptor synaptic levels under basal conditions.

  3. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  4. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  5. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

    PubMed Central

    Chattopadhyay, Nibedita; Berger, Allison J.; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  6. KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism.

    PubMed

    Chattopadhyay, Nibedita; Berger, Allison J; Koenig, Erik; Bannerman, Bret; Garnsey, James; Bernard, Hugues; Hales, Paul; Maldonado Lopez, Angel; Yang, Yu; Donelan, Jill; Jordan, Kristen; Tirrell, Stephen; Stringer, Bradley; Xia, Cindy; Hather, Greg; Galvin, Katherine; Manfredi, Mark; Rhodes, Nelson; Amidon, Ben

    2015-01-01

    In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition. PMID:26709701

  7. 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

  8. 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

  9. 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

  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. 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.

  12. A novel injectable BRET-based in vivo imaging probe for detecting the activity of hypoxia-inducible factor regulated by the ubiquitin-proteasome system

    PubMed Central

    Kuchimaru, Takahiro; Suka, Tomoya; Hirota, Keisuke; Kadonosono, Tetsuya; Kizaka-Kondoh, Shinae

    2016-01-01

    The ubiquitin-proteasome system (UPS) is a selective protein degradation system that plays a critical role in many essential biological processes by regulating the existence of various cellular proteins. The target proteins of UPS are recognized and tagged with polyubiquitin chains by E3 ubiquitin ligases, which have high substrate-specific activities. Here we present a novel injectable imaging probe POL-N that can detect the UPS-regulated hypoxia-inducible factor (HIF) activity in vivo. Because the luciferase is fused to the E3 ligase-recognition domain of the HIF-1α, POL-N is intact only in the HIFα-overexpressing cells, that is, HIF-active cells, generating signals via an intramolecular bioluminescence resonance energy transfer (BRET) between luciferase and a near-infrared (NIR) fluorescent dye at the C-terminal end of the probe. Off-target signals of the NIR-BRET were so low that we could achieve highly sensitive and fast detection of intratumoral HIF-activity. Notably, we successfully detected hypoxic liver metastasis, which is extremely difficult to detect by injectable imaging probes due to strong off-target signals, as early as 1 h after systemic injection of POL-N. Our probe design can be widely adapted to UPS-target proteins and may contribute to the exploration of their roles in animal disease models. PMID:27698477

  13. HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest*

    PubMed Central

    Romani, Bizhan; Shaykh Baygloo, Nima; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2015-01-01

    Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP. PMID:26032416

  14. HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest.

    PubMed

    Romani, Bizhan; Shaykh Baygloo, Nima; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2015-07-10

    Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G2/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G2/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G2/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G2/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.

  15. 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.

  16. A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells.

    PubMed Central

    Shumway, Stuart D; Miyamoto, Shigeki

    2004-01-01

    Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway. PMID:14763901

  17. A novel Bruton's tyrosine kinase inhibitor CC-292 in combination with the proteasome inhibitor carfilzomib impacts the bone microenvironment in a multiple myeloma model with resultant antimyeloma activity.

    PubMed

    Eda, H; Santo, L; Cirstea, D D; Yee, A J; Scullen, T A; Nemani, N; Mishima, Y; Waterman, P R; Arastu-Kapur, S; Evans, E; Singh, J; Kirk, C J; Westlin, W F; Raje, N S

    2014-09-01

    Bruton's tyrosine kinase (Btk) modulates B-cell development and activation and has an important role in antibody production. Interestingly, Btk may also affect human osteoclast (OC) function; however, the mechanism was unknown. Here we studied a potent and specific Btk inhibitor, CC-292, in multiple myeloma (MM). In this report, we demonstrate that, although CC-292 increased OC differentiation, it inhibited OC function via inhibition of c-Src, Pyk2 and cortactin, all involved in OC-sealing zone formation. As CC-292 did not show potent in vitro anti-MM activity, we next evaluated it in combination with the proteasome inhibitor, carfilzomib. We first studied the effect of carfilzomib on OC. Carfilzomib did not have an impact on OC-sealing zone formation but significantly inhibited OC differentiation. CC-292 combined with carfilzomib inhibited both sealing zone formation and OC differentiation, resulting in more profound inhibition of OC function than carfilzomib alone. Moreover, the combination treatment in an in vivo MM mouse model inhibited tumor burden compared with CC-292 alone; it also increased bone volume compared with carfilzomib alone. These results suggest that CC-292 combined with carfilzomib augments the inhibitory effects against OC within the bone microenvironment and has promising therapeutic potential for the treatment of MM and related bone disease.

  18. The pan-HDAC inhibitor vorinostat potentiates the activity of the proteasome inhibitor carfilzomib in human DLBCL cells in vitro and in vivo.

    PubMed

    Dasmahapatra, Girija; Lembersky, Dmitry; Kramer, Lora; Fisher, Richard I; Friedberg, Jonathan; Dent, Paul; Grant, Steven

    2010-06-01

    Interactions between histone deacetylase inhibitors (HDACIs) and the novel proteasome inhibitor carfilzomib (CFZ) were investigated in GC- and activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells. Coadministration of subtoxic or minimally toxic concentrations of CFZ) with marginally lethal concentrations of HDACIs (vorinostat, SNDX-275, or SBHA) synergistically increased mitochondrial injury, caspase activation, and apoptosis in both GC- and ABC-DLBCL cells. These events were associated with Jun NH2-terminal kinase (JNK) and p38MAPK activation, abrogation of HDACI-mediated nuclear factor-kappaB activation, AKT inactivation, Ku70 acetylation, and induction of gammaH2A.X. Genetic or pharmacologic JNK inhibition significantly diminished CFZ/vorinostat lethality. CFZ/vorinostat induced pronounced lethality in 3 primary DLBCL specimens but minimally affected normal CD34(+) hematopoietic cells. Bortezomib-resistant GC (SUDHL16) and ABC (OCI-LY10) cells exhibited partial cross-resistance to CFZ. However, CFZ/vorinostat dramatically induced resistant cell apoptosis, accompanied by increased JNK activation and gammaH2A.X expression. Finally, subeffective vorinostat doses markedly increased CFZ-mediated tumor growth suppression and apoptosis in a murine xenograft OCI-LY10 model. These findings indicate that HDACIs increase CFZ activity in GC- and ABC-DLBCL cells sensitive or resistant to bortezomib through a JNK-dependent mechanism in association with DNA damage and inhibition of nuclear factor-kappaB activation. Together, they support further investigation of strategies combining CFZ and HDACIs in DLBCL. PMID:20233973

  19. The pan-HDAC inhibitor vorinostat potentiates the activity of the proteasome inhibitor carfilzomib in human DLBCL cells in vitro and in vivo

    PubMed Central

    Dasmahapatra, Girija; Lembersky, Dmitry; Kramer, Lora; Fisher, Richard I.; Friedberg, Jonathan; Dent, Paul

    2010-01-01

    Interactions between histone deacetylase inhibitors (HDACIs) and the novel proteasome inhibitor carfilzomib (CFZ) were investigated in GC- and activated B-cell–like diffuse large B-cell lymphoma (ABC-DLBCL) cells. Coadministration of subtoxic or minimally toxic concentrations of CFZ) with marginally lethal concentrations of HDACIs (vorinostat, SNDX-275, or SBHA) synergistically increased mitochondrial injury, caspase activation, and apoptosis in both GC- and ABC-DLBCL cells. These events were associated with Jun NH2-terminal kinase (JNK) and p38MAPK activation, abrogation of HDACI-mediated nuclear factor-κB activation, AKT inactivation, Ku70 acetylation, and induction of γH2A.X. Genetic or pharmacologic JNK inhibition significantly diminished CFZ/vorinostat lethality. CFZ/vorinostat induced pronounced lethality in 3 primary DLBCL specimens but minimally affected normal CD34+ hematopoietic cells. Bortezomib-resistant GC (SUDHL16) and ABC (OCI-LY10) cells exhibited partial cross-resistance to CFZ. However, CFZ/vorinostat dramatically induced resistant cell apoptosis, accompanied by increased JNK activation and γH2A.X expression. Finally, subeffective vorinostat doses markedly increased CFZ-mediated tumor growth suppression and apoptosis in a murine xenograft OCI-LY10 model. These findings indicate that HDACIs increase CFZ activity in GC- and ABC-DLBCL cells sensitive or resistant to bortezomib through a JNK-dependent mechanism in association with DNA damage and inhibition of nuclear factor-κB activation. Together, they support further investigation of strategies combining CFZ and HDACIs in DLBCL. PMID:20233973

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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.

