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Sample records for 26s proteasome pathway

  1. Compensatory role of the Nrf2-ARE pathway against paraquat toxicity: Relevance of 26S proteasome activity.

    PubMed

    Izumi, Yasuhiko; Yamamoto, Noriyuki; Matsushima, Sayaka; Yamamoto, Takamori; Takada-Takatori, Yuki; Akaike, Akinori; Kume, Toshiaki

    2015-11-01

    Oxidative stress and the ubiquitin-proteasome system play a key role in the pathogenesis of Parkinson disease. Although the herbicide paraquat is an environmental factor that is involved in the etiology of Parkinson disease, the role of 26S proteasome in paraquat toxicity remains to be determined. Using PC12 cells overexpressing a fluorescent protein fused to the proteasome degradation signal, we report here that paraquat yielded an inhibitory effect on 26S proteasome activity without an obvious decline in 20S proteasome activity. Relative low concentrations of proteasome inhibitors caused the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is targeted to the ubiquitin-proteasome system, and activated the antioxidant response element (ARE)-dependent transcription. Paraquat also upregulated the protein level of Nrf2 without increased expression of Nrf2 mRNA, and activated the Nrf2-ARE pathway. Consequently, paraquat induced expression of Nrf2-dependent ARE-driven genes, such as γ-glutamylcysteine synthetase, catalase, and hemeoxygenase-1. Knockdown of Nrf2 or inhibition of γ-glutamylcysteine synthetase and catalase exacerbated paraquat-induced toxicity, whereas suppression of hemeoxygenase-1 did not. These data indicate that the compensatory activation of the Nrf2-ARE pathway via inhibition of 26S proteasome serves as part of a cellular defense mechanism to protect against paraquat toxicity.

  2. Attenuation of glucocorticoid signaling through targeted degradation of p300 via the 26S proteasome pathway.

    PubMed

    Li, Qiao; Su, Anna; Chen, Jihong; Lefebvre, Yvonne A; Haché, Robert J G

    2002-12-01

    The effects of acetylation on gene expression are complex, with changes in chromatin accessibility intermingled with direct effects on transcriptional regulators. For the nuclear receptors, both positive and negative effects of acetylation on specific gene transcription have been observed. We report that p300 and steroid receptor coactivator 1 interact transiently with the glucocorticoid receptor and that the acetyltransferase activity of p300 makes an important contribution to glucocorticoid receptor-mediated transcription. Treatment of cells with the deacetylase inhibitor, sodium butyrate, inhibited steroid-induced transcription and altered the transient association of glucocorticoid receptor with p300 and steroid receptor coactivator 1. Additionally, sustained sodium butyrate treatment induced the degradation of p300 through the 26S proteasome pathway. Treatment with the proteasome inhibitor MG132 restored both the level of p300 protein and the transcriptional response to steroid over 20 h of treatment. These results reveal new levels for the regulatory control of gene expression by acetylation and suggest feedback control on p300 activity.

  3. Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome

    PubMed Central

    He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

    2016-01-01

    The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

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

    PubMed

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

    2011-01-01

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

  5. Aging perturbs 26S proteasome assembly in Drosophila melanogaster

    PubMed Central

    Vernace, Vita A.; Arnaud, Lisette; Schmidt-Glenewinkel, Thomas; Figueiredo-Pereira, Maria E.

    2012-01-01

    Aging is associated with loss of quality control in protein turnover. The ubiquitin-proteasome pathway is critical to this quality control process as it degrades mutated and damaged proteins. We identified a unique aging-dependent mechanism that contributes to proteasome dysfunction in Drosophila melanogaster. Our studies are the first to show that the major proteasome form in old (43–47 days old) female and male flies is the weakly active 20S core particle, while in younger (1–32 days old) flies highly active 26S proteasomes are preponderant. Old (43–47 days) flies of both genders also exhibit a decline (~50%) in ATP levels, which is relevant to 26S proteasomes, as their assembly is ATP-dependent. The steep declines in 26S proteasome and ATP levels were observed at an age (43–47 days) when the flies exhibited a marked drop in locomotor performance, attesting that these are “old age” events. Remarkably, treatment with a proteasome inhibitor increases ubiquitinated protein levels and shortens the life span of old but not young flies. In conclusion, our data reveal a previously unknown mechanism that perturbs proteasome activity in “old-age” female and male Drosophila most likely depriving them of the ability to effectively cope with proteotoxic damages caused by environmental and/or genetic factors. PMID:17413001

  6. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

    SciTech Connect

    Kwak, Mi-Kyoung . E-mail: mkwak@yumail.ac.kr; Kensler, Thomas W.

    2006-07-14

    The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.

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

    PubMed

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

    2015-12-07

    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.

  8. Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

    PubMed Central

    Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

    2015-01-01

    Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

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

    PubMed Central

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

    2010-01-01

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

  10. The Regulatory Complex of Drosophila melanogaster 26s Proteasomes

    PubMed Central

    Hölzl, Harald; Kapelari, Barbara; Kellermann, Josef; Seemüller, Erika; Sümegi, Máté; Udvardy, Andor; Medalia, Ohad; Sperling, Joseph; Müller, Shirley A.; Engel, Andreas; Baumeister, Wolfgang

    2000-01-01

    Drosophila melanogaster embryos are a source for homogeneous and stable 26S proteasomes suitable for structural studies. For biochemical characterization, purified 26S proteasomes were resolved by two-dimensional (2D) gel electrophoresis and subunits composing the regulatory complex (RC) were identified by amino acid sequencing and immunoblotting, before corresponding cDNAs were sequenced. 17 subunits from Drosophila RCs were found to have homologues in the yeast and human RCs. An additional subunit, p37A, not yet described in RCs of other organisms, is a member of the ubiquitin COOH-terminal hydrolase family (UCH). Analysis of EM images of 26S proteasomes-UCH-inhibitor complexes allowed for the first time to localize one of the RC's specific functions, deubiquitylating activity. The masses of 26S proteasomes with either one or two attached RCs were determined by scanning transmission EM (STEM), yielding a mass of 894 kD for a single RC. This value is in good agreement with the summed masses of the 18 identified RC subunits (932 kD), indicating that the number of subunits is complete. PMID:10893261

  11. Conformational switching of the 26S proteasome enables substrate degradation.

    PubMed

    Matyskiela, Mary E; Lander, Gabriel C; Martin, Andreas

    2013-07-01

    The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the 26S proteasome structure from Saccharomyces cerevisiae during substrate degradation, showing that the regulatory particle switches from a preengaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Notably, Rpn11 moves from an occluded position to directly above the central pore, thus facilitating substrate deubiquitination concomitant with translocation.

  12. 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.

  13. [Structures and functions of the 26S proteasome Rpn10 family].

    PubMed

    Kawahara, Hiroyuki

    2002-09-01

    The ubiquitin-dependent proteolytic pathway is thought to be one of the vital systems for cellular regulations, including control of the cell cycle, differentiation and apoptosis. In this pathway, poly-ubiquitinated proteins are selectively degraded by the 26S proteasome, a multisubunit proteolytic machinery. Recognition of the poly-ubiquitin chain by the 26S proteasome should be a key step leading to the selective degradation of target proteins, and the Rpn10 subunit of the 26S proteasome has been shown to preferentially bind the poly-ubiquitin chain in vitro. We previously reported that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated, alternative splicing. To determine whether such alternative splicing mechanisms occur in organisms other than the mouse, we searched for Rpn10 isoforms in various species. Here we summarize the gene organization of the Rpn10 in lower species and provide evidence that the competence for generating all distinct forms of Rpn10 alternative splicing has expanded through evolution. Some of the Rpn10 family genes were found to be expressed in distinct developmental stages, suggesting that they have distinct functions during embryogenesis. For example, Rpn10c and Rpn10e were exclusively expressed at specific developmental stages and in specific tissues, while Rpn10a was expressed constitutively. Our experimental results indicate that the respective Rpn10 proteins possess distinct roles in the progression of development. Furthermore, some of the Rpn10 variants specifically interacted with important developmental regulators.

  14. D1 dopamine receptor stimulation impairs striatal proteasome activity in Parkinsonism through 26S proteasome disassembly.

    PubMed

    Barroso-Chinea, Pedro; Thiolat, Marie-Laure; Bido, Simone; Martinez, Audrey; Doudnikoff, Evelyne; Baufreton, Jérôme; Bourdenx, Mathieu; Bloch, Bertrand; Bezard, Erwan; Martin-Negrier, Marie-Laure

    2015-06-01

    Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization.

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

    PubMed

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

    2012-01-31

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

  16. KIAA0368-deficiency affects disassembly of 26S proteasome under oxidative stress condition.

    PubMed

    Haratake, Kousuke; Sato, Akitsugu; Tsuruta, Fuminori; Chiba, Tomoki

    2016-06-01

    Many cellular stresses cause damages of intracellular proteins, which are eventually degraded by the ubiquitin and proteasome system. The proteasome is a multicatalytic protease complex composed of 20S core particle and the proteasome activators that regulate the proteasome activity. Extracellular mutants 29 (Ecm29) is a 200 kDa protein encoded by KIAA0368 gene, associates with the proteasome, but its role is largely unknown. Here, we generated KIAA0368-deficient mice and investigated the function of Ecm29 in stress response. KIAA0368-deficient mice showed normal peptidase activity and proteasome formation at normal condition. Under stressed condition, 26S proteasome dissociates in wild-type cells, but not in KIAA0368(-/-) cells. This response was correlated with efficient degradation of damaged proteins and resistance to oxidative stress of KIAA0368(-/-) cells. Thus, Ecm29 is involved in the dissociation process of 26S proteasome, providing clue to analyse the mechanism of proteasomal degradation under various stress condition.

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

    PubMed Central

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

    2016-01-01

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

  18. Inherent asymmetry in the 26S proteasome is defined by the ubiquitin receptor RPN13.

    PubMed

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

    2014-02-28

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

  19. Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome.

    PubMed

    Pack, Chan-Gi; Yukii, Haruka; Toh-e, Akio; Kudo, Tai; Tsuchiya, Hikaru; Kaiho, Ai; Sakata, Eri; Murata, Shigeo; Yokosawa, Hideyoshi; Sako, Yasushi; Baumeister, Wolfgang; Tanaka, Keiji; Saeki, Yasushi

    2014-03-06

    The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells. Here, we investigate the absolute concentration, spatio-temporal dynamics and complex formation of the proteasome in living cells using fluorescence correlation spectroscopy. We find that the 26S proteasome complex is highly mobile, and that almost all proteasome subunits throughout the cell are stably incorporated into 26S proteasomes. The interaction between 19S and 20S particles is stable even in an importin-α mutant, suggesting that the 26S proteasome is assembled in the cytoplasm. Furthermore, a genetically stabilized 26S proteasome mutant is able to enter the nucleus. These results suggest that the 26S proteasome completes its assembly process in the cytoplasm and translocates into the nucleus through the nuclear pore complex as a holoenzyme.

  20. The Cdc48-Vms1 complex maintains 26S proteasome architecture.

    PubMed

    Tran, Joseph R; Brodsky, Jeffrey L

    2014-03-15

    The 26S proteasome is responsible for most regulated protein turnover and for the degradation of aberrant proteins in eukaryotes. The assembly of this ~2.5 MDa multicatalytic protease requires several dedicated chaperones and, once assembled, substrate selectivity is mediated by ubiquitin conjugation. After modification with ubiquitin, substrates are escorted to the proteasome by myriad factors, including Cdc48 (cell-division cycle 48). Cdc48 also associates with numerous cofactors, but, to date, it is unclear whether each cofactor facilitates proteasome delivery. We discovered that yeast lacking a conserved Cdc48 cofactor, Vms1 [VCP (valosin-containing protein)/Cdc48-associated mitochondrial stress-responsive], accumulate proteasome-targeted ubiquitinated proteins. Vms1 mutant cells also contain elevated levels of unassembled 20S proteasome core particles and select 19S cap subunits. In addition, we found that the ability of Vms1 to support 26S proteasome assembly requires Cdc48 interaction, and that the loss of Vms1 reduced 26S proteasome levels and cell viability after prolonged culture in the stationary phase. The results of the present study highlight an unexpected link between the Cdc48-Vms1 complex and the preservation of proteasome architecture, and indicate how perturbed proteasome assembly affects the turnover of ubiquitinated proteins and maintains viability in aging cells.

  1. The Cdc48–Vms1 complex maintains 26S proteasome architecture

    PubMed Central

    Tran, Joseph R.; Brodsky, Jeffrey L.

    2014-01-01

    The 26S proteasome is responsible for most regulated protein turnover and for the degradation of aberrant proteins in eukaryotes. The assembly of this ~2.5 MDa multicatalytic protease requires several dedicated chaperones and, once assembled, substrate selectivity is mediated by ubiquitin conjugation. After modification with ubiquitin, substrates are escorted to the proteasome by myriad factors, including Cdc48 (cell-division cycle 48). Cdc48 also associates with numerous cofactors, but, to date, it is unclear whether each cofactor facilitates proteasome delivery. We discovered that yeast lacking a conserved Cdc48 cofactor, Vms1 [VCP (valosin-containing protein)/Cdc48-associated mitochondrial stress-responsive], accumulate proteasome-targeted ubiquitinated proteins. Vms1 mutant cells also contain elevated levels of unassembled 20S proteasome core particles and select 19S cap subunits. In addition, we found that the ability of Vms1 to support 26S proteasome assembly requires Cdc48 interaction, and that the loss of Vms1 reduced 26S proteasome levels and cell viability after prolonged culture in the stationary phase. The results of the present study highlight an unexpected link between the Cdc48–Vms1 complex and the preservation of proteasome architecture, and indicate how perturbed proteasome assembly affects the turnover of ubiquitinated proteins and maintains viability in aging cells. PMID:24351022

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

    PubMed Central

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

    2009-01-01

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

  3. 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.

  4. The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome*

    PubMed Central

    Jacobson, Andrew D.; Zhang, Nan-Yan; Xu, Ping; Han, Ke-Jun; Noone, Seth; Peng, Junmin; Liu, Chang-Wei

    2009-01-01

    The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates. PMID:19858201

  5. Differential expression of 26S proteasome subunits and functional activity during neonatal development.

    PubMed

    Claud, Erika C; McDonald, Julie A K; He, Shu-Mei; Yu, Yueyue; Duong, Lily; Sun, Jun; Petrof, Elaine O

    2014-08-29

    Proteasomes regulate many essential cellular processes by degrading intracellular proteins. While aging is known to be associated with dysfunction of the proteasome, there are few reports detailing activity and function of proteasomes in the early stages of life. To elucidate the function and development of mammalian proteasomes, 26S proteasomes were affinity-purified from rat intestine, spleen and liver. The developmental expression of core, regulatory and immunoproteasome subunits was analyzed by immunoblotting and reverse-transcriptase PCR of mRNA subunits, and proteasome catalytic function was determined by fluorogenic enzymatic assays. The expression of core (β2, β5, α7 and β1) and regulatory (Rpt5) subunits was found to be present at low levels at birth and increased over time particularly at weaning. In contrast, while gradual developmental progression of proteasome structure was also seen with the immunoproteasome subunits (β1i, β5i, and β2i), these were not present at birth. Our studies demonstrate a developmental pattern to 26S proteasome activity and subunit expression, with low levels of core proteasome components and absence of immunoproteasomes at birth followed by increases at later developmental stages. This correlates with findings from other studies of a developmental hyporesponsiveness of the adaptive immune system to allow establishment of microbial colonization immediately after birth.

  6. Structure of an endogenous yeast 26S proteasome reveals two major conformational states.

    PubMed

    Luan, Bai; Huang, Xiuliang; Wu, Jianping; Mei, Ziqing; Wang, Yiwei; Xue, Xiaobin; Yan, Chuangye; Wang, Jiawei; Finley, Daniel J; Shi, Yigong; Wang, Feng

    2016-03-08

    The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.

  7. 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

  8. Structure of an endogenous yeast 26S proteasome reveals two major conformational states

    PubMed Central

    Luan, Bai; Huang, Xiuliang; Wu, Jianping; Mei, Ziqing; Wang, Yiwei; Xue, Xiaobin; Yan, Chuangye; Wang, Jiawei; Finley, Daniel J.; Shi, Yigong; Wang, Feng

    2016-01-01

    The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function. PMID:26929360

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 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

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

    PubMed

    Sihn, Choong-Ryoul; Cho, Si Young; Lee, Jeong Ho; Lee, Tae Ryong; Kim, Sang Hoon

    2007-04-27

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

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

    SciTech Connect

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

    2013-05-31

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

  13. Precise assembly and regulation of 26S proteasome and correlation between proteasome dysfunction and neurodegenerative diseases

    PubMed Central

    Im, Eunju; Chung, Kwang Chul

    2016-01-01

    Neurodegenerative diseases (NDs) often involve the formation of abnormal and toxic protein aggregates, which are thought to be the primary factor in ND occurrence and progression. Aged neurons exhibit marked increases in aggregated protein levels, which can lead to increased cell death in specific brain regions. As no specific drugs/therapies for treating the symptoms or/and progression of NDs are available, obtaining a complete understanding of the mechanism underlying the formation of protein aggregates is needed for designing a novel and efficient removal strategy. Intracellular proteolysis generally involves either the lysosomal or ubiquitin-proteasome system. In this review, we focus on the structure and assembly of the proteasome, proteasome-mediated protein degradation, and the multiple dynamic regulatory mechanisms governing proteasome activity. We also discuss the plausibility of the correlation between changes in proteasome activity and the occurrence of NDs. [BMB Reports 2016; 49(9): 459-473] PMID:27312603

  14. Precise assembly and regulation of 26S proteasome and correlation between proteasome dysfunction and neurodegenerative diseases.

    PubMed

    Im, Eunju; Chung, Kwang Chul

    2016-09-01

    Neurodegenerative diseases (NDs) often involve the formation of abnormal and toxic protein aggregates, which are thought to be the primary factor in ND occurrence and progression. Aged neurons exhibit marked increases in aggregated protein levels, which can lead to increased cell death in specific brain regions. As no specific drugs/therapies for treating the symptoms or/and progression of NDs are available, obtaining a complete understanding of the mechanism underlying the formation of protein aggregates is needed for designing a novel and efficient removal strategy. Intracellular proteolysis generally involves either the lysosomal or ubiquitin-proteasome system. In this review, we focus on the structure and assembly of the proteasome, proteasome-mediated protein degradation, and the multiple dynamic regulatory mechanisms governing proteasome activity. We also discuss the plausibility of the correlation between changes in proteasome activity and the occurrence of NDs. [BMB Reports 2016; 49(9): 459-473].

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-08

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

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

    PubMed Central

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

    2016-01-01

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

  18. Structural insights into the functional cycle of the ATPase module of the 26S proteasome.

    PubMed

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

    2017-02-07

    In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA(+) ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA(+) ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Formation of an intricate helical bundle dictates the assembly of the 26S proteasome lid.

    PubMed

    Estrin, Eric; Lopez-Blanco, José Ramón; Chacón, Pablo; Martin, Andreas

    2013-09-03

    The 26S proteasome is the major ATP-dependent protease in eukaryotes and thus involved in regulating a diverse array of vital cellular processes. Three subcomplexes form this massive degradation machine: the lid, the base, and the core. While assembly of base and core has been well-studied, the detailed molecular mechanisms involved in formation of the nine-subunit lid remain largely unknown. Here, we reveal that helices found at the C terminus of each lid subunit form a helical bundle that directs the ordered self-assembly of the lid subcomplex. Furthermore, we use an integrative modeling approach to gain critical insights into the bundle topology and provide an important structural framework for our biochemical data. We show that the helical bundle serves as a hub through which the last-added subunit Rpn12 monitors proper lid assembly before incorporation into the proteasome. Finally, we predict that the assembly of the COP9 signalosome depends on a similar helical bundle.

  4. Structural insights into the functional cycle of the ATPase module of the 26S proteasome

    PubMed Central

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

    2017-01-01

    In eukaryotic cells, the ubiquitin–proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis. PMID:28115689

  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. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

    PubMed Central

    Braten, Ori; Livneh, Ido; Ziv, Tamar; Admon, Arie; Kehat, Izhak; Caspi, Lilac H.; Gonen, Hedva; Bercovich, Beatrice; Godzik, Adam; Jahandideh, Samad; Jaroszewski, Lukasz; Sommer, Thomas; Kwon, Yong Tae; Guharoy, Mainak; Tompa, Peter; Ciechanover, Aaron

    2016-01-01

    The “canonical” proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established—in both human and yeast cells—a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes. PMID:27385826

  7. The RPN1 Subunit of the 26S Proteasome in Arabidopsis Is Essential for Embryogenesis

    PubMed Central

    Brukhin, Vladimir; Gheyselinck, Jacqueline; Gagliardini, Valeria; Genschik, Pascal; Grossniklaus, Ueli

    2005-01-01

    The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates. PMID:16169895

  8. An evolutionarily conserved pathway controls proteasome homeostasis

    PubMed Central

    Rousseau, Adrien; Bertolotti, Anne

    2016-01-01

    The proteasome is essential for the selective degradation of most cellular proteins but how cells maintain adequate amounts of proteasome is unclear. Here we found an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1 whose inhibition 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, ensured that the supply of RACs and proteasome subunits increased under challenging conditions 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 to the rising needs. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation. PMID:27462806

  9. 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.

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

    PubMed

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

    2015-09-22

    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.

  11. Evidence that the Arabidopsis Ubiquitin C-terminal Hydrolases 1 and 2 associate with the 26S proteasome and the TREX-2 complex.

    PubMed

    Tian, Gang; Lu, Qing; Kohalmi, Susanne E; Rothstein, Steven J; Cui, Yuhai

    2012-11-01

    The 26S proteasome interacts with a number of different proteins, while the TREX-2 complex is an important component of the mRNA export machinery. In animals and yeast, members of the Ubiquitin C-terminal Hydrolase 37 (UCH37) family are found to associate with the 26S proteasome, but this has not been demonstrated in plants. The Arabidopsis UCH1 and UCH2 are orthologous to UCH37. Here, we show that UCH1 and UCH2 interact with the 26S proteasome lid subunits. In addition, the two UCHs also interact with TREX-2 components. Our data suggest that Arabidopsis UCHs may serve as a link between the 26S proteasome lid complex and the TREX-2 complex.

  12. The role of ubiquitin and the 26S proteasome in plant abiotic stress signaling

    PubMed Central

    Stone, Sophia L.

    2014-01-01

    Ubiquitin is a small, highly conserved, ubiquitously expressed eukaryotic protein with immensely important and diverse regulatory functions. A well-studied function of ubiquitin is its role in selective proteolysis by the ubiquitin-proteasome system (UPS). The UPS has emerged as an integral player in plant response and adaptation to environmental stresses such as drought, salinity, cold and nutrient deprivation. The UPS has also been shown to influence the production and signal transduction of stress-related hormones such as abscisic acid. Understanding UPS function has centered mainly on defining the role of E3 ubiquitin ligases, which are the substrate-recruiting component of the ubiquitination pathway. The recent identification of stress signaling/regulatory proteins that are the subject of ubiquitin-dependent degradation has increased our knowledge of how the UPS facilitates responses to adverse environmental conditions. A brief overview is provided on role of the UPS in modulating protein stability during abiotic stress signaling. E3 ubiquitin ligases for which stress-related substrate proteins have been identified are discussed. PMID:24795732

  13. Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

    PubMed Central

    Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

    2015-01-01

    ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome

  14. Noncovalent inhibitors of human 20S and 26S proteasome based on trypsin inhibitor SFTI-1.

    PubMed

    Dębowski, Dawid; Cichorek, Mirosława; Lubos, Marta; Wójcik, Sławomir; Łęgowska, Anna; Rolka, Krzysztof

    2016-09-01

    Sunflower trypsin inhibitor (SFTI-1) is recognized as an attractive scaffold to designed potent inhibitors of various proteases. We have recently found that its analogues inhibit noncovalently both human and yeast 20S proteasomes. Here, a set of novel and more potent in vitro inhibitors is presented. The inhibitory potency of the peptides was assessed with human 20S proteasome in the presence or absence of sodium dodecyl sulfate and with human 26 proteasome. Their antiproliferative action against tumor (human melanoma cells A375) and normal cells (46 BR.1N human fibroblasts and HaCaT keratinocytes) was determined. The selected fluoresceine-labeled inhibitors were able to internalize into A375 cells and were sometimes present as foci in the cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 685-696, 2016.

  15. Structural insights into the COP9 signalosome and its common architecture with the 26S proteasome lid and eIF3.

    PubMed

    Enchev, Radoslav I; Schreiber, Anne; Beuron, Fabienne; Morris, Edward P

    2010-03-14

    The evolutionary conserved COP9 signalosome (CSN), a large multisubunit complex, plays a central role in regulating ubiquitination and cell signaling. Here we report recombinant insect cell expression and two-step purification of human CSN and demonstrate its functional assembly. We further obtain a three-dimensional structure of both native and recombinant CSN using electron microscopy and single particle analysis. Antibody labeling of CSN5 and segmentation of the structure suggest a likely subunit distribution and the architecture of its helical repeat subunits is revealed. We compare the structure of CSN with its homologous complexes, the 26S proteasome lid and eIF3, and propose a conserved architecture implying similar assembly pathways and/or conserved substrate interaction modes.

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

    SciTech Connect

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

    2012-09-07

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

  17. The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

    PubMed

    Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

    2016-02-01

    The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

  18. Using siRNA techniques to dissect proteasome assembly pathways in mammalian cells.

    PubMed

    Kaneko, Takeumi; Murata, Shigeo

    2012-01-01

    The 26S proteasome is an ATP-dependent protease known to collaborate with ubiquitin, the polymerization of which acts as a marker for protein degradation in eukaryotic cells, and is involved in a diverse array of biological processes, such as the cell-cycle progression, DNA repair, apoptosis, immune response, signal transduction, transcription, metabolism, protein quality control, and developmental program. The 26S proteasome is a huge protease complex and consists of one catalytic core called the 20S proteasome (or 20S core particle) and one or two 19S regulatory particles (19S RP), which include 14 and 19 different subunits, respectively. Recent studies have revealed that the proteasome formation requires multiple assembly factors and that the assembly pathways are highly conserved between yeast and mammalian cells. This chapter is focused on experimental approaches to reveal the assembly pathways of the proteasome using small interfering RNA techniques in mammalian cells. Knockdown of a proteasome subunit causes arrest of the assembly process before incorporation of the targeted subunit and accumulation of a specific intermediate.

  19. A pilot study to investigate the role of the 26S proteasome in radiotherapy resistance and loco-regional recurrence following breast conserving therapy for early breast cancer.

    PubMed

    Elfadl, Dalia; Hodgkinson, Victoria C; Long, Ervine D; Scaife, Lucy; Drew, Philip J; Lind, Michael J; Cawkwell, Lynn

    2011-08-01

    Breast conserving therapy is a currently accepted method for managing patients with early stage breast cancer. However, approximately 7% of patients may develop loco-regional tumour recurrence within 5 years. We previously reported that expression of the 26S proteasome may be associated with radio-resistance. Here we aimed to analyse the 26S proteasome in a pilot series of early breast cancers and correlate the findings with loco-regional recurrence. Fourteen patients with early breast cancer who developed loco-regional recurrence within 4 years of completing breast conserving therapy were selected according to strict criteria and compared with those from 14 patients who were disease-free at 10 years. Decreased expression of the 26S proteasome was significantly associated with radio-resistance, manifested as the development of a loco-regional recurrence within 4 years of breast conserving therapy (p = 0.018). This small pilot study provides further suggestion that the 26S proteasome may be associated with response to radiotherapy.

  20. Inhibition of the 26S proteasome by peptide mimics of the coiled-coil region of its ATPase subunits.

    PubMed

    Inobe, Tomonao; Genmei, Reiko

    Regulation of proteasomal degradation is an indispensable tool for biomedical studies. Thus, there is demand for novel proteasome inhibitors. Proteasomal degradation requires formation of coiled-coil structure by the N-terminal region of ATPase subunits of the proteasome cap. Here we show that peptides that mimic the N-terminal coiled-coil region of ATPase subunits interfere with proteasome function. These results suggest that coiled-coil peptides represent promising new proteasome inhibitors and that N-terminal coiled-coil regions of ATPase subunits are targets for proteasome inhibition.

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

    PubMed Central

    Wang, Feng; Deng, Xing Wang

    2011-01-01

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

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

    PubMed

    Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu; Kim, Ki-Jeong; Park, Chang-Jin; Kim, Young-Jin; Park, Ohkmae K; Paek, Kyung-Hee

    2006-12-15

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

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

    SciTech Connect

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

    2006-12-15

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

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

    PubMed Central

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

    2010-01-01

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

  5. The effect of temperature adaptation on the ubiquitin-proteasome pathway in notothenioid fishes.

    PubMed

    Todgham, Anne E; Crombie, Timothy A; Hofmann, Gretchen E

    2017-02-01

    There is an accumulating body of evidence suggesting that the sub-zero Antarctic marine environment places physiological constraints on protein homeostasis. Levels of ubiquitin (Ub)-conjugated proteins, 20S proteasome activity and mRNA expression of many proteins involved in both the Ub tagging of damaged proteins as well as the different complexes of the 26S proteasome were measured to examine whether there is thermal compensation of the Ub-proteasome pathway in Antarctic fishes to better understand the efficiency of the protein degradation machinery in polar species. Both Antarctic (Trematomus bernacchii, Pagothenia borchgrevinki) and non-Antarctic (Notothenia angustata, Bovichtus variegatus) notothenioids were included in this study to investigate the mechanisms of cold adaptation of this pathway in polar species. Overall, there were significant differences in the levels of Ub-conjugated proteins between the Antarctic notothenioids and B. variegatus, with N. angustata possessing levels very similar to those of the Antarctic fishes. Proteasome activity in the gills of Antarctic fishes demonstrated a high degree of temperature compensation such that activity levels were similar to activities measured in their temperate relatives at ecologically relevant temperatures. A similar level of thermal compensation of proteasome activity was not present in the liver of two Antarctic fishes. Higher gill proteasome activity is likely due in part to higher cellular levels of proteins involved in the Ub-proteasome pathway, as evidenced by high mRNA expression of relevant genes. Reduced activity of the Ub-proteasome pathway does not appear to be the mechanism responsible for elevated levels of denatured proteins in Antarctic fishes, at least in the gills.

  6. 26S and PA28-20S Proteasome Activity in Cytosolic Extracts from Embryonic Stem Cells.

    PubMed

    Hernebring, Malin

    2016-01-01

    The proteasome is a complex multisubunit protease that plays a major role in the degradation of proteins in eukaryotic cells. Proteasome function is one of the key players regulating the proteome and it is vital for many cellular processes. The method described here makes it possible to assay the proteolytic capacities of proteasome complexes separately in crude cytosolic extracts from ES cells. The method is based on hydrolysis of a fluorogenic peptide substrate in lysates prepared under conditions that favor the interactions of the 20S proteasomal catalytical core with either the 19S or the PA28αβ proteasome regulator.