  5. 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

  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. 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

  8. 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.

  9. Enhanced NIF neutron activation diagnostics

    SciTech Connect

    Yeamans, C. B.; Bleuel, D. L.; Bernstein, L. A.

    2012-10-15

    The NIF neutron activation diagnostic suite relies on removable activation samples, leading to operational inefficiencies and a fundamental lower limit on the half-life of the activated product that can be observed. A neutron diagnostic system measuring activation of permanently installed samples could remove these limitations and significantly enhance overall neutron diagnostic capabilities. The physics and engineering aspects of two proposed systems are considered: one measuring the {sup 89}Zr/{sup 89m}Zr isomer ratio in the existing Zr activation medium and the other using potassium zirconate as the activation medium. Both proposed systems could improve the signal-to-noise ratio of the current system by at least a factor of 5 and would allow independent measurement of fusion core velocity and fuel areal density.

  10. Enhanced NIF neutron activation diagnostics.

    PubMed

    Yeamans, C B; Bleuel, D L; Bernstein, L A

    2012-10-01

    The NIF neutron activation diagnostic suite relies on removable activation samples, leading to operational inefficiencies and a fundamental lower limit on the half-life of the activated product that can be observed. A neutron diagnostic system measuring activation of permanently installed samples could remove these limitations and significantly enhance overall neutron diagnostic capabilities. The physics and engineering aspects of two proposed systems are considered: one measuring the (89)Zr/(89 m)Zr isomer ratio in the existing Zr activation medium and the other using potassium zirconate as the activation medium. Both proposed systems could improve the signal-to-noise ratio of the current system by at least a factor of 5 and would allow independent measurement of fusion core velocity and fuel areal density.

  11. Enhanced NIF neutron activation diagnosticsa)

    NASA Astrophysics Data System (ADS)

    Yeamans, C. B.; Bleuel, D. L.; Bernstein, L. A.

    2012-10-01

    The NIF neutron activation diagnostic suite relies on removable activation samples, leading to operational inefficiencies and a fundamental lower limit on the half-life of the activated product that can be observed. A neutron diagnostic system measuring activation of permanently installed samples could remove these limitations and significantly enhance overall neutron diagnostic capabilities. The physics and engineering aspects of two proposed systems are considered: one measuring the 89Zr/89mZr isomer ratio in the existing Zr activation medium and the other using potassium zirconate as the activation medium. Both proposed systems could improve the signal-to-noise ratio of the current system by at least a factor of 5 and would allow independent measurement of fusion core velocity and fuel areal density.

  12. Proteasome-mediated degradation of IκBα and processing of p105 in Crohn disease and ulcerative colitis

    PubMed Central

    Visekruna, Alexander; Joeris, Thorsten; Seidel, Daniel; Kroesen, Anjo; Loddenkemper, Christoph; Zeitz, Martin; Kaufmann, Stefan H.E.; Schmidt-Ullrich, Ruth; Steinhoff, Ulrich

    2006-01-01

    Enhanced NF-κB activity is involved in the pathology of both forms of inflammatory bowel disease (IBD), Crohn disease (CD) and ulcerative colitis (UC). Here we analyzed the mechanism of proteasome-mediated NF-κB activation in CD and UC. Our studies demonstrate that the subunit composition and the proteolytic function of proteasomes differ between UC and CD. High expression of the immunoproteasome subunits β1i and β2i is characteristic of the inflamed mucosa of CD. In line with this, we found enhanced processing of NF-κB precursor p105 and degradation of inhibitor of NF-κB, IκBα, by immunoproteasomes isolated from the mucosa of CD patients. In comparison with healthy controls and CD patients, UC patients exhibited an intermediate phenotype regarding the proteasome-mediated processing/degradation of NF-κB components. Finally, increased expression of the NF-κB family member c-Rel in the inflamed mucosa of CD patients suggests that p50/c-Rel is important for IFN-γ–mediated induction of immunoproteasomes via IL-12–driven Th1 responses. These findings suggest that distinct proteasome subunits influence the intensity of NF-κB–mediated inflammation in IBD patients. PMID:17124531

  13. 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.

  14. 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

  15. 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.

  16. Oxidative stress and proteasome inhibitors in multiple myeloma.

    PubMed

    Lipchick, Brittany C; Fink, Emily E; Nikiforov, Mikhail A

    2016-03-01

    Multiple myeloma is a form of plasma cell neoplasm that accounts for approximately 10% of all hematological malignancies. Recently, several novel drugs have been discovered that almost doubled the overall survival of multiple myeloma patients. One of these drugs, the first-in-class proteasome inhibitor bortezomib (Velcade) has demonstrated remarkable response rates in multiple myeloma patients, and yet, currently this disease remains incurable. The major factor undermining the success of multiple myeloma treatment is a rapidly emerging resistance to the available therapy. Thus, the development of stand-alone or adjuvant anti-myeloma agents becomes of paramount importance. Overproduction of intracellular reactive oxygen species (ROS) often accompanies malignant transformation due to oncogene activation and/or enhanced metabolism in tumor cells. As a result, these cells possess higher levels of ROS and lower levels of antioxidant molecules compared to their normal counterparts. Unbalanced production of ROS leads to oxidative stress which, if left unchecked, could be toxic for the cell. In multiple myeloma cells where high rates of immunoglobulin synthesis is an additional factor contributing to overproduction of ROS, further induction of oxidative stress can be an effective strategy to cope with this disease. Here we will review the available data on the role of oxidative stress in the cytotoxicity of proteasome inhibitors and the use of ROS-inducing compounds as anti-myeloma agents. PMID:26827824

  17. 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.

  18. Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes.

    PubMed

    Liepe, Juliane; Holzhütter, Hermann-Georg; Bellavista, Elena; Kloetzel, Peter M; Stumpf, Michael P H; Mishto, Michele

    2015-01-01

    Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage.

  19. Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes.

    PubMed

    Liepe, Juliane; Holzhütter, Hermann-Georg; Bellavista, Elena; Kloetzel, Peter M; Stumpf, Michael P H; Mishto, Michele

    2015-01-01

    Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage. PMID:26393687

  20. 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.

  1. Proteasome function is not impaired in healthy aging of the lung.

    PubMed

    Caniard, Anne; Ballweg, Korbinian; Lukas, Christina; Yildirim, Ali Ö; Eickelberg, Oliver; Meiners, Silke

    2015-10-01

    Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging. PMID:26540298

  2. 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.

  3. Proteasome function is not impaired in healthy aging of the lung.

    PubMed

    Caniard, Anne; Ballweg, Korbinian; Lukas, Christina; Yildirim, Ali Ö; Eickelberg, Oliver; Meiners, Silke

    2015-10-01

    Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging.

  4. 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

  5. Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells

    SciTech Connect

    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. -- Highlights: ► Nrf2 silencing in pancreatic β-cells enhanced tunicamycin-mediated ER stress. ► Expression of the proteasome was inducible by Nrf2 signaling. ► Nrf2 activator D3T protected β-cells from tunicamycin-mediated ER stress. ► Protective effect of D3T was associated with Nrf2-dependent proteasome

  6. Synergistic anti-proliferative and pro-apoptotic activity of combined therapy with bortezomib, a proteasome inhibitor, with anti-epidermal growth factor receptor (EGFR) drugs in human cancer cells.

    PubMed

    Cascone, Tina; Morelli, Maria Pia; Morgillo, Floriana; Kim, Woo-Young; Rodolico, Gabriella; Pepe, Stefano; Tortora, Giampaolo; Berrino, Liberato; Lee, Ho-Young; Heymach, John V; Ciardiello, Fortunato

    2008-09-01

    The proteasome plays a pivotal role in the turnover of regulatory transduction proteins induced by activated cell membrane growth factor receptors. The epidermal growth factor receptor (EGFR) pathway is crucial in the development and progression of human epithelial cancers. Proteasome inhibition may sensitize human cancer cell lines to EGFR inhibitors. We investigated the growth inhibitory and pro-apoptotic effects of the proteasome inhibitor bortezomib in combination with anti-EGFR drugs, such as gefitinib, vandetanib, and cetuximab in EGFR-expressing human cancer cell lines. Bortezomib determined dose-dependent growth inhibition in a nine cancer cell line panel (IC(50) values, range 6-42 nM). A significant synergistic growth inhibitory effect was observed with the combination of bortezomib and each EGFR inhibitor in all cell lines (combination index, CI, range 0.10-0.55), which was accompanied by a significant induction in apoptosis by the combined treatment with bortezomib, cetuximab and vandetanib. In HCT-116 colon cancer and A549 lung adenocarcinoma cells, bortezomib plus EGFR inhibitor treatment induced a more effective inhibition of EGFR-activated down-stream signals, including a marked suppression in activated, phosphorylated Akt (P-Akt). In contrast, overexpression of a constitutively active P-Akt protected A549 cells by cell growth inhibition and apoptosis following treatment with bortezomib and EGFR inhibitors. The combined treatment with bortezomib and EGFR inhibitors has a synergistic growth inhibitory and pro-apoptotic activity in different human cancer cells which possess a functional EGFR-dependent autocrine growth pathway through to a more efficient and sustained inhibition of Akt.