  7. Gankyrin is an ankyrin-repeat oncoprotein that interacts with CDK4 kinase and the S6 ATPase of the 26 S proteasome.

    PubMed

    Dawson, Simon; Apcher, Sebastien; Mee, Maureen; Higashitsuji, Hiroaki; Baker, Rohan; Uhle, Stefan; Dubiel, Wolfgang; Fujita, Jun; Mayer, R John

    2002-03-29

    A yeast two-hybrid screen with the human S6 (TBP7, RPT3) ATPase of the 26 S proteasome has identified gankyrin, a liver oncoprotein, as an interacting protein. Gankyrin interacts with both free and regulatory complex-associated S6 ATPase and is not stably associated with the 26 S particle. Deletional mutagenesis shows that the C-terminal 78 amino acids of the S6 ATPase are necessary and sufficient to mediate the interaction with gankyrin. Deletion of an orthologous gene in Saccharomyces cerevisiae suggests that it is dispensable for cell growth and viability. Overexpression and precipitation of tagged gankyrin from cultured cells detects a complex containing co-transfected tagged S6 ATPase (or endogenous S6) and endogenous cyclin D-dependent kinase CDK4. The proteasomal ATPases are part of the AAA (ATPases associated with diverse cellular activities) family, members of which are molecular chaperones; gankyrin complexes may therefore influence CDK4 function during oncogenesis.

  8. New insights into the role of the ubiquitin-proteasome pathway in the regulation of apoptosis.

    PubMed

    Liu, Cui-Hua; Goldberg, Alfred L; Qiu, Xiao-Bo

    2007-01-01

    The ubiquitin-proteasome pathway (UPP) is the major system responsible for degradation of intracellular proteins in eukaryotes. By controlling the levels of key proteins, it regulates almost all of the cellular activities, including cell cycle progression, DNA replication and repair, transcription, protein quality control, immune response, and apoptosis. UPP is composed of the ubiquitination system that marks proteins for degradation and the proteasome which degrades the ubiquitinated proteins. The 26S proteasome is a 2400 kDa complex consisting of more than 40 subunits. Following ubiquitination catalyzed by the ubiquitin activating enzyme (El), a ubiquitin-carrier protein (E2), and one of the cell's many ubiquitin-protein ligases (E3s), the protein substrates are targeted to the proteasome for degradation into small peptides. E3s regulate the degradation of protein substrates indirectly by determining both the specificity and timing of substrate ubiquitination, whereas the deubiquitinating enzymes can inhibit this process by releasing ubiquitin from substrates. In this review, we attempt to highlight the recent progress in research on UPP and its role in the regulation of apoptosis by focusing on several of its important components, including the ubiqutin ligase Nrdp 1, which regulates ErbB/EGFR family of receptor tyrosine kinases, the ubiquitin-carrier protein BRUCE/Apollon (an Inhibitor of Apoptosis Protein), and the novel proteasome subunit hRpnl3 (a binding site for the deubiquitinating enzyme, UCH37).

  9. Combination of quercetin and tannic acid in inhibiting 26S proteasome affects S5a and 20S expression, and accumulation of ubiquitin resulted in apoptosis in cancer chemoprevention.

    PubMed

    Chang, Tsui-Ling; Wang, Chi-Hsien

    2013-04-01

    To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or β-casein, and (5) inhibit β-casein degradation by the 26S proteasome in vitro. Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination.

  10. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  11. PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana.

    PubMed

    Cho, Seok Keun; Bae, Hansol; Ryu, Moon Young; Wook Yang, Seong; Kim, Woo TaeK

    2015-09-04

    Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress.

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

    SciTech Connect

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

    2007-04-10

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

  13. Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

    ERIC Educational Resources Information Center

    Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

    2006-01-01

    This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

  14. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    ERIC Educational Resources Information Center

    Hegde, Ashok N.

    2010-01-01

    Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for…

  15. 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

  16. Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

    PubMed Central

    Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

    2013-01-01

    Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

  17. Stress-responsive mitogen-activated protein kinases interact with the EAR motif of a poplar zinc finger protein and mediate its degradation through the 26S proteasome.

    PubMed

    Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand

    2011-11-01

    Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

  18. Activity-based profiling of the proteasome pathway during hepatitis C virus infection.

    PubMed

    Nasheri, Neda; Ning, Zhibin; Figeys, Daniel; Yao, Shao; Goto, Natalie K; Pezacki, John Paul

    2015-11-01

    Hepatitis C virus (HCV) infection often leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The stability of the HCV proteins is controlled by ubiquitin-dependent and ubiquitin-independent proteasome pathways. Many viruses modulate proteasome function for their propagation. To examine the interrelationship between HCV and the proteasome pathways we employed a quantitative activity-based protein profiling method. Using this approach we were able to quantify the changes in the activity of several proteasome subunits and found that proteasome activity is drastically reduced by HCV replication. The results imply a link between the direct downregulation of the activity of this pathway and chronic HCV infection.

  19. Yeast counterparts of subunits S5a and p58 (S3) of the human 26S proteasome are encoded by two multicopy suppressors of nin1-1.

    PubMed Central

    Kominami, K; Okura, N; Kawamura, M; DeMartino, G N; Slaughter, C A; Shimbara, N; Chung, C H; Fujimuro, M; Yokosawa, H; Shimizu, Y; Tanahashi, N; Tanaka, K; Toh-e, A

    1997-01-01

    Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome. Images PMID:9017604

  20. Ubiquitin-proteasome pathway components as therapeutic targets for CNS maladies.

    PubMed

    Upadhya, Sudarshan C; Hegde, Ashok N

    2005-01-01

    In the central nervous system (CNS), abnormal deposition of insoluble protein aggregates or inclusion bodies within nerve cells is commonly observed in association with several neurodegenerative diseases. The ubiquitinated protein aggregates are believed to result from malfunction or overload of the ubiquitin-proteasome pathway or from structural changes in the protein substrates which prevent their recognition and degradation by the ubiquitin-proteasome pathway. Impaired proteolysis might also contribute to the synaptic dysfunction seen early in neurodegenerative diseases because the ubiquitin-proteasome pathway is known to play a role in normal functioning of synapses. Because specificity of the ubiquitin proteasome mediated proteolysis is determined by specific ubiquitin ligases (E3s), identification of specific E3s and their allosteric modulators are likely to provide effective therapeutic targets for the treatment of several CNS disorders. Another unexplored area for the discovery of drug targets is the proteasome. Although many inhibitors of the proteasome are available, no effective drugs exist that can stimulate the proteasome. Since abnormal protein aggregation is a common feature of different neurodegenerative diseases, enhancement of proteasome activity might be an efficient way to remove the aggregates that accumulate in the brain. In this review, we discuss how the components of the ubiquitin-proteasome pathway could be potential targets for therapy of CNS diseases and disorders.

  1. N-Myristoylation of the Rpt2 subunit of the yeast 26S proteasome is implicated in the subcellular compartment-specific protein quality control system.

    PubMed

    Kimura, Ayuko; Kurata, Yoichi; Nakabayashi, Jun; Kagawa, Hiroyuki; Hirano, Hisashi

    2016-01-01

    Ubiquitination is the posttranslational modification of a protein by covalent attachment of ubiquitin. Controlled proteolysis via the ubiquitin-proteasome system (\\UPS) alleviates cellular stress by clearing misfolded proteins. In budding yeast, UPS within the nucleus degrades the nuclear proteins as well as proteins imported from the cytoplasm. While the predominantly nuclear localization of the yeast proteasome is maintained by the importin-mediated transport, N-myristoylation of the proteasome subunit Rpt2 was indicated to cause dynamic nucleo-cytoplasmic localization of proteasomes. Here, we quantitatively analyzed the ubiquitinated peptides using anti-K-ε-GG antibody in yeast cell lines with or without a mutation in the N-myristoylation site of Rpt2 and detected upregulated ubiquitination of proteins with nucleo-cytoplasmic localizations in the mutant strains. Moreover, both the protein and ubiquitinated peptide levels of two Hsp70 family chaperones involved in the nuclear import of misfolded proteins, Ssa and Sse1, were elevated in the mutant strains, whereas levels of an Hsp70 family chaperone involved in the nuclear export, Ssb, were reduced. Taken together, our results indicate that N-myristoylation of Rpt2 is involved in controlled proteolysis via regulation of the nucleo-cytoplasmic localization of the yeast proteasome.

  2. Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae.

    PubMed

    Fukunaga, Keisuke; Kudo, Tai; Toh-e, Akio; Tanaka, Keiji; Saeki, Yasushi

    2010-06-11

    The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Delta rpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.

  3. Purification, crystallization and preliminary X-ray data collection of the N-terminal domain of the 26S proteasome regulatory subunit p27 and its complex with the ATPase domain of Rpt5 from Mus musculus.

    PubMed

    Diao, Wentao; Yang, Xue; Zhou, Hao

    2014-05-01

    The protein 26S proteasome regulatory subunit p27 is one of the four chaperones that help in the assembly of the 19S regulatory particle (RP) of the 26S proteasome. In the present work, the N-terminus of p27 (residues 1-128) from Mus musculus was cloned, expressed, purified and crystallized alone and in complex with the C-terminal ATPase domain of Rpt5 (residues 173-442). The crystals of p27((1-128)) diffracted to 1.7 Å resolution and belonged to space group P212121, with unit-cell parameters a = 26.79, b = 30.39, c = 145.06 Å. Resolution-dependent Matthews coefficient probability analysis suggested the presence of only one molecule per asymmetric unit, with 40.5% solvent content and a VM value of 2.02 Å(3) Da(-1). The crystal of the p27((1-128))-Rpt5((173-442)) complex diffracted to 4 Å resolution and belonged to space group P222, with unit-cell parameters a = 75.93, b = 76.08, c = 336.85 Å. The presence of four heterodimers in the asymmetric unit with 53.2% solvent content and a VM value of 2.63 Å(3) Da(-1) or five heterodimers in the asymmetric unit with 41.5% solvent content and a VM value of 2.10 Å(3) Da(-1) is assumed.

  4. The mechanism for molecular assembly of the proteasome.

    PubMed

    Sahara, Kazutaka; Kogleck, Larissa; Yashiroda, Hideki; Murata, Shigeo

    2014-01-01

    In eukaryotic cells, the ubiquitin proteasome system plays important roles in diverse cellular processes. The 26S proteasome is a large enzyme complex that degrades ubiquitinated proteins. It consists of 33 different subunits that form two subcomplexes, the 20S core particle and the 19S regulatory particle. Recently, several chaperones dedicated to the accurate assembly of this protease complex have been identified, but the complete mechanism of the 26S proteasome assembly is still unclear. In this review, we summarize what is known about the assembly of proteasome to date and present our group's recent findings on the role of the GET pathway in the assembly of the 26S proteasome, in addition to its role in mediating the insertion of tail-anchored (TA) proteins into the ER membrane.

  5. High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

    PubMed

    Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

    2007-02-01

    To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

  6. Proteasome Dysfunction Activates Autophagy and the Keap1-Nrf2 Pathway*

    PubMed Central

    Kageyama, Shun; Sou, Yu-shin; Uemura, Takefumi; Kametaka, Satoshi; Saito, Tetsuya; Ishimura, Ryosuke; Kouno, Tsuguka; Bedford, Lynn; Mayer, R. John; Lee, Myung-Shik; Yamamoto, Masayuki; Waguri, Satoshi; Tanaka, Keiji; Komatsu, Masaaki

    2014-01-01

    The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis. PMID:25049227

  7. Molecular Pathways: Turning Proteasomal Protein Degradation into a Unique Treatment Approach

    PubMed Central

    Stintzing, Sebastian; Lenz, Heinz-Josef

    2015-01-01

    Cancer treatment regimens have evolved from single cytotoxic substances affecting all proliferative tissues towards antibodies and kinase inhibitors targeting tumor specific pathways. Treatment efficacy and cancer survival has overall improved and side effects have become less frequent. The ubiquitin proteasome system (UPS) mediated proteasomal protein degradation is the most critical pathway to regulate the quantity of signal proteins involved in carcinogenesis and tumor progression. These processes are, as well as protein recycling, highly regulated and offer targets for biomarker and drug development. Unspecific proteasome inhibitors such as bortezomib and carfilzomib have shown clinical efficacy and are approved for clinical use. Inhibitors of more substrate specific enzymes of degradation processes are developed and in early clinical trials. The novel compounds focus on the degradation of key regulatory proteins such as p53, p27Kip1 and β-catenin, and inhibitors specific for growth factor receptor kinases turnover are in pre-clinical testing. PMID:24756373

  8. Proteasomal Dysfunction Induced By Diclofenac Engenders Apoptosis Through Mitochondrial Pathway.

    PubMed

    Amanullah, Ayeman; Upadhyay, Arun; Chhangani, Deepak; Joshi, Vibhuti; Mishra, Ribhav; Yamanaka, Koji; Mishra, Amit

    2017-05-01

    Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. J. Cell. Biochem. 118: 1014-1027, 2017. © 2016 Wiley Periodicals, Inc.

  9. Impairment of the ubiquitin-proteasome pathway in RPE alters the expression of inflammation related genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. Retinal pigment epithelial cells (RPE) are a major source of ocular inflammatory cytokines. In this work we determined the relationship between impairment of the UPP and expression of inflammation-related f...

  10. Regulation of the retinoblastoma-E2F pathway by the ubiquitin-proteasome system.

    PubMed

    Sengupta, Satyaki; Henry, R William

    2015-10-01

    The retinoblastoma tumor suppressor (RB) and its related family members p107 and p130 regulate cell proliferation through the transcriptional repression of genes involved in cellular G1 to S phase transition. However, RB proteins are functionally versatile, and numerous genetic and biochemical studies point to expansive roles in cellular growth control, pluripotency, and apoptotic response. For the vast majority of genes, RB family members target the E2F family of transcriptional activators as an integral component of its gene regulatory mechanism. These interactions are regulated via reversible phosphorylation by Cyclin/Cyclin-dependent kinase (Cdk) complexes, a major molecular mechanism that regulates transcriptional output of RB/E2F target genes. Recent studies indicate an additional level of regulation involving the ubiquitin-proteasome system that renders pervasive control over each component of the RB pathway. Disruption of the genetic circuitry for proteasome-mediated targeting of the RB pathway has serious consequences on development and cellular transformation, and is associated with several forms of human cancer. In this review, we discuss the role of the ubiquitin-proteasome system in proteolytic control of RB-E2F pathway components, and recent data that points to surprising non-proteolytic roles for the ubiquitin-proteasome system in novel transcriptional regulatory mechanisms.

  11. Age-related dysfunctions of the autophagy lysosomal pathway in hippocampal pyramidal neurons under proteasome stress.

    PubMed

    Gavilán, Elena; Pintado, Cristina; Gavilan, Maria P; Daza, Paula; Sánchez-Aguayo, Inmaculada; Castaño, Angélica; Ruano, Diego

    2015-05-01

    Autophagy plays a key role in the maintenance of cellular homeostasis, and autophagy deregulation gives rise to severe disorders. Many of the signaling pathways regulating autophagy under stress conditions are still poorly understood. Using a model of proteasome stress in rat hippocampus, we have characterized the functional crosstalk between the ubiquitin proteasome system and the autophagy-lysosome pathway, identifying also age-related modifications in the crosstalk between both proteolytic systems. Under proteasome inhibition, both autophagy activation and resolution were efficiently induced in young but not in aged rats, leading to restoration of protein homeostasis only in young pyramidal neurons. Importantly, proteasome stress inhibited glycogen synthase kinase-3β in young but activated in aged rats. This age-related difference could be because of a dysfunction in the signaling pathway of the insulin growth factor-1 under stress situations. Present data highlight the potential role of glycogen synthase kinase-3β in the coordination of both proteolytic systems under stress situation, representing a key molecular target to sort out this deleterious effect.

  12. THE PROTEASOME REGULATES BACTERIAL CpG DNA-INDUCED SIGNALING PATHWAYS IN MURINE MACROPHAGES

    PubMed Central

    Gao, Jian Jun; Shen, Jing; Kolbert, Christopher; Raghavakaimal, Sreekumar; Papasian, Christopher J.; Qureshi, Asaf A.; Vogel, Stefanie N.; Morrison, David C.; Qureshi, Nilofer

    2010-01-01

    Our previous work has provided strong evidence that the proteasome is central to the vast majority of genes induced in mouse macrophages in response to lipopolysaccharide (LPS) stimulation. In the studies presented here, we evaluated the role of the macrophage proteasome in response to a second microbial product CpG DNA (unmethylated bacterial DNA). For these studies, we applied Affymetrix microarray analysis of RNA derived from murine macrophages stimulated with CpG DNA in the presence or absence of proteasome inhibitor, lactacystin. The results of these studies revealed that similar to LPS, a vast majority of those macrophage genes regulated by CpG DNA are also under the control of the proteasome at 4 h. In contrast to LPS stimulation, however, many of these genes were induced much later than 4 h, at 18 h, in response to CpG DNA. Lactacystin treatment of macrophages completely blocked the CpG DNA-induced gene expression of TNF-α and other genes involved in production of inflammatory mediators. These data strongly support the conclusion that, similar to LPS, the macrophage proteasome is a key regulator of CpG DNA-induced signaling pathways. PMID:20160661

  13. Regulating the ubiquitin/proteasome pathway via cAMP-signaling: neuroprotective potential

    PubMed Central

    Huang, He; Wang, Hu; Figueiredo-Pereira, Maria E.

    2013-01-01

    The cAMP-signaling pathway has been under intensive investigation for decades. It is a wonder that such a small simple molecule like cAMP can modulate a vast number of diverse processes in different types of cells. The ubiquitous involvement of cAMP-signaling in a variety of cellular events requires tight spatial and temporal control of its generation, propagation, compartmentalization, and elimination. Among the various steps of the cAMP-signaling pathway, G-protein coupled receptors, adenylate cyclases, phosphodiesterases, the two major cAMP targets, i.e. protein kinase A and exchange protein activated by cAMP, as well as the A-kinase anchoring proteins, are potential targets for drug development. Herein we review the recent progress on the regulation and manipulation of different steps of the cAMP-signaling pathway. We end by focusing on the emerging role of cAMP-signaling in modulating protein degradation via the ubiquitin/proteasome pathway. New discoveries on the regulation of the ubiquitin/proteasome pathway by cAMP-signaling support the development of new therapeutic approaches to prevent proteotoxicity in chronic neurodegenerative disorders and other human disease conditions associated with impaired protein turnover by the ubiquitin/proteasome pathway and the accumulation of ubiquitin-protein aggregates. PMID:23686612

  14. Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius.

    PubMed

    Anjum, Rana S; Bray, Sian M; Blackwood, John K; Kilkenny, Mairi L; Coelho, Matthew A; Foster, Benjamin M; Li, Shurong; Howard, Julie A; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J; Robinson, Nicholas P

    2015-09-08

    In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

  15. Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

    NASA Astrophysics Data System (ADS)

    Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

    2015-09-01

    In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

  16. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    NASA Technical Reports Server (NTRS)

    Jagoe, R. T.; Goldberg, A. L.

    2001-01-01

    Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

  17. Regulation of Bovine Papillomavirus Replicative Helicase E1 by the Ubiquitin-Proteasome Pathway

    PubMed Central

    Malcles, Marie-Helene; Cueille, Nathalie; Mechali, Francisca; Coux, Olivier; Bonne-Andrea, Catherine

    2002-01-01

    Papillomaviruses maintain their genomes in a relatively constant copy number as stable extrachromosomal plasmids in the nuclei of dividing host cells. The viral initiator of replication, E1, is not detected in papillomavirus-infected cells. Here, we present evidence that E1 encoded by bovine papillomavirus type 1 is an unstable protein that is degraded through the ubiquitin-proteasome pathway. In a cell-free system derived from Xenopus egg extracts, E1 degradation is regulated by both cyclin E/Cdk2 binding and E1 replication activity. Free E1 is readily ubiquitinated and degraded by the proteasome, while it becomes resistant to this degradation pathway when bound to cyclin E/Cdk2 complexes before the start of DNA synthesis. This stabilization is reversed in a process involving E1-dependent replication activity. In transiently transfected cells, E1 is also polyubiquitinated and accumulates when proteasome activity is inhibited. Thus, the establishment and maintenance of a stable number of papillomavirus genomes in latently infected cells are in part a function of regulated ubiquitin-mediated degradation of E1. PMID:12388695

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

    PubMed

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

    2014-01-01

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

  19. Reciprocal regulation of cilia and autophagy via the MTOR and proteasome pathways.

    PubMed

    Wang, Shixuan; Livingston, Man J; Su, Yunchao; Dong, Zheng

    2015-04-03

    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.

  20. Stimulation of ubiquitin-proteasome pathway through the expression of amidohydrolase for N-terminal asparagine (Ntan1) in cultured rat hippocampal neurons exposed to static magnetism.

    PubMed

    Hirai, Takao; Taniura, Hideo; Goto, Yasuaki; Ogura, Masato; Sng, Judy C G; Yoneda, Yukio

    2006-03-01

    In order to elucidate mechanisms underlying modulation by static magnetism of the cellular functionality and/or integrity in the brain, we screened genes responsive to brief magnetism in cultured rat hippocampal neurons using differential display analysis. We have for the first time cloned and identified Ntan1 (amidohydrolase for N-terminal asparagine) as a magnetism responsive gene in rat brain. Ntan1 is an essential component of a protein degradation signal, which is a destabilizing N-terminal residue of a protein, in the N-end rule. In situ hybridization histochemistry revealed abundant expression of Ntan1 mRNA in hippocampal neurons in vivo. Northern blot analysis showed that Ntan1 mRNA was increased about three-fold after 3 h in response to brief magnetism. Brief magnetism also increased the transcriptional activity of Ntan1 promoter by luciferase reporter assay. Brief magnetism induced degradation of microtubule-associated protein 2 (MAP2) without affecting cell morphology and viability, which was prevented by a selective inhibitor of 26S proteasome in hippocampal neurons. Overexpression of Ntan1 using recombinant Ntan1 adenovirus vector resulted in a marked decrease in the MAP2 protein expression in hippocampal neurons. Our results suggest that brief magnetism leads to the induction of Ntan1 responsible for MAP2 protein degradation through ubiquitin-proteasome pathway in rat hippocampal neurons.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  4. REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

    SciTech Connect

    Chai, Fan; Liang, Yan; Bi, Jiong; Chen, Li; Zhang, Fan; Cui, Youhong; Jiang, Jun

    2015-01-02

    Highlights: • High expression of REGγ is correlated with ERα status and poor clinical features. • Cell growth, mobility and invasion are significantly impaired by REGγ knockdown. • REGγ indirectly regulates ERα protein expression. - Abstract: REGγ is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REGγ is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REGγ was highly expressed in breast cancers, and the expression of REGγ was positively correlated with breast cancer patient estrogen receptor alpha (ERα) status. Moreover, the expression of REGγ was found positively associated with poor clinical features and low survival rates in ERα positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REGγ knockdown. Lastly, we demonstrated that REGγ indirectly regulates the degradation of ERα protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REGγ expression was positively correlated with ERα status and poor clinical prognosis in ERα positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.

  5. New insights to the ubiquitin-proteasome pathway (UPP) mechanism during spermatogenesis.

    PubMed

    Hou, Cong-Cong; Yang, Wan-Xi

    2013-04-01

    Spermatogenesis is a complicated and highly ordered process which begins with the differentiation of spermatogonial stem cells and ends with the formation of mature sperm. After meiosis, several morphological changes occur during spermatogenesis. During spermatogenesis, many proteins and organelles are degraded, and the ubiquitin-proteasome pathway (UPP) plays a key role in the process which facilitates the formation of condensed sperm. UPP contains various indispensable components: ubiquitin, ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin ligase enzyme E3 and proteasomes. At some key stages of spermatogenesis, such as meiosis, acrosome biogenesis, and spermatozoa maturation, the ubiquitin-related components (including deubiquitination enzymes) exert positive and active functions. Generally speaking, deficient UPP will block spermatogenesis which may induce infertility at various degrees. Although ubiquitination during spermatogenesis has been widely investigated, further detailed aspects such as the mechanism of ubiquitination during the formation of midpiece and acrosome morphogenesis still remains unknown. The present review will overview current progress on ubiquitination during spermatogenesis, and will provide some suggestions for future studies on the functions of UPP components during spermatogenesis.

  6. Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius

    PubMed Central

    Anjum, Rana S.; Bray, Sian M.; Blackwood, John K.; Kilkenny, Mairi L.; Coelho, Matthew A.; Foster, Benjamin M.; Li, Shurong; Howard, Julie A.; Pellegrini, Luca; Albers, Sonja-Verena; Deery, Michael J.; Robinson, Nicholas P.

    2015-01-01

    In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved. PMID:26348592

  7. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway in the pathology of calpainopathy.

    PubMed

    Rajakumar, Dhanarajan; Alexander, Mathew; Oommen, Anna

    2013-10-01

    The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-κB signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-κB/IKK β signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-κB signaling could be considered for treatment of LGMD 2A.

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

    SciTech Connect

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

    2011-01-14

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

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

    PubMed

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

    2011-01-14

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

  10. Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles.

    PubMed

    Waite, Kenrick A; De-La Mota-Peynado, Alina; Vontz, Gabrielle; Roelofs, Jeroen

    2016-02-12

    The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well characterized transcriptional up-regulation of genes encoding proteasome subunits, cells are also capable of down-regulating cellular levels of proteasomes through proteaphagy.

  11. Selective overproduction of the proteasome inhibitor salinosporamide A via precursor pathway regulation

    PubMed Central

    Lechner, Anna; Eustáquio, Alessandra S.; Gulder, Tobias A. M.; Hafner, Mathias; Moore, Bradley S.

    2011-01-01

    The chlorinated natural product salinosporamide A is a potent 20S proteasome inhibitor currently in clinical trials as an anticancer agent. To deepen our understanding of salinosporamide biosynthesis, we investigated the function of a LuxR-type pathway-specific regulatory gene, salR2, and observed a selective effect on the production of salinosporamide A over its less active aliphatic analogs. SalR2 was shown to specifically activate genes involved in the biosynthesis of the halogenated precursor chloroethylmalonyl-CoA, which is a dedicated precursor of salinosporamide A. Specifically, SalR2 activates transcription of two divergent operons – one of which contains the unique S-adenosyl-L-methionine-dependent chlorinase encoding gene salL. By applying this knowledge towards rational engineering, we were able to selectively double salinosporamide A production. This study exemplifies the specialized regulation of a polyketide precursor pathway and its application to the selective overproduction of a specific natural product congener. PMID:22195555

  12. Autophagy and proteasome interconnect to coordinate cross-presentation through MHC class I pathway in B cells.

    PubMed

    Dasari, Vijayendra; Rehan, Sweera; Tey, Siok-Keen; Smyth, Mark J; Smith, Corey; Khanna, Rajiv

    2016-11-01

    Cross-presentation of exogenous protein antigens by B cells through the major histocompatibility complex (MHC) class I pathway in lymphoid malignancies, and transplant setting has been recognised as an important mediator of immune pathogenesis and T cell-mediated immune regulation. However, the precise mechanism of cross-presentation of exogenous antigens in B cells has remained unresolved. Here we have delineated a novel pathway for cross-presentation in B cells, which involves synergistic cooperation of the proteasome and autophagy. After endocytosis, protein antigen is processed through an autophagy- and proteasome-dependent pathway and CD8(+) T-cell epitopes are loaded onto MHC class I molecules within the autophagolysomal compartment rather than the conventional secretory pathway, which requires transporters associated with antigen processing-dependent transport. Interestingly, this cross-presentation was critically dependent on valosin-containing protein (VCP)/p97 ATPase through its participation in autophagy. Loss of VCP/p97 ATPase was coincident with accumulation of LC3-II and marked reduction in antigen presentation. These observations provide unique insight on how the autophagy and proteasomal degradation systems interconnect to coordinate MHC class I-restricted cross-presentation in B cells.

  13. Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

    PubMed Central

    Choy, Milly M.; Sessions, October M.; Gubler, Duane J.; Ooi, Eng Eong

    2015-01-01

    Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector. PMID:26566123

  14. Contributions of the ubiquitin-proteasome pathway and apoptosis to human skeletal muscle wasting with age.

    PubMed

    Whitman, Samantha A; Wacker, Michael J; Richmond, Scott R; Godard, Michael P

    2005-09-01

    The primary mechanism that contributes to decreasing skeletal muscle strength and size with healthy aging is not presently known. This study examined the contribution of the ubiquitin-proteasome pathway and apoptosis to skeletal muscle wasting in older adults (n = 21; mean age = 72.76 +/- 8.31 years) and young controls (n = 21; mean age = 21.48 +/- 2.93 years). Subjects underwent a percutaneous muscle biopsy of the vastus lateralis to determine: (1) ubiquitin ligase gene expression (MAFbx and MuRF1); (2) frequency of apoptosis; and (3) individual fiber type and cross-sectional area. In addition, a whole muscle strength test was also performed. A one-way ANOVA revealed significant increases in the number of positive TUNEL cells in older adults (87%; p < 0.05), although no significant increase in caspase-3/7 activity was detected. Additionally, ubiquitin ligase gene expression, individual muscle fiber type and CSA were not different between old and young subjects. Muscle strength was also significantly lower in old compared to young subjects (p < 0.05). In conclusion, this study indicates a preferential role for apoptosis contributing to decreases in muscle function with age.

  15. Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway.

    PubMed

    He, Q; Liu, Y

    2005-11-01

    Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) promotes its degradation through the ubiquitin-proteasome pathway. Ubiquitination of FRQ requires FWD-1 (F-box/WD-40 repeat-containing protein-1), which is the substrate-recruiting subunit of an SCF (SKP/Cullin/F-box)-type ubiquitin ligase. In the fwd-1 mutant strains, FRQ degradation is defective, resulting in the accumulation of hyperphosphorylated FRQ and the loss of the circadian rhythmicities. The CSN (COP9 signalosome) promotes the function of SCF complexes in vivo. But in vitro, deneddylation of cullins by CSN inhibits SCF activity. In Neurospora, the disruption of the csn-2 subunit impairs FRQ degradation and compromises the normal circadian functions. These defects are due to the dramatically reduced levels of FWD-1 in the csn-2 mutant, a result of its rapid degradation. Other components of the SCF(FWD-1) complex, SKP-1 and CUL-1 are also unstable in the mutant. These results establish important roles for SCF(FWD-1) and CSN in the circadian clock of Neurospora and suggest that they are conserved components of the eukaryotic circadian clocks. In addition, these findings resolve the CSN paradox and suggest that the major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

  16. 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

  17. Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways.

    PubMed

    Wills, Jonathan; Credle, Joel; Oaks, Adam W; Duka, Valeriy; Lee, Jae-Hoon; Jones, Jessica; Sidhu, Anita

    2012-01-01

    SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau.

  18. Proteasome inhibition enhances resistance to DNA damage via upregulation of Rpn4-dependent DNA repair genes.

    PubMed

    Karpov, Dmitry S; Spasskaya, Daria S; Tutyaeva, Vera V; Mironov, Alexander S; Karpov, Vadim L

    2013-09-17

    The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.

  19. Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway.

    PubMed

    Padrão, Juliana da Cruz; Cabral, Gabriel Rabello de Abreu; da Silva, Maria de Fátima Sarro; Seabra, Sergio Henrique; DaMatta, Renato Augusto

    2014-10-01

    Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.

  20. Regulation of ubiquitin proteasome pathway molecular markers in response to endurance and resistance exercise and training.

    PubMed

    Stefanetti, Renae J; Lamon, Séverine; Wallace, Marita; Vendelbo, Mikkel H; Russell, Aaron P; Vissing, Kristian

    2015-07-01

    Knowledge on the effects of divergent exercise on ostensibly protein degradation pathways may be valuable for counteracting muscle wasting and for understanding muscle remodelling. This study examined mRNA and/or protein levels of molecular markers of the ubiquitin proteasome pathway (UPP), including FBXO32 (atrogin-1), MURF-1, FBXO40, FOXO1 and FOXO3. Protein substrates of atrogin-1-including EIF3F, MYOG and MYOD1-and of MURF-1-including PKM and MHC-were also measured. Subjects completed 10 weeks of endurance training (ET) or resistance training (RT) followed by a single-bout of endurance exercise (EE) or resistance exercise (RE). Following training, atrogin-1, FBXO40, FOXO1 and FOXO3 mRNA increased independently of exercise mode, whereas MURF-1 mRNA and FOXO3 protein increased following ET only. No change in other target proteins occurred post-training. In the trained state, single-bout EE, but not RE, increased atrogin-1, MURF-1, FBXO40, FOXO1, FOXO3 mRNA and FOXO3 protein. In contrast to EE, FBXO40 mRNA and protein decreased following single-bout RE. MURF-1 and FOXO1 protein levels as well as the protein substrates of atrogin-1 and MURF-1 were unchanged following training and single-bout exercise. This study demonstrates that the intracellular signals elicited by ET and RT result in an upregulation of UPP molecular markers, with a greater increase following ET. However, in the trained state, the expression levels of UPP molecular markers are increased following single-bout EE, but are less responsive to single-bout RE. This suggests that adaptations following endurance exercise training are more reliant on protein UPP degradation processes than adaptations following resistance exercise training.

  1. Intracellular composition of fatty acid affects the processing and function of tyrosinase through the ubiquitin–proteasome pathway

    PubMed Central

    Ando, Hideya; Wen, Zhi-Ming; Kim, Hee-Yong; Valencia, Julio C.; Costin, Gertrude-E.; Watabe, Hidenori; Yasumoto, Ken-ichi; Niki, Yoko; Kondoh, Hirofumi; Ichihashi, Masamitsu; Hearing, Vincent J.

    2005-01-01

    Proteasomes are multicatalytic proteinase complexes within cells that selectively degrade ubiquitinated proteins. We have recently demonstrated that fatty acids, major components of cell membranes, are able to regulate the proteasomal degradation of tyrosinase, a critical enzyme required for melanin biosynthesis, in contrasting manners by relative increases or decreases in the ubiquitinated tyrosinase. In the present study, we show that altering the intracellular composition of fatty acids affects the post-Golgi degradation of tyrosinase. Incubation with linoleic acid (C18:2) dramatically changed the fatty acid composition of cultured B16 melanoma cells, i.e. the remarkable increase in polyunsaturated fatty acids such as linoleic acid and arachidonic acid (C20:4) was compensated by the decrease in monounsaturated fatty acids such as oleic acid (C18:1) and palmitoleic acid (C16:1), with little effect on the proportion of saturated to unsaturated fatty acid. When the composition of intracellular fatty acids was altered, tyrosinase was rapidly processed to the Golgi apparatus from the ER (endoplasmic reticulum) and the degradation of tyrosinase was increased after its maturation in the Golgi. Retention of tyrosinase in the ER was observed when cells were treated with linoleic acid in the presence of proteasome inhibitors, explaining why melanin synthesis was decreased in cells treated with linoleic acid and a proteasome inhibitor despite the abrogation of tyrosinase degradation. These results suggest that the intracellular composition of fatty acid affects the processing and function of tyrosinase in connection with the ubiquitin–proteasome pathway and suggest that this might be a common physiological approach to regulate protein degradation. PMID:16232122

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

    PubMed

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

    2015-11-01

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

  3. 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

  4. Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells

    PubMed Central

    Felley-Bosco, Emanuela; Bender, Florent C.; Courjault-Gautier, Françoise; Bron, Claude; Quest, Andrew F. G.

    2000-01-01

    To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway. PMID:11114180

  5. Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells.

    PubMed

    Felley-Bosco, E; Bender, F C; Courjault-Gautier, F; Bron, C; Quest, A F

    2000-12-19

    To investigate whether caveolin-1 (cav-1) may modulate inducible nitric oxide synthase (iNOS) function in intact cells, the human intestinal carcinoma cell lines HT29 and DLD1 that have low endogenous cav-1 levels were transfected with cav-1 cDNA. In nontransfected cells, iNOS mRNA and protein levels were increased by the addition of a mix of cytokines. Ectopic expression of cav-1 in both cell lines correlated with significantly decreased iNOS activity and protein levels. This effect was linked to a posttranscriptional mechanism involving enhanced iNOS protein degradation by the proteasome pathway, because (i) induction of iNOS mRNA by cytokines was not affected and (ii) iNOS protein levels increased in the presence of the proteasome inhibitors N-acetyl-Leu-Leu-Norleucinal and lactacystin. In addition, a small amount of iNOS was found to cofractionate with cav-1 in Triton X-100-insoluble membrane fractions where also iNOS degradation was apparent. As has been described for endothelial and neuronal NOS isoenzymes, direct binding between cav-1 and human iNOS was detected in vitro. Taken together, these results suggest that cav-1 promotes iNOS presence in detergent-insoluble membrane fractions and degradation there via the proteasome pathway.

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

    PubMed Central

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

    2010-01-01

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

  7. Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The accumulation, aggregation, and precipitation of proteins are etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome p...

  8. Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea

    PubMed Central

    Fu, Xian; Liu, Rui; Sanchez, Iona; Silva-Sanchez, Cecilia; Hepowit, Nathaniel L.; Cao, Shiyun; Chen, Sixue

    2016-01-01

    ABSTRACT The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself was not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis. PMID:27190215

  9. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  10. 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.

  11. 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.

  12. Synergistic effects of the SAPK/JNK and the proteasome pathway on glial fibrillary acidic protein (GFAP) accumulation in Alexander disease.

    PubMed

    Tang, Guomei; Xu, Zhiheng; Goldman, James E

    2006-12-15

    Protein aggregates in astrocytes that contain glial fibrillary acidic protein (GFAP), small heat shock proteins, and ubiquitinated proteins are termed Rosenthal fibers and characterize Alexander disease, a leukodystrophy caused by heterozygous mutations in GFAP. The mechanisms responsible for the massive accumulation of GFAP in Alexander disease remain unclear. In this study, we show that overexpression of both wild type and R239C mutant human GFAP led to cytoplasmic inclusions. GFAP accumulation also led to a decrease of proteasome activity and an activation of the MLK2-JNK pathway. In turn, the expression of activated mixed lineage kinases (MLKs) induced JNK activation and increased GFAP accumulation, whereas blocking the JNK pathway decreased GFAP accumulation. Activated MLK also inhibited proteasome function. A direct inhibition of proteasome function pharmacologically further activated JNK. Our data suggest a synergistic interplay between the proteasome and the SAPK/JNK pathway in the context of GFAP accumulation. Feedback interactions among GFAP accumulation, SAPK/JNK activation, and proteasomal hypofunction cooperate to produce further protein accumulation and cellular stress responses.

  13. The proteasome of malaria parasites: A multi-stage drug target for chemotherapeutic intervention?

    PubMed Central

    Aminake, Makoah Nigel; Arndt, Hans-Dieter; Pradel, Gabriele

    2012-01-01

    The ubiquitin/proteasome system serves as a regulated protein degradation pathway in eukaryotes, and is involved in many cellular processes featuring high protein turnover rates, such as cell cycle control, stress response and signal transduction. In malaria parasites, protein quality control is potentially important because of the high replication rate and the rapid transformations of the parasite during life cycle progression. The proteasome is the core of the degradation pathway, and is a major proteolytic complex responsible for the degradation and recycling of non-functional ubiquitinated proteins. Annotation of the genome for Plasmodium falciparum, the causative agent of malaria tropica, revealed proteins with similarity to human 26S proteasome subunits. In addition, a bacterial ClpQ/hslV threonine peptidase-like protein was identified. In recent years several independent studies indicated an essential function of the parasite proteasome for the liver, blood and transmission stages. In this review, we compile evidence for protein recycling in Plasmodium parasites and discuss the role of the 26S proteasome as a prospective multi-stage target for antimalarial drug discovery programs. PMID:24533266

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

    PubMed

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

    2015-01-27

    26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid subcomplexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base subcomplexes, and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Rpn11 in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11-m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of the 5 module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Reintroducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11-Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine, and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.

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

    PubMed

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

    2015-01-27

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

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

    PubMed Central

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

    2015-01-01

    26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes. PMID:26182356

  17. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  18. Targeted therapy of the XIAP/proteasome pathway overcomes TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent 'type I' mode.

    PubMed

    Gillissen, B; Richter, A; Richter, A; Overkamp, T; Essmann, F; Hemmati, P G; Preissner, R; Belka, C; Daniel, P T

    2013-05-23

    TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.

  19. Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

    PubMed Central

    Kubota, Toru; Kita, Shunsuke; Nakatsu, Yuichiro; Aoki, Natsuko; Mori, Yoshio; Maenaka, Katsumi; Takeda, Makoto; Kidokoro, Minoru

    2014-01-01

    ABSTRACT Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection. PMID:25552722

  20. CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

    PubMed Central

    Schubert, Ulrich; Antón, Luis C.; Bačík, Igor; Cox, Josephine H.; Bour, Stéphane; Bennink, Jack R.; Orlowski, Marian; Strebel, Klaus; Yewdell, Jonathan W.

    1998-01-01

    ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked. PMID:9499087

  1. 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

  2. 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

  3. Proteasome dynamics.

    PubMed

    Enenkel, Cordula

    2014-01-01

    Proteasomes are highly conserved multisubunit protease complexes and occur in the cyto- and nucleoplasm of eukaryotic cells. In dividing cells proteasomes exist as holoenzymes and primarily localize in the nucleus. During quiescence they dissociate into proteolytic core and regulatory complexes and are sequestered into motile cytosolic clusters. Proteasome clusters rapidly clear upon the exit from quiescence, where proteasome core and regulatory complexes reassemble and localize to the nucleus again. The mechanisms underlying proteasome transport and assembly are not yet understood. Here, I summarize our present knowledge about nuclear transport and assembly of proteasomes in yeast and project our studies in this eukaryotic model organism to the mammalian cell system. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

  4. The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway.

    PubMed Central

    Bailey, J L; Wang, X; England, B K; Price, S R; Ding, X; Mitch, W E

    1996-01-01

    Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF. PMID:8617877

  5. Time course of ubiquitin-proteasome and macroautophagy-lysosome pathways in skeletal muscle in rats with heart failure.

    PubMed

    Fujita, Naoto; Fujino, Hidemi; Sakamoto, Hiroki; Takegaki, Jyunya; Deie, Masataka

    2015-01-01

    Patients with heart failure have limited exercise capacity due to not only the myocardial dysfunction but also skeletal muscle atrophy. However, the mechanisms and time course of protein degradation in skeletal muscle during heart failure remain unclear, and there is no established standard treatment. The purpose of the present study was to investigate the time course of major protein degradation pathways in skeletal muscle during heart failure. Four-week-old male Wistar rats were randomly assigned to heart failure induced by monocrotaline or control groups. At 14 and 21 days after monocrotaline injection, the lungs, heart, and gastrocnemius and soleus muscles were removed and analyzed. There was no significant difference in body weight between the groups at 14 days after monocrotaline injection. Although there were no morphological changes in the skeletal muscle of the monocrotaline group at this time point, ubiquitin-proteasome and macroautophagylysosome pathways were activated in the monocrotaline group. Additionally, the pathways were less strongly activated in the soleus muscle than in the gastrocnemius muscle. These results suggest that physical exercise that shifts to slow muscle characteristics should begin when there is no indication of skeletal muscle atrophy to prevent exercise intolerance with heart failure.

  6. Newcastle disease virus degrades HIF-1α through proteasomal pathways independent of VHL and p53

    PubMed Central

    Abd-Aziz, Noraini; Stanbridge, Eric J.

    2016-01-01

    Newcastle disease virus (NDV) is a candidate agent for oncolytic virotherapy. Despite its potential, the exact mechanism of its oncolysis is still not known. Recently, we reported that NDV exhibited an increased oncolytic activity in hypoxic cancer cells. These types of cells negatively affect therapeutic outcome by overexpressing pro-survival genes under the control of the hypoxia-inducible factor (HIF). HIF-1 is a heterodimeric transcriptional factor consisting of a regulated α (HIF-1α) and a constitutive β subunit (HIF-1β). To investigate the effects of NDV infection on HIF-1α in cancer cells, the osteosarcoma (Saos-2), breast carcinoma (MCF-7), colon carcinoma (HCT116) and fibrosarcoma (HT1080) cell lines were used in the present study. Data obtained showed that a velogenic NDV infection diminished hypoxia-induced HIF-1α accumulation, leading to a decreased activation of its downstream target gene, carbonic anhydrase 9. This NDV-induced downregulation of HIF-1α occurred post-translationally and was partially abrogated by proteasomal inhibition. The process appeared to be independent of the tumour suppressor protein p53. These data revealed a correlation between NDV infection and HIF-1α downregulation, which highlights NDV as a promising agent to eliminate hypoxic cancer cells. PMID:27902314

  7. Newcastle disease virus degrades HIF-1α through proteasomal pathways independent of VHL and p53.

    PubMed

    Abd-Aziz, Noraini; Stanbridge, Eric J; Shafee, Norazizah

    2016-12-01

    Newcastle disease virus (NDV) is a candidate agent for oncolytic virotherapy. Despite its potential, the exact mechanism of its oncolysis is still not known. Recently, we reported that NDV exhibited an increased oncolytic activity in hypoxic cancer cells. These types of cells negatively affect therapeutic outcome by overexpressing pro-survival genes under the control of the hypoxia-inducible factor (HIF). HIF-1 is a heterodimeric transcriptional factor consisting of a regulated α (HIF-1α) and a constitutive β subunit (HIF-1β). To investigate the effects of NDV infection on HIF-1α in cancer cells, the osteosarcoma (Saos-2), breast carcinoma (MCF-7), colon carcinoma (HCT116) and fibrosarcoma (HT1080) cell lines were used in the present study. Data obtained showed that a velogenic NDV infection diminished hypoxia-induced HIF-1α accumulation, leading to a decreased activation of its downstream target gene, carbonic anhydrase 9. This NDV-induced downregulation of HIF-1α occurred post-translationally and was partially abrogated by proteasomal inhibition. The process appeared to be independent of the tumour suppressor protein p53. These data revealed a correlation between NDV infection and HIF-1α downregulation, which highlights NDV as a promising agent to eliminate hypoxic cancer cells.

  8. The PUB domain: a putative protein-protein interaction domain implicated in the ubiquitin-proteasome pathway.

    PubMed

    Suzuki, T; Park, H; Till, E A; Lennarz, W J

    2001-10-12

    Cytoplasmic peptide:N-glycanase (PNGase) is a de-N-glycosylating enzyme which may be involved in the proteasome-dependent pathway for degradation of misfolded glycoproteins formed in the endoplasmic reticulum (ER) that are exported into the cytoplasm. A cytoplasmic PNGase found in Saccharomyces cerevisiae, Png1p, is widely distributed in higher eukaryotes as well as in yeast (Suzuki, T., et al. J. Cell Biol. 149, 1039-1051, 2000). The recently uncovered complete genome sequence of Arabidopsis thaliana prompted us to search for the protein homologue of Png1p in this organism. Interestingly, when the mouse Png1p homologue sequence was used as a query, not only a Png1p homologue containing a transglutaminase-like domain that is believed to contain a catalytic triad for PNGase activity, but also four proteins which had a domain of 46 amino acids in length that exhibited significant similarity to the N-terminus of mouse Png1p were identified. Moreover, three of these homologous proteins were also found to possess a UBA or UBX domain, which are found in various proteins involved in the ubiquitin-related pathway. We name this newly found homologous region the PUB (Peptide:N-glycanase/UBA or UBX-containing proteins) domain and propose that this domain may mediate protein-protein interactions.

  9. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice

    PubMed Central

    Rodriguez, Karl A.; Dodds, Sherry G.; Strong, Randy; Galvan, Veronica; Sharp, Z. D.; Buffenstein, Rochelle

    2014-01-01

    Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24 mg/kg (14 ppm) rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS), heat shock factor 1 (HSF1), and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome. PMID:25414638

  10. Divergent tissue and sex effects of rapamycin on the proteasome-chaperone network of old mice.

    PubMed

    Rodriguez, Karl A; Dodds, Sherry G; Strong, Randy; Galvan, Veronica; Sharp, Z D; Buffenstein, Rochelle

    2014-01-01

    Rapamycin, an allosteric inhibitor of the mTOR kinase, increases longevity in mice in a sex-specific manner. In contrast to the widely accepted theory that a loss of proteasome activity is detrimental to both life- and healthspan, biochemical studies in vitro reveal that rapamycin inhibits 20S proteasome peptidase activity. We tested if this unexpected finding is also evident after chronic rapamycin treatment in vivo by measuring peptidase activities for both the 26S and 20S proteasome in liver, fat, and brain tissues of old, male and female mice fed encapsulated chow containing 2.24 mg/kg (14 ppm) rapamycin for 6 months. Further we assessed if rapamycin altered expression of the chaperone proteins known to interact with the proteasome-mediated degradation system (PMDS), heat shock factor 1 (HSF1), and the levels of key mTOR pathway proteins. Rapamycin had little effect on liver proteasome activity in either gender, but increased proteasome activity in female brain lysates and lowered its activity in female fat tissue. Rapamycin-induced changes in molecular chaperone levels were also more substantial in tissues from female animals. Furthermore, mTOR pathway proteins showed more significant changes in female tissues compared to those from males. These data show collectively that there are divergent tissue and sex effects of rapamycin on the proteasome-chaperone network and that these may be linked to the disparate effects of rapamycin on males and females. Further our findings suggest that rapamycin induces indirect regulation of the PMDS/heat-shock response through its modulation of the mTOR pathway rather than via direct interactions between rapamycin and the proteasome.

  11. 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

  12. Fundamental Reaction Pathway for Peptide Metabolism by Proteasome: Insights from First-principles Quantum Mechanical/Molecular Mechanical Free Energy Calculations

    PubMed Central

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

    2013-01-01

    Proteasome is the major component of the crucial nonlysosomal protein degradation pathway in the cells, but the detailed reaction pathway is unclear. In this study, first-principles quantum mechanical/molecular mechanical free energy calculations have been performed to explore, for the first time, possible reaction pathways for proteasomal proteolysis/hydrolysis of a representative peptide, succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin (Suc-LLVY-AMC). The computational results reveal that the most favorable reaction pathway consists of six steps. The first is a water-assisted proton transfer within proteasome, activating Thr1-Oγ. The second is a nucleophilic attack on the carbonyl carbon of a Tyr residue of substrate by the negatively charged Thr1-Oγ, followed by the dissociation of the amine AMC (third step). The fourth step is a nucleophilic attack on the carbonyl carbon of the Tyr residue of substrate by a water molecule, accompanied by a proton transfer from the water molecule to Thr1-Nz. Then, Suc-LLVY is dissociated (fifth step), and Thr1 is regenerated via a direct proton transfer from Thr1-Nz to Thr1-Oγ. According to the calculated energetic results, the overall reaction energy barrier of the proteasomal hydrolysis is associated with the transition state (TS3b) for the third step involving a water-assisted proton transfer. The determined most favorable reaction pathway and the rate-determining step have provided a reasonable interpretation of the reported experimental observations concerning the substituent and isotopic effects on the kinetics. The calculated overall free energy barrier of 18.2 kcal/mol is close to the experimentally-derived activation free energy of ~18.3–19.4 kcal/mol, suggesting that the computational results are reasonable. PMID:24111489

  13. Neuroinflammation and J2 prostaglandins: linking impairment of the ubiquitin-proteasome pathway and mitochondria to neurodegeneration

    PubMed Central

    Figueiredo-Pereira, Maria E.; Rockwell, Patricia; Schmidt-Glenewinkel, Thomas; Serrano, Peter

    2015-01-01

    The immune response of the CNS is a defense mechanism activated upon injury to initiate repair mechanisms while chronic over-activation of the CNS immune system (termed neuroinflammation) may exacerbate injury. The latter is implicated in a variety of neurological and neurodegenerative disorders such as Alzheimer and Parkinson diseases, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain injury, HIV dementia, and prion diseases. Cyclooxygenases (COX-1 and COX-2), which are key enzymes in the conversion of arachidonic acid into bioactive prostanoids, play a central role in the inflammatory cascade. J2 prostaglandins are endogenous toxic products of cyclooxygenases, and because their levels are significantly increased upon brain injury, they are actively involved in neuronal dysfunction induced by pro-inflammatory stimuli. In this review, we highlight the mechanisms by which J2 prostaglandins (1) exert their actions, (2) potentially contribute to the transition from acute to chronic inflammation and to the spreading of neuropathology, (3) disturb the ubiquitin-proteasome pathway and mitochondrial function, and (4) contribute to neurodegenerative disorders such as Alzheimer and Parkinson diseases, and amyotrophic lateral sclerosis, as well as stroke, traumatic brain injury (TBI), and demyelination in Krabbe disease. We conclude by discussing the therapeutic potential of targeting the J2 prostaglandin pathway to prevent/delay neurodegeneration associated with neuroinflammation. In this context, we suggest a shift from the traditional view that cyclooxygenases are the most appropriate targets to treat neuroinflammation, to the notion that J2 prostaglandin pathways and other neurotoxic prostaglandins downstream from cyclooxygenases, would offer significant benefits as more effective therapeutic targets to treat chronic neurodegenerative diseases, while minimizing adverse side effects. PMID:25628533

  14. Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway.

    PubMed

    Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong

    2009-07-01

    Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP.

  15. Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

    PubMed Central

    Geddes, J. M. H.; Caza, M.; Croll, D.; Stoynov, N.; Foster, L. J.

    2016-01-01

    ABSTRACT The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis. PMID:26758180

  16. 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.

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

    PubMed

    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

    2015-09-01

    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.

  18. 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.

  19. Murraya koenigii leaf extract inhibits proteasome activity and induces cell death in breast cancer cells

    PubMed Central

    2013-01-01

    Background Inhibition of the proteolytic activity of 26S proteasome, the protein-degrading machine, is now considered a novel and promising approach for cancer therapy. Interestingly, proteasome inhibitors have been demonstrated to selectively kill cancer cells and also enhance the sensitivity of tumor cells to chemotherapeutic agents. Recently, polyphenols/flavonoids have been reported to inhibit proteasome activity. Murraya koenigii Spreng, a medicinally important herb of Indian origin, has been used for centuries in the Ayurvedic system of medicine. Here we show that Murraya koenigii leaves (curry leaves), a rich source of polyphenols, inhibit the proteolytic activity of the cancer cell proteasome, and cause cell death. Methods Hydro-methanolic extract of curry leaves (CLE) was prepared and its total phenolic content [TPC] determined by, the Folin-Ciocalteau’s method. Two human breast carcinoma cell lines: MCF-7 and MDA-MB-231 and a normal human lung fibroblast cell line, WI-38 were used for the studies. Cytotoxicity of the CLE was assessed by the MTT assay. We studied the effect of CLE on growth kinetics using colony formation assay. Growth arrest was assessed by cell cycle analysis and apoptosis by Annexin-V binding using flow cytometry. Inhibition of the endogenous 26S proteasome was studied in intact cells and cell extracts using substrates specific to 20S proteasomal enzymes. Results CLE decreased cell viability and altered the growth kinetics in both the breast cancer cell lines in a dose-dependent manner. It showed a significant arrest of cells in the S phase albeit in cancer cells only. Annexin V binding data suggests that cell death was via the apoptotic pathway in both the cancer cell lines. CLE treatment significantly decreased the activity of the 26S proteasome in the cancer but not normal cells. Conclusions Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification

  20. LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

    PubMed Central

    Doeppner, Thorsten R.; Niu, Feng; Li, Qiaochuan; Yang, Yanping; Kuckelkorn, Ulrike; Hagemann, Nina; Li, Wei; Hermann, Dirk M.; Dai, Yun; Zhou, Wen; Jin, Fengyan

    2015-01-01

    Since disturbance of angiogenesis predisposes to ischemic injuries, attempts to promote angiogenesis have been made to improve clinical outcomes of patients with many ischemic disorders. While hypoxia inducible factors (HIFs) stimulate vascular remodeling and angiogenesis, hyperlipidemia impairs angiogenesis in response to various pro-angiogenic factors. However, it remains uncertain how HIFs regulate angiogenesis under hyperlipidemia. Here, we report that exposure to low-density lipoprotein (LDL) suppressed in vitro angiogenesis of human brain microvascular endothelial cells. Whereas LDL exposure diminished expression of HIF-1α and HIF-2α induced by hypoxia, it inhibited DMOG- and TNFα-induced HIF-1α and HIF-2α expression in normoxia. Notably, in both hypoxia and normoxia, LDL markedly reduced expression of HIF-1β, a constitutively stable HIF subunit, an event associated with NF-κB inactivation. Moreover, knockdown of HIF-1β down-regulated HIF-1α and HIF-2α expression, in association with increased HIF-1α hydroxylation and 20S proteasome activity after LDL exposure. Significantly, the proteasome inhibitor BSc2118 prevented angiogenesis attenuation by LDL through restoring expression of HIFs. Together, these findings argue that HIF-1β might act as a novel cross-link between the HIF and NF-κB pathways in suppression of angiogenesis by LDL, while proteasome inhibitors might promote angiogenesis by reactivating this signaling cascade under hyperlipidemia. PMID:26388611

  1. Hypoxia and Hypoxia Mimetics Decrease Aquaporin 5 (AQP5) Expression through Both Hypoxia Inducible Factor-1α and Proteasome-Mediated Pathways

    PubMed Central

    Kawedia, Jitesh D.; Yang, Fan; Sartor, Maureen A.; Gozal, David; Czyzyk-Krzeska, Maria; Menon, Anil G.

    2013-01-01

    The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels. PMID:23469202

  2. Tert-butylhydroquinone lowers blood pressure in AngII-induced hypertension in mice via proteasome-PTEN-Akt-eNOS pathway

    PubMed Central

    Xu, Bing-Can; Long, Hui-Bao; Luo, Ke-Qin

    2016-01-01

    Tert-butylhydroquinone (tBHQ), as an antioxidant, has been widely used for many years to prevent oxidization of food products. The aim of this study was to investigate whether tBHQ activates endothelial nitric oxide synthase (eNOS) to prevent endothelial dysfunction and lower blood pressure. The role of Akt in tBHQ-induced eNOS phosphorylation was examined in human umbilical vein endothelial cells (HUVEC) or in mice. tBHQ treatment of HUVEC increased both Akt-Ser473 phosphorylation, accompanied with increased eNOS-Ser1177 phosphorylation and NO release. Mechanically, pharmacologic or genetic inhibition of Akt abolished tBHQ-enhanced NO release and eNOS phosphorylation in HUVEC. Gain-function of PTEN or inhibition of 26S proteasome abolished tBHQ-enhanced Akt phosphorylation in HUVEC. Ex vivo analysis indicated that tBHQ improved Ach-induced endothelium-dependent relaxation in LPC-treated mice aortic arteries, which were abolished by inhibition of Akt or eNOS. In animal study, administration of tBHQ significantly increased eNOS-Ser1177 phosphorylation and acetylcholine-induced vasorelaxation, and lowered AngII-induced hypertension in wildtype mice, but not in mice deficient of Akt or eNOS. In conclusion, tBHQ via proteasome-dependent degradation of PTEN increases Akt phosphorylation, resulting in upregulation of eNOS-derived NO production and consequent improvement of endothelial function in vivo. In this way, tBHQ lowers blood pressure in hypertensive mice. PMID:27435826

  3. Emerging Mechanistic Insights into AAA Complexes Regulating Proteasomal Degradation

    PubMed Central

    Förster, Friedrich; Schuller, Jan M.; Unverdorben, Pia; Aufderheide, Antje

    2014-01-01

    The 26S proteasome is an integral element of the ubiquitin-proteasome system (UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells. It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates. The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases, shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship. Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS). PMID:25102382

  4. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-14

    Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  5. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-21

    Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  6. Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway.

    PubMed Central

    Oberdorf, J; Webster, J M; Zhu, C C; Luo, S G; Wojcikiewicz, R J

    1999-01-01

    Activation of certain phosphoinositidase-C-linked cell-surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and, thus, lead to Ins(1,4,5)P3-receptor down-regulation. In the current study we have sought to determine whether the ubiquitin/proteasome pathway is involved in this adaptive response. The data presented show (i) that activation of phosphoinositidase-C-linked receptors causes Ins(1,4,5)P3-receptor ubiquitination in a range of cell types (AR4-2J cells, INS-1 cells and rat cerebellar granule cells), (ii) that the Ins(1,4,5)P3-receptor down-regulation induced by activation of these receptors is blocked by proteasome inhibitors, (iii) that all known Ins(1,4,5)P3 receptors (types I, II and III) are substrates for ubiquitination, (iv) that ubiquitination occurs while Ins(1,4,5)P3 receptors are membrane-bound, (v) that Ins(1,4, 5)P3-receptor ubiquitination and down-regulation are stimulated only by those agonists that elevate Ins(1,4,5)P3 concentration persistently, and (vi) that a portion of cellular Ins(1,4,5)P3 receptors (those that are not type-I-receptor-associated) can be resistant to ubiquitination and degradation. In total these data indicate that the ubiquitin/proteasome pathway mediates Ins(1,4, 5)P3-receptor down-regulation and suggest that ubiquitination is stimulated by the binding of Ins(1,4,5)P3 to its receptor. PMID:10191279

  7. Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro.

    PubMed

    Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

    2012-01-01

    The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant-pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro - PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.

  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. 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.

  10. 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

  11. Processing and MHC class II presentation of exogenous soluble antigen involving a proteasome-dependent cytosolic pathway in CD40-activated B cells.

    PubMed

    Becker, Hans Jiro; Kondo, Eisei; Shimabukuro-Vornhagen, Alexander; Theurich, Sebastian; von Bergwelt-Baildon, Michael S

    2016-08-01

    Activated B cells have the capacity to present antigen and induce immune responses as potent antigen-presenting cells (APCs). As in other APCs, antigen presentation by B cells involves antigen internalization, antigen processing, and peptide loading onto MHC molecules. However, while the mechanism of antigen processing has been studied extensively in other APCs, this pathway remains elusive in B cells. The aim of this study was to investigate the MHC class II processing pathway in CD40-activated B cells (CD40Bs), as a model for activated, antigen-presenting B cells. Using CMV pp65 as a model antigen, we evaluated processing and presentation of the CD4 + T-cell epitope 509-523 (K509) by human CD40Bs in ELISPOT assays. As expected, stimulation of specific CD4 + T-cell clones was attenuated after pretreatment of CD40Bs with inhibitors of classic class II pathway components. However, proteasome inhibitors such as epoxomicin limited antigen presentation as well. This suggests that the antigen is processed in a non-classical, cytosolic MHC class II pathway. Further experiments with truncated protein variants revealed involvement of the proteasome in processing of the N and C extensions of the epitope. Access to the cytosol was shown to be size dependent. Epoxomicin sensitivity exclusively in CD40B cells, but not in dendritic cells, suggests a novel processing mechanism unique to this APC. Our data suggest that B cells process antigen using a distinct, non-classical class II pathway.

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

    PubMed

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

    2015-12-01

    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.

  13. 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

  14. Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway.