  7. 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.

  8. 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

  9. Post-translational modification of cardiac proteasomes: functional delineation enabled by proteomics

    PubMed Central

    Scruggs, Sarah B.; Zong, Nobel C.; Wang, Ding; Stefani, Enrico

    2012-01-01

    Proteasomes are ubiquitously expressed multicatalytic complexes that serve as key regulators of protein homeostasis. There are several lines of evidence indicating that proteasomes exist in heterogeneous subpopulations in cardiac muscle, differentiated, in part, by post-translational modifications (PTMs). PTMs regulate numerous facets of proteasome function, including catalytic activities, complex assembly, interactions with associating partners, subcellular localization, substrate preference, and complex turnover. Classical technologies used to identify PTMs on proteasomes have lacked the ability to determine site specificity, quantify stoichiometry, and perform large-scale, multi-PTM analysis. Recent advancements in proteomic technologies have largely overcome these limitations. We present here a discussion on the importance of PTMs in modulating proteasome function in cardiac physiology and pathophysiology, followed by the presentation of a state-of-the-art proteomic workflow for identifying and quantifying PTMs of cardiac proteasomes. PMID:22523251

  10. Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells

    PubMed Central

    Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark

    2016-01-01

    Summary Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here we report formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, which bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of ‘α4-α4’ proteasomes depends on the relative cellular levels of α4 and α3, and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of a novel mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses. PMID:26997268

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Long-term morphine treatment enhances proteasome-dependent degradation of G beta in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization.

    PubMed

    Moulédous, Lionel; Neasta, Jérémie; Uttenweiler-Joseph, Sandrine; Stella, Alexandre; Matondo, Mariette; Corbani, Maïthé; Monsarrat, Bernard; Meunier, Jean-Claude

    2005-08-01

    The initial aim of this study was to identify protein changes associated with long-term morphine treatment in a recombinant human neuroblastoma SH-SY5Y clone (sc2) stably overexpressing the human mu-opioid (MOP) receptor. In MOP receptor-overexpressing sc2 cells, short-term morphine exposure was found to be much more potent and efficacious in inhibiting forskolin-elicited production of cAMP, and long-term morphine exposure was shown to induce a substantially higher degree of opiate dependence, as reflected by adenylate cyclase sensitization, than it did in wild-type neuroblastoma cells. Differential proteomic analysis of detergent-resistant membrane rafts isolated from untreated and chronically morphine-treated sc2 cells revealed long-term morphine exposure to have reliably induced a 30 to 40% decrease in the abundance of five proteins, subsequently identified by mass spectrometry as G protein subunits alphai(2), alphai(3), beta(1), and beta(2), and prohibitin. Quantitative Western blot analyses of whole-cell extracts showed that long-term morphine treatment-induced down-regulation of Gbeta but not of the other proteins is highly correlated (r(2) = 0.96) with sensitization of adenylate cyclase. Down-regulation of Gbeta and adenylate cyclase sensitization elicited by long-term morphine treatment were suppressed in the presence of carbobenzoxy-l-leucyl-l-leucyl-l-norvalinal (MG-115) or lactacystin. Thus, sustained activation of the MOP receptor by morphine in sc2 cells seems to promote proteasomal degradation of Gbeta to sensitize adenylate cyclase. Together, our data suggest that the long-term administration of opiates may elicit dependence by altering the neuronal balance of heterotrimeric G proteins and adenylate cyclases, with the ubiquitin-proteasome pathway playing a pivotal role. PMID:15901846

  16. Centrosomal localisation of the cancer/testis (CT) antigens NY-ESO-1 and MAGE-C1 is regulated by proteasome activity in tumour cells.

    PubMed

    Pagotto, Anna; Caballero, Otavia L; Volkmar, Norbert; Devalle, Sylvie; Simpson, Andrew J G; Lu, Xin; Christianson, John C

    2013-01-01

    The Cancer/Testis (CT) antigen family of genes are transcriptionally repressed in most human tissues but are atypically re-expressed in many malignant tumour types. Their restricted expression profile makes CT antigens ideal targets for cancer immunotherapy. As little is known about whether CT antigens may be regulated by post-translational processing, we investigated the mechanisms governing degradation of NY-ESO-1 and MAGE-C1 in selected cancer cell lines. Inhibitors of proteasome-mediated degradation induced the partitioning of NY-ESO-1 and MAGE-C1 into a detergent insoluble fraction. Moreover, this treatment also resulted in increased localisation of NY-ESO-1 and MAGE-C1 at the centrosome. Despite their interaction, relocation of either NY-ESO-1 or MAGE-C1 to the centrosome could occur independently of each other. Using a series of truncated fragments, the regions corresponding to NY-ESO-1(91-150) and MAGE-C1(900-1116) were established as important for controlling both stability and localisation of these CT antigens. Our findings demonstrate that the steady state levels of NY-ESO-1 and MAGE-C1 are regulated by proteasomal degradation and that both behave as aggregation-prone proteins upon accumulation. With proteasome inhibitors being increasingly used as front-line treatment in cancer, these data raise issues about CT antigen processing for antigenic presentation and therefore immunogenicity in cancer patients.

  17. 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

  18. Eucommia ulmoides Oliver Extract, Aucubin, and Geniposide Enhance Lysosomal Activity to Regulate ER Stress and Hepatic Lipid Accumulation

    PubMed Central

    Lee, Hwa-Young; Lee, Geum-Hwa; Lee, Mi-Rin; Kim, Hye-Kyung; Kim, Nan-young; Kim, Seung-Hyun; Lee, Yong-Chul; Kim, Hyung-Ryong; Chae, Han-Jung

    2013-01-01

    Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE) on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER) stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia. PMID:24349058

  19. 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

  20. 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

  1. Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma

    PubMed Central

    Vandewynckel, Yves-Paul; Coucke, Céline; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Vandierendonck, Astrid; Raevens, Sarah; Verhelst, Xavier; Van Steenkiste, Christophe; Libbrecht, Louis; Geerts, Anja; Van Vlierberghe, Hans

    2016-01-01

    Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators. PMID:27167000

  2. 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

  3. 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

  4. Ras enhances Myc protein stability.

    PubMed

    Sears, R; Leone, G; DeGregori, J; Nevins, J R

    1999-02-01

    Various experiments have demonstrated a collaborative action of Myc and Ras, both in normal cell growth control as well as during oncogenesis. We now show that Ras enhances the accumulation of Myc activity by stabilizing the Myc protein. Whereas Myc has a very short half-life when produced in the absence of mitogenic signals, due to degradation by the 26S proteasome, the half-life of Myc increases markedly in growth-stimulated cells. This stabilization is dependent on the Ras/Raf/MAPK pathway and is not augmented by proteasome inhibition, suggesting that Ras inhibits the proteasome-dependent degradation of Myc. We propose that one aspect of Myc-Ras collaboration is an ability of Ras to enhance the accumulation of transcriptionally active Myc protein.

  5. Secomycalolide A: A New Proteasome Inhibitor Isolated from a Marine Sponge of the Genus Mycale

    PubMed Central

    Tsukamoto, Sachiko; Koimaru, Keiichirou; Ohta, Tomihisa

    2005-01-01

    A new oxazole-containing proteasome inhibitor, secomycalolide A, together with known mycalolide A and 30-hydroxymycalolide A, was isolated from a marine sponge of the genus Mycale. They showed proteasome inhibitory activities with IC50 values of 11–45 μg/mL.

  6. 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

  7. 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.

  8. 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

  9. 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

  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. 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

  12. 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

  13. 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

  14. 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

  15. Mouse zygote-specific proteasome assembly chaperone important for maternal-to-zygotic transition

    PubMed Central

    Shin, Seung-Wook; Shimizu, Natsumi; Tokoro, Mikiko; Nishikawa, Satoshi; Hatanaka, Yuki; Anzai, Masayuki; Hamazaki, Jun; Kishigami, Satoshi; Saeki, Kazuhiro; Hosoi, Yoshihiko; Iritani, Akira; Murata, Shigeo; Matsumoto, Kazuya

    2013-01-01

    Summary During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin–proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT. PMID:23429752

  16. 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.