    PubMed

    Dong, Bin; Li, Hai; Singh, Amar Bahadur; Cao, Aiqin; Liu, Jingwen

    2015-02-13

    Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α.

  15. Aβ40 Reduces P-Glycoprotein at the Blood–Brain Barrier through the Ubiquitin–Proteasome Pathway

    PubMed Central

    Zhong, Yu; Wolf, Andrea; LeVine, Harry; Miller, David S.; Bauer, Björn

    2016-01-01

    Failure to clear amyloid-β (Aβ) from the brain is in part responsible for Aβ brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aβ across the blood–brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood–brain barrier in AD, which has been shown to be associated with Aβ brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We exposed isolated rat brain capillaries to 100 nm Aβ40, Aβ40, aggregated Aβ40, and Aβ42. We observed that only Aβ40 triggered reduction of P-gp protein expression and transport activity levels; this occurred in a dose- and time-dependent manner. To identify the steps involved in Aβ-mediated P-gp reduction, we inhibited protein ubiquitination, protein trafficking, and the ubiquitin–proteasome system, and monitored P-gp protein expression, transport activity, and P-gp-ubiquitin levels. Thus, exposing brain capillaries to Aβ40 triggers ubiquitination, internalization, and proteasomal degradation of P-gp. These findings may provide potential therapeutic targets within the blood–brain barrier to limit P-gp degradation in AD and improve Aβ brain clearance. SIGNIFICANCE STATEMENT The mechanism reducing blood–brain barrier P-glycoprotein (P-gp) in Alzheimer's disease is poorly understood. In the present study, we focused on defining this mechanism. We demonstrate that Aβ40 drives P-gp ubiquitination, internalization, and proteasome-dependent degradation, reducing P-gp protein expression and transport activity in isolated brain capillaries. These findings may provide potential therapeutic avenues within the blood–brain barrier to limit P-gp degradation in Alzheimer's disease and improve Aβ brain clearance. PMID:26865616

  16. Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

    PubMed

    Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M; Jones, John E; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R; Eisner, Gilbert; Gildea, John J; Felder, Robin A; Jose, Pedro A

    2008-06-01

    Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

  17. Progressive skeletal muscle weakness in transgenic mice expressing CTG expansions is associated with the activation of the ubiquitin-proteasome pathway.

    PubMed

    Vignaud, Alban; Ferry, Arnaud; Huguet, Aline; Baraibar, Martin; Trollet, Capucine; Hyzewicz, Janek; Butler-Browne, Gillian; Puymirat, Jack; Gourdon, Genevieve; Furling, Denis

    2010-05-01

    Myotonic dystrophy type 1 (DM1) is a neuromuscular disease caused by the expansion of a CTG repeat in the DMPK gene and characterised by progressive skeletal muscle weakness and wasting. To investigate the effects of the CTG expansion on the physiological function of the skeletal muscles, we have used a transgenic mouse model carrying the human DM1 region with 550 expanded CTG repeats. Maximal force is reduced in the skeletal muscles of 10-month-old but not in 3-month-old DM1 mice when compared to age-matched non-transgenic littermates. The progressive weakness observed in the DM1 mice is directly related to the reduced muscle mass and muscle fibre size. A significant increase in trypsin-like proteasome activity and Fbxo32 expression is also measured in the DM1 muscles indicating that an atrophic process mediated by the ubiquitin-proteasome pathway may contribute to the progressive muscle wasting and weakness in the DM1 mice.

  18. ATPase and ubiquitin-binding proteins of the yeast proteasome.

    PubMed

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

    1997-03-01

    The 26S proteasome is a 2-Megadalton proteolytic complex with over 30 distinct subunits. The 19S particle, a subcomplex of the 26S proteasome, is thought to confer ATP-dependence and ubiquitin-dependence on the proteolytic core particle of the proteasome. Given the complexity of the 19S particle, genetic approaches are likely to play an important role in its analysis. We have initiated biochemical and genetic studies of the 19S particle in Saccharomyces cerevisiae. Here we describe the localization to the proteasome of several ATPases that were previously proposed to be involved in transcription. Independent studies indicate that the mammalian 26S proteasome contains closely related ATPases. We have also found that the multiubiquitin chain binding protein Mcb1, a homolog of the mammalian S5a protein, is a subunit of the yeast proteasome. However, contrary to expectation, MCB1 is not an essential gene in yeast. The mcb1 mutant grows at a nearly wild-type rate, and the breakdown of most ubiquitin-protein conjugates is unaffected in this strain. One substrate, Ub-Proline-beta gal, was found to require MCB1 for its breakdown, but it remains unclear whether Mcb1 serves as a ubiquitin receptor in this process. Our data suggest that the recognition of ubiquitin conjugates by the proteasome is a complex process which must involve proteins other than Mcb1.

  19. The role of the ubiquitin-proteasome system in the response of the ligninolytic fungus Trametes versicolor to nitrogen deprivation.

    PubMed

    Staszczak, Magdalena

    2008-03-01

    The white rot fungus Trametes versicolor is an efficient lignin degrader with ecological significance and industrial applications. Lignin-modifying enzymes of white rot fungi are mainly produced during secondary metabolism triggered in these microorganisms by nutrient deprivation. Selective ubiquitin/proteasome-mediated proteolysis is known to play a crucial role in the response of cells to various stresses such as nutrient limitation, heat shock, and heavy metal exposure. Previous studies from our laboratory demonstrated that proteasomal degradation of intracellular proteins is involved in the regulation of laccase, a major ligninolytic enzyme of T. versicolor, in response to cadmium. In the present study, it was found that the 6-h nitrogen starvation leads to depletion of intracellular free ubiquitin pool in T. versicolor. The difference in the intracellular level of free monomeric ubiquitin observed between the mycelium extract from the nitrogen-deprived and that from the nitrogen-sufficient culture was accompanied by the different pattern of ubiquitin-dependent degradation. Furthermore, it was found that nitrogen deprivation affected 26S proteasome activities of T. versicolor. Proteasome inhibition by lactacystin beta-lactone, a highly specific agent, increased laccase activity in nitrogen-deprived cultures, but not in nitrogen-sufficient cultures. The present study implicates the ubiquitin/proteasome-mediated proteolytic pathway in the response of T. versicolor to nitrogen deprivation.

  20. The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Binds to HIF-1α and Causes Its Degradation via the Proteasome Pathway

    PubMed Central

    Kousoulas, Konstantin G.

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761–6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway. PMID:23221556

  1. Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast

    SciTech Connect

    Mannen, Taro; Andoh, Tomoko; Tani, Tokio

    2008-01-25

    Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1{sup +} gene ({delta}dss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A){sup +} RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in {delta}dss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1{sup +} gene, which encodes a component of the 26S proteasome, as a suppressor for the ts{sup -} phenotype of {delta}dss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.

  2. 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.

  3. Proteomic Analysis Implicates Dominant Alterations of RNA Metabolism and the Proteasome Pathway in the Cellular Response to Carbon-Ion Irradiation

    PubMed Central

    Xie, Da-Fei; Xie, Yi; Liu, Xiao-Dan; Wang, Qi; Sui, Li; Song, Man; Zhang, Hong; Zhou, Jianhua; Zhou, Ping-Kun

    2016-01-01

    Radiotherapy with heavy ions is considered advantageous compared to irradiation with photons due to the characteristics of the Braggs peak and the high linear energy transfer (LET) value. To understand the mechanisms of cellular responses to different LET values and dosages of heavy ion radiation, we analyzed the proteomic profiles of mouse embryo fibroblast MEF cells exposed to two doses from different LET values of heavy ion 12C. Total proteins were extracted from these cells and examined by Q Exactive with Liquid Chromatography (LC)—Electrospray Ionization (ESI) Tandem MS (MS/MS). Using bioinformatics approaches, differentially expressed proteins with 1.5 or 2.0-fold changes between different dosages of exposure were compared. With the higher the dosage and/or LET of ion irradiation, the worse response the cells were in terms of protein expression. For instance, compared to the control (0 Gy), 771 (20.2%) proteins in cells irradiated at 0.2 Gy of carbon-ion radiation with 12.6 keV/μm, 313 proteins (8.2%) in cells irradiated at 2 Gy of carbon-ion radiation with 12.6 keV/μm, and 243 proteins (6.4%) in cells irradiated at 2 Gy of carbon-ion radiation with 31.5 keV/μm exhibited changes of 1.5-fold or greater. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Munich Information Center for Protein Sequences (MIPS) analysis, and BioCarta analysis all indicated that RNA metabolic processes (RNA splicing, destabilization and deadenylation) and proteasome pathways may play key roles in the cellular response to heavy-ion irradiation. Proteasome pathways ranked highest among all biological processes associated with heavy carbon-ion irradiation. In addition, network analysis revealed that cellular pathways involving proteins such as Col1a1 and Fn1 continued to respond to high dosages of heavy-ion irradiation, suggesting that these pathways still protect cells against damage. However, pathways such as those involving Ikbkg1 responded

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

    SciTech Connect

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

    2013-07-05

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

  5. Autophagy and ubiquitin–proteasome system contribute to sperm mitophagy after mammalian fertilization

    PubMed Central

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

    2016-01-01

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

  6. Proteasome stress responses in Schistosoma mansoni.

    PubMed

    de Paula, Renato Graciano; de Magalhães Ornelas, Alice Maria; Morais, Enyara Rezende; de Souza Gomes, Matheus; de Paula Aguiar, Daniela; Magalhães, Lizandra Guidi; Rodrigues, Vanderlei

    2015-05-01

    The proteasome proteolytic system is the major ATP-dependent protease in eukaryotic cells responsible for intracellular protein turnover. Schistosoma mansoni has been reported to contain an ubiquitin-proteasome proteolytic pathway, and many studies have suggested a biological role of proteasomes in the development of this parasite. Additionally, evidence has suggested diversity in proteasome composition under several cellular conditions, and this might contribute to the regulation of its function in this parasite. The proteasomal system has been considered important to support the protein homeostasis during cellular stress. In this study, we described in vitro effects of oxidative stress, heat shock, and chemical stress on S. mansoni adults. Our findings showed that chemical stress induced with curcumin, IBMX, and MG132 modified the gene expression of the proteasomal enzymes SmHul5 and SmUbp6. Likewise, the expression of these genes was upregulated during oxidative stress and heat shock. Analyses of the S. mansoni life cycle showed differential gene expression in sporocysts, schistosomulae, and miracidia. These results suggested that proteasome accessory proteins participate in stress response during the parasite development. The expression level of SmHul5 and SmUbp6 was decreased by 16-fold and 9-fold, respectively, by the chemical stress induced with IBMX, which suggests proteasome disassembly. On the other hand, curcumin, MG132, oxidative stress, and heat shock increased the expression of these genes. Furthermore, the gene expression of maturation proteasome protein (SmPOMP) was increased in stress conditions induced by curcumin, MG132, and H₂O₂, which could be related to the synthesis of new proteasomes. S. mansoni adult worms were found to utilize similar mechanisms to respond to different conditions of stress. Our results demonstrated that oxidative stress, heat shock, and chemical stress modified the expression profile of genes related to the ubiquitin-proteasome

  7. Phosphorylation regulates mycobacterial proteasome.

    PubMed

    Anandan, Tripti; Han, Jaeil; Baun, Heather; Nyayapathy, Seeta; Brown, Jacob T; Dial, Rebekah L; Moltalvo, Juan A; Kim, Min-Seon; Yang, Seung Hwan; Ronning, Donald R; Husson, Robert N; Suh, Joowon; Kang, Choong-Min

    2014-09-01

    Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

  8. Molecular mechanisms of proteasome plasticity in aging.

    PubMed

    Rodriguez, Karl A; Gaczynska, Maria; Osmulski, Pawel A

    2010-02-01

    The ubiquitin-proteasome pathway plays a crucial role in regulation of intracellular protein turnover. Proteasome, the central protease of the pathway, encompasses multi-subunit 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.

  9. The Proteasome Inhibitor Bortezomib Affects Chondrosarcoma Cells via the Mitochondria-Caspase Dependent Pathway and Enhances Death Receptor Expression and Autophagy

    PubMed Central

    Lohberger, Birgit; Steinecker-Frohnwieser, Bibiane; Stuendl, Nicole; Kaltenegger, Heike; Leithner, Andreas; Rinner, Beate

    2016-01-01

    High grade chondrosarcoma is characterized by its lack of response to conventional cytotoxic chemotherapy, the tendency to develop lung metastases, and low survival rates. Research within the field prioritizes the development and expansion of new treatment options for dealing with unresectable or metastatic diseases. Numerous clinical trials using the proteasome inhibitor bortezomib have shown specific efficacy as an active antitumor agent for treating a variety of solid tumors. However, as of yet the effect of bortezomib on chondrosarcoma has not been investigated. In our study, bortezomib decreased cell viability and proliferation in two different chondrosarcoma cell lines in a time- and dose dependent manner. FACS analysis, mRNA- and protein expression studies illustrated that induction of apoptosis developed through the intrinsic mitochondria-caspase dependent pathway. Furthermore, bortezomib treatment significantly increased expression of the death receptors TRAILR-1 and TRAILR-2 in chondrosarcoma cells. An increased expression of the autophagy markers Atg5/12, Beclin, and LC3BI-II supports the interpretation that bortezomib functions as a trigger for autophagy. Our results demonstrated for the first time that bortezomib reduced viability and proliferation of chondrosarcoma cells, induced apoptosis via the mitochondria-caspase dependent pathway and enhanced death receptor expression and autophagy. PMID:27978543

  10. Molecular and cellular roles of PI31 (PSMF1) protein in regulation of proteasome function.

    PubMed

    Li, Xiaohua; Thompson, David; Kumar, Brajesh; DeMartino, George N

    2014-06-20

    We investigated molecular features and cellular roles of PI31 (PSMF1) on regulation of proteasome function. PI31 has a C-terminal HbYX (where Hb is a hydrophobic amino acid, Y is tyrosine, and X is any amino acid) motif characteristic of several proteasome activators. Peptides corresponding to the PI31 C terminus also bind to and activate the 20 S proteasome in an HbYX-dependent manner, but intact PI31protein inhibits in vitro 20 S activity. Binding to and inhibition of the proteasome by PI31 are conferred by the HbYX-containing proline-rich C-terminal domain but do not require HbYX residues. Thus, multiple regions of PI31 bind independently to the proteasome and collectively determine effects on activity. PI31 blocks the ATP-dependent in vitro assembly of 26 S proteasome from 20 S proteasome and PA700 subcomplexes but has no effect on in vitro activity of the intact 26 S proteasome. To determine the physiologic significance of these in vitro effects, we assessed multiple aspects of cellular proteasome content and function after altering PI31 levels. We detected no change in overall cellular proteasome content or function when PI31 levels were either increased by moderate ectopic overexpression or decreased by RNA interference (RNAi). We also failed to identify a role of PI31 ADP-ribosylation as a mechanism for regulation of overall 26 S proteasome content and function, as recently proposed. Thus, despite its in vitro effects on various proteasome activities and its structural relationship to established proteasome regulators, cellular roles and mechanisms of PI31 in regulation of proteasome function remain unclear and require future definition.

  11. Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway.

    PubMed

    Lucas, Christopher D; Allen, Keith C; Dorward, David A; Hoodless, Laura J; Melrose, Lauren A; Marwick, John A; Tucker, Carl S; Haslett, Christopher; Duffin, Rodger; Rossi, Adriano G

    2013-03-01

    Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time- and concentration-dependent neutrophil apoptosis (apigenin, EC=12.2 μM; luteolin, EC=14.6 μM; and wogonin, EC=28.9 μM). Induction of apoptosis was caspase dependent, as it was blocked by the broad-spectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents.

  12. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

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

  13. 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

  14. Identification of proteasome subunit beta type 2 associated with deltamethrin detoxification in Drosophila Kc cells by cDNA microarray analysis and bioassay analyses.

    PubMed

    Hu, Junli; Jiao, Dongxu; Xu, Qin; Ying, Xiaoli; Liu, Wei; Chi, Qingping; Ye, Yuting; Li, Xueyu; Cheng, Luogen

    2016-05-10

    Insecticide deltamethrin resistance has presented a difficult obstacle for pest control and the resistance development is complex and associated with many genes. To better understand the possible molecular mechanisms involved in DM stress, in this study, cDNA microarray analysis was employed. 448 differentially expressed genes with at least a 2-fold expression difference were identified in Drosophila cells after DM exposure. Moreover, some genes were confirmed with qPCR, which yielded results consistent with the microarray analysis. Three members of the ubiquitin-proteasome system were significantly elevated in DM-stressed cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM detoxification. The proteasome beta2 subunit (Prosbeta2) is a member of 20S proteasome subunit family, which forms the proteolytic core of 26S proteasome. Whether Prosbeta2 participates in DM detoxification requires further study. RNAi and heterologous expression were conducted to investigate the contribution of Prosbeta2 in DM detoxification. The results revealed Prosbeta2 knockdown significantly reduce the level of DM detoxification in RNAi-treated cells after 48 h. Overexpression of Prosbeta2 increased cellular viability. These detoxification results represent the first evidence that Prosbeta2 plays a role in the detoxification of DM, which may provide new idea and target for studying the molecular mechanisms of insect resistance.

  15. 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

  16. Evolution of proteasome regulators in eukaryotes.

    PubMed

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

    2015-05-04

    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.

  17. 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.

  18. Effects of Radiation on Proteasome Function in Prostate Cancer Cells

    DTIC Science & Technology

    2010-02-01

    proteasomes from irradiated and un-irradiated samples were loaded on a 11cm agarose strip (BioRad) for their separation based on their isoelectric point (pI...25micrograms of pure 26S preparations from PC-3 ZsGreenODC-negative or PC3 ZsGreenODC-positive sorted cells were focused on 11cm strips for the

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

    PubMed

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

    2014-09-15

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

  20. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system

    PubMed Central

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-01

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. PMID:26732515

  1. The transcriptional repressor protein PRH interacts with the proteasome.

    PubMed Central

    Bess, Kirstin L; Swingler, Tracey E; Rivett, A Jennifer; Gaston, Kevin; Jayaraman, Padma-Sheela

    2003-01-01

    PRH (proline-rich homeodomain protein)/Hex is important in the control of cell proliferation and differentiation. We have shown previously that PRH contains two domains that can bring about transcriptional repression independently; the PRH homeodomain represses transcription by binding to TATA box sequences, whereas the proline-rich N-terminal domain can repress transcription by interacting with members of the Groucho/TLE (transducin-like enhancer of split) family of co-repressor proteins. The proteasome is a multi-subunit protein complex involved in the processing and degradation of proteins. Some proteasome subunits have been suggested to play a role in the regulation of transcription. In the present study, we show that PRH interacts with the HC8 subunit of the proteasome in the context of both 20 and 26 S proteasomes. Moreover, we show that PRH is associated with the proteasome in haematopoietic cells and that the proline-rich PRH N-terminal domain is responsible for this interaction. Whereas PRH can be cleaved by the proteasome, it does not appear to be degraded rapidly in vitro or in vivo, and the proteolytic activity of the proteasome is not required for transcriptional repression by PRH. However, proteasomal digestion of PRH can liberate truncated PRH proteins that retain the ability to bind to DNA. We discuss these findings in terms of the biological role of PRH in gene regulation and the control of cell proliferation. PMID:12826010

  2. Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation in Saccharomyces cerevisiae

    PubMed Central

    Kelly, Shane P.; Bedwell, David M.

    2015-01-01

    Protein turnover is an important regulatory mechanism that facilitates cellular adaptation to changing environmental conditions. Previous studies have shown that ribosome abundance is reduced during nitrogen starvation by a selective autophagy mechanism termed ribophagy, which is dependent upon the deubiquitinase Ubp3p. In this study, we asked whether the abundance of various translation and RNA turnover factors are reduced following the onset of nitrogen starvation in Saccharomyces cerevisiae. We found distinct differences in the abundance of the proteins tested following nitrogen starvation: (1) The level of some did not change; (2) others were reduced with kinetics similar to ribophagy, and (3) a few proteins were rapidly depleted. Furthermore, different pathways differentially degraded the various proteins upon nitrogen starvation. The translation factors eRF3 and eIF4GI, and the decapping enhancer Pat1p, required an intact autophagy pathway for their depletion. In contrast, the deadenylase subunit Pop2p and the decapping enzyme Dcp2p were rapidly depleted by a proteasome-dependent mechanism. The proteasome-dependent depletion of Dcp2p and Pop2p was also induced by rapamycin, suggesting that the TOR1 pathway influences this pathway. Like ribophagy, depletion of eIF4GI, eRF3, Dcp2p, and Pop2p was dependent upon Ubp3p to varying extents. Together, our results suggest that the autophagy and proteasomal pathways degrade distinct translation and RNA turnover factors in a Ubp3p-dependent manner during nitrogen starvation. While ribophagy is thought to mediate the reutilization of scarce resources during nutrient limitation, our results suggest that the selective degradation of specific proteins could also facilitate a broader reprogramming of the post-transcriptional control of gene expression. PMID:25795416

  3. Ubiquitin-independent proteasomal degradation of Fra-1 is antagonized by Erk1/2 pathway-mediated phosphorylation of a unique C-terminal destabilizer.

    PubMed

    Basbous, Jihane; Chalbos, Dany; Hipskind, Robert; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2007-06-01

    Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G(0)/G(1)-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins.

  4. APC/C and retinoblastoma interaction: cross-talk of retinoblastoma protein with the ubiquitin proteasome pathway

    PubMed Central

    Ramanujan, Ajeena; Tiwari, Swati

    2016-01-01

    The ubiquitin (Ub) ligase anaphase promoting complex/cyclosome (APC/C) and the tumour suppressor retinoblastoma protein (pRB) play key roles in cell cycle regulation. APC/C is a critical regulator of mitosis and G1-phase of the cell cycle whereas pRB keeps a check on proliferation by inhibiting transition to the S-phase. APC/C and pRB interact with each other via the co-activator of APC/C, FZR1, providing an alternative pathway of regulation of G1 to S transition by pRB using a post-translational mechanism. Both pRB and FZR1 have complex roles and are implicated not only in regulation of cell proliferation but also in differentiation, quiescence, apoptosis, maintenance of chromosomal integrity and metabolism. Both are also targeted by transforming viruses. We discuss recent advances in our understanding of the involvement of APC/C and pRB in cell cycle based decisions and how these insights will be useful for development of anti-cancer and anti-viral drugs. PMID:27402801

  5. Cloning and partial characterization of the proteasome S4 ATPase from Plasmodium falciparum.

    PubMed

    Certad, G; Abrahem, A; Georges, E

    1999-11-01

    Certad, G., Abrahem, A., and Georges, E. 1999. Cloning and Partial characterization of the proteasome S4 ATPase from Plasmodium falciparum. Experimental Parasitology 93, 123-131. The ATP-ubiquitin-proteasome pathway mediates the nonlysosomal degradation of cytosolic proteins in eukaryotic cells. The activities of this pathway have been shown to regulate cell growth and differentiation through modulation of regulatory proteins. The proteasome is a large complex consisting of two multisubunit structures, the 20S and 19S(PA700) or P28 complexes, that combine to form the 26S particles. In this study, we describe the cloning of a cDNA encoding the proteasome subunit 4 ATPase homologue from Plasmodium falciparum (PFS4). Analysis of the PFS4 cDNA sequence shows an open reading frame encoding a deduced protein of 455 amino acids. Moreover, comparison of PFS4 cDNA sequence to that of genomic fragments encoding PFS4 showed identical sequences with no detectable introns. Database searches revealed a high sequence identity to those of rice, yeast, mouse, Drosophila, and human S4 ATPases. However, PFS4 contains two unique inserts of nine and seven amino acid residues in the N-terminal domain. Interestingly, only the rice S4 contains the latter (seven amino acids) insert with four identical amino acids. In vitro expression of the full-length cDNA encoding the PFS4, using a transcription-translation-coupled reticulocyte lysate, shows a 50-kDa [(35)S]methionine-labeled protein which was immunoprecipitated with PFS4 anti-peptide antiserum. Southern blot analysis of genomic DNA digests shows a single gene copy of PFS4 in P. falciparum. Of interest was the effect of the proteasome-specific natural product, lactacystin, on the growth of the parasite, with IC(50) values of 0.6-0.92 microM. The latter IC(50) values of lactacystin for different clones of P. falciparum are comparable to those obtained for mammalian cell lines (0.65 microM), suggesting the presence of a conserved

  6. Proteasome activator 200: the heat is on...

    PubMed

    Savulescu, Anca F; Glickman, Michael H

    2011-05-01

    Proteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1-6), attaches to the same surface of the CP ring, and likewise, induces pore opening.

  7. Mammalian proteasome subtypes: Their diversity in structure and function.

    PubMed

    Dahlmann, Burkhardt

    2016-02-01

    The 20S proteasome is a multicatalytic proteinase catalysing the degradation of the majority of intracellular proteins. Thereby it is involved in almost all basic cellular processes, which is facilitated by its association with various regulator complexes so that it appears in different disguises like 26S proteasome, hybrid-proteasome and others. The 20S proteasome has a cylindrical structure built up by four stacked rings composed of α- and β-subunits. Since the three active site-containing β-subunits can all or in part be replaced by immuno-subunits, three main subpopulations exist, namely standard-, immuno- and intermediate-proteasomes. Due to posttranslational modifications or/and genetic variations all α- and β-subunits occur in multiple iso- or proteoforms. This leads to the fact that each of the three subpopulations is composed of a variety of 20S proteasome subtypes. This review summarizes the knowledge of proteasome subtypes in mammalian cells and tissues and their possible biological and medical relevancy.

  8. Increased proteasome activity determines human embryonic stem cell identity

    PubMed Central

    Vilchez, David; Boyer, Leah; Morantte, Ianessa; Lutz, Margaret; Merkwirth, Carsten; Joyce, Derek; Spencer, Brian; Page, Lesley; Masliah, Eliezer; Berggren, W. Travis; Gage, Fred H.; Dillin, Andrew

    2016-01-01

    Embryonic stem cells are able to replicate continuously in the absence of senescence and, therefore, are immortal in culture1,2. While genome stability is central for survival of stem cells; proteome stability may play an equally important role in stem cell identity and function. Additionally, with the asymmetric divisions invoked by stem cells, the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. We hypothesized that stem cells have an increased proteostasis ability compared to their differentiated counterparts and asked whether proteasome activity differed among human embryonic stem cells (hESCs). Notably, hESC populations exhibit a high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11/RPN-63–5 and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. Proteasome inhibition affects pluripotency of hESCs inducing differentiation towards specific cell lineages. FOXO4, an insulin/IGF-1 responsive transcription factor associated with long lifespan in invertebrates6,7, regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Our results establish a novel regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates with hESC function and identity. PMID:22972301

  9. Rice ROOT ARCHITECTURE ASSOCIATED1 Binds the Proteasome Subunit RPT4 and Is Degraded in a D-Box and Proteasome-Dependent Manner1[W][OA

    PubMed Central

    Han, Ye; Cao, Hong; Jiang, Jiafu; Xu, Yunyuan; Du, Jizhou; Wang, Xin; Yuan, Ming; Wang, Zhiyong; Xu, Zhihong; Chong, Kang

    2008-01-01

    Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle. PMID:18701670

  10. Small acidic protein 1 and SCF(TIR)(1) ubiquitin proteasome pathway act in concert to induce 2,4-dichlorophenoxyacetic acid-mediated alteration of actin in Arabidopsis roots.

    PubMed

    Takahashi, Maho; Umetsu, Kana; Oono, Yutaka; Higaki, Takumi; Blancaflor, Elison B; Rahman, Abidur

    2017-03-01

    2,4-Dichlorophenoxyacetic acid (2,4-D), a functional analogue of auxin, is used as an exogenous source of auxin as it evokes physiological responses like the endogenous auxin, indole-3-acetic acid (IAA). Previous molecular analyses of the auxin response pathway revealed that IAA and 2,4-D share a common mode of action to elicit downstream physiological responses. However, recent findings with 2,4-D-specific mutants suggested that 2,4-D and IAA might also use distinct pathways to modulate root growth in Arabidopsis. Using genetic and cellular approaches, we demonstrate that the distinct effects of 2,4-D and IAA on actin filament organization partly dictate the differential responses of roots to these two auxin analogues. 2,4-D but not IAA altered the actin structure in long-term and short-term assays. Analysis of the 2,4-D-specific mutant aar1-1 revealed that small acidic protein 1 (SMAP1) functions positively to facilitate the 2,4-D-induced depolymerization of actin. The ubiquitin proteasome mutants tir1-1 and axr1-12, which show enhanced resistance to 2,4-D compared with IAA for inhibition of root growth, were also found to have less disrupted actin filament networks after 2,4-D exposure. Consistently, a chemical inhibitor of the ubiquitin proteasome pathway mitigated the disrupting effects of 2,4-D on the organization of actin filaments. Roots of the double mutant aar1-1 tir1-1 also showed enhanced resistance to 2,4-D-induced inhibition of root growth and actin degradation compared with their respective parental lines. Collectively, these results suggest that the effects of 2,4-D on actin filament organization and root growth are mediated through synergistic interactions between SMAP1 and SCF(TIR)(1) ubiquitin proteasome components.

  11. Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

    PubMed Central

    Ma, Yi-ming; Han, Wei; Li, Jia; Hu, Li-hong; Zhou, Yu-bo

    2015-01-01

    Aim: To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms. Methods: Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye. Results: Physalin B inhibited the viability of HCT116 cells with an IC50 value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5–10 μmol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis. Conclusion: Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro. PMID:25832431

  12. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    SciTech Connect

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

    2014-03-25

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

  13. Proteasomes and protein conjugation across domains of life.

    PubMed

    Maupin-Furlow, Julie

    2011-12-19

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes.

  14. 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.

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

    SciTech Connect

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

    2010-01-01

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

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

    SciTech Connect

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

    2010-06-16

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

  17. The role of the proteasome in AML

    PubMed Central

    Csizmar, C M; Kim, D-H; Sachs, Z

    2016-01-01

    Acute myeloid leukemia (AML) is deadly hematologic malignancy. Despite a well-characterized genetic and molecular landscape, targeted therapies for AML have failed to significantly improve clinical outcomes. Over the past decade, proteasome inhibition has been demonstrated to be an effective therapeutic strategy in several hematologic malignancies. Proteasome inhibitors, such as bortezomib and carfilzomib, have become mainstays of treatment for multiple myeloma and mantle cell lymphoma. In light of this success, there has been a surge of literature exploring both the role of the proteasome and the effects of proteasome inhibition in AML. Pre-clinical studies have demonstrated that proteasome inhibition disrupts proliferative cell signaling pathways, exhibits cytotoxic synergism with other chemotherapeutics and induces autophagy of cancer-related proteins. Meanwhile, clinical trials incorporating bortezomib into combination chemotherapy regimens have reported a range of responses in AML patients, with complete remission rates >80% in some cases. Taken together, this preclinical and clinical evidence suggests that inhibition of the proteasome may be efficacious in this disease. In an effort to focus further investigation into this area, these recent studies and their findings are reviewed here. PMID:27911437

  18. Subunit-selective proteasome activity profiling uncovers uncoupled proteasome subunit activities during bacterial infections.

    PubMed

    Misas-Villamil, Johana C; van der Burgh, Aranka M; Grosse-Holz, Friederike; Bach-Pages, Marcel; Kovács, Judit; Kaschani, Farnusch; Schilasky, Sören; Emon, Asif Emran Khan; Ruben, Mark; Kaiser, Markus; Overkleeft, Hermen S; van der Hoorn, Renier A L

    2017-01-24

    The proteasome is a nuclear - cytoplasmic proteolytic complex involved in nearly all regulatory pathways in plant cells. The three different catalytic activities of the proteasome can have different functions but tools to monitor and control these subunits selectively are not yet available in plant science. Here, we introduce subunit-selective inhibitors and dual-color fluorescent activity-based probes for studying two of the three active catalytic subunits of the plant proteasome. We validate these tools in two model plants and use this to study the proteasome during plant-microbe interactions. Our data reveals that Nicotiana benthamiana incorporates two different paralogs of each catalytic subunit into active proteasomes. Interestingly, both β1 and β5 activities are significantly increased upon infection with pathogenic Pseudomonas syringae pv. tomato DC3000 lacking hopQ1-1 (PtoDC3000(ΔhQ)) whilst the activity profile of the β1 subunit changes. Infection with wild-type PtoDC3000 causes proteasome activities that range from strongly induced β1 and β5 activities to strongly suppressed β5 activities, revealing that β1 and β5 activities can be uncoupled during bacterial infection. These selective probes and inhibitors are now available to the plant science community and can be widely and easily applied to study the activity and role of the different catalytic subunits of the proteasome in different plant species. This article is protected by copyright. All rights reserved.

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

    PubMed Central

    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

  20. INT6 May Influence Breast Cancer Formation by Regulating the 26S Proteasome

    DTIC Science & Technology

    2007-04-01

    critically reading the manuscript. We also thank Nick Rhind, Chris Norbury, Roger Tsien , and Colin Gordon for kindly providing materials that are...Yarbrough, C. A., Tsien , R. Y., and Remington, S. J. (2006) Biochemistry 45(32), 9639-9647 20. Bähler, J., Wu, J. Q., Longtine, M. S., Shah, N. G

  1. INT6 May Influence Breast Cancer Formation by Regulating the 26S Proteasome

    DTIC Science & Technology

    2006-04-01

    have shown that int6kd cells display mitotic abnormalities in that they become multinucleated and contain abnormal spindles (see also Figure 1A-D). In...Int6∆C, but not wild type Int6, will lead to formation of larger acini (Figure 3B), suggesting that mInt6, like Int6∆C, might cause abnormal cell...functions in Int6-reduced HeLa cells and found evidence of abnormal mitosis due to the presence of tri-polar spindles (as reported in 2004). 4. I

  2. Regulation of the Response to Radiotherapy and Hyperthermia in Prostate Cancer by the 26s Proteasome

    DTIC Science & Technology

    2003-04-01

    and hair follicles, undergo rapid ’interphase death’ within hours of irradiation. Interphase death is now acknowledged to represent rapid apoptosis...any. Gynaecomastia caused by HAART has been reported Received: 10 January 2002;’acc ted: 15 January 2002. for several drugs, e.g. efavirenz

  3. Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

    PubMed Central

    Brooks, P; Fuertes, G; Murray, R Z; Bose, S; Knecht, E; Rechsteiner, M C; Hendil, K B; Tanaka, K; Dyson, J; Rivett, J

    2000-01-01

    Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes. PMID:10657252

  4. 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

  5. Ubiquitin-proteasome system and hereditary cardiomyopathies.

    PubMed

    Schlossarek, Saskia; Frey, Norbert; Carrier, Lucie

    2014-06-01

    Adequate protein turnover is essential for cardiac homeostasis. Different protein quality controls are involved in the maintenance of protein homeostasis, including molecular chaperones and co-chaperones, the autophagy-lysosomal pathway, and the ubiquitin-proteasome system (UPS). In the last decade, a series of evidence has underlined a major function of the UPS in cardiac physiology and disease. Particularly, recent studies have shown that dysfunctional proteasomal function leads to cardiac disorders. Hypertrophic and dilated cardiomyopathies are the two most prevalent inherited cardiomyopathies. Both are primarily transmitted as an autosomal-dominant trait and mainly caused by mutations in genes encoding components of the cardiac sarcomere, including a relevant striated muscle-specific E3 ubiquitin ligase. A growing body of evidence indicates impairment of the UPS in inherited cardiomyopathies as determined by measurement of the level of ubiquitinated proteins, the activities of the proteasome and/or the use of fluorescent UPS reporter substrates. The present review will propose mechanisms of UPS impairment in inherited cardiomyopathies, summarize the potential consequences of UPS impairment, including activation of the unfolded protein response, and underline some therapeutic options available to restore proteasome function and therefore cardiac homeostasis and function. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy".

  6. 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

  7. Recognition and Cleavage of Related to Ubiquitin 1 (Rub1) and Rub1-Ubiquitin Chains by Components of the Ubiquitin-Proteasome System*

    PubMed Central

    Singh, Rajesh K.; Zerath, Sylvia; Kleifeld, Oded; Scheffner, Martin; Glickman, Michael H.; Fushman, David

    2012-01-01

    Of all ubiquitin-like proteins, Rub1 (Nedd8 in mammals) is the closest kin of ubiquitin. We show via NMR that structurally, Rub1 and ubiquitin are fundamentally similar as well. Despite these profound similarities, the prevalence of Rub1/Nedd8 and of ubiquitin as modifiers of the proteome is starkly different, and their attachments to specific substrates perform different functions. Recently, some proteins, including p53, p73, EGFR, caspase-7, and Parkin, have been shown to be modified by both Rub1/Nedd8 and ubiquitin within cells. To understand whether and how it might be possible to distinguish among the same target protein modified by Rub1 or ubiquitin or both, we examined whether ubiquitin receptors can differentiate between Rub1 and ubiquitin. Surprisingly, Rub1 interacts with proteasome ubiquitin-shuttle proteins comparably to ubiquitin but binds more weakly to a proteasomal ubiquitin receptor Rpn10. We identified Rub1-ubiquitin heteromers in yeast and Nedd8-Ub heteromers in human cells. We validate that in human cells and in vitro, human Rub1 (Nedd8) forms chains with ubiquitin where it acts as a chain terminator. Interestingly, enzymatically assembled K48-linked Rub1-ubiquitin heterodimers are recognized by various proteasomal ubiquitin shuttles and receptors comparably to K48-linked ubiquitin homodimers. Furthermore, these heterologous chains are cleaved by COP9 signalosome or 26S proteasome. A derubylation function of the proteasome expands the repertoire of its enzymatic activities. In contrast, Rub1 conjugates may be somewhat resilient to the actions of other canonical deubiquitinating enzymes. Taken together, these findings suggest that once Rub1/Nedd8 is channeled into ubiquitin pathways, it is recognized essentially like ubiquitin. PMID:23105008

  8. The proteasome: mechanisms of biology and markers of activity and response to treatment in multiple myeloma.

    PubMed

    Manasanch, Elisabet E; Korde, Neha; Zingone, Adriana; Tageja, Nishant; Fernandez de Larrea, Carlos; Bhutani, Manisha; Wu, Peter; Roschewski, Mark; Landgren, Ola

    2014-08-01

    Since the early 1990s, the synthesis and subsequent clinical application of small molecule inhibitors of the ubiquitin proteasome pathway (UPP) has revolutionized the treatment and prognosis of multiple myeloma. In this review, we summarize important aspects of the biology of the UPP with a focus on its structure and key upstream/downstream regulatory components. We then review current knowledge of plasma cell sensitivity to proteasome inhibition and highlight new proteasome inhibitors that have recently entered clinical development. Lastly, we address the putative role of circulating proteasomes as a novel biomarker in multiple myeloma and provide guidance for future clinical trials using proteasome inhibitors.

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

    PubMed Central

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

    2013-01-01

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

  10. Structure of a proteasome Pba1-Pba2 complex: implications for proteasome assembly, activation, and biological function.

    PubMed

    Stadtmueller, Beth M; Kish-Trier, Erik; Ferrell, Katherine; Petersen, Charisse N; Robinson, Howard; Myszka, David G; Eckert, Debra M; Formosa, Tim; Hill, Christopher P

    2012-10-26

    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.

  11. Denervation-Induced Activation of the Standard Proteasome and Immunoproteasome

    PubMed Central

    Ferrington, Deborah A.; Baumann, Cory W.; Thompson, LaDora V.

    2016-01-01

    The standard 26S proteasome is responsible for the majority of myofibrillar protein degradation leading to muscle atrophy. The immunoproteasome is an inducible form of the proteasome. While its function has been linked to conditions of atrophy, its contribution to muscle proteolysis remains unclear. Therefore, the purpose of this study was to determine if the immunoproteasome plays a role in skeletal muscle atrophy induced by denervation. Adult male C57BL/6 wild type (WT) and immunoproteasome knockout lmp7-/-/mecl-1-/- (L7M1) mice underwent tibial nerve transection on the left hindlimb for either 7 or 14 days, while control mice did not undergo surgery. Proteasome activity (caspase-, chymotrypsin-, and trypsin- like), protein content of standard proteasome (β1, β5 and β2) and immunoproteasome (LMP2, LMP7 and MECL-1) catalytic subunits were determined in the gastrocnemius muscle. Denervation induced significant atrophy and was accompanied by increased activities and protein content of the catalytic subunits in both WT and L7M1 mice. Although denervation resulted in a similar degree of muscle atrophy between strains, the mice lacking two immunoproteasome subunits showed a differential response in the extent and duration of proteasome features, including activities and content of the β1, β5 and LMP2 catalytic subunits. The results indicate that immunoproteasome deficiency alters the proteasome’s composition and activities. However, the immunoproteasome does not appear to be essential for muscle atrophy induced by denervation. PMID:27875560

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

    PubMed Central

    Jazirehi, Ali R.; Economou, James S.

    2012-01-01

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

  13. 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...

  14. 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...

  15. Proteasome dysfunction induces muscle growth defects and protein aggregation

    PubMed Central

    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

    2014-01-01

    ABSTRACT 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. PMID:25380823

  16. Emerging therapies targeting the ubiquitin proteasome system in cancer

    PubMed Central

    Weathington, Nathaniel M.; Mallampalli, Rama K.

    2014-01-01

    The ubiquitin proteasome system (UPS) is an essential metabolic constituent of cellular physiology that tightly regulates cellular protein concentrations with specificity and precision to optimize cellular function. Inhibition of the proteasome has proven very effective in the treatment of multiple myeloma, and this approach is being tested for utility in other malignancies. New pharmaceuticals targeting the proteasome itself or specific proximal pathways of the UPS are in development as antiproliferatives or immunomodulatory agents. In this article, we discuss the biology of UPS-targeting drugs, their use as therapy for neoplasia, and the state of clinical and preclinical development for emerging therapeutics. PMID:24382383

  17. Emerging therapies targeting the ubiquitin proteasome system in cancer.

    PubMed

    Weathington, Nathaniel M; Mallampalli, Rama K

    2014-01-01

    The ubiquitin proteasome system (UPS) is an essential metabolic constituent of cellular physiology that tightly regulates cellular protein concentrations with specificity and precision to optimize cellular function. Inhibition of the proteasome has proven very effective in the treatment of multiple myeloma, and this approach is being tested for utility in other malignancies. New pharmaceuticals targeting the proteasome itself or specific proximal pathways of the UPS are in development as antiproliferatives or immunomodulatory agents. In this article, we discuss the biology of UPS-targeting drugs, their use as therapy for neoplasia, and the state of clinical and preclinical development for emerging therapeutics.

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

    PubMed

    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

    2015-04-07

    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.

  19. 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.

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

    DOE PAGES

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; ...

    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

  1. A leucine-rich diet modulates the tumor-induced down-regulation of the MAPK/ERK and PI3K/Akt/mTOR signaling pathways and maintains the expression of the ubiquitin-proteasome pathway in the placental tissue of NMRI mice.

    PubMed

    Viana, Laís Rosa; Gomes-Marcondes, Maria Cristina Cintra

    2015-02-01

    Placental tissue injury is concomitant with tumor development. We investigated tumor-driven placental damage by tracing certain steps of the protein synthesis and degradation pathways under leucine-rich diet supplementation in MAC16 tumor-bearing mice. Cell signaling and ubiquitin-proteasome pathways were assessed in the placental tissues of pregnant mice, which were distributed into three groups on a control diet (pregnant control, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid) and three other groups on a leucine-rich diet (pregnant, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid). MAC tumor growth down-regulated the cell-signaling pathways of the placental tissue and decreased the levels of IRS-1, Akt/PKB, Erk/MAPK, mTOR, p70S6K, STAT3, and STAT6 phosphorylated proteins, as assessed by the multiplex Millipore Luminex assay. Leucine supplementation maintained the levels of these proteins within the established cell-signaling pathways. In the tumor-bearing group (MAC) only, the placental tissue showed increased PC5 mRNA expression, as assessed by quantitative RT-PCR, decreased 19S and 20S protein expression, as assessed by Western blot analysis, and decreased placental tyrosine levels, likely reflecting up-regulation of the ubiquitin-proteasome pathway. Similar effects were found in the pregnant injected with MAC-ascitic fluid group, confirming that the effects of the tumor were mimicked by MAC-ascitic fluid injection. Although tumor progression occurred, the degradation pathway-related protein levels were modulated under leucine-supplementation conditions. In conclusion, tumor evolution reduced the protein expression of the cell-signaling pathway associated with elevated protein degradation, thereby jeopardizing placental activity. Under the leucine-rich diet, the impact of cancer on placental function could be minimized by improving the cell-signaling activity and reducing the proteolytic process.

  2. 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.

  3. Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites.

    PubMed

    Muñoz, Christian; San Francisco, Juan; Gutiérrez, Bessy; González, Jorge

    2015-01-01

    In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target.

  4. Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites

    PubMed Central

    Muñoz, Christian; San Francisco, Juan; Gutiérrez, Bessy; González, Jorge

    2015-01-01

    In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target. PMID:26090380

  5. Proteasome Regulation of ULBP1 Transcription

    PubMed Central

    Butler, James E.; Moore, Mikel B.; Presnell, Steven R.; Chan, Huei-Wei; Chalupny, N. Jan; Lutz, Charles T.

    2009-01-01

    Killer lymphocytes recognize stress-activated NKG2D ligands on tumors. We examined NKG2D ligand expression in head and neck squamous cell carcinoma (HNSCC) cells and other cell lines. HNSCC cells typically expressed MHC class I chain-related gene A (MICA), MICB, UL16-binding protein (ULBP)2, and ULBP3, but they were uniformly negative for cell surface ULBP1 and ULBP4. We then studied how cancer treatments affected NKG2D ligand expression. NKG2D ligand expression was not changed by most cancer-relevant treatments. However, bortezomib and other proteasome inhibitor drugs with distinct mechanisms of action dramatically and specifically up-regulated HNSCC ULBP1 mRNA and cell surface protein. Proteasome inhibition also increased RNA for ULBP1 and other NKG2D ligands in nontransformed human keratinocytes. Proteasome inhibitor drugs increased ULBP1 transcription by acting at a site in the 522-bp ULBP1 promoter. Although the DNA damage response pathways mediated by ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) signaling had been reported to up-regulate NKG2D ligand expression, we found that ULBP1 up-regulation was not inhibited by caffeine and wortmannin, inhibitors of ATM/ATR signaling. ULBP1 expression in HNSCC cells was not increased by several ATM/ATR activating treatments, including bleomycin, cisplatin, aphidicolin, and hydroxyurea. Ionizing radiation caused ATM activation in HNSCC cells, but high-level ULBP1 expression was not induced by gamma radiation or UV radiation. Thus, ATM/ATR signaling was neither necessary nor sufficient for high-level ULBP1 expression in human HNSCC cell lines and could not account for the proteasome effect. The selective induction of ULBP1 expression by proteasome inhibitor drugs, along with variable NKG2D ligand expression by human tumor cells, indicates that NKG2D ligand genes are independently regulated. PMID:19414815

  6. Synthetic analogs of green tea polyphenols as proteasome inhibitors.

    PubMed Central

    Smith, David M.; Wang, Zhigang; Kazi, Aslamuzzaman; Li, Lian-Hai; Chan, Tak-Hang; Dou, Q. Ping

    2002-01-01

    BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing

  7. Assembly mechanisms of specialized core particles of the proteasome.

    PubMed

    Bai, Minghui; Zhao, Xian; Sahara, Kazutaka; Ohte, Yuki; Hirano, Yuko; Kaneko, Takeumi; Yashiroda, Hideki; Murata, Shigeo

    2014-07-16

    The 26S proteasome has a highly complicated structure comprising the 20S core particle (CP) and the 19S regulatory particle (RP). Along with the standard CP in all eukaryotes, vertebrates have two more subtypes of CP called the immunoproteasome and the thymoproteasome. The immunoproteasome has catalytic subunits β1i, β2i, and β5i replacing β1, β2, and β5 and enhances production of major histocompatibility complex I ligands. The thymoproteasome contains thymus-specific subunit β5t in place of β5 or β5i and plays a pivotal role in positive selection of CD8+ T cells. Here we investigate the assembly pathways of the specialized CPs and show that β1i and β2i are incorporated ahead of all the other β-subunits and that both β5i and β5t can be incorporated immediately after the assembly of β3 in the absence of β4, distinct from the assembly of the standard CP in which β-subunits are incorporated in the order of β2, β3, β4, β5, β6, β1, and β7. The propeptide of β5t is a key factor for this earlier incorporation, whereas the body sequence seems to be important for the earlier incorporation of β5i. This unique feature of β5t and β5i may account for preferential assembly of the immunoproteasome and the thymoproteasome over the standard type even when both the standard and specialized subunits are co-expressed.

  8. Structural Analysis of the Bacterial Proteasome Activator Bpa in Complex with the 20S Proteasome.

    PubMed

    Bolten, Marcel; Delley, Cyrille L; Leibundgut, Marc; Boehringer, Daniel; Ban, Nenad; Weber-Ban, Eilika

    2016-12-06

    Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex.

  9. Peptide-based proteasome inhibitors in anticancer drug design.

    PubMed

    Micale, Nicola; Scarbaci, Kety; Troiano, Valeria; Ettari, Roberta; Grasso, Silvana; Zappalà, Maria

    2014-09-01

    The identification of the key role of the eukaryotic 26S proteasome in regulated intracellular proteolysis and its importance as a target in many pathological conditions wherein the proteasomal activity is defective (e.g., malignancies, autoimmune diseases, neurodegenerative diseases, etc.) prompted several research groups to the development of specific inhibitors of this multicatalytic complex with the aim of obtaining valid drug candidates. In regard to the anticancer therapy, the peptide boronate bortezomib (Velcade®) represents the first molecule approved by FDA for the treatment of multiple myeloma in 2003 and mantle cell lymphoma in 2006. Since then, a plethora of molecules targeting the proteasome have been identified as potential anticancer agents and a few of them reached clinical trials or are already in the market (i.e., carfilzomib; Kyprolis®). In most cases, the design of new proteasome inhibitors (PIs) takes into account a proven peptide or pseudopeptide motif as a base structure and places other chemical entities throughout the peptide skeleton in such a way to create an efficacious network of interactions within the catalytic sites. The purpose of this review is to provide an in-depth look at the current state of the research in the field of peptide-based PIs, specifically those ones that might find an application as anticancer agents.

  10. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin.

    PubMed Central

    Eytan, E; Ganoth, D; Armon, T; Hershko, A

    1989-01-01

    Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway. Images PMID:2554287

  11. Computational Approaches for the Discovery of Human Proteasome Inhibitors: An Overview.

    PubMed

    Guedes, Romina A; Serra, Patrícia; Salvador, Jorge A R; Guedes, Rita C

    2016-07-16

    Proteasome emerged as an important target in recent pharmacological research due to its pivotal role in degrading proteins in the cytoplasm and nucleus of eukaryotic cells, regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription, immune response, and signaling processes. The last two decades witnessed intensive efforts to discover 20S proteasome inhibitors with significant chemical diversity and efficacy. To date, the US FDA approved to market three proteasome inhibitors: bortezomib, carfilzomib, and ixazomib. However new, safer and more efficient drugs are still required. Computer-aided drug discovery has long being used in drug discovery campaigns targeting the human proteasome. The aim of this review is to illustrate selected in silico methods like homology modeling, molecular docking, pharmacophore modeling, virtual screening, and combined methods that have been used in proteasome inhibitors discovery. Applications of these methods to proteasome inhibitors discovery will also be presented and discussed to raise improvements in this particular field.

  12. 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.

  13. Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming.

    PubMed

    Riz, Irene; Hawley, Teresa S; Marsal, Jeffrey W; Hawley, Robert G

    2016-10-11

    Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62. The eukaryotic translation initiation factor gene EIF4E3 was among the Nrf2 target genes upregulated in LP-1/Cfz cells, suggesting existence of a positive feedback loop. In line with this, we found that siRNA knockdown of eIF4E3 decreased Nrf2 protein levels. On the other hand, elevated SQSTM1/p62 levels were due at least in part to activation of the PERK-eIF2α pathway. LP-1/Cfz cells had decreased levels of reactive oxygen species as well as elevated levels of fatty acid oxidation and prosurvival autophagy. Genetic and pharmacologic inhibition of the Nrf2-EIF4E3 axis or the PERK-eIF2α pathway, disruption of redox homeostasis or inhibition of fatty acid oxidation or autophagy conferred sensitivity to carfilzomib. Our findings were supported by clinical data where increased EIF4E3 expression was predictive of Nrf2 target gene upregulation in a subgroup of patients with chemoresistant minimal residual disease and relapsed/refractory MM. Thus, our data offer a preclinical rationale for including inhibitors of the SQSTM1/p62-Nrf2 pathway to the treatment regimens for certain advanced stage MM patients.

  14. Noncanonical SQSTM1/p62-Nrf2 pathway activation mediates proteasome inhibitor resistance in multiple myeloma cells via redox, metabolic and translational reprogramming

    PubMed Central

    Riz, Irene; Hawley, Teresa S.; Marsal, Jeffrey W.; Hawley, Robert G.

    2016-01-01

    Multiple Myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal plasma cells in the bone marrow, with drug resistance being a major cause of therapeutic failure. We established a carfilzomib-resistant derivative of the LP-1 MM cell line (LP-1/Cfz) and found that the transcription factor NF-E2 p45-related factor 2 (Nrf2; gene symbol NFE2L2) contributes to carfilzomib resistance. The mechanism of Nrf2 activation involved enhanced translation of Nrf2 as well as its positive regulator, the autophagy receptor sequestosome 1 (SQSTM1)/p62. The eukaryotic translation initiation factor gene EIF4E3 was among the Nrf2 target genes upregulated in LP-1/Cfz cells, suggesting existence of a positive feedback loop. In line with this, we found that siRNA knockdown of eIF4E3 decreased Nrf2 protein levels. On the other hand, elevated SQSTM1/p62 levels were due at least in part to activation of the PERK-eIF2α pathway. LP-1/Cfz cells had decreased levels of reactive oxygen species as well as elevated levels of fatty acid oxidation and prosurvival autophagy. Genetic and pharmacologic inhibition of the Nrf2-EIF4E3 axis or the PERK-eIF2α pathway, disruption of redox homeostasis or inhibition of fatty acid oxidation or autophagy conferred sensitivity to carfilzomib. Our findings were supported by clinical data where increased EIF4E3 expression was predictive of Nrf2 target gene upregulation in a subgroup of patients with chemoresistant minimal residual disease and relapsed/refractory MM. Thus, our data offer a preclinical rationale for including inhibitors of the SQSTM1/p62-Nrf2 pathway to the treatment regimens for certain advanced stage MM patients. PMID:27626179

  15. Pesticides that inhibit the ubiquitin-proteasome system: effect measure modification by genetic variation in SKP1 in Parkinson׳s disease.

    PubMed

    Rhodes, Shannon L; Fitzmaurice, Arthur G; Cockburn, Myles; Bronstein, Jeff M; Sinsheimer, Janet S; Ritz, Beate

    2013-10-01

    Cytoplasmic inclusions known as Lewy bodies, a hallmark of Parkinson's disease (PD) pathology, may protect against cytotoxic proteins. Since the ubiquitin-proteasome system (UPS) degrades cytotoxic proteins, dysfunction in the UPS may contribute to PD etiology. Our goal in this study was to screen pesticides for proteasome inhibition and investigate (i) whether ambient exposures to pesticides that inhibit the UPS increase PD risk and (ii) whether genetic variation in candidate genes of the UPS pathway modify those increased risks. We assessed 26S UPS activity in SK-N-MC(u) cells by fluorescence. We recruited idiopathic PD cases (n=360) and population-based controls (n=816) from three counties in California with considerable commercial agriculture. We determined ambient pesticide exposure by our validated GIS-based model utilizing residential and workplace address histories. We limited effect measure modification assessment to Caucasians (287 cases, 453 controls). Eleven of 28 pesticides we screened inhibited 26S UPS activity at 10 µM. Benomyl, cyanazine, dieldrin, endosulfan, metam, propargite, triflumizole, and ziram were associated with increased PD risk. We estimated an odds ratio of 2.14 (95% CI: 1.42, 3.22) for subjects with ambient exposure to any UPS-inhibiting pesticide at both residential and workplace addresses; this association was modified by genetic variation in the s-phase kinase-associated protein 1 gene (SKP1; interaction p-value=0.005). Our results provide evidence that UPS-inhibiting pesticides play a role in the etiology of PD and suggest that genetic variation in candidate genes involved in the UPS pathway might exacerbate the toxic effects of pesticide exposures.

  16. Sperm-surface ATP in boar spermatozoa is required for fertilization: relevance to sperm proteasomal function.

    PubMed

    Yi, Young-Joo; Park, Chang-Sik; Kim, Eui-Sook; Song, Eun-Sook; Jeong, Ji-Hyeon; Sutovsky, Peter

    2009-01-01

    Extracellular ATP has been implicated in a number of cellular events, including mammalian sperm function. The complement of ATP-dependent sperm proteins includes six subunits of the 26S proteasome, a multi-subunit protease specific to ubiquitinated substrate-proteins. Proteolysis of ubiquitinated proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis (AE) and sperm-zona pellucida (ZP) penetration. The 26S proteasome is uniquely present on the sperm acrosomal surface during mammalian, ascidian, and invertebrate fertilization. The proteasome is a multi-subunit protease complex of approximately 2 MDa composed of the 19S regulatory complex and a 20S proteolytic core. Integrity of the 19S complex is maintained by six 19S ATPase subunits (PSMC1 through PSMC6). Consequently, we hypothesized that fertilization will be blocked by the depletion of sperm-surface associated ATP (ssATP). Depletion of ssATP by the Solanum tuberosum apyrase, a 49 kDa, non-cell permeant enzyme, significantly reduced the ATP content measured by an adapted luminescence-ATP assay from which all permeabilizing agents were excluded. Addition of active apyrase to porcine in vitro fertilization (IVF) medium caused a concentration dependent reduction in the overall fertilization rate. No such outcomes were observed in control groups using heat-inactivated apyrase. Apyrase treatment altered the band pattern of 19S ATPase subunits PSMC1 (Rpt2) and PSMC4 (Rpt3) in Western blotting, suggesting that it had an effect on the integrity of the sperm proteasomal 19S complex. Apyrase only altered the proteasomal core activities slightly, since these activities are not directly dependent on external ATP. In contrast, sperm treatment with MG132, a specific inhibitor of the proteasomal core chymotrypsin-like activity, inhibited the target proteolytic activity, but also induced a compensatory elevation in proteasomal peptidyl

  17. 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.

  18. 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

  19. The ubiquitin proteasome system in neuropathology.

    PubMed

    Lehman, Norman L

    2009-09-01

    The ubiquitin proteasome system (UPS) orchestrates the turnover of innumerable cellular proteins. In the process of ubiquitination the small protein ubiquitin is attached to a target protein by a peptide bond. The ubiquitinated target protein is subsequently shuttled to a protease complex known as the 26S proteasome and subjected to degradative proteolysis. The UPS facilitates the turnover of proteins in several settings. It targets oxidized, mutant or misfolded proteins for general proteolytic destruction, and allows for the tightly controlled and specific destruction of proteins involved in development and differentiation, cell cycle progression, circadian rhythms, apoptosis, and other biological processes. In neuropathology, alteration of the UPS, or mutations in UPS target proteins may result in signaling abnormalities leading to the initiation or progression of tumors such as astrocytomas, hemangioblastomas, craniopharyngiomas, pituitary adenomas, and medulloblastomas. Dysregulation of the UPS may also contribute to tumor progression by perturbation of DNA replication and mitotic control mechanisms, leading to genomic instability. In neurodegenerative diseases caused by the expression of mutant proteins, the cellular accumulation of these proteins may overload the UPS, indirectly contributing to the disease process, e.g., sporadic Parkinsonism and prion diseases. In other cases, mutation of UPS components may directly cause pathological accumulation of proteins, e.g., autosomal recessive Parkinsonism and spinocerebellar ataxias. Defects or dysfunction of the UPS may also underlie cognitive disorders such as Angelman syndrome, Rett syndrome and autism, and muscle and nerve diseases, e.g., inclusion body myopathy and giant axon neuropathy. This paper describes the basic biochemical mechanisms comprising the UPS and reviews both its theoretical and proven involvement in neuropathological diseases. The potential for the UPS as a target of pharmacological therapy

  20. 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

  1. The Proteasome Inhibition Model of Parkinson’s Disease

    PubMed Central

    Bentea, Eduard; Verbruggen, Lise; Massie, Ann

    2016-01-01

    The pathological hallmarks of Parkinson’s disease are the progressive loss of nigral dopaminergic neurons and the formation of intracellular inclusion bodies, termed Lewy bodies, in surviving neurons. Accumulation of proteins in large insoluble cytoplasmic aggregates has been proposed to result, partly, from a failure in the function of intracellular protein degradation pathways. Evidence in support for such a hypothesis emerged in the beginning of the years 2000 with studies demonstrating structural and functional deficits in the ubiquitin-proteasome pathway in post-mortem nigral tissue of patients with Parkinson’s disease. These fundamental findings have inspired the development of a new generation of animal models based on the use of proteasome inhibitors to disturb protein homeostasis and trigger nigral dopaminergic neurodegeneration. In this review, we provide an updated overview of the current approaches in employing proteasome inhibitors to model Parkinson’s disease, with particular emphasis on rodent studies. In addition, the mechanisms underlying proteasome inhibition-induced cell death and the validity criteria (construct, face and predictive validity) of the model will be critically discussed. Due to its distinct, but highly relevant mechanism of inducing neuronal death, the proteasome inhibition model represents a useful addition to the repertoire of toxin-based models of Parkinson’s disease that might provide novel clues to unravel the complex pathogenesis of this disorder. PMID:27802243

  2. Effects of Hydroxy Groups in the A-Ring on the Anti-proteasome Activity of Flavone.

    PubMed

    Nakamura, Kasumi; Yang, Jia-Hua; Sato, Eiji; Miura, Naoyuki; Wu, Yi-Xin

    2015-01-01

    The ubiquitin-proteasome pathway plays an important role in regulating apoptosis and the cell cycle. Recently, proteasome inhibitors have been shown to have antitumor effects and have been used in anticancer therapy for several cancers such as multiple myeloma. Although some flavones, such as apigenin, chrysin and luteolin, have a specific role in the inhibition of proteasome activity and induced apoptosis in some reports, these findings did not address all flavone types. To further investigate the proteasome-inhibitory mechanism of flavonoids, we examined the inhibitory activity of 5,6,7-trihydroxyflavone, baicalein and 5,6,7,4'-tetrahydroxyflavone, scutellarein on extracted proteasomes from mice and cancer cells. Unlike the other flavones, baicalein and scutellarein did not inhibit proteasome activity or accumulate levels of ubiquitinated proteins. These results indicate that flavones with hydroxy groups at positions 5, 6 and 7 of the A-ring lack the anti-proteasome function.