  17. Polycarboxylates Enhance Beetle Antifreeze Protein Activity

    PubMed Central

    Amornwittawat, Natapol; Wang, Sen; Duman, John G.; Wen, Xin

    2008-01-01

    Summary Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N′,N″,N‴,N‴-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn2+ coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed. PMID:18620083

  18. Structural Insights into the Regulatory Particle of the Proteasome from Methanocaldococcus jannaschii

    SciTech Connect

    Zhang, F.; Hu, M; Tian, G; Zhang, P; Finley, D; Jeffrey, P; Shi, Y

    2009-01-01

    Eukaryotic proteasome consists of a core particle (CP), which degrades unfolded protein, and a regulatory particle (RP), which is responsible for recognition, ATP-dependent unfolding, and translocation of polyubiquitinated substrate protein. In the archaea Methanocaldococcus jannaschii, the RP is a homohexameric complex of proteasome-activating nucleotidase (PAN). Here, we report the crystal structures of essential elements of the archaeal proteasome: the CP, the ATPase domain of PAN, and a distal subcomplex that is likely the first to encounter substrate. The distal subcomplex contains a coiled-coil segment and an OB-fold domain, both of which appear to be conserved in the eukaryotic proteasome. The OB domains of PAN form a hexameric ring with a 13 A pore, which likely constitutes the outermost constriction of the substrate translocation channel. These studies reveal structural codes and architecture of the complete proteasome, identify potential substrate-binding sites, and uncover unexpected asymmetry in the RP of archaea and eukaryotes.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. Proteasome function shapes innate and adaptive immune responses.

    PubMed

    Kammerl, Ilona E; Meiners, Silke

    2016-08-01

    The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively. PMID:27343191

  5. Proteasome function shapes innate and adaptive immune responses.

    PubMed

    Kammerl, Ilona E; Meiners, Silke

    2016-08-01

    The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively.

  6. 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

  7. 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

  8. Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription?

    PubMed

    Nawaz, Zafar; O'Malley, Bert W

    2004-03-01

    The importance of the ubiquitin proteasome pathway in higher eukaryotes has been well established in cell cycle regulation, signal transduction, and cell differentiation, but has only recently been linked to nuclear hormone receptor-regulated gene transcription. Characterization of a number of ubiquitin proteasome pathway enzymes as coactivators and observations that several nuclear receptors are ubiquitinated and degraded in the course of their nuclear activities provide evidence that ubiquitin proteasome-mediated protein degradation plays an integral role in eukaryotic transcription. In addition to receptors, studies have revealed that coactivators are ubiquitinated and degraded via the proteasome. The notion that the ubiquitin proteasome pathway is involved in gene transcription is further strengthened by the fact that ubiquitin proteasome pathway enzymes are recruited to the promoters of target genes and that proteasome-dependent degradation of nuclear receptors is required for efficient transcriptional activity. These findings suggest that protein degradation is coupled with nuclear receptor coactivation activity. It is possible that the ubiquitin proteasome pathway modulates transcription by promoting remodeling and turnover of the nuclear receptor-transcription complex. In this review, we discus the possible role of the ubiquitin proteasome pathway in nuclear hormone receptor-regulated gene transcription.

  9. 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.

  10. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis

    SciTech Connect

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment

  11. Blm10 facilitates nuclear import of proteasome core particles

    PubMed Central

    Weberruss, Marion H; Savulescu, Anca F; Jando, Julia; Bissinger, Thomas; Harel, Amnon; Glickman, Michael H; Enenkel, Cordula

    2013-01-01

    Short-lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT-like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP-CP assemblies are primarily nuclear and promote cell division. During quiescence, RP-CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10-bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG-rich nucleoporins and is dissociated from the CP by Ran-GTP. Thus, Blm10 represents the first CP-dedicated nuclear import receptor in yeast. PMID:23982732

  12. A potent and selective inhibitor for the UBLCP1 proteasome phosphatase

    PubMed Central

    He, Yantao; Guo, Xing; Yu, Zhi-Hong; Wu, Li; Gunawan, Andrea M.; Zhang, Yan; Dixon, Jack E.; Zhang, Zhong-Yin

    2015-01-01

    The ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1) has been implicated as a negative regulator of the proteasome, a key mediator in the ubiquitin-dependent protein degradation. Small molecule inhibitors that block UBLCP1 activity would be valuable as research tools and potential therapeutics for human diseases caused by the cellular accumulation of misfold/damaged proteins. We report a salicylic acid fragment-based library approach aimed at targeting both the phosphatase active site and its adjacent binding pocket for enhanced affinity and selectivity. Screening of the focused libraries led to the identification of the first potent and selective UBLCP1 inhibitor 13. Compound 13 exhibits an IC50 of 1.0 μM for UBLCP1 and greater than 5-fold selectivity against a large panel of protein phosphatases from several distinct families. Importantly, the inhibitor possesses efficacious cellular activity and is capable of inhibiting UBLCP1 function in cells, which in turn up-regulates nuclear proteasome activity. These studies set the groundwork for further developing compound 13 into chemical probes or potential therapeutic agents targeting the UBLCP1 phosphatase. PMID:25907364

  13. 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

  14. 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

  15. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis.

    PubMed

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli-germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli-germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. PMID:25886977

  16. 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

  17. Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria

    PubMed Central

    Blouin, Jean-Marc; Duchartre, Yann; Costet, Pierre; Lalanne, Magalie; Ged, Cécile; Lain, Ana; Millet, Oscar; de Verneuil, Hubert; Richard, Emmanuel

    2013-01-01

    Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROSC73R mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROSP248Q mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROSC73R and UROSP248Q are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (UrosP248Q/P248Q) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones. PMID:24145442

  18. Therapeutic potential of proteasome inhibitors in congenital erythropoietic porphyria.

    PubMed

    Blouin, Jean-Marc; Duchartre, Yann; Costet, Pierre; Lalanne, Magalie; Ged, Cécile; Lain, Ana; Millet, Oscar; de Verneuil, Hubert; Richard, Emmanuel

    2013-11-01

    Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS(C73R) mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS(P248Q) mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS(C73R) and UROS(P248Q) are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros(P248Q/P248Q)) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones. PMID:24145442

  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. Two-substrate association with the 20S proteasome at single-molecule level.

    PubMed

    Hutschenreiter, Silke; Tinazli, Ali; Model, Kirstin; Tampé, Robert

    2004-07-01

    The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a 'dead-end' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule cross-correlation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity. PMID:15175655

  1. Two-substrate association with the 20S proteasome at single-molecule level.

    PubMed

    Hutschenreiter, Silke; Tinazli, Ali; Model, Kirstin; Tampé, Robert

    2004-07-01

    The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a 'dead-end' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule cross-correlation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity.

  2. Two-substrate association with the 20S proteasome at single-molecule level

    PubMed Central

    Hutschenreiter, Silke; Tinazli, Ali; Model, Kirstin; Tampé, Robert

    2004-01-01

    The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal-chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End-on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a ‘dead-end' proteasome can process only one substrate at a time. In contrast, the side-on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single-molecule cross-correlation spectroscopy. Thus, the two-stroke engine offers the advantage of speeding up degradation without enhancing complexity. PMID:15175655

  3. 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

  4. Proteasome Dysfunction Mediates High Glucose-Induced Apoptosis in Rodent Beta Cells and Human Islets

    PubMed Central

    Broca, Christophe; Varin, Elodie; Armanet, Mathieu; Tourrel-Cuzin, Cécile; Bosco, Domenico; Dalle, Stéphane; Wojtusciszyn, Anne

    2014-01-01

    The ubiquitin/proteasome system (UPS), a major cellular protein degradation machinery, plays key roles in the regulation of many cell functions. Glucotoxicity mediated by chronic hyperglycaemia is detrimental to the function and survival of pancreatic beta cells. The aim of our study was to determine whether proteasome dysfunction could be involved in beta cell apoptosis in glucotoxic conditions, and to evaluate whether such a dysfunction might be pharmacologically corrected. Therefore, UPS activity was measured in GK rats islets, INS-1E beta cells or human islets after high glucose and/or UPS inhibitor exposure. Immunoblotting was used to quantify polyubiquitinated proteins, endoplasmic reticulum (ER) stress through CHOP expression, and apoptosis through the cleavage of PARP and caspase-3, whereas total cell death was detected through histone-associated DNA fragments measurement. In vitro, we found that chronic exposure of INS-1E cells to high glucose concentrations significantly decreases the three proteasome activities by 20% and leads to caspase-3-dependent apoptosis. We showed that pharmacological blockade of UPS activity by 20% leads to apoptosis in a same way. Indeed, ER stress was involved in both conditions. These results were confirmed in human islets, and proteasome activities were also decreased in hyperglycemic GK rats islets. Moreover, we observed that a high glucose treatment hypersensitized beta cells to the apoptotic effect of proteasome inhibitors. Noteworthily, the decreased proteasome activity can be corrected with Exendin-4, which also protected against glucotoxicity-induced apoptosis. Taken together, our findings reveal an important role of proteasome activity in high glucose-induced beta cell apoptosis, potentially linking ER stress and glucotoxicity. These proteasome dysfunctions can be reversed by a GLP-1 analog. Thus, UPS may be a potent target to treat deleterious metabolic conditions leading to type 2 diabetes. PMID:24642635

  5. Complement modulates the function of the ubiquitin-proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells.

    PubMed

    Kitzler, Thomas M; Papillon, Joan; Guillemette, Julie; Wing, Simon S; Cybulsky, Andrey V

    2012-05-01

    In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin-proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFP(u) (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFP(u) levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFP(u) was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFP(u) degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Lifelong maintenance of composition, function and cellular/subcellular distribution of proteasomes in human liver.