  3. Regulation of Akt-mTOR, ubiquitin-proteasome and autophagy-lysosome pathways in response to formoterol administration in rat skeletal muscle.

    PubMed

    Joassard, Olivier Roger; Amirouche, Adel; Gallot, Yann Simon; Desgeorges, Marine Maud; Castells, Josiane; Durieux, Anne-Cécile; Berthon, Phanélie; Freyssenet, Damien Gilles

    2013-11-01

    Administration of β2-agonists triggers skeletal muscle anabolism and hypertrophy. We investigated the time course of the molecular events responsible for rat skeletal muscle hypertrophy in response to 1, 3 and 10 days of formoterol administration (i.p. 2000μg/kg/day). A marked hypertrophy of rat tibialis anterior muscle culminated at day 10. Phosphorylation of Akt, ribosomal protein S6, 4E-BP1 and ERK1/2 was increased at day 3, but returned to control level at day 10. This could lead to a transient increase in protein translation and could explain previous studies that reported increase in protein synthesis following β2-agonist administration. Formoterol administration was also associated with a significant reduction in MAFbx/atrogin-1 mRNA level (day 3), suggesting that formoterol can also affect protein degradation of MAFbx/atrogin1 targeted substrates, including MyoD and eukaryotic initiation factor-3f (eIF3-f). Surprisingly, mRNA level of autophagy-related genes, light chain 3 beta (LC3b) and gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1), as well as lysosomal hydrolases, cathepsin B and cathepsin L, was significantly and transiently increased after 1 and/or 3 days, suggesting that autophagosome formation would be increased in response to formoterol administration. However, this has to be relativized since the mRNA level of Unc-51-like kinase1 (Ulk1), BCL2/adenovirus E1B interacting protein3 (Bnip3), and transcription factor EB (TFEB), as well as the protein content of Ulk1, Atg13, Atg5-Atg12 complex and p62/Sqstm1 remained unchanged or was even decreased in response to formoterol administration. These results demonstrate that the effects of formoterol are mediated, in part, through the activation of Akt-mTOR pathway and that other signaling pathways become more important in the regulation of skeletal muscle mass with chronic administration of β2-agonists.

  4. Radiosensitizing effect of PSMC5, a 19S proteasome ATPase, in H460 lung cancer cells.

    PubMed

    Yim, Ji-Hye; Yun, Hong Shik; Lee, Su-Jae; Baek, Jeong-Hwa; Lee, Chang-Woo; Song, Ji-Young; Um, Hong-Duck; Park, Jong Kuk; Kim, Jae-Sung; Park, In-Chul; Hwang, Sang-Gu

    2016-01-01

    The function of PSMC5 (proteasome 26S subunit, ATPase 5) in tumors, particularly with respect to cancer radioresistance, is not known. Here, we identified PSMC5 as a novel radiosensitivity biomarker, demonstrating that radiosensitive H460 cells were converted to a radioresistance phenotype by PSMC5 depletion. Exposure of H460 cells to radiation induced a marked accumulation of cell death-promoting reactive oxygen species, but this effect was blocked in radiation-treated H460 PSMC5-knockdown cells through downregulation of the p53-p21 pathway. Interestingly, PSMC5 depletion in H460 cells enhanced both AKT activation and MDM2 transcription, thereby promoting the degradation of p53 and p21 proteins. Furthermore, specific inhibition of AKT with triciribine or knockdown of MDM2 with small interfering RNA largely restored p21 expression in PSMC5-knockdown H460 cells. Our data suggest that PSMC5 facilitates the damaging effects of radiation in radiation-responsive H460 cancer cells and therefore may serve as a prognostic indicator for radiotherapy and molecular targeted therapy in lung cancer patients.

  5. Natural Compounds with Proteasome Inhibitory Activity for Cancer Prevention and Treatment

    PubMed Central

    Yang, H; Landis-Piwowar, KR.; Chen, D; Milacic, V; Dou, QP

    2012-01-01

    The proteasome is a multicatalytic protease complex that degrades most endogenous proteins including misfolded or damaged proteins to ensure normal cellular function. The ubiquitin-proteasome degradation pathway plays an essential role in multiuple cellular processes, including cell cycle progression, proliferation, apoptosis and angiogenesis. It has been shown that human cancer cells are more sensitive to proteasome inhibition than normal cells, indicating that a proteasome inhibitor could be used as a novel anticancer drug. Indeed, this idea has been supported by the encouraging results of the clinical trials using the proteasome inhibitor Bortezomib (Velcade, PS-341), a drug approved by the US Food and Drug Administration (FDA). Several natural compounds, including the microbial metabolite lactacystin, green tea polyphenols, and traditional medicinal triterpenes, have been shown to be potent proteasome inhibitors. These findings suggest the potential use of natural proteasome inhibitors as not only chemopreventive and chemotherapeutic agents, but also tumor sensitizers to conventional radiotherapy and chemotherapy. In this review, we will summarize the structure and biological activities of the proteasome and several natural compounds with proteasome inhibitory activity, and will discuss the potential use of these compounds for the prevention and treatment of human cancers. PMID:18537678

  6. 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

  7. The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome

    PubMed Central

    Reeg, Sandra; Jung, Tobias; Castro, José P.; Davies, Kelvin J.A.; Henze, Andrea; Grune, Tilman

    2016-01-01

    One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome. PMID:27498116

  8. Proteasomes play an essential role in thymocyte apoptosis.

    PubMed Central

    Grimm, L M; Goldberg, A L; Poirier, G G; Schwartz, L M; Osborne, B A

    1996-01-01

    Cell death in many different organisms requires the activation of proteolytic cascades involving cytosolic proteases. Here we describe a novel requirement in thymocyte cell death for the 20S proteasome, a highly conserved multicatalytic protease found in all eukaryotes. Specific inhibitors of proteasome function blocked cell death induced by ionizing radiation, glucocorticoids or phorbol ester. In addition to inhibiting apoptosis, these signals prevented the cleavage of poly(ADP-ribose) polymerase that accompanies many cell deaths. Since overall rates of protein degradation were not altered significantly during cell death in thymocytes, these results suggest that the proteasome may either degrade regulatory protein(s) that normally inhibit the apoptotic pathway or may proteolytically activate protein(s) than promote cell death. Images PMID:8670888

  9. The capture proteasome assay (CAPA) to evaluate subtype-specific proteasome inhibitors.

    PubMed

    Vigneron, Nathalie; Abi Habib, Joanna; Van den Eynde, Benoît J

    2015-09-01

    We recently developed a new assay to measure proteasome activity in vitro (CAPA for capture proteasome assay) [1], based on proteasome capture on an antibody-coated plate. When used with lysates originating from cells expressing either standard proteasome, immunoproteasome or intermediate proteasomes β5i or β1i-β5i, this assay allows the individual monitoring of the chymotrypsin-like, trypsin-like and caspase-like activities of the corresponding proteasome subtypes. The efficiency and specificity of four proteasome inhibitors were studied using the CAPA assay, demonstrating the potential of this assay for the development of subtype-specific proteasome inhibitors.

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

    PubMed Central

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

    2015-01-01

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

  11. Proteasome 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

  12. Characterization of the proteasome interaction network using a QTAX-based tag-team strategy and protein interaction network analysis.

    PubMed

    Guerrero, Cortnie; Milenkovic, Tijana; Przulj, Natasa; Kaiser, Peter; Huang, Lan

    2008-09-09

    Quantitative analysis of tandem-affinity purified cross-linked (x) protein complexes (QTAX) is a powerful technique for the identification of protein interactions, including weak and/or transient components. Here, we apply a QTAX-based tag-team mass spectrometry strategy coupled with protein network analysis to acquire a comprehensive and detailed assessment of the protein interaction network of the yeast 26S proteasome. We have determined that the proteasome network is composed of at least 471 proteins, significantly more than the total number of proteins identified by previous reports using proteasome subunits as baits. Validation of the selected proteasome-interacting proteins by reverse copurification and immunoblotting experiments with and without cross-linking, further demonstrates the power of the QTAX strategy for capturing protein interactions of all natures. In addition, >80% of the identified interactions have been confirmed by existing data using protein network analysis. Moreover, evidence obtained through network analysis links the proteasome to protein complexes associated with diverse cellular functions. This work presents the most complete analysis of the proteasome interaction network to date, providing an inclusive set of physical interaction data consistent with physiological roles for the proteasome that have been suggested primarily through genetic analyses. Moreover, the methodology described here is a general proteomic tool for the comprehensive study of protein interaction networks.

  13. 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

  14. 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

  15. Role of proteasomes in disease

    PubMed Central

    Dahlmann, Burkhardt

    2007-01-01

    A functional ubiquitin proteasome system is essential for all eukaryotic cells and therefore any alteration to its components has potential pathological consequences. Though the exact underlying mechanism is unclear, an age-related decrease in proteasome activity weakens cellular capacity to remove oxidatively modified proteins and favours the development of neurodegenerative and cardiac diseases. Up-regulation of proteasome activity is characteristic of muscle wasting conditions including sepsis, cachexia and uraemia, but may not be rate limiting. Meanwhile, enhanced presence of immunoproteasomes in aging brain and muscle tissue could reflect a persistent inflammatory defence and anti-stress mechanism, whereas in cancer cells, their down-regulation reflects a means by which to escape immune surveillance. Hence, induction of apoptosis by synthetic proteasome inhibitors is a potential treatment strategy for cancer, whereas for other diseases such as neurodegeneration, the use of proteasome-activating or -modulating compounds could be more effective. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; ). PMID:18047740

  16. High-resolution cryo-EM structure of the proteasome in complex with ADP-AlFx.

    PubMed

    Ding, Zhanyu; Fu, Zhenglin; Xu, Cong; Wang, Yifan; Wang, Yanxing; Li, Junrui; Kong, Liangliang; Chen, Jinhuan; Li, Na; Zhang, Rongguang; Cong, Yao

    2017-03-01

    The 26S proteasome is an ATP-dependent dynamic 2.5 MDa protease that regulates numerous essential cellular functions through degradation of ubiquitinated substrates. Here we present a near-atomic-resolution cryo-EM map of the S. cerevisiae 26S proteasome in complex with ADP-AlFx. Our biochemical and structural data reveal that the proteasome-ADP-AlFx is in an activated state, displaying a distinct conformational configuration especially in the AAA-ATPase motor region. Noteworthy, this map demonstrates an asymmetric nucleotide binding pattern with four consecutive AAA-ATPase subunits bound with nucleotide. The remaining two subunits, Rpt2 and Rpt6, with empty or only partially occupied nucleotide pocket exhibit pronounced conformational changes in the AAA-ATPase ring, which may represent a collective result of allosteric cooperativity of all the AAA-ATPase subunits responding to ATP hydrolysis. This collective motion of Rpt2 and Rpt6 results in an elevation of their pore loops, which could play an important role in substrate processing of proteasome. Our data also imply that the nucleotide occupancy pattern could be related to the activation status of the complex. Moreover, the HbYX tail insertion may not be sufficient to maintain the gate opening of 20S core particle. Our results provide new insights into the mechanisms of nucleotide-driven allosteric cooperativity of the complex and of the substrate processing by the proteasome.

  17. Regulation of Feedback between Protein Kinase A and the Proteasome System Worsens Huntington's Disease

    PubMed Central

    Lin, Jiun-Tsai; Chang, Wei-Cheng; Chen, Hui-Mei; Lai, Hsing-Lin; Chen, Chih-Yeh; Tao, Mi-Hua

    2013-01-01

    Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a CAG repeat in the Huntingtin (HTT) gene. Abnormal regulation of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway occurs during HD progression. Here we found that lower PKA activity was associated with proteasome impairment in the striatum for two HD mouse models (R6/2 and N171-82Q) and in mutant HTT (mHTT)-expressing striatal cells. Because PKA regulatory subunits (PKA-Rs) are proteasome substrates, the mHTT-evoked proteasome impairment caused accumulation of PKA-Rs and subsequently inhibited PKA activity. Conversely, activation of PKA enhanced the phosphorylation of Rpt6 (a component of the proteasome), rescued the impaired proteasome activity, and reduced mHTT aggregates. The dominant-negative Rpt6 mutant (Rpt6S120A) blocked the ability of a cAMP-elevating reagent to enhance proteasome activity, whereas the phosphomimetic Rpt6 mutant (Rpt6S120D) increased proteasome activity, reduced HTT aggregates, and ameliorated motor impairment. Collectively, our data demonstrated that positive feedback regulation between PKA and the proteasome is critical for HD pathogenesis. PMID:23275441

  18. Targeting the ubiquitin–proteasome system for cancer therapy

    PubMed Central

    Shen, Min; Schmitt, Sara; Buac, Daniela; Dou, Q Ping

    2013-01-01

    Introduction The ubiquitin–proteasome system (UPS) degrades 80 – 90% of intracellular proteins. Cancer cells take advantage of the UPS for their increased growth and decreased apoptotic cell death. Thus, the components that make up the UPS represent a diverse group of potential anti-cancer targets. The success of the first-in-class proteasome inhibitor bortezomib not only proved that the proteasome is a feasible and valuable anti-cancer target, but also inspired researchers to extensively explore other potential targets of this pathway. Areas covered This review provides a broad overview of the UPS and its role in supporting cancer development and progression, especially in aspects of p53 inactivation, p27 turnover and NF-κB activation. Also, efforts toward the development of small molecule inhibitors (SMIs) targeting different steps in this pathway for cancer treatment are reviewed and discussed. Expert opinion Whereas some of the targets in the UPS, such as the 20S pro-teasome, Nedd8 activating enzyme and HDM2, have been well-established and validated, there remains a large pool of candidates waiting to be investigated. Development of SMIs targeting the UPS has been largely facilitated by state-of-the-art technologies such as high-throughput screening and computer-assisted drug design, both of which require a better understanding of the targets of interest. PMID:23822887

  19. 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.…

  20. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene.

    PubMed

    Czub, Barbara; Shah, Amna Z; Alfano, Giovanna; Kruczek, Przemysław M; Chakarova, Christina F; Bhattacharya, Shomi S

    2016-01-01

    The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.

  1. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene

    PubMed Central

    Czub, Barbara; Shah, Amna Z.; Alfano, Giovanna; Kruczek, Przemysław M.; Chakarova, Christina F.; Bhattacharya, Shomi S.

    2016-01-01

    The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS. PMID:26872363

  2. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  3. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  4. Lysine 419 targets human glucocorticoid receptor for proteasomal degradation.

    PubMed

    Wallace, Andrew D; Cao, Yan; Chandramouleeswaran, Sindhu; Cidlowski, John A

    2010-12-01

    Glucocorticoid receptors (GRs) are members of a highly conserved family of ligand dependent transcription factors which following hormone binding undergo homologous down-regulation reducing the levels of receptor protein. This decline in human GR (hGR) is due in part to a decrease in protein receptor stability that may limit cellular responsiveness to ligand. To examine the role of the proteasome protein degradation pathway in steroid-dependent hGR responsiveness, we utilized the proteasomal inhibitors MG-132, beta-lactone, and epoxomicin. HeLa cells and COS cells were treated with proteasome inhibitors in the presence of the GR agonist dexamethasone (Dex), or were pretreated with proteasomal inhibitor and then Dex. Dexamethasone induced glucocorticoid responsive reporter activity significantly over untreated controls, whereas cells treated with proteasomal inhibitors and Dex together showed 2-3-fold increase in activity. Protein sequence analysis of the hGR protein identified several candidate protein degradation motifs including a PEST element. Mutagenesis of this element at lysine 419 was done and mutant K419A hGR failed to undergo ligand dependent down-regulation. Mutant K419A hGR displayed 2-3-fold greater glucocorticoid responsive reporter activity in the presence of Dex than wild type hGR. These differences in transcriptional activity were not due to altered subcellular localization, since when the mutant K419A hGR was fused with the green fluorescent protein (GFP) it was found to move in and out of the nucleus similarly to wild type hGR. Together these results suggest that the proteasome and the identified PEST degradation motif limit steroid-dependent human glucocorticoid receptor signaling.

  5. K(ATP) channel block prevents proteasome inhibitor-induced apoptosis in differentiated PC12 cells.

    PubMed

    Nam, Yoon Jeong; Lee, Da Hee; Lee, Min Sung; Lee, Chung Soo

    2015-10-05

    Dysfunction of the proteasome system has been suggested to be implicated in neuronal degeneration. Modulation of KATP channels appears to affect the viability of neuronal cells exposed to toxic insults. However, the effect of KATP channel blockers on the neuronal cell death mediated by proteasome inhibition has not been studied. The present study investigated the effect of KATP channel blockers on proteasome inhibitor-induced apoptosis in differentiated PC12 cells and SH-SY5Y cells. 5-Hydroxydecanoate (a selective KATP channel blocker) and glibenclamide (a cell surface and mitochondrial KATP channel inhibitor) reduced the proteasome inhibitor-induced apoptosis. Addition of the KATP channel blockers attenuated the proteasome inhibitor-induced changes in the levels of apoptosis-related proteins, the loss of the mitochondrial transmembrane potential, the increase in the formation of reactive oxygen species and the depletion of glutathione in both cell lines. The results show that KATP channel blockers may attenuate proteasome inhibitor-induced apoptosis in PC12 cells by suppressing activation of the mitochondrial pathway and of the caspase-8- and Bid-dependent pathways. The preventive effect appears to be associated with the inhibition of the formation of reactive oxygen species and the depletion of glutathione. KATP channel blockade appears to prevent proteasome inhibition-induced neuronal cell death.

  6. Proteasome inhibitors in cancer therapy: Treatment regimen and peripheral neuropathy as a side effect.

    PubMed

    Kaplan, Gulce Sari; Torcun, Ceyda Corek; Grune, Tilman; Ozer, Nesrin Kartal; Karademir, Betul

    2017-02-01

    Proteasomal system plays an important role in protein turnover, which is essential for homeostasis of cells. Besides degradation of oxidized proteins, it is involved in the regulation of many different signaling pathways. These pathways include mainly cell differentiation, proliferation, apoptosis, transcriptional activation and angiogenesis. Thus, proteasomal system is a crucial target for treatment of several diseases including neurodegenerative diseases, cystic fibrosis, atherosclerosis, autoimmune diseases, diabetes and cancer. Over the last fifteen years, proteasome inhibitors have been tested to highlight their mechanisms of action and used in the clinic to treat different types of cancer. Proteasome inhibitors are mainly used in combinational therapy along with classical chemo-radiotherapy. Several studies have proved their significant effects but serious side effects such as peripheral neuropathy, limits their use in required effective doses. Recent studies focus on peripheral neuropathy as the primary side effect of proteasome inhibitors. Therefore, it is important to delineate the underlying mechanisms of peripheral neuropathy and develop new inhibitors according to obtained data. This review will detail the role of proteasome inhibition in cancer therapy and development of peripheral neuropathy as a side effect. Additionally, new approaches to prevent treatment-limiting side effects will be discussed in order to help researchers in developing effective strategies to overcome side effects of proteasome inhibitors.

  7. 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

    an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus.

  8. 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

    an accumulation of ubiquitinated proteins and caused increase in the amount of endoplasmic reticulum membranes in the parasite. Taken together, our results suggest that the ubiquitin-proteasome pathway is required for cell cycle and EFF transformation in T. foetus. PMID:26047503

  9. Altered composition of liver proteasome assemblies contributes to enhanced proteasome activity in the exceptionally long-lived naked mole-rat.

    PubMed

    Rodriguez, Karl A; Edrey, Yael H; Osmulski, Pawel; Gaczynska, Maria; Buffenstein, Rochelle

    2012-01-01

    The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life.

  10. Extracellular proteasome-osteopontin circuit regulates cell migration with implications in multiple sclerosis.

    PubMed

    Dianzani, Chiara; Bellavista, Elena; Liepe, Juliane; Verderio, Claudia; Martucci, Morena; Santoro, Aurelia; Chiocchetti, Annalisa; Luca Gigliotti, Casimiro; Boggio, Elena; Ferrara, Benedetta; Riganti, Loredana; Keller, Christin; Janek, Katharina; Niewienda, Agathe; Fenoglio, Chiara; Sorosina, Melissa; Cantello, Roberto; Kloetzel, Peter M; Stumpf, Michael P H; Paul, Friedemann; Ruprecht, Klemens; Galimberti, Daniela; Martinelli Boneschi, Filippo; Comi, Cristoforo; Dianzani, Umberto; Mishto, Michele

    2017-03-09

    Osteopontin is a pleiotropic cytokine that is involved in several diseases including multiple sclerosis. Secreted osteopontin is cleaved by few known proteases, modulating its pro-inflammatory activities. Here we show by in vitro experiments that secreted osteopontin can be processed by extracellular proteasomes, thereby producing fragments with novel chemotactic activity. Furthermore, osteopontin reduces the release of proteasomes in the extracellular space. The latter phenomenon seems to occur in vivo in multiple sclerosis, where it reflects the remission/relapse alternation. The extracellular proteasome-mediated inflammatory pathway may represent a general mechanism to control inflammation in inflammatory diseases.

  11. Extracellular proteasome-osteopontin circuit regulates cell migration with implications in multiple sclerosis

    PubMed Central

    Dianzani, Chiara; Bellavista, Elena; Liepe, Juliane; Verderio, Claudia; Martucci, Morena; Santoro, Aurelia; Chiocchetti, Annalisa; Luca Gigliotti, Casimiro; Boggio, Elena; Ferrara, Benedetta; Riganti, Loredana; Keller, Christin; Janek, Katharina; Niewienda, Agathe; Fenoglio, Chiara; Sorosina, Melissa; Cantello, Roberto; Kloetzel, Peter M.; Stumpf, Michael P. H.; Paul, Friedemann; Ruprecht, Klemens; Galimberti, Daniela; Martinelli Boneschi, Filippo; Comi, Cristoforo; Dianzani, Umberto; Mishto, Michele

    2017-01-01

    Osteopontin is a pleiotropic cytokine that is involved in several diseases including multiple sclerosis. Secreted osteopontin is cleaved by few known proteases, modulating its pro-inflammatory activities. Here we show by in vitro experiments that secreted osteopontin can be processed by extracellular proteasomes, thereby producing fragments with novel chemotactic activity. Furthermore, osteopontin reduces the release of proteasomes in the extracellular space. The latter phenomenon seems to occur in vivo in multiple sclerosis, where it reflects the remission/relapse alternation. The extracellular proteasome-mediated inflammatory pathway may represent a general mechanism to control inflammation in inflammatory diseases. PMID:28276434

  12. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    PubMed

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-02-23

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  13. 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.

  14. Development and Characterization of Proteasome Inhibitors

    PubMed Central

    Kim, Kyung Bo; Fonseca, Fabiana N.; Crews, Craig M.

    2008-01-01

    Although many proteasome inhibitors have been either synthesized or identified from natural sources, the development of more sophisticated, selective proteasome inhibitors is important for a detailed understanding of proteasome function. We have found that antitumor natural product epoxomicin and eponemycin, both of which are linear peptides containing a α,β-epoxyketone pharmacophore, target proteasome for their antitumor activity. Structural studies of the proteasome–epoxomicin complex revealed that the unique specificity of the natural product toward proteasome is due to the α,β-epoxyketone pharmacophore, which forms an unusual six-membered morpholino ring with the amino terminal catalytic Thr-1 of the 20S proteasome. Thus, we believe that a facile synthetic approach for α,β-epoxyketone linear peptides provides a unique opportunity to develop proteasome inhibitors with novel activities. In this chapter, we discuss the detailed synthetic procedure of the α′,β′-epoxyketone natural product epoxomicin and its derivatives. PMID:16338383

  15. Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria.

    PubMed

    Mookerjee, Shona A; Brand, Martin D

    2011-11-01

    Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5-4h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10-20mV from its resting value, and was not significant below about 120mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded.

  16. 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

  17. Circulating extracellular proteasome in the cerebrospinal fluid: a study on concentration and proteolytic activity.

    PubMed

    Mueller, Oliver; Anlasik, Timur; Wiedemann, Jonas; Thomassen, Jan; Wohlschlaeger, Jeremias; Hagel, Vincent; Keyvani, Kathy; Schwieger, Isabel; Dahlmann, Burkhardt; Sure, Ulrich; Sixt, Stephan Urs

    2012-03-01

    Alterations of the intracellular ubiquitin-proteasome pathway are found in neurodegenerative and inflammatory disorders of the central nervous system, as well as in its malignancies. Inhibitory substrates of the proteasomes represent promising approaches to control autoimmune inflammations and induction of apoptosis in cancer cells. Extracellular circulating proteasomes are positively correlated to outcome prognosis in hematogenic neoplasias and the outcome in critically ill patients. Previously, we reported raised levels of proteolytic active 20S proteasomes in the extracellular alveolar space in patients with acute respiratory distress syndrome (ARDS). For the cerebrospinal fluid, we assumed that extracellular circulating proteasomes with enzymatic activity can be found, too. Cerebrospinal fluid (CSF) samples of twenty-six patients (14 females, 12 males), who underwent diagnostic spinal myelography, were analyzed for leukocyte cell count, total protein content, lactate and interleukine-6 (Il-6) concentrations. CSF samples were analyzed for concentration and enzymatic activity of extracellular 20S proteasomes (fluorescenic substrate cleavage; femtokatal). Blood samples were analyzed with respect to concentration of extracellular circulating proteasomes. Choroidal plexus was harvested at autopsies and examined with immunoelectron microscopy (EM) for identification of possible transportation mechanisms. Statistical analysis was performed using SPSS (18.0.3). In all patients, extracellular proteasome was found in the CSF. The mean concentration was 24.6 ng/ml. Enzymatic activity of the 20S subunits of proteasomes was positively identified by the fluorescenic subtrate cleavage at a mean of 8.5 fkat/ml. Concentrations of extracellular proteasomes in the CSF, total protein content and Il-6 were uncorrelated. Immunoelectron microscopy revealed merging vesicles of proteasomes with the outer cell membrane suggestive of an exozytic transport mechanism. For the first time

  18. The capture proteasome assay: A method to measure proteasome activity in vitro.

    PubMed

    Vigneron, Nathalie; Abi Habib, Joanna; Van den Eynde, Benoît J

    2015-08-01

    Because of its crucial role in various cellular processes, the proteasome is the focus of intensive research for the development of proteasome inhibitors to treat cancer and autoimmune diseases. Here, we describe a new and easy assay to measure the different proteasome activities in vitro (chymotrypsin-like, caspase-like, and trypsin-like) based on proteasome capture on antibody-coated plates, namely the capture proteasome assay (CAPA). Applying the CAPA to lysates from cells expressing standard proteasome, immunoproteasome, or intermediate proteasomes β5i or β1i-β5i, we can monitor the activity of the four proteasome subtypes. The CAPA provided similar results as the standard whole-cell proteasome-Glo assay without the problem of contaminating proteases requiring inhibitors. However, the profile of trypsin-like activity differed between the two assays. This could be partly explained by the presence of MgSO4 in the proteasome-Glo buffer, which inhibits the trypsin-like activity of the proteasome. The CAPA does not need MgSO4 and, therefore, provides a more precise measurement of the trypsin-like activity. The CAPA provides a quick and accurate method to measure proteasome activity in vitro in a very specific manner and should be useful for the development of proteasome inhibitors.

  19. 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

  20. 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

    2013-11-05

    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.

  1. 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

  2. Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum.

    PubMed

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2008-10-15

    To determine whether protein degradation plays a role in the endoplasmic reticulum (ER) retention of cytochromes P450, the effects of proteasomal inhibitors on the expression and distribution of green fluorescent protein chimeras of CYP2C2 and related proteins was examined. In transfected cells, expression levels of chimeras of full-length CYP2C2 and its cytosolic domain, but not its N-terminal transmembrane sequence, were increased by proteasomal inhibition. Redistribution of all three chimeras from the reticular ER into a perinuclear compartment and, in a subset of cells, also to the cell surface was observed after proteasomal inhibition. Redistribution was blocked by the microtubular inhibitor, nocodazole, suggesting that redistribution to the cell surface followed the conventional vesicular transport pathway. Similar redistributions were detected for BAP31, a CYP2C2 binding chaperone; CYP2E1 and CYP3A4, which are also degraded by the proteasomal pathway; and for cytochrome P450 reductase, which does not undergo proteasomal degradation; but not for the ER membrane proteins, sec61 and calnexin. Redistribution does not result from saturation of an ER retention "receptor" since in some cases protein levels were unaffected. Proteasomal inhibition may, therefore, alter ER retention by affecting a protein critical for ER retention, either directly, or indirectly by affecting the composition of the ER membranes.

  3. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    SciTech Connect

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    2009-10-16

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic {alpha}- and {beta}-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.

  4. De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder.

    PubMed

    Küry, Sébastien; Besnard, Thomas; Ebstein, Frédéric; Khan, Tahir N; Gambin, Tomasz; Douglas, Jessica; Bacino, Carlos A; Sanders, Stephan J; Lehmann, Andrea; Latypova, Xénia; Khan, Kamal; Pacault, Mathilde; Sacharow, Stephanie; Glaser, Kimberly; Bieth, Eric; Perrin-Sabourin, Laurence; Jacquemont, Marie-Line; Cho, Megan T; Roeder, Elizabeth; Denommé-Pichon, Anne-Sophie; Monaghan, Kristin G; Yuan, Bo; Xia, Fan; Simon, Sylvain; Bonneau, Dominique; Parent, Philippe; Gilbert-Dussardier, Brigitte; Odent, Sylvie; Toutain, Annick; Pasquier, Laurent; Barbouth, Deborah; Shaw, Chad A; Patel, Ankita; Smith, Janice L; Bi, Weimin; Schmitt, Sébastien; Deb, Wallid; Nizon, Mathilde; Mercier, Sandra; Vincent, Marie; Rooryck, Caroline; Malan, Valérie; Briceño, Ignacio; Gómez, Alberto; Nugent, Kimberly M; Gibson, James B; Cogné, Benjamin; Lupski, James R; Stessman, Holly A F; Eichler, Evan E; Retterer, Kyle; Yang, Yaping; Redon, Richard; Katsanis, Nicholas; Rosenfeld, Jill A; Kloetzel, Peter-Michael; Golzio, Christelle; Bézieau, Stéphane; Stankiewicz, Paweł; Isidor, Bertrand

    2017-02-02

    Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

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

    PubMed Central

    Svozil, Julia; Gruissem, Wilhelm; Baerenfaller, Katja

    2015-01-01

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

  6. Transgenic pig carrying green fluorescent proteasomes

    PubMed Central

    Miles, Edward L.; O’Gorman, Chad; Zhao, Jianguo; Samuel, Melissa; Walters, Eric; Yi, Young-Joo; Prather, Randall S.; Wells, Kevin D.; Sutovsky, Peter

    2013-01-01

    Among its many functions, the ubiquitin–proteasome system regulates substrate-specific proteolysis during the cell cycle, apoptosis, and fertilization and in pathologies such as Alzheimer’s disease, cancer, and liver cirrhosis. Proteasomes are present in human and boar spermatozoa, but little is known about the interactions of proteasomal subunits with other sperm proteins or structures. We have created a transgenic boar with green fluorescent protein (GFP) tagged 20S proteasomal core subunit α-type 1 (PSMA1-GFP), hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes. Using direct epifluorescence imaging and indirect immunofluorescence detection, we have confirmed the presence of PSMA1-GFP in the sperm acrosome. Western blotting revealed a protein band corresponding to the predicted mass of PSMA1-GFP fusion protein (57 kDa) in transgenic spermatozoa. Transgenic boar fertility was confirmed by in vitro fertilization, resulting in transgenic blastocysts, and by mating, resulting in healthy transgenic offspring. Immunoprecipitation and proteomic analysis revealed that PSMA1-GFP copurifies with several acrosomal membrane-associated proteins (e.g., lactadherin/milk fat globule E8 and spermadhesin alanine-tryptophan-asparagine). The interaction of MFGE8 with PSMA1-GFP was confirmed through cross-immunoprecipitation. The identified proteasome-interacting proteins may regulate sperm proteasomal activity during fertilization or may be the substrates of proteasomal proteolysis during fertilization. Proteomic analysis also confirmed the interaction/coimmunoprecipitation of PSMA1-GFP with 13/14 proteasomal core subunits. These results demonstrate that the PSMA1-GFP was incorporated in the assembled sperm proteasomes. This mammal carrying green fluorescent proteasomes will be useful for studies of fertilization and wherever the ubiquitin–proteasome system plays a role in cellular function or pathology. PMID:23550158

  7. Proteasome Modulates Mitochondrial Function During Cellular Senescence

    PubMed Central

    Torres, Claudio A.; Perez, Viviana I.

    2009-01-01

    Proteasome plays fundamental roles in the removal of oxidized proteins and in the normal degradation of short-lived proteins. Previously we have provided evidences that the impairment in proteasome observed during the replicative senescence of human fibroblasts has significant effects on MAPK signaling, proliferation, life span, senescent phenotype and protein oxidative status. These studies have demonstrated that proteasome inhibition and replicative senescence caused accumulation of intracellular protein carbonyl content. In this study, we have investigated the mechanisms by which proteasome dysfunction modulates protein oxidation during cellular senescence. The results indicate that proteasome inhibition during replicative senescence have significant effects on the intra and extracellular ROS production in vitro. The data also show that ROS impaired the proteasome function, which is partially reversible by antioxidants. Increases in ROS after proteasome inhibition correlated with a significant negative effect on the activity of most mitochondrial electron transporters. We propose that failures in proteasome during cellular senescence lead to mitochondrial dysfunction, ROS production and oxidative stress. Furthermore, it is likely that changes in proteasome dynamics could generate a pro-oxidative condition at the immediate extracellular microenvironment that could cause tissue injury during aging, in vivo. PMID:17976388

  8. Molecular shredders: how proteasomes fulfill their role.

    PubMed

    Groll, Michael; Clausen, Tim

    2003-12-01

    The 20S proteasome is a large, cylinder-shaped protease that is found in all domains of life and plays a crucial role in cellular protein turnover. It has multiple catalytic centers located within the hollow cavity of a molecular cage. This architecture prevents unwanted degradation of endogenous proteins and promotes processive degradation of substrates by restricting the dissociation of partially digested polypeptides. Although this kind of self-compartmentalization is generally conserved, the proteasomes of bacteria, archaea and eukaryotes show many differences in architecture, subunit composition and regulation. The structure of the 20S proteasome and its inherent role in the regulation of proteasome function are gradually being elucidated.

  9. Similarities between methamphetamine toxicity and proteasome inhibition.

    PubMed

    Fornai, F; Lenzi, P; Gesi, M; Ferrucci, M; Lazzeri, G; Capobianco, L; de Blasi, A; Battaglia, G; Nicoletti, F; Ruggieri, S; Paparelli, A

    2004-10-01

    The monoamine neurotoxin methamphetamine (METH) is commonly used as an experimental model for Parkinson's disease (PD). In fact, METH-induced striatal dopamine (DA) loss is accompanied by damage to striatal nerve endings arising from the substantia nigra. On the other hand, PD is characterized by neuronal inclusions within nigral DA neurons. These inclusions contain alpha-synuclein, ubiquitin, and various components of a metabolic pathway named the ubiquitin-proteasome (UP) system, while mutation of genes coding for various components of the UP system is responsible for inherited forms of PD. In this presentation we demonstrate for the first time the occurrence of neuronal inclusions in vivo in the nigrostriatal system of the mouse following administration of METH. We analyzed, in vivo and in vitro, the shape and the fine structure of these neuronal bodies by using transmission electron microscopy. Immunocytochemical investigation showed that these METH-induced cytosolic inclusions stain for ubiquitin, alpha-synuclein, and UP-related molecules, thus sharing similar components with Lewy bodies occurring in PD, with an emphasis on enzymes belonging to the UP system. In line with this, blockade of this multicatalytic pathway by the selective inhibitor epoxomycin produced cell inclusions with similar features. Moreover, using a multifaceted pharmacological approach, we could demonstrate the need for endogenous DA in order to form neuronal inclusions.

  10. Both PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2 Promote Seedling Photomorphogenesis in Multiple Light Signaling Pathways1[C][W][OPEN

    PubMed Central

    Zhou, Peng; Song, Meifang; Yang, Qinghua; Su, Liang; Hou, Pei; Guo, Lin; Zheng, Xu; Xi, Yulin; Meng, Fanhua; Xiao, Yang; Yang, Li; Yang, Jianping

    2014-01-01

    Arabidopsis (Arabidopsis thaliana) seedlings undergo photomorphogenesis in the light and etiolation in the dark. Light-activated photoreceptors transduce the light signals through a series of photomorphogenesis promoting or repressing factors to modulate many developmental processes in plants, such as photomorphogenesis and shade avoidance. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a conserved RING finger E3 ubiquitin ligase, which mediates degradation of several photomorphogenesis promoting factors, including ELONGATED HYPOCOTYL5 (HY5) and LONG HYPOCOTYL IN FAR-RED1 (HFR1), through a 26S proteasome-dependent pathway. PHYTOCHROME RAPIDLY REGULATED1 (PAR1) was first detected as an early repressed gene in both phytochrome A (phyA)-mediated far-red and phyB-mediated red signaling pathways, and subsequent studies showed that both PAR1 and PAR2 are negative factors of shade avoidance in Arabidopsis. However, the role of PAR1 and PAR2 in seedling deetiolation, and their relationships with other photomorphogenesis promoting and repressing factors are largely unknown. Here, we confirmed that both PAR1 and PAR2 redundantly enhance seedling deetiolation in multiple photoreceptor signaling pathways. Their transcript abundances are repressed by phyA, phyB, and cryptochrome1 under far-red, red, and blue light conditions, respectively. Both PAR1 and PAR2 act downstream of COP1, and COP1 mediates the degradation of PAR1 and PAR2 through the 26S proteasome pathway. Both PAR1 and PAR2 act in a separate pathway from HY5 and HFR1 under different light conditions, except for sharing in the same pathway with HFR1 under far-red light. Together, our results substantiate that PAR1 and PAR2 are positive factors functioning in multiple photoreceptor signaling pathways during seedling deetiolation. PMID:24335334

  11. Rapid Turnover of Extracellular Signal-Regulated Kinase 3 by the Ubiquitin-Proteasome Pathway Defines a Novel Paradigm of Mitogen-Activated Protein Kinase Regulation during Cellular Differentiation

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Pelletier, Stéphane; Pellerin, Johanne; Meloche, Sylvain

    2003-01-01

    Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G1 arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability. PMID:12808096

  12. Effects of an Anticarcinogenic Bowman-Birk Protease Inhibitor on Purified 20S Proteasome and MCF-7 Breast Cancer Cells

    PubMed Central

    Souza, Larissa da Costa; Camargo, Ricardo; Demasi, Marilene; Santana, Jaime Martins; de Freitas, Sonia Maria

    2014-01-01

    Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis. PMID:24475156

  13. 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

  14. Ubiquitin/proteasome-mediated proteolysis is involved in the response to flooding stress in soybean roots, independent of oxygen limitation.

    PubMed

    Yanagawa, Yuki; Komatsu, Setsuko

    2012-04-01

    Ubiquitin/proteasome-mediated proteolysis plays an important role in the response to several environmental stresses. Here, we described the relationship of the proteolysis in the flooding stress in soybean (Glycine max L. cultivar Enrei). Immunoblot analyses were performed using antibodies against two subunits of 26S proteasome, Rpt5 and Rpn10, 20S proteasome and two subunits of COP9 signalosome (CSN), CSN4 and CSN5, to compare between flooded and untreated roots. We also examined their protein amounts in the condition of low oxygen. Moreover, crude extracts from flooded or untreated roots incubated with or without a proteasome inhibitor MG132 were analyzed by proteomics technique. We revealed that the amount of ubiquitinated proteins in soybean roots decreased after flooding treatment and increased to levels similar to controls after de-submergence. Both CSN4 and CSN5 accumulated following flooding treatment, although no significant difference was observed in proteasome. Low oxygen had no effect on the amount of ubiquitinated proteins or CSN4. By 2D-PAGE, the amount of 6 proteins changed significantly following MG132 treatment in flooding stressed plants. We conclude that the accumulation of CSN proteins might enhance the degradation of ubiquitinated proteins independent of hypoxia caused by flooding, thereby lowering their abundance during flooding stress.

  15. The N-End Rule Pathway

    PubMed Central

    Tasaki, Takafumi; Sriram, Shashikanth M.; Park, Kyong Soo; Kwon, Yong Tae

    2013-01-01

    The N-end rule pathway is a proteolytic system in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as essential components of degrons, called N-degrons. Known N-recognins in eukaryotes mediate protein ubiquitylation and selective proteolysis by the 26S proteasome. Substrates of N-recognins can be generated when normally embedded destabilizing residues are exposed at the N terminus by proteolytic cleavage. N-degrons can also be generated through modifications of posttranslationally exposed pro-N-degrons of otherwise stable proteins; such modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Although there are variations in components, degrons, and hierarchical structures, the proteolytic systems based on generation and recognition of N-degrons have been observed in all eukaryotes and prokaryotes examined thus far. The N-end rule pathway regulates homeostasis of various physiological processes, in part, through interaction with small molecules. Here, we review the biochemical mechanisms, structures, physiological functions, and small-molecule-mediated regulation of the N-end rule pathway. PMID:22524314

  16. 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.

  17. The proteasome complex and the maintenance of pluripotency: sustain the fate by mopping up?

    PubMed

    Schröter, Friederike; Adjaye, James

    2014-02-18

    The proteasome is a multi-enzyme complex responsible for orchestrating protein quality control by degrading misfolded, damaged, abnormal and foreign proteins. Studies related to the association of the proteasomal system in the preservation of self-renewal in both human and mouse pluripotent cells are sparse, and therefore a clear indication of the emergence of a new and important field of research. Under specific conditions the standard proteasome switches to the newly synthesized immunoproteasome, a catalytically active protein chamber also involved in the regulation of protein homeostasis, cell signaling and gene expression. Herein we review recent data to help elucidate and highlight the pivotal role of the proteasome complex, constitutive as well as inducible, in the regulation of self-renewal, pluripotency and differentiation of both embryonic and induced pluripotent stem cells. The proteasome that is endowed with enhanced proteolytic activity maintains self-renewal by regulating gene expression. In addition to protein degradation, the proteasome activator PA28, compartments of the 19S regulatory particle and key members of the ubiquitin pathway dictate the fate of a pluripotent stem cell. We anticipate that our observations will stimulate active research in this new and emerging theme related to stem cell biology, disease and regenerative medicine.

  18. Proteasome inhibitor MG-132 lowers gastric adenocarcinoma TMK1 cell proliferation via bone morphogenetic protein signaling

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Jao Yiu; Yu Le; Cho, C.H.

    2008-06-27

    Proteasome inhibitor is a novel class of cancer therapeutics, of which the mechanism of action is not fully understood. It is reported that proteasome inhibitor enhances bone morphogenetic protein (BMP) signaling in osteoblasts to stimulate bone formation. BMP signaling is also an important tumor-suppressing pathway in gastric carcinogenesis. We therefore sought to determine the anti-mitogenic effect of proteasome inhibition in relation to BMP signaling in gastric cancer cells. Results showed that proteasome inhibitor MG-132 significantly suppressed the proliferation and the colony-forming ability of gastric cancer TMK1 cells. In this connection, MG-132 activated BMP signaling, manifested as an increase in Smad1/5/8 phosphorylation and up-regulation of p21{sup Waf1/Cip1} mRNA and protein expression. Knockdown of BMP receptor II by RNA interference abolished Smad1/5/8 phosphorylation, p21{sup Waf1/Cip1} induction, and the inhibition of cell proliferation induced by MG-132. Further analysis revealed that MG-132 up-regulated the expression of BMP1 and BMP4 and suppressed the expression of Smad6. Knockdown of Smad6 also mimicked the effect of MG-132 on BMP signaling. Collectively, these findings suggest that inhibition of proteasome suppresses gastric cancer cell proliferation via activation of BMP signaling. This discovery may open up a novel therapeutic avenue to proteasome inhibitors for the management of gastric cancer.

  19. 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.

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

    PubMed

    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

    2016-02-19

    Hundreds of pathways for degradation converge at ubiquitin recognition by a proteasome. Here, we found that the five known proteasomal ubiquitin receptors in yeast are collectively nonessential for ubiquitin recognition and identified a sixth receptor, Rpn1. A site ( T1: ) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like ( UBL: ) domains of substrate shuttling factors. T1 structures with monoubiquitin or lysine 48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for lysine 48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site ( T2: ) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus, a two-site recognition domain intrinsic to the proteasome uses distinct ubiquitin-fold ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme.

  1. Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome.

    PubMed

    Witkowska, Julia; Karpowicz, Przemysław; Gaczynska, Maria; Osmulski, Pawel A; Jankowska, Elżbieta

    2014-08-01

    Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators.

  2. Fos family protein degradation by the proteasome.

    PubMed

    Gomard, Tiphanie; Jariel-Encontre, Isabelle; Basbous, Jihane; Bossis, Guillaume; Moquet-Torcy, Gabriel; Mocquet-Torcy, Gabriel; Piechaczyk, Marc

    2008-10-01

    c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

  3. Caspase activation inhibits proteasome function during apoptosis.

    PubMed

    Sun, Xiao-Ming; Butterworth, Michael; MacFarlane, Marion; Dubiel, Wolfgang; Ciechanover, Aaron; Cohen, Gerald M

    2004-04-09

    The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.

  4. 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

  5. Dysregulation of Ubiquitin-Proteasome System in Neurodegenerative Diseases

    PubMed Central

    Zheng, Qiuyang; Huang, Timothy; Zhang, Lishan; Zhou, Ying; Luo, Hong; Xu, Huaxi; Wang, Xin

    2016-01-01

    The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways, where abnormal UPS function has been observed in cancer and neurological diseases. Many neurodegenerative diseases share a common pathological feature, namely intracellular ubiquitin-positive inclusions formed by aggregate-prone neurotoxic proteins. This suggests that dysfunction of the UPS in neurodegenerative diseases contributes to the accumulation of neurotoxic proteins and to instigate neurodegeneration. Here, we review recent findings describing various aspects of UPS dysregulation in neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. PMID:28018215

  6. 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.

  7. Cereblon inhibits proteasome activity by binding to the 20S core proteasome subunit beta type 4.

    PubMed

    Lee, Kwang Min; Lee, Jongwon; Park, Chul-Seung

    2012-10-26

    In humans, mutations in the gene encoding cereblon (CRBN) are associated with mental retardation. Although CRBN has been investigated in several cellular contexts, its function remains unclear. Here, we demonstrate that CRBN plays a role in regulating the ubiquitin-proteasome system (UPS). Heterologous expression of CRBN inhibited proteasome activity in a human neuroblastoma cell line. Furthermore, proteasome subunit beta type 4 (PSMB4), the β7 subunit of the 20S core complex, was identified as a direct binding partner of CRBN. These findings suggest that CRBN may modulate proteasome activity by directly interacting with the β7 subunit.

  8. Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.

    PubMed

    Eide, Erik J; Woolf, Margaret F; Kang, Heeseog; Woolf, Peter; Hurst, William; Camacho, Fernando; Vielhaber, Erica L; Giovanni, Andrew; Virshup, David M

    2005-04-01

    The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

  9. Proteasomal dysfunction in sporadic Parkinson's disease.

    PubMed

    McNaught, Kevin St P; Jackson, Tehone; JnoBaptiste, Ruth; Kapustin, Alexander; Olanow, C Warren

    2006-05-23

    The cause and mechanism of neuronal death in sporadic Parkinson's disease (PD) continue to elude investigators. Recently, alterations in proteasomal function have been detected in the brain of patients with the illness. The biochemical basis of the defect and its relevance to the disease process are now being studied. The available results suggest that proteasomal dysfunction could underlie protein accumulation, Lewy body formation, and neuron death in PD. The cause of proteasomal dysfunction is unknown at present, but this could relate to gene mutations, oxidative damage, ATP depletion, or the actions of environmental toxins. It remains to be established if proteasomal dysfunction plays a primary or a secondary role in the initiation or progression of the neurodegenerative process in PD.

  10. Reactive nucleolar and Cajal body responses to proteasome inhibition in sensory ganglion neurons.

    PubMed

    Palanca, Ana; Casafont, Iñigo; Berciano, María T; Lafarga, Miguel

    2014-06-01

    The dysfunction of the ubiquitin proteasome system has been related to a broad array of neurodegenerative disorders in which the accumulation of misfolded protein aggregates causes proteotoxicity. The ability of proteasome inhibitors to induce cell cycle arrest and apoptosis has emerged as a powerful strategy for cancer therapy. Bortezomib is a proteasome inhibitor used as an antineoplastic drug, although its neurotoxicity frequently causes a severe sensory peripheral neuropathy. In this study we used a rat model of bortezomib treatment to study the nucleolar and Cajal body responses to the proteasome inhibition in sensory ganglion neurons that are major targets of bortezomib-induced neurotoxicity. Treatment with bortezomib induced dose-dependent dissociation of protein synthesis machinery (chromatolysis) and nuclear retention of poly(A) RNA granules resulting in neuronal dysfunction. However, as a compensatory response to the proteotoxic stress, both nucleoli and Cajal bodies exhibited reactive changes. These include an increase in the number and size of nucleoli, strong nucleolar incorporation of the RNA precursor 5'-fluorouridine, and increased expression of both 45S rRNA and genes encoding nucleolar proteins UBF, fibrillarin and B23. Taken together, these findings appear to reflect the activation of the nucleolar transcription in response to proteotoxic stress Furthermore, the number of Cajal bodies, a parameter related to transcriptional activity, increases upon proteasome inhibition. We propose that nucleoli and Cajal bodies are important targets in the signaling pathways that are activated by the proteotoxic stress response to proteasome inhibition. The coordinating activity of these two organelles in the production of snRNA, snoRNA and rRNA may contribute to neuronal survival after proteasome inhibition. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

  11. 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.

  12. Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins

    PubMed Central

    Dunys, Julie; Kawarai, Toshitaka; Wilk, Sherwin; St. George-Hyslop, Peter; Alves Da Costa, Cristine; Checler, Frédéric

    2005-01-01

    PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S

  13. 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.

  14. Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

    PubMed

    Vannini, Candida; Bracale, Marcella; Crinelli, Rita; Marconi, Valerio; Campomenosi, Paola; Marsoni, Milena; Scoccianti, Valeria

    2014-01-01

    Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

  15. Flavanonol taxifolin attenuates proteasome inhibition-induced apoptosis in differentiated PC12 cells by suppressing cell death process.

    PubMed

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

    2015-03-01

    The proteasomal dysfunction and mitochondrial impairment has been implicated in neuronal degeneration. Taxifolin has antioxidant and anti-inflammatory effects. However, the effect of taxifolin on the neuronal cell death induced by proteasome inhibition has not been studied. Therefore, in the respect of cell death process, we assessed the effect of taxifolin on the proteasome inhibition-induced apoptosis in neuronal cell injury using differentiated PC12 cells. The proteasome inhibitors MG132 and MG115 induced a decrease in Bid, Bcl-2, and survivin protein levels, an increase in Bax, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases(-8, -9 and -3), an increase in the tumor suppressor p53 levels and cleavage of PARP-1. The addition of taxifolin attenuated the proteasome inhibitor-induced changes in the apoptosis-related protein levels, formation of reactive oxygen species, depletion and oxidation of GSH, formations of malondialdehyde and carbonyls, and cell death. The results show that taxifolin may attenuate the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The preventive effect of taxifolin appears to be attributed to its inhibitory effect on the formation of reactive oxygen species, and depletion and oxidation of GSH.

  16. Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening.

    PubMed

    Sadre-Bazzaz, Kianoush; Whitby, Frank G; Robinson, Howard; Formosa, Tim; Hill, Christopher P

    2010-03-12

    The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here, we report a 3.4 A resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture and the observation that Blm10 induces a disordered proteasome gate structure challenge the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.

  17. Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

    PubMed Central

    Cheng, Jinglei; Fujita, Akikazu; Tokumoto, Toshinobu

    2006-01-01

    Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make “ApoB-crescents.” ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50–100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12–24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways. PMID:16597703

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

    Bardag-Gorce, Fawzia

    2009-01-01

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

  20. Measuring activity in the ubiquitin-proteasome system: from large scale discoveries to single cells analysis.

    PubMed

    Melvin, Adam T; Woss, Gregery S; Park, Jessica H; Waters, Marcey L; Allbritton, Nancy L

    2013-09-01

    The ubiquitin-proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS has provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington's disease. These reporters, usually consisting of a recognition sequence fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes. This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, a recent study is presented highlighting the development of a novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples.

  1. Proapoptotic fibronectin fragment induces the degradation of ubiquitinated p53 via proteasomes in periodontal ligament cells

    PubMed Central

    Ghosh, Abhijit; Joo, Nam Eok; Chen, Tina Chunyuan; Kapila, Yvonne L.

    2009-01-01

    Background and Objective The extracellular matrix (ECM) plays a key role in signaling necessary for tissue remodeling and cell survival. However, signals from disease-altered ECMs, as that present in inflammatory diseases like periodontitis and arthritis, may lead to apoptosis or programmed cell death of resident cells. Previously, we found that a disease-associated fibronectin fragment triggers apoptosis of primary human periodontal ligament (PDL) cells via a novel apoptotic pathway in which the tumor suppressor, p53, is transcriptionally downregulated. Materials and Methods We used immunofluorescence, transfection assays, western blotting and ELISAs to show that p53 is degraded by a proteasomal pathway in response to a proapoptotic disease-associated fibronectin fragment. Results We now show that under these same apoptotic conditions p53 is further downregulated by post-translational ubiquitination and subsequent targeting to the proteasome for degradation. Pretreatment of cells with the proteasomal inhibitors MG132 and lactacystin rescued the cells from apoptosis. p53 levels in cells transfected with ubiquitin siRNA were resistant to degradation induced by the proapoptotic fibronectin fragment, showing that ubiquitination is important for the proapoptotic fibronectin fragment-induced degradation of p53. Conclusions These data show that a proapoptotic fibronectin matrix induces ubiquitination and degradation of p53 in the proteasome as part of a novel mechanism of apoptosis associated with inflammatory diseases. PMID:20337881

  2. Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program.

    PubMed

    Matsushita, Akane; Inoue, Haruhiko; Goto, Shingo; Nakayama, Akira; Sugano, Shoji; Hayashi, Nagao; Takatsuji, Hiroshi

    2013-01-01

    The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin-proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis.

  3. Rationale and efficacy of proteasome inhibitor combined with arsenic trioxide in the treatment of acute promyelocytic leukemia

    PubMed Central

    Ganesan, S; Alex, A A; Chendamarai, E; Balasundaram, N; Palani, H K; David, S; Kulkarni, U; Aiyaz, M; Mugasimangalam, R; Korula, A; Abraham, A; Srivastava, A; Padua, R A; Chomienne, C; George, B; Balasubramanian, P; Mathews, V

    2016-01-01

    Arsenic trioxide (ATO) mediates PML-RARA (promyelocytic leukemia–retinoic acid receptor-α) oncoprotein degradation via the proteasome pathway and this degradation appears to be critical for achieving cure in acute promyeloytic leukemia (APL). We have previously demonstrated significant micro-environment-mediated drug resistance (EMDR) to ATO in APL. Here we demonstrate that this EMDR could be effectively overcome by combining a proteasome inhibitor (bortezomib) with ATO. A synergistic effect on combining these two agents in vitro was noted in both ATO-sensitive and ATO-resistant APL cell lines. The mechanism of this synergy involved downregulation of the nuclear factor-κB pathway, increase in unfolded protein response (UPR) and an increase in reactive oxygen species generation in the malignant cell. We also noted that PML-RARA oncoprotein is effectively cleared with this combination in spite of proteasome inhibition by bortezomib, and that this clearance is mediated through a p62-dependent autophagy pathway. We further demonstrated that proteasome inhibition along with ATO had an additive effect in inducing autophagy. The beneficial effect of this combination was further validated in an animal model and in an on-going clinical trial. This study raises the potential of a non-myelotoxic proteasome inhibitor replacing anthracyclines in the management of high-risk and relapsed APL. PMID:27560113

  4. Terminal functionalized thiourea-containing dipeptides as multidrug-resistance reversers that target 20S proteasome and cell proliferation.

    PubMed

    Qin, Jian-Mei; Huang, Ri-Zhen; Yao, Gui-Yang; Liao, Zhi-Xin; Pan, Ying-Ming; Wang, Heng-Shan

    2017-01-27

    A series of inhibitors of 20S proteasome based on terminal functionalized dipeptide derivatives containing the thiourea moiety were synthesized and evaluated for inhibition of 20S proteasome and the effects of multidrug-resistance reversers. These compounds exhibited significant selectivity to the β5-subunit of the human 20S proteasome with IC50 values at submicromolar concentrations. A docking study of the most active compound 6i revealed key interactions between 6i and the active site of the 20S proteasome in which the thiourea moiety and a nitro group were important for improving activity. In particular, compound 6i appeared to be the most potent compound against the NCI-H460 cell line, and displayed similar efficiency in drug-sensitive versus drug-resistant cancer cell lines, at least partly, by inhibition of the activity of 20S proteasome and induce apoptosis. In addition, 6i-induced apoptosis was significantly facilitated in NCI-H460/DOX cells that had been pretreated with inhibitors of P-gp. Mechanistically, compound 6i might trigger apoptotic signalling pathway. Thus, we conclude that dipeptide derivatives containing the thiourea moiety may be the potential inhibitors of proteasome with the ability to reverse multidrug resistance.

  5. 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.

  6. The Proline/Arginine Dipeptide from Hexanucleotide Repeat Expanded C9ORF72 Inhibits the Proteasome

    PubMed Central

    Lan, Matthews; Mojsilovic-Petrovic, Jelena; Choi, Won Hoon; Safren, Nathaniel; Barmada, Sami

    2017-01-01

    Abstract An intronic hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in ∼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR20) and glycine/arginine repeated 20 times (GR20)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR20 kills motor neurons with an LD50 of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR20 does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR20 does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR20, however, does not have these effects. The effects of PR20 on the proteasome are likely to be direct because (1) PR20 physically associates with proteasomes in biochemical assays, and (2) PR20 inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR20 on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with C9ORF72 HRE. PMID:28197542

  7. The proteasome inhibitor, PS-341, causes cytokeratin aggresome formation.

    PubMed

    Bardag-Gorce, Fawzia; Riley, Nora E; Nan, Li; Montgomery, Rosalyn O; Li, Jun; French, Barbara A; Lue, Yan H; French, Samuel W

    2004-02-01

    Mallory body (MB) experimental induction takes 10 weeks of drug ingestion. Therefore, it is difficult to study the dynamics and mechanisms involved in vivo. Consequently, an in vitro study was done using primary tissue culture of hepatocytes from drug-primed mice livers in which MBs had already formed. The hypothesis to be tested was that MBs are cytokeratin aggresomes, which form when hepatocytes have a defective ubiquitin-proteasome pathway by which turnover of cytokeratin proteins is prevented. To test this hypothesis, primary tissue cultures of the hepatocytes from normal and MB-forming livers were incubated with the proteasome inhibitor PS-341 and then the cytokeratin filaments and the filament connecting proteins, that is, beta-actin, and ZO1, were visualized by immunofluorescence microscopy. PS-341 caused detachment of the cytokeratins from the cell surface plasma membrane. The cytokeratin filaments retracted toward the nucleus and cytokeratin aggresomes formed. In human livers, MBs showed colocalization of cytokeratin-8 (CK-8) with ubiquitin but not with beta-actin or ZO1. Mouse hepatoma cell lines were studied using PS-341 to induce cytokeratin aggresome formation. In these cell lines, the cytokeratin filaments first retracted toward the nucleus then formed cytokeratin-ubiquitin aggresomes polarized at one side of the nucleus. At the same time, the cells became dissociated from each other, however. The results simulated MB formation. MBs differ from cytokeratin aggresomes both morphologically and in ultrastructure.

  8. The interplay between autophagy and the ubiquitin-proteasome system in cardiac proteotoxicity

    PubMed Central

    Wang, Changhua; Wang, Xuejun

    2014-01-01

    Proteotoxicity refers to the detrimental effects of damaged/misfolded proteins on the cell. Cardiac muscle is particularly susceptible to proteotoxicity because sustained and severe proteotoxic stress leads to cell death and cardiac muscle has very limited self-renewal capacity. The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway (ALP) are two major pathways responsible for degradation of most cellular proteins. Alterations of UPS and ALP functions are associated with the accumulation of proteotoxic species in the heart, a key pathological feature of common forms of heart disease including idiopathic, ischemic, and pressure-overloaded cardiomyopathies and a large subset of congestive heart failure. Emerging evidence suggests proteasome inhibition or impairment activates autophagy and conversely, acute ALP inhibition may sometimes increase intrinsic proteasome peptidase activities but chronic ALP inhibition hinders UPS performance in ubiquitinated protein degradation. The exact molecular basis on which the two degradative pathways interact remains largely undefined. Here we review current understanding of the roles of the UPS and autophagy in the control of cardiac proteotoxicity, with a specific focus on the crosstalk between the two pathways. PMID:25092168

  9. Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

    PubMed Central

    2010-01-01

    Background Proteasomes play a central role in the major histocompatibility class I (MHCI) antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site). There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC), optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07) and the immunoproteasome (MCC = 0.36 ± 0.06) were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific to predict cleavage by

  10. Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer.

    PubMed

    Walerych, Dawid; Lisek, Kamil; Sommaggio, Roberta; Piazza, Silvano; Ciani, Yari; Dalla, Emiliano; Rajkowska, Katarzyna; Gaweda-Walerych, Katarzyna; Ingallina, Eleonora; Tonelli, Claudia; Morelli, Marco J; Amato, Angela; Eterno, Vincenzo; Zambelli, Alberto; Rosato, Antonio; Amati, Bruno; Wiśniewski, Jacek R; Del Sal, Giannino

    2016-08-01

    In cancer, the tumour suppressor gene TP53 undergoes frequent missense mutations that endow mutant p53 proteins with oncogenic properties. Until now, a universal mutant p53 gain-of-function program has not been defined. By means of multi-omics: proteome, DNA interactome (chromatin immunoprecipitation followed by sequencing) and transcriptome (RNA sequencing/microarray) analyses, we identified the proteasome machinery as a common target of p53 missense mutants. The mutant p53-proteasome axis globally affects protein homeostasis, inhibiting multiple tumour-suppressive pathways, including the anti-oncogenic KSRP-microRNA pathway. In cancer cells, p53 missense mutants cooperate with Nrf2 (NFE2L2) to activate proteasome gene transcription, resulting in resistance to the proteasome inhibitor carfilzomib. Combining the mutant p53-inactivating agent APR-246 (PRIMA-1MET) with the proteasome inhibitor carfilzomib is effective in overcoming chemoresistance in triple-negative breast cancer cells, creating a therapeutic opportunity for treatment of solid tumours and metastasis with mutant p53.