    PubMed

    Bellavista, Elena; Martucci, Morena; Vasuri, Francesco; Santoro, Aurelia; Mishto, Michele; Kloss, Alexander; Capizzi, Elisa; Degiovanni, Alessio; Lanzarini, Catia; Remondini, Daniel; Dazzi, Alessandro; Pellegrini, Sara; Cescon, Matteo; Capri, Miriam; Salvioli, Stefano; D'Errico-Grigioni, Antonia; Dahlmann, Burkhardt; Grazi, Gian Luca; Franceschi, Claudio

    2014-01-01

    Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of β5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation.

  16. 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

  17. Colorectal Carcinogenesis, Radiation Quality, and the Ubiquitin-Proteasome Pathway

    PubMed Central

    Datta, Kamal; Suman, Shubhankar; Kumar, Santosh; Fornace, Albert J

    2016-01-01

    Adult colorectal epithelium undergoes continuous renewal and maintains homeostatic balance through regulated cellular proliferation, differentiation, and migration. The canonical Wnt signaling pathway involving the transcriptional co-activator β-catenin is important for colorectal development and normal epithelial maintenance, and deregulated Wnt/β-catenin signaling has been implicated in colorectal carcinogenesis. Colorectal carcinogenesis has been linked to radiation exposure, and radiation has been demonstrated to alter Wnt/β-catenin signaling, as well as the proteasomal pathway involved in the degradation of the signaling components and thus regulation of β-catenin. The current review discusses recent progresses in our understanding of colorectal carcinogenesis in relation to different types of radiation and roles that radiation quality plays in deregulating β-catenin and ubiquitin-proteasome pathway (UPP) for colorectal cancer initiation and progression. PMID:26819641

  18. Enhancement of deoxyribozyme activity by cationic copolymers.

    PubMed

    Gao, Jueyuan; Shimada, Naohiko; Maruyama, Atsushi

    2015-02-01

    Deoxyribozymes, or DNAzymes, are DNA molecules with enzymatic activity. DNAzymes with ribonuclease activity have various potential applications in biomedical and bioanalytical fields; however, most constructs have limited turnover despite optimization of reaction conditions and DNAzyme structures. A cationic comb-type copolymer accelerates DNA hybridization and strand exchange rates, and we hypothesized that the copolymer would enhance deoxyribozyme activity by promoting turnover. The copolymer did not change DNAzyme activity under single-turnover conditions, suggesting that the copolymer affects neither the folding structure of DNAzyme nor the association of a divalent cation, a catalytic cofactor, to DNAzyme. The copolymer enhanced activity of the evaluated DNAzyme over a wide temperature range under multiple-turnover conditions. The copolymer increased the DNAzyme kcat/KM by fifty-fold at 50 °C, the optimal temperature for the DNAzyme in the absence of the copolymer. The acceleration effect was most significant when the reaction temperature was slightly higher than the melting temperature of the enzyme/substrate complex; acceleration of two orders of magnitude was observed. We concluded that the copolymer accelerated the turnover step without influencing the chemical cleavage step. In contrast to the copolymer, a cationic surfactant, CTAB, strongly inhibited the DNAzyme activity under either single- or multiple-turnover conditions. PMID:26218121

  19. Enhanced multistatic active sonar signal processing.

    PubMed

    Zhao, Kexin; Liang, Junli; Karlsson, Johan; Li, Jian

    2013-07-01

    Multistatic active sonar systems involve the transmission and reception of multiple probing sequences and can achieve significantly enhanced performance of target detection and localization through exploiting spatial diversity. This paper mainly focuses on two signal processing aspects of such systems, namely, enhanced range-Doppler imaging and improved target parameter estimation. The main contributions of this paper are (1) a hybrid dense-sparse method is proposed to generate range-Doppler images with both low sidelobe levels and high accuracy; (2) a generalized K-Means clustering (GKC) method for target association is developed to associate the range measurements from different transmitter-receiver pairs, which is actually a range fitting procedure; (3) the extended invariance principle-based weighted least-squares method is developed for accurate target position and velocity estimation. The effectiveness of the proposed multistatic active sonar signal processing techniques is verified using numerical examples.

  20. 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

  1. 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.

  2. 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.

  3. Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals

    PubMed Central

    Postiglione, Ilaria; Chiaviello, Angela; Barra, Federica; Roscetto, Emanuela; Soriano, Amata A.; Catania, Maria Rosaria; Palumbo, Giuseppe; Pierantoni, Giovanna Maria

    2015-01-01

    Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53+/+) and H1299 (p53−/−) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis. PMID:26343643

  4. 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

  5. The role of allostery in the ubiquitin-proteasome system

    PubMed Central

    Liu, Jin; Nussinov, Ruth

    2012-01-01

    The Ubiquitin-Proteasome System is involved in many cellular processes including protein degradation. Degradation of a protein via this system involves two successive steps: ubiquitination and degradation. Ubiquitination tags the target protein with ubiquitin-like proteins, such as ubiquitin, SUMO and NEDD8, via a cascade involving three enzymes: activating enzyme E1, conjugating enzyme E2, and E3 ubiquitin ligases. The proteasomes recognize the ubiquitin-like protein tagged substrate proteins and degrade them. Accumulating evidence indicates that allostery is a central player in the regulation of ubiquitination, as well as deubiquitination and degradation. Here, we provide an overview of the key mechanistic roles played by allostery in all steps of these processes, and highlight allosteric drugs targeting them. Throughout the review, we emphasize the crucial mechanistic role played by linkers in allosterically controlling the Ubiquitin-Proteasome System action by biasing the sampling of the conformational space, which facilitate the catalytic reactions of the ubiquitination and degradation. Finally, we propose that allostery may similarly play key roles in the regulation of molecular machines in the cell, and as such allosteric drugs can be expected to be increasingly exploited in therapeutic regimes. PMID:23234564

  6. 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.

  7. Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

    PubMed

    Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

    2015-05-01

    The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21. PMID:24975575

  8. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

    PubMed Central

    Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

    2016-01-01

    Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

  9. 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.

  10. Synthesis and Biological Evaluation of Naphthoquinone Analogs as a Novel Class of Proteasome Inhibitors

    PubMed Central

    Lawrence, Harshani R.; Kazi, Aslamuzzaman; Luo, Yunting; Kendig, Robert; Ge, Yiyu; Jain, Sanjula; Daniel, Kenyon; Santiago, Daniel; Guida, Wayne C.; Sebti, Saïd M.

    2012-01-01

    Screening of the NCI Diversity Set-1 identified PI-083 (NSC-45382) a proteasome inhibitor selective for cancer over normal cells. Focused libraries of novel compounds based on PI-083 chloronaphthoquinone and sulfonamide moieties were synthesized to gain a better understanding of the structure activity relationship responsible for chymotrypsin-like proteasome inhibitory activity. This led to the demonstration that the chloronaphthoquinone and the sulfonamide moieties are critical for inhibitory activity. The pyridyl group in PI-083 can be replaced with other heterocyclic groups without significant loss of activity. Molecular modeling studies were also performed to explore the detailed interactions of PI-083 and its derivatives with the β5 and β6 subunits of the 20S proteasome. The refined model showed an H-bond interaction between the Asp-114 and the sulfonamide moiety of the PI-083 in the β6 subunit. PMID:20621484

  11. 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

  12. 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

  13. Detecting insider activity using enhanced directory virtualization.

    SciTech Connect

    Shin, Dongwan; Claycomb, William R.

    2010-07-01

    Insider threats often target authentication and access control systems, which are frequently based on directory services. Detecting these threats is challenging, because malicious users with the technical ability to modify these structures often have sufficient knowledge and expertise to conceal unauthorized activity. The use of directory virtualization to monitor various systems across an enterprise can be a valuable tool for detecting insider activity. The addition of a policy engine to directory virtualization services enhances monitoring capabilities by allowing greater flexibility in analyzing changes for malicious intent. The resulting architecture is a system-based approach, where the relationships and dependencies between data sources and directory services are used to detect an insider threat, rather than simply relying on point solutions. This paper presents such an architecture in detail, including a description of implementation results.

  14. Serine deprivation enhances antineoplastic activity of biguanides.

    PubMed

    Gravel, Simon-Pierre; Hulea, Laura; Toban, Nader; Birman, Elena; Blouin, Marie-José; Zakikhani, Mahvash; Zhao, Yunhua; Topisirovic, Ivan; St-Pierre, Julie; Pollak, Michael

    2014-12-15

    Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

  15. Role of the ubiquitin-proteasome pathway and some peptidases during seed germination and copper stress in bean cotyledons.

    PubMed

    Karmous, Inès; Chaoui, Abdelilah; Jaouani, Khadija; Sheehan, David; El Ferjani, Ezzedine; Scoccianti, Valeria; Crinelli, Rita

    2014-03-01

    The role of the ubiquitin (Ub)-proteasome pathway and some endo- and aminopeptidases (EPs and APs, respectively) was studied in cotyledons of germinating bean seeds (Phaseolus vulgaris L.). The Ub system appeared to be important both in the early (3 days) and late (9 days) phases of germination. In the presence of copper, an increase in protein carbonylation and a decrease in reduced -SH pool occurred, indicating protein damage. This was associated with an enhancement in accumulation of malondialdehyde, a major product of lipid peroxidation, and an increase in content of hydrogen peroxide (H2O2), showing oxidative stress generation. Moreover, copper induced inactivation of the Ub-proteasome (EC 3.4.25) pathway and inhibition of leucine and proline aminopeptidase activities (EC 3.4.11.1 and EC 3.4.11.5, respectively), thus limiting their role in modulating essential metabolic processes, such as the removal of regulatory and oxidatively-damaged proteins. By contrast, total trypsin and chymotrypsin-like activities (EC 3.4.21.4 and EC 3.4.21.1, respectively) increased after copper exposure, in parallel with a decrease in their inhibitor capacities (i.e. trypsin inhibitor and chymotrypsin inhibitor activity), suggesting that these endoproteases are part of the protective mechanisms against copper stress.

  16. 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

  17. Dual targeting of glioblastoma multiforme with a proteasome inhibitor (Velcade) and a phosphatidylinositol 3-kinase inhibitor (ZSTK474).

    PubMed

    Lin, Lehang; Gaut, Daria; Hu, Kaishun; Yan, Haiyan; Yin, Dong; Koeffler, H Phillip

    2014-02-01

    Proteasome inhibitors have been proven to be effective anticancer compounds in many tumor models, including glioblastoma multiforme (GBM). In this study, we found that the proteasome inhibitor Velcade (PS-341/bortezomib) caused GBM cell death while simultaneously activating the PI3K/Akt pathway. Therefore, we sought to investigate if the PI3K inhibitor ZSTK474 would enhance the effectiveness of Velcade in anticancer therapy. Two GBM cell lines were used to detect the effects of Velcade and ZSTK474 alone or in combination in vitro. The combination of Velcade and ZSTK474 synergistically inhibited the proliferation of GBM cell lines. Cell apoptosis was increased when exposed to Velcade and ZSTK474 in combination as shown by Annexin V analysis. Treatment with both drugs led to downregulation of the p-Akt, p-4EBP1 and p-mTOR proteins as determined by western blot analysis. The anticancer ability of Velcade for glioblastoma multiforme was, therefore, enhanced by combination with the PI3K pathway inhibitor ZSTK474 in glioblastoma multiforme.

  18. HSP90 protects the human T-cell leukemia virus type 1 (HTLV-1) tax oncoprotein from proteasomal degradation to support NF-κB activation and HTLV-1 replication.

    PubMed

    Gao, Linlin; Harhaj, Edward William

    2013-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients.

  19. Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System.

    PubMed

    Matilainen, Olli; Jha, Sweta; Holmberg, Carina I

    2016-01-01

    The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions. PMID:27613038

  20. Enhanced interleukin activity following asbestos inhalation.

    PubMed Central

    Hartmann, D P; Georgian, M M; Oghiso, Y; Kagan, E

    1984-01-01

    Asbestos inhalation can cause pulmonary fibrosis and is associated with a variety of immunological abnormalities. The purpose of this study was to evaluate the effects of asbestos inhalation on interleukin-1 (IL-1) and interleukin-2 (IL-2) production in a rodent model. Two groups of rats were exposed, by intermittent inhalation, to either amphibole (crocidolite) or serpentine (chrysotile) asbestos. A third (control) group of rats was sham exposed to clean air. Animals from the three exposure groups were thereafter immunized (or not immunized) with fetal calf serum antigens. In order to assay interleukin activity, supernatants were generated from cultures containing alveolar macrophages and autologous splenic lymphocytes, and from cultures containing alveolar macrophages alone. Using assay systems designed to detect IL-1 and IL-2 functional activity, the supernatants were evaluated for their capacity to stimulate lymphoproliferation and fibroblast DNA synthesis. Macrophage-lymphocyte co-culture supernatants, when obtained from immunized, asbestos exposed rats, contained greater IL-1 and IL-2 activity than identical supernatants from immunized, sham exposed animals. These between group differences were not, however, observed in supernatants from unimmunized rats, or when supernatants were generated in the absence of immune lymphocytes. These observations suggest that asbestos exposure is associated with enhanced activation of lymphocytes by antigens. The possible relevance of these findings to asbestos related fibrogenesis and immunological stimulation is discussed. PMID:6608427

  1. : Synthesis, Characterization, and Enhanced Photocatalytic Activity

    NASA Astrophysics Data System (ADS)

    Gao, Xiaoming; Fu, Feng; Li, Wenhong

    2014-12-01

    3D hierarchical microspheres of Cu-loaded Bi2WO6 are successfully prepared by the hydrothermal synthesis method on a large scale. The as-prepared samples are characterized by UV-Vis DRS, BET, XRD, XPS, and SEM. The results reveal that the light absorption of Cu-loaded Bi2WO6 has higher intensity in the visible range and a bathochromic shift of the absorption edge compared to that of pure Bi2WO6. The photocatalytic activity is evaluated by phenol removal from aqueous solution under visible-light irradiation. The results demonstrate that loaded Cu significantly enhances the photocatalytic activity of Bi2WO6, for the loaded Cu acts as the electron receptor on the surface of Bi2WO6, and inhibits the recombination of photogenerated electron-hole. The content of loaded Cu has an impact on the catalytic activity, and the 1.0 wt.% Cu-loaded Bi2WO6 exhibits the best photocatalytic activity in the degradation of phenol. Furthermore, the reaction kinetics of phenol removal from aqueous solution over the Cu-loaded Bi2WO6 is established by the way of the Langmuir-Hinshelwood model. The results indicate that the process of photodegradation of phenol on Cu-loaded Bi2WO6 match the Langmuir-Hinshelwood kinetic model.

  2. Proteasome inhibitor MG-132 enhances histone deacetylase inhibitor SAHA-induced cell death of chronic myeloid leukemia cells by an ROS-mediated mechanism and downregulation of the Bcr-Abl fusion protein

    PubMed Central

    ZHOU, WENJING; ZHU, WEIWEI; MA, LIYA; XIAO, FENG; QIAN, WENBIN

    2015-01-01

    Recently, there has been progress in the treatment of chronic myeloid leukemia (CML). However, novel therapeutic strategies are required in order to address the emerging problem of imatinib resistance. Histone deacetylase inhibitors (HDACi) and proteasome inhibitors are promising alternatives, and may be amenable to integration with current therapeutic approaches. However, the mechanisms underlying the interaction between these two agents remain unclear. The present study assessed the cytotoxic effect of the HDACi, suberoylanilide hydroxamic acid (SAHA), in combination with the proteasome inhibitor, MG-132, in imatinib-sensitive K562 and imatinib-resistant K562G cells, and investigated the mechanism underlying this effect. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and protein expression levels were determined by western blotting. Reactive oxygen species (ROS) generation levels were observed under a fluorescence microscope The results indicated that SAHA and MG-132 act in a synergistic manner to induce cell death in K562 and K562G cells. This effect was associated with Bcr-Abl downregulation and the production of ROS. Notably, the ROS scavenger, N-acetyl-L-cysteine, almost fully reversed the cell death and Bcr-Abl downregulation that was induced by the combination of SAHA and MG-132. By contrast, the pan-caspase inhibitor, z-VAD-fmk, only partially reversed the cell death induced by these two drugs in CML cells. These results indicated that increased intracellular ROS levels are important in the induction of cell death and the downregulation of Bcr-Abl. In conclusion, the present results suggested that combined SAHA and MG-132 may be a promising treatment for CML. PMID:26722260

  3. 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

  4. 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

  5. Systemic Administration of a Proteasome Inhibitor Does Not Cause Nigrostriatal Dopamine Degeneration

    PubMed Central

    Mathur, Brian N.; Neely, M. Diana; Dyllick-Brenzinger, Melanie; Tandon, Anurag; Deutch, Ariel Y.

    2007-01-01

    Proteasomal dysfunction has been suggested to contribute to the degeneration of nigrostriatal dopamine neurons in Parkinson’s disease. A recent study reported that systemic treatment of rats with the proteasome inhibitor Z-lle-Glu(OtBu)-Ala-Leu-al (PSI) causes a slowly progressive degeneration of nigrostriatal dopamine neurons, the presence of inclusion bodies in dopamine neurons, and motor impairment. We examined in vitro and in vivo the effects of PSI on nigrostriatal dopamine neurons. Mass spectrometric analysis was employed to verify the authenticity of the PSI compound. PSI was non-specifically toxic to neurons in ventral mesencephalic organotypic slice cultures, indicating that impairment of proteasome function in vitro is toxic. Moreover, systemic administration of PSI transiently decreased brain proteasome activity. Systemic treatment of rats with PSI did not, however, result in any biochemical or anatomical evidence of lesions of nigrostriatal dopamine neurons, nor were any changes in locomotor activity observed. These data suggest that systemic administration of proteasome inhibitors to normal adult rats does not reliably cause an animal model of parkinsonism. PMID:17706185

  6. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  7. How Soluble GARP Enhances TGFβ Activation

    PubMed Central

    Fridrich, Sven; Hahn, Susanne A.; Linzmaier, Marion; Felten, Matthias; Zwarg, Jenny; Lennerz, Volker; Tuettenberg, Andrea; Stöcker, Walter

    2016-01-01

    GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo. PMID:27054568

  8. How Soluble GARP Enhances TGFβ Activation.

    PubMed

    Fridrich, Sven; Hahn, Susanne A; Linzmaier, Marion; Felten, Matthias; Zwarg, Jenny; Lennerz, Volker; Tuettenberg, Andrea; Stöcker, Walter

    2016-01-01

    GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo. PMID:27054568

  9. Titanium surface hydrophilicity enhances platelet activation.

    PubMed

    Alfarsi, Mohammed A; Hamlet, Stephen M; Ivanovski, Saso

    2014-01-01

    Titanium implant surface modification is a key strategy used to enhance osseointegration. Platelets are the first cells that interact with the implant surface whereupon they release a wide array of proteins that influence the subsequent healing process. This study therefore investigated the effect of titanium surface modification on the attachment and activation of human platelets. The surface characteristics of three titanium surfaces: smooth (SMO), micro-rough (SLA) and hydrophilic micro-rough (SLActive) and the subsequent attachment and activation of platelets following exposure to these surfaces were determined. The SLActive surface showed the presence of significant nanoscale topographical features. While attached platelets appeared to be morphologically similar, significantly fewer platelets attached to the SLActive surface compared to both the SMO and SLA surfaces. The SLActive surface however induced the release of the higher levels of chemokines β-thromboglobulin and platelet factor 4 from platelets. This study shows that titanium surface topography and chemistry have a significant effect on platelet activation and chemokine release.

  10. 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

  11. 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.

  12. 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.

  13. Roles of the ubiquitin proteasome system in the effects of drugs of abuse.

    PubMed

    Massaly, Nicolas; Francès, Bernard; Moulédous, Lionel

    2014-01-01

    Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS) plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However, the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors. PMID:25610367

  14. "Depupylation" of Prokaryotic Ubiquitin-like Protein from Mycobacterial Proteasome Substrates

    SciTech Connect

    Burns, K.E.; Li, H.; Cerda-Maira, F. A.; Wang, T.; Bishai, W. R.; Darwin, K. H.

    2010-09-10

    Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb), as it dooms proteins to the Mtb proteasome. Studies suggest that Pup and Ub do not share similar mechanisms of activation and conjugation to target proteins. Dop (deamidase of Pup; Mtb Rv2112c/MT2172) deamidates the C-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). While studies have shed light on the conjugation of Pup to proteins, it was not known if Pup could be removed from substrates in a manner analogous to the deconjugation of Ub from eukaryotic proteins. Here, we show that Mycobacteria have a depupylase activity provided by Dop. The discovery of a depupylase strengthens the parallels between the Pup- and Ub-tagging systems of prokaryotes and eukaryotes, respectively.

  15. Roles of the ubiquitin proteasome system in the effects of drugs of abuse

    PubMed Central

    Massaly, Nicolas; Francès, Bernard; Moulédous, Lionel

    2015-01-01

    Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS) plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However, the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors. PMID:25610367

  16. Optimization and Evaluation of 5-Styryl-Oxathiazol-2-one Mycobacterium tuberculosis Proteasome Inhibitors as Potential Antitubercular Agents

    PubMed Central

    Russo, Francesco; Gising, Johan; Åkerbladh, Linda; Roos, Annette K; Naworyta, Agata; Mowbray, Sherry L; Sokolowski, Anders; Henderson, Ian; Alling, Torey; Bailey, Mai A; Files, Megan; Parish, Tanya; Karlén, Anders; Larhed, Mats

    2015-01-01

    This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure–activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs. PMID:26246997

  17. JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis

    PubMed Central

    Deng, Shulin; Jang, In-Cheol; Su, Linlin; Xu, Jun; Chua, Nam-Hai

    2016-01-01

    H3K9 methylation is usually associated with DNA methylation, and together they symbolize transcriptionally silenced heterochromatin. A number of proteins involved in epigenetic processes have been characterized. However, how the stability of these proteins is regulated at the post-translational level is largely unknown. Here, we show that an Arabidopsis JmjC domain protein, JMJ24, possesses ubiquitin E3 ligase activity. JMJ24 directly targets a DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), for proteasomal degradation to initiate destabilization of the heterochromatic state of endogenous silenced loci. Our results uncover an additional connection between two conserved epigenetic modifications: histone modification and DNA methylation. PMID:26798133

  18. 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

  19. 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...

  20. 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...

  1. 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

  2. Exploiting nature's rich source of proteasome inhibitors as starting points in drug development.

    PubMed

    Gräwert, Melissa Ann; Groll, Michael

    2012-02-01

    Cancer is the No. 2 cause of death in the Western world and one of the most expensive diseases to treat. Thus, it is not surprising, that every major pharmaceutical and biotechnology company has a blockbuster oncology product. In 2003, Millennium Pharmaceuticals entered the race with Velcade®, a first-in-class proteasome inhibitor that has been approved by the FDA for treatment of multiple myeloma and its sales have passed the billion dollar mark. Velcade®'s extremely toxic boronic acid pharmacophore, however, contributes to a number of severe side effects. Nevertheless, the launching of this product has validated the proteasome as a target in fighting cancer and further proteasome inhibitors have entered the market as anti-cancer drugs. Additionally, proteasome inhibitors have found application as crop protection agents, anti-parasitics, immunosuppressives, as well as in new therapies for muscular dystrophies and inflammation. Many of these compounds are based on microbial metabolites. In this review, we emphasize the important role of the structural elucidation of the various unique binding mechanisms of these compounds that have been optimized throughout evolution to target the proteasome. Based on this knowledge, medicinal chemists have further optimized these natural products, resulting in potential drugs with reduced off-target activities.

  3. Proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in H9c2 cells.

    PubMed

    Lee, Hyunjung; Park, Jinyoung; Kim, Eunice EunKyeong; Yoo, Young Sook; Song, Eun Joo

    2016-05-01

    The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol- induced cardiac hypertrophy. We demonstrated that cholesterol- induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol- induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure. [BMB Reports 2016; 49(5): 270-275]. PMID:26592933

  4. 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

  5. Proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in H9c2 cells

    PubMed Central

    Lee, Hyunjung; Park, Jinyoung; Kim, Eunice EunKyeong; Yoo, Young Sook; Song, Eun Joo

    2016-01-01

    The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol-induced cardiac hypertrophy. We demonstrated that cholesterol-induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol-induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure. [BMB Reports 2016; 49(5): 270-275] PMID:26592933

  6. The Role of the Ubiquitin Proteasome System in Ischemia and Ischemic Tolerance

    PubMed Central

    Meller, Robert

    2010-01-01

    Ubiquitin modification targets a protein for rapid degradation by the proteasome. However, poly-ubiquitination of proteins can result in multiple functions depending on the topology of the ubiquitin chain. Therefore ubiquitin signaling offers a more complex and versatile biology compared to many other post translational modifications. One area of potential for the application of this knowledge is the field of ischemia-induced brain damage, as occurs following a stroke. The ubiquitin proteasome system may exert a dual role on neuronal outcome following ischemia. Harmful ischemia results in an overload of the ubiquitin proteasome system, and blocking the proteasome reduces brain infarction following ischemia. However, the rapid and selective degradation of proteins following brief ischemia results in endogenous protection against ischemia. Therefore further understanding of the molecular signaling mechanisms which regulate the ubiquitin proteasome system may reveal novel therapeutic targets to reduce brain damage when ischemia is predicted, or to reduce the activation of the cell death mechanisms and the inflammatory response following stroke. The aim of this review is to discuss some of the recent advances in the understanding of protein ubiquitination and its implications for novel stroke therapies. PMID:19181875

  7. 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

  8. 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.

  9. Pri sORF peptides induce selective proteasome-mediated protein processing.

    PubMed

    Zanet, J; Benrabah, E; Li, T; Pélissier-Monier, A; Chanut-Delalande, H; Ronsin, B; Bellen, H J; Payre, F; Plaza, S

    2015-09-18

    A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

  10. 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

  11. Proteasome Impairment Induces Recovery of Mitochondrial Membrane Potential and an Alternative Pathway of Mitochondrial Fusion

    PubMed Central

    Shirozu, Ryohei; Yashiroda, Hideki

    2015-01-01

    Mitochondria are vital and highly dynamic organelles that continuously fuse and divide to maintain mitochondrial quality. Mitochondrial dysfunction impairs cellular integrity and is known to be associated with various human diseases. However, the mechanism by which the quality of mitochondria is maintained remains largely unexplored. Here we show that impaired proteasome function recovers the growth of yeast cells lacking Fzo1, a pivotal protein for mitochondrial fusion. Decreased proteasome activity increased the mitochondrial oxidoreductase protein Mia40 and the ratio of the short isoform of mitochondrial intermembrane protein Mgm1 (s-Mgm1) to the long isoform (l-Mgm1). The increase in Mia40 restored mitochondrial membrane potential, while the increase in the s-Mgm1/l-Mgm1 ratio promoted mitochondrial fusion in an Fzo1-independent manner. Our findings demonstrate a new pathway for mitochondrial quality control that is induced by proteasome impairment. PMID:26552703

  12. Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells.

    PubMed

    Lee, Jin-Gu; Takahama, Shokichi; Zhang, Guofeng; Tomarev, Stanislav I; Ye, Yihong

    2016-07-01

    To safeguard proteomic integrity, cells rely on the proteasome to degrade aberrant polypeptides, but it is unclear how cells remove defective proteins that have escaped degradation owing to proteasome insufficiency or dysfunction. Here we report a pathway termed misfolding-associated protein secretion, which uses the endoplasmic reticulum (ER)-associated deubiquitylase USP19 to preferentially export aberrant cytosolic proteins. Intriguingly, the catalytic domain of USP19 possesses an unprecedented chaperone activity, allowing recruitment of misfolded proteins to the ER surface for deubiquitylation. Deubiquitylated cargos are encapsulated into ER-associated late endosomes and secreted to the cell exterior. USP19-deficient cells cannot efficiently secrete unwanted proteins, and grow more slowly than wild-type cells following exposure to a proteasome inhibitor. Together, our findings delineate a protein quality control (PQC) pathway that, unlike degradation-based PQC mechanisms, promotes protein homeostasis by exporting misfolded proteins through an unconventional protein secretion process. PMID:27295555

  13. The ubiquitin-proteasomal system is critical for multiple myeloma: implications in drug discovery

    PubMed Central

    Cao, Biyin; Mao, Xinliang

    2011-01-01

    Bortezomib is a specific inhibitor of proteasomes, the most important protease complexes in protein degradation. Bortezomib can induce apoptosis of a variety of cancer cells, including leukemia, lymphoma, multiple myeloma, breast cancers, prostate cancers, lung cancers, and so on. However, extensive studies and overall evaluation suggested that multiple myeloma is the most sensitive and the best responsive disease which was later approved by Food and Drug Administration for bortezomib treatment. Because proteasomes are an essential component in the ubiquitin-proteasomal protein degradation pathway, the discovery of bortezomib implicates that the UPS is critical for myeloma pathophysiology. The UPS also contains ubiquitin, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), ubiquitin ligases (E3) and deubiquitinases (Dubs). In this review, we examined and analyzed the recent advancements of the UPS components in multiple myeloma and its implications in drug discovery for myeloma treatment. PMID:22432065

  14. 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

  15. Enhanced fluoride sorption by mechanochemically activated kaolinites.

    PubMed

    Meenakshi, S; Sundaram, C Sairam; Sukumar, Rugmini

    2008-05-01

    Kaolinite clay obtained from the mines was processed and studied for its fluoride sorption capacity. The surface area of the clay mineral was increased from 15.11 m(2)/g (raw) to 32.43 m(2)/g (activated) by mechanochemical activation. Batch adsorption studies were conducted to optimize various equilibrating conditions like the effect of contact time, dosage, pH for both raw and micronized kaolinites (RK and MK). The effect of other interfering anions on the defluoridation capacity (DC) of the sorbents was studied. Sorption of fluoride by the sorbents was observed over a wide pH range of 3-11. The studies revealed there is an enhanced fluoride sorption on MK. FTIR and XRD were used for the characterization of the sorbent. The surface morphology of the clay material was observed using SEM. The adsorption of fluoride was studied at three different temperatures, viz., 303, 313 and 323 K. The sorption data obtained at optimized conditions were subjected to Freundlich and Langmuir isotherms. Sorption intensity (1/n) (0.770-0.810) has been evaluated using Freundlich isotherm, whereas the values of sorption capacity Q(0) (0.609, 0.714 and 0.782 mg/g) and binding energy b (0.158, 0.145 and 0.133 L/mg) at three different temperatures have been estimated using Langmuir isotherm. Adsorption process was found to be controlled by both Freundlich and Langmuir isotherms. Thermodynamic studies revealed that the sorption of fluoride on MK is endothermic and a spontaneous process. The kinetic studies indicate that the sorption of fluoride on MK follows pseudo-first-order and intraparticle diffusion models.

  16. 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

  17. Stiff substrates enhance cultured neuronal network activity

    PubMed Central

    Zhang, Quan-You; Zhang, Yan-Yan; Xie, Jing; Li, Chen-Xu; Chen, Wei-Yi; Liu, Bai-Lin; Wu, Xiao-an; Li, Shu-Na; Huo, Bo; Jiang, Lin-Hua; Zhao, Hu-Cheng

    2014-01-01

    The mechanical property of extracellular matrix and cell-supporting substrates is known to modulate neuronal growth, differentiation, extension and branching. Here we show that substrate stiffness is an important microenvironmental cue, to which mouse hippocampal neurons respond and integrate into synapse formation and transmission in cultured neuronal network. Hippocampal neurons were cultured on polydimethylsiloxane substrates fabricated to have similar surface properties but a 10-fold difference in Young's modulus. Voltage-gated Ca2+ channel currents determined by patch-clamp recording were greater in neurons on stiff substrates than on soft substrates. Ca2+ oscillations in cultured neuronal network monitored using time-lapse single cell imaging increased in both amplitude and frequency among neurons on stiff substrates. Consistently, synaptic connectivity recorded by paired recording was enhanced between neurons on stiff substrates. Furthermore, spontaneous excitatory postsynaptic activity became greater and more frequent in neurons on stiff substrates. Evoked excitatory transmitter release and excitatory postsynaptic currents also were heightened at synapses between neurons on stiff substrates. Taken together, our results provide compelling evidence to show that substrate stiffness is an important biophysical factor modulating synapse connectivity and transmission in cultured hippocampal neuronal network. Such information is useful in designing instructive scaffolds or supporting substrates for neural tissue engineering. PMID:25163607

  18. 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 stu