  11. The proteasome activator 11 S REG (PA28) and class I antigen presentation.

    PubMed Central

    Rechsteiner, M; Realini, C; Ustrell, V

    2000-01-01

    There are two immune responses in vertebrates: humoral immunity is mediated by circulating antibodies, whereas cytotoxic T lymphocytes (CTL) confer cellular immunity. CTL lyse infected cells upon recognition of cell-surface MHC Class I molecules complexed with foreign peptides. The displayed peptides are produced in the cytosol by degradation of host proteins or proteins from intracellular pathogens that might be present. Proteasomes are cylindrical multisubunit proteases that generate many of the peptides eventually transferred to the cell surface for immune surveillance. In mammalian proteasomes, six active sites face a central chamber. As this chamber is sealed off from the enzyme's surface, there must be mechanisms to promote entry of substrates. Two protein complexes have been found to bind the ends of the proteasome and activate it. One of the activators is the 19 S regulatory complex of the 26 S proteasome; the other activator is '11 S REG' [Dubiel, Pratt, Ferrell and Rechsteiner (1992) J. Biol. Chem. 267, 22369-22377] or 'PA28' [Ma, Slaughter and DeMartino (1992) J. Biol. Chem. 267, 10515-10523]. During the past 7 years, our understanding of the structure of REG molecules has increased significantly, but much less is known about their biological functions. There are three REG subunits, namely alpha, beta and gamma. Recombinant REGalpha forms a ring-shaped heptamer of known crystal structure. 11 S REG is a heteroheptamer of alpha and beta subunits. REGgamma is also presumably a heptameric ring, and it is found in the nuclei of the nematode work Caenorhabditis elegans and higher organisms, where it may couple proteasomes to other nuclear components. REGalpha and REGbeta, which are abundant in vertebrate immune tissues, are located mostly in the cytoplasm. Synthesis of REG alpha and beta subunits is induced by interferon-gamma, and this has led to the prevalent hypothesis that REG alpha/beta hetero-oligomers play an important role in Class I antigen

  12. Functional asymmetries of proteasome translocase pore.

    PubMed

    Erales, Jenny; Hoyt, Martin A; Troll, Fabian; Coffino, Philip

    2012-05-25

    Degradation by proteasomes involves coupled translocation and unfolding of its protein substrates. Six distinct but paralogous proteasome ATPase proteins, Rpt1 to -6, form a heterohexameric ring that acts on substrates. An axially positioned loop (Ar-Φ loop) moves in concert with ATP hydrolysis, engages substrate, and propels it into a proteolytic chamber. The aromatic (Ar) residue of the Ar-Φ loop in all six Rpts of S. cerevisiae is tyrosine; this amino acid is thought to have important functional contacts with substrate. Six yeast strains were constructed and characterized in which Tyr was individually mutated to Ala. The mutant cells were viable and had distinct phenotypes. rpt3, rpt4, and rpt5 Tyr/Ala mutants, which cluster on one side of the ATPase hexamer, were substantially impaired in their capacity to degrade substrates. In contrast, rpt1, rpt2, and rpt6 mutants equaled or exceeded wild type in degradation activity. However, rpt1 and rpt6 mutants had defects that limited cell growth or viability under conditions that stressed the ubiquitin proteasome system. In contrast, the rpt3 mutant grew faster than wild type and to a smaller size, a defect that has previously been associated with misregulation of G1 cyclins. This rpt3 phenotype probably results from altered degradation of cell cycle regulatory proteins. Finally, mutation of five of the Rpt subunits increased proteasome ATPase activity, implying bidirectional coupling between the Ar-Φ loop and the ATP hydrolysis site. The present observations assign specific functions to individual Rpt proteins and provide insights into the diverse roles of the axial loops of individual proteasome ATPases.

  13. 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

  14. Incorporation of the Rpn12 subunit couples completion of proteasome regulatory particle lid assembly to lid-base joining.

    PubMed

    Tomko, Robert J; Hochstrasser, Mark

    2011-12-23

    The 26S proteasome, the central eukaryotic protease, comprises a core particle capped by a 19S regulatory particle (RP). The RP is divisible into base and lid subcomplexes. Lid biogenesis and incorporation into the RP remain poorly understood. We report several lid intermediates, including the free Rpn12 subunit and a lid particle (LP) containing the remaining eight subunits, LP2. Rpn12 binds LP2 in vitro, and each requires the other for assembly into 26S proteasomes. Stable Rpn12 incorporation depends on all other lid subunits, indicating that Rpn12 distinguishes LP2 from smaller lid subcomplexes. The highly conserved C terminus of Rpn12 bridges the lid and base, mediating both stable binding to LP2 and lid-base joining. Our data suggest a hierarchical assembly mechanism where Rpn12 binds LP2 only upon correct assembly of all other lid subunits, and the Rpn12 tail then helps drive lid-base joining. Rpn12 incorporation thus links proper lid assembly to subsequent assembly steps.

  15. 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

    2014-01-03

    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.

  16. HDAC and Proteasome Inhibitors Synergize to Activate Pro-Apoptotic Factors in Synovial Sarcoma

    PubMed Central

    Barrott, Jared J.; Yao, Ren Jie; Poulin, Neal M.; Brodin, Bertha A.; Jones, Kevin B.; Underhill, T. Michael; Nielsen, Torsten O.

    2017-01-01

    Conventional cytotoxic therapies for synovial sarcoma provide limited benefit, and no drugs specifically targeting its driving SS18-SSX fusion oncoprotein are currently available. Patients remain at high risk for early and late metastasis. A high-throughput drug screen consisting of over 900 tool compounds and epigenetic modifiers, representing over 100 drug classes, was undertaken in a panel of synovial sarcoma cell lines to uncover novel sensitizing agents and targetable pathways. Top scoring drug categories were found to be HDAC inhibitors and proteasomal targeting agents. We find that the HDAC inhibitor quisinostat disrupts the SS18-SSX driving protein complex, thereby reestablishing expression of EGR1 and CDKN2A tumor suppressors. In combination with proteasome inhibition, HDAC inhibitors synergize to decrease cell viability and elicit apoptosis. Quisinostat inhibits aggresome formation in response to proteasome inhibition, and combination treatment leads to elevated endoplasmic reticulum stress, activation of pro-apoptotic effector proteins BIM and BIK, phosphorylation of BCL-2, increased levels of reactive oxygen species, and suppression of tumor growth in a murine model of synovial sarcoma. This study identifies and provides mechanistic support for a particular susceptibility of synovial sarcoma to the combination of quisinostat and proteasome inhibition. PMID:28056055

  17. Production of proteasome inhibitor syringolin A by the endophyte Rhizobium sp. strain AP16.

    PubMed

    Dudnik, Alexey; Bigler, Laurent; Dudler, Robert

    2014-06-01

    Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts.

  18. The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

    PubMed Central

    Pinto, Maria J.; Alves, Pedro L.; Martins, Luís; Pedro, Joana R.; Ryu, Hyun R.; Jeon, Noo Li; Taylor, Anne M.

    2016-01-01

    Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin–proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses. PMID:27022091

  19. Proteolytic Pathways Induced by Herbicides That Inhibit Amino Acid Biosynthesis

    PubMed Central

    Zulet, Amaia; Gil-Monreal, Miriam; Villamor, Joji Grace; Zabalza, Ana; van der Hoorn, Renier A. L.; Royuela, Mercedes

    2013-01-01

    Background The herbicides glyphosate (Gly) and imazamox (Imx) inhibit the biosynthesis of aromatic and branched-chain amino acids, respectively. Although these herbicides inhibit different pathways, they have been reported to show several common physiological effects in their modes of action, such as increasing free amino acid contents and decreasing soluble protein contents. To investigate proteolytic activities upon treatment with Gly and Imx, pea plants grown in hydroponic culture were treated with Imx or Gly, and the proteolytic profile of the roots was evaluated through fluorogenic kinetic assays and activity-based protein profiling. Results Several common changes in proteolytic activity were detected following Gly and Imx treatment. Both herbicides induced the ubiquitin-26 S proteasome system and papain-like cysteine proteases. In contrast, the activities of vacuolar processing enzymes, cysteine proteases and metacaspase 9 were reduced following treatment with both herbicides. Moreover, the activities of several putative serine protease were similarly increased or decreased following treatment with both herbicides. In contrast, an increase in YVADase activity was observed under Imx treatment versus a decrease under Gly treatment. Conclusion These results suggest that several proteolytic pathways are responsible for protein degradation upon herbicide treatment, although the specific role of each proteolytic activity remains to be determined. PMID:24040092

  20. 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

  1. Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum

    PubMed Central

    Verchot, Jeanmarie

    2016-01-01

    The endoplasmic reticulum (ER) is central to plant virus replication, translation, maturation, and egress. Ubiquitin modification of ER associated cellular and viral proteins, alongside the actions of the 26S proteasome, are vital for the regulation of infection. Viruses can arrogate ER associated ubiquitination as well as cytosolic ubiquitin ligases with the purpose of directing the ubiquitin proteasome system (UPS) to new targets. Such targets include necessary modification of viral proteins which may stabilize certain complexes, or modification of Argonaute to suppress gene silencing. The UPS machinery also contributes to the regulation of effector triggered immunity pattern recognition receptor immunity. Combining the results of unrelated studies, many positive strand RNA plant viruses appear to interact with cytosolic Ub-ligases to provide novel avenues for controlling the deleterious consequences of disease. Viral interactions with the UPS serve to regulate virus infection in a manner that promotes replication and movement, but also modulates the levels of RNA accumulation to ensure successful biotrophic interactions. In other instances, the UPS plays a central role in cellular immunity. These opposing roles are made evident by contrasting studies where knockout mutations in the UPS can either hamper viruses or lead to more aggressive diseases. Understanding how viruses manipulate ER associated post-translational machineries to better manage virus–host interactions will provide new targets for crop improvement. PMID:27869775

  2. Solution structure of yeast Rpn9: insights into proteasome lid assembly.

    PubMed

    Hu, Yunfei; Wu, Yujie; Li, Qianwen; Zhang, Wenbo; Jin, Changwen

    2015-03-13

    The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive. Recent cryo-EM studies at near-atomic resolution provided invaluable information on the RP architecture in different functional states. Nevertheless, atomic resolution structural information on the RP is still limited, and deeper understanding of RP assembly mechanism requires further studies on the structures and interactions of individual subunits or subcomplexes. Herein we report the high-resolution NMR structures of the PCI-containing subunit Rpn9 from Saccharomyces cerevisiae. The 45-kDa protein contains an all-helical N-terminal domain and a C-terminal PCI domain linked via a semiflexible hinge. The N-terminal domain mediates interaction with the ubiquitin receptor Rpn10, whereas the PCI domain mediates interaction with the neighboring PCI subunit Rpn5. The Rpn9-Rpn5 interface highlights two structural motifs on the winged helix module forming a hydrophobic center surrounded by ionic pairs, which is a common pattern for all PCI-PCI interactions in the lid. The results suggest that divergence in surface composition among different PCI pairs may contribute to the modulation of lid assembly.

  3. Role of ubiquitin-proteasome in protein quality control and signaling: implication in the pathogenesis of eye diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin–proteasome pathway (UPP) plays important roles in many cellular functions, such as protein quality control, cell cycle control, and signal transduction. The selective degradation of aberrant proteins by the UPP is essential for the timely removal of potential cytotoxic damaged or other...

  4. 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

  5. MM-1 facilitates degradation of c-Myc by recruiting proteasome and a novel ubiquitin E3 ligase.

    PubMed

    Kimura, Yumiko; Nagao, Arisa; Fujioka, Yuko; Satou, Akiko; Taira, Takahiro; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2007-10-01

    We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc by recruiting the HDAC1 complex via TIF1beta/KAP1, a transcriptional corepressor. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, we found that MM-1 was bound to a component of proteasome and stimulated degradation of c-Myc in human cells. Knockdown of endogenous MM-1 in human HeLa cells by introduction of siRNA against MM-1 stabilized the endogenous c-Myc. To identify proteins that participate in c-Myc degradation by MM-1, in vivo and in vitro binding assays were carried out. The results showed that MM-1 directly bound to Rpt3, a subunit of 26S proteasome, and that c-Myc directly bound to Skp2, which recruited ElonginC, ElonginB and Cullin2, thereby forming a novel ubiquitin E3 ligase. Knockdown of endogenous Cullin2 stabilized the endogenous c-Myc. Thus, MM-1 is a factor that connects c-Myc to the ubiquitin E3 ligase and the proteasome.

  6. 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.

  7. The effect of peptidic and non-peptidic proteasome inhibitors on the biological properties of Acanthamoeba castellanii belonging to the T4 genotype.

    PubMed

    Siddiqui, Ruqaiyyah; Saleem, Sahreena; Khan, Naveed Ahmed

    2016-09-01

    The treatment of Acanthamoeba infections remains problematic, suggesting that new targets and/or chemotherapeutic agents are needed. Bioassay-guided screening of drugs that are clinically-approved for non-communicable diseases against opportunistic eukaryotic pathogens is a viable strategy. With known targets and mode of action, such drugs can advance to clinical trials at a faster pace. Recently Bortezomib (proteasome inhibitor) has been approved by FDA in the treatment of multiple myeloma. As proteasomal pathways are well known regulators of a variety of eukaryotic cellular functions, the overall aim of the present study was to study the effects of peptidic and non-peptidic proteasome inhibitors on the biology and pathogenesis of Acanthamoeba castellanii of the T4 genotype, in vitro. Zymographic assays revealed that inhibition of proteasome had detrimental effects on the extracellular proteolytic activities of A. castellanii. Proteasome inhibition affected A. castellanii growth (using amoebistatic assays), but not viability of A. castellanii. Importantly, proteasome inhibitors affected encystation as determined by trophozoite transformation into the cyst form, as well as excystation, as determined by cyst transformation into the trophozoite form. The ability of proteasome inhibitor to block Acanthamoeba differentiation is significant, as it presents a major challenge in the successful treatment of Acanthamoeba infection. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic infections.

  8. Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.

    PubMed

    Livinskaya, Veronika A; Barlev, Nickolai A; Nikiforov, Andrey A

    2014-05-01

    The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells.

  9. Subcellular distribution and dynamics of active proteasome complexes unraveled by a workflow combining in vivo complex cross-linking and quantitative proteomics.

    PubMed

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

    2013-03-01

    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.

  10. Cks1, Cdk1, and the 19S proteasome collaborate to regulate gene induction-dependent nucleosome eviction in yeast.

    PubMed

    Chaves, Susana; Baskerville, Chris; Yu, Veronica; Reed, Steven I

    2010-11-01

    Cks1, Cdk1 (Cdc28), and the proteasome are required for efficient transcriptional induction of GAL1 and other genes in Saccharomyces cerevisiae. We show here that one function of these proteins is to reduce nucleosome density on chromatin in a gene induction-specific manner. The transcriptional requirement for Cks1 can be bypassed if nucleosome density is reduced by an alternative pathway, indicating that this is the primary function of Cks1 in the context of gene induction. We further show that Cks1, Cdk1, and the 19S subunit of the proteasome are recruited to chromatin by binding directly to the histone H4 amino-terminal tail. However, this activity of the proteasome does not require the protease activity associated with the 20S subunit. These data suggest a model where binding of a complex consisting of Cks1, Cdk1, and the 19S proteasome to histone H4 leads to removal of nucleosomes via a nonproteolytic activity of the proteasome.

  11. A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster

    PubMed Central

    Pickering, Andrew M.; Staab, Trisha A.; Tower, John; Sieburth, Derek; Davies, Kelvin J. A.

    2013-01-01

    SUMMARY In mammalian cells, hydrogen peroxide (H2O2)-induced adaptation to oxidative stress is strongly dependent on an Nrf2 transcription factor-mediated increase in the 20S proteasome. Here, we report that both Caenorhabditis elegans nematode worms and Drosophila melanogaster fruit flies are also capable of adapting to oxidative stress with H2O2 pre-treatment. As in mammalian cells, this adaptive response in worms and flies involves an increase in proteolytic activity and increased expression of the 20S proteasome, but not of the 26S proteasome. We also found that the increase in 20S proteasome expression in both worms and flies, as in mammalian cells, is important for the adaptive response, and that it is mediated by the SKN-1 and CNC-C orthologs of the mammalian Nrf2 transcription factor, respectively. These studies demonstrate that stress mechanisms operative in cell culture also apply in disparate intact organisms across a wide biological diversity. PMID:23038734

  12. 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

  13. BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

    PubMed

    Wiggins, Ceri M; Tsvetkov, Peter; Johnson, Mark; Joyce, Claire L; Lamb, Christopher A; Bryant, Nia J; Komander, David; Shaul, Yosef; Cook, Simon J

    2011-03-15

    BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

  14. Activating Cell Death Ligand Signaling Through Proteasome Inhibition

    DTIC Science & Technology

    2009-05-01

    Activating Cell Death Ligand Signaling Through Proteasome Inhibition PRINCIPAL INVESTIGATOR: Steven R Schwarze...SUBTITLE Activating Cell Death Ligand Signaling Through 5a. CONTRACT NUMBER Proteasome Inhibition 5b. GRANT NUMBER W81XWH-08-1-0392 5c...proteasome inhibition can act as an anti-neoplastic agent in vivo by sensitizing cancer cells to cell death ligands in the tumor microenvironment

  15. Inhibition of proteasome activity is involved in cobalt-induced apoptosis of human alveolar macrophages.

    PubMed

    Araya, Jun; Maruyama, Muneharu; Inoue, Akira; Fujita, Tadashi; Kawahara, Junko; Sassa, Kazuhiko; Hayashi, Ryuji; Kawagishi, Yukio; Yamashita, Naohiro; Sugiyama, Eiji; Kobayashi, Masashi

    2002-10-01

    Inhalation of particulate cobalt has been known to induce interstitial lung disease. There is growing evidence that apoptosis plays a crucial role in physiological and pathological settings and that the ubiquitin-proteasome system is involved in the regulation of apoptosis. Cadmium, the same transitional heavy metal as cobalt, has been reported to accumulate ubiquitinated proteins in neuronal cells. On the basis of these findings, we hypothesized that cobalt would induce apoptosis in the lung by disturbance of the ubiquitin-proteasome pathway. To evaluate this, we exposed U-937 cells and human alveolar macrophages (AMs) to cobalt chloride (CoCl(2)) and examined their apoptosis by DNA fragmentation assay, 4',6-diamidino-2'-phenylindol dihydrochloride staining, and Western blot analysis. CoCl(2) induced apoptosis and accumulated ubiquitinated proteins. Exposure to CoCl(2) inhibited proteasome activity in U-937 cells. Cobalt-induced apoptosis was mediated via mitochondrial pathway because CoCl(2) released cytochrome c from mitochondria. These results suggest that cobalt-induced apoptosis of AMs may be one of the mechanisms for cobalt-induced lung injury and that the accumulation of ubiquitinated proteins might be involved in this apoptotic process.

  16. 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.

  17. The proteasome and the degradation of oxidized proteins: Part III—Redox regulation of the proteasomal system

    PubMed Central

    Höhn, Tobias Jung Annika; Grune, Tilman

    2014-01-01

    Here, we review shortly the current knowledge on the regulation of the proteasomal system during and after oxidative stress. After addressing the components of the proteasomal system and the degradation of oxidatively damaged proteins in part I and II of this series, we address here which changes in activity undergo the proteasome and the ubiquitin-proteasomal system itself under oxidative conditions. While several components of the proteasomal system undergo direct oxidative modification, a number of redox-regulated events are modulating the proteasomal activity in a way it can address the major tasks in an oxidative stress situation: the removal of oxidized proteins and the adaptation of the cellular metabolism to the stress situation. PMID:24563857

  18. The ubiquitin-proteasome system in cardiac proteinopathy: a quality control perspective

    PubMed Central

    Su, Huabo; Wang, Xuejun

    2010-01-01

    Protein quality control (PQC) depends on elegant collaboration between molecular chaperones and targeted proteolysis in the cell. The latter is primarily carried out by the ubiquitin-proteasome system, but recent advances in this area of research suggest a supplementary role for the autophagy-lysosomal pathway in PQC-related proteolysis. The (patho)physiological significance of PQC in the heart is best illustrated in cardiac proteinopathy, which belongs to a family of cardiac diseases caused by expression of aggregation-prone proteins in cardiomyocytes. Cardiac proteasome functional insufficiency (PFI) is best studied in desmin-related cardiomyopathy, a bona fide cardiac proteinopathy. Emerging evidence suggests that many common forms of cardiomyopathy may belong to proteinopathy. This review focuses on examining current evidence, as it relates to the hypothesis that PFI impairs PQC in cardiomyocytes and contributes to the progression of cardiac proteinopathies to heart failure. PMID:19696071

  19. ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51.

    PubMed

    Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Yasui, Hironobu; Inanami, Osamu

    2013-10-11

    In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.

  20. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease

    PubMed Central

    Nath, Samir R.; Lieberman, Andrew P.

    2017-01-01

    Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology. PMID:28381987

  1. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease.

    PubMed

    Nath, Samir R; Lieberman, Andrew P

    2017-01-01

    Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology.

  2. Phylogenetic analysis of nematodes of the genus Pratylenchus using nuclear 26S rDNA.

    PubMed

    Al-Banna, L; Williamson, V; Gardner, S L

    1997-02-01

    We used nucleotide sequences of the large subunit ribosomal genes (26S rDNA) to examine evolutionary relationships among species of the genus Pratylenchus (Order: Tylenchida, Family: Pratylenchidae), commonly known as root-lesion nematodes. Ten species of Pratylenchus were studied including, P. penetrans, P. crenatus, P. minyus, P. vulnus, P. thornei, P. musicola, P. coffeae, P. hexincisus, P. scribneri, and P. brachyurus. The species Hirschmanniella belli, Meloidogyne javanica, Heterorhabditis bacteriophora, Nacobbus aberrans, Radopholus similis, and Xiphinema index were used as outgroups. Based on parsimony analyses of approximately 307 aligned nucleotides of the D3 expansion region of the 26S rDNA, it is clear that species of Pratylenchus are a paraphyletic assemblage. The outgroup taxon H. belli shares a common ancestor with the clade that includes P. vulnus and P. crenatus while N. aberrans and R. similis share a common ancestor with 5 other species included in this study.

  3. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  4. Evidence for an Independent 26-s Microseismic Source near the Vanuatu Islands

    NASA Astrophysics Data System (ADS)

    Zeng, Xiangfang; Ni, Sidao

    2014-09-01

    The 26 s peak in the ambient seismic noise spectrum is persistently excited and observed at stations globally. Using noise cross-correlation functions (NCFs), the location suggests that the source could be situated in the Gulf of Guinea and Fiji Basin. However, the Fiji Basin was proposed to be the mirror site (near antipode) of the Gulf of Guinea source instead of an independent source, assuming that the surface waves more efficiently propagate along the major-arc paths of oceanic movements. To investigate the propagation of the Rayleigh waves along continental and oceanic paths, we analyzed the surface wave data recorded from an earthquake near the Gulf of Guinea and found that Rayleigh waves travel along continental minor-arc paths more efficiently than along oceanic major-arc paths. We then located the source in the western Pacific Ocean from group velocities measured with earthquake data by using the travel time misfit in NCFs after calibration and concluded that the source is in the Vanuatu Islands. Moreover, the temporal variation of the 26 s microseismic peak observed in the western Pacific seismic stations is very different from that in stations near the Gulf of Guinea, which suggests that they are excited by independent sources. Therefore, the Vanuatu source should be an independent microseismic source. As it is close to volcanoes in the Vanuatu islands, the Pacific 26 s microseismic source might be excited by magmatic processes, which are also responsible for very-long-period volcanic tremors.

  5. Ubiquitin proteasome system research in gastrointestinal cancer

    PubMed Central

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-01-01

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design. PMID:26909134

  6. Ubiquitin proteasome system research in gastrointestinal cancer.

    PubMed

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-02-15

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design.

  7. Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly*

    PubMed Central

    Li, Xia; Li, Yanjie; Arendt, Cassandra S.; Hochstrasser, Mark

    2016-01-01

    Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (β1, β2, and β5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, β5 (Doa3/Pre2) has a 75-residue propeptide, β5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the β5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although β5pro is functionally linked to the Ump1 assembly factor, trans-expressed β5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed β5pro mutants; the strongest encoded the β7 proteasome subunit. Previous data suggested that β7 and β5pro have overlapping roles in bringing together two half-proteasomes, but the timing of β7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind β7 incorporation into the half-mer. Our results suggest that β7 insertion precedes half-mer dimerization, and the β7 tail and β5 propeptide have unequal roles in half-mer joining. PMID:26627836

  8. Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly.

    PubMed

    Li, Xia; Li, Yanjie; Arendt, Cassandra S; Hochstrasser, Mark

    2016-01-22

    Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (β1, β2, and β5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, β5 (Doa3/Pre2) has a 75-residue propeptide, β5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the β5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although β5pro is functionally linked to the Ump1 assembly factor, trans-expressed β5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed β5pro mutants; the strongest encoded the β7 proteasome subunit. Previous data suggested that β7 and β5pro have overlapping roles in bringing together two half-proteasomes, but the timing of β7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind β7 incorporation into the half-mer. Our results suggest that β7 insertion precedes half-mer dimerization, and the β7 tail and β5 propeptide have unequal roles in half-mer joining.

  9. 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.

  10. Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F

    PubMed Central

    Bosen, Felicitas; Celli, Anna; Crumrine, Debra; vom Dorp, Katharina; Ebel, Philipp; Jastrow, Holger; Dörmann, Peter; Winterhager, Elke; Mauro, Theodora; Willecke, Klaus

    2016-01-01

    The keratitis–ichthyosis–deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome. PMID:26070424

  11. Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F.

    PubMed

    Bosen, Felicitas; Celli, Anna; Crumrine, Debra; vom Dorp, Katharina; Ebel, Philipp; Jastrow, Holger; Dörmann, Peter; Winterhager, Elke; Mauro, Theodora; Willecke, Klaus

    2015-07-08

    The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome.

  12. 15-Deoxy-Δ12,14-Prostaglandin J2 Modifies Components of the Proteasome and Inhibits Inflammatory Responses in Human Endothelial Cells

    PubMed Central

    Marcone, Simone; Evans, Paul; Fitzgerald, Desmond J.

    2016-01-01

    15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an electrophilic lipid mediator derived from PGD2 with potent anti-inflammatory effects. These are likely to be due to the covalent modification of cellular proteins, via a reactive α,β-unsaturated carbonyl group in its cyclopentenone ring. This study was carried out to identify novel cellular target(s) for covalent modification by 15d-PGJ2 and to investigate the anti-inflammatory effects of the prostaglandin on endothelial cells (EC). The data presented here show that 15d-PGJ2 modifies and inhibits components of the proteasome and consequently inhibits the activation of the NF-κB pathway in response to TNF-α. This, in turn, inhibits the adhesion and migration of monocytes toward activated EC, by reducing the expression of adhesion molecules and chemokines in the EC. The effects are consistent with the covalent modification of 13 proteins in the 19S particle of the proteasome identified by mass spectrometry and the suppression of proteasome function, and were similar to the effects seen with a known proteasome inhibitor (MG132). The ubiquitin–proteasome system has been implicated in the regulation of several inflammatory processes and the observation that 15d-PGJ2 profoundly affects the proteasome functions in human EC suggests that 15d-PGJ2 may regulate the progression of inflammatory disorders such as atherosclerosis. PMID:27833612

  13. 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.

  14. 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

  15. Farnesyl transferase inhibitors, autophagy, and proteasome inhibition: synergy for all the right reasons.

    PubMed

    Lonial, Sagar; Boise, Lawrence H

    2011-04-01

    The increasing appreciation of the importance of autophagy as consequence of cancer therapy or underlying disease biology is illustrated by the large number of papers that are evaluating autophagy as a cancer target. While autophagy is often linked to the generation of metabolic precursors, it is also important in diseases where protein production is a hallmark of the disease itself, such as pancreatic cancer and multiple myeloma. Multiple myeloma is characterized by ongoing autophagy as a consequence of constitutive immunoglobulin production, which creates the need for efficient transfer and disposal of misfolded or unfolded proteins. In order to survive this cellular stress, plasma cells depend on proteasomal degradation of the large volume of misfolded proteins as well as the autophagy pathway. It has previously been suggested that the excess proteins not targeted to the proteasome, or that accumulate when the proteasome is inhibited through the use of chemically active agents such as bortezomib, are linked to impaired cell survival, and that their packaging in the form of an aggresome somehow minimizes their 'proteotoxicity' allowing these toxic proteins to be sequestered away from normal cellular machinery.

  16. The genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors

    PubMed Central

    Schorn, Michelle; Zettler, Judith; Noel, Joseph P.; Dorrestein, Pieter C.; Moore, Bradley S.; Kaysser, Leonard

    2013-01-01

    The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α′,β′-epoxyketone pharmacophore allows binding to the catalytic β-subunits of the proteasome with extraordinary specificity. Here we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anti-cancer drug Kyprolis™, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid non-ribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation. PMID:24168704

  17. 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

  18. Proteasome Inhibition and Combination Therapy for Non-Hodgkin's Lymphoma: From Bench to Bedside

    PubMed Central

    Feldman, Tatyana; Goy, André

    2012-01-01

    Although patients with B-cell non-Hodgkin's lymphoma (NHL) usually respond to initial conventional chemotherapy, they often relapse and mortality has continued to increase over the last three decades in spite of salvage therapy or high dose therapy and stem cell transplantation. Outcomes vary by subtype, but there continues to be a need for novel options that can help overcome chemotherapy resistance, offer new options as consolidation or maintenance therapy postinduction, and offer potentially less toxic combinations, especially in the elderly population. The bulk of these emerging novel agents for cancer treatment target important biological cellular processes. Bortezomib is the first in the class of proteasome inhibitors (PIs), which target the critical process of intracellular protein degradation or recycling and editing through the proteasome. Bortezomib is approved for the treatment of relapsed or refractory mantle cell lymphoma. The mechanisms of proteasome inhibition are very complex by nature (because they affect many pathways) and not fully understood. However, mechanisms of action shared by bortezomib and investigational PIs such as carfilzomib, marizomib, ONX-0912, and MLN9708 are distinct from those of other NHL treatments, making them attractive options for combination therapy. Preclinical evidence suggests that the PIs have additive and/or synergistic activity with a large number of agents both in vitro and in vivo, from cytotoxics to new biologicals, supporting a growing number of combination studies currently underway in NHL patients, as reviewed in this article. The results of these studies will help our understanding about how to best integrate proteasome inhibition in the management of NHL and continue to improve patient outcomes. PMID:22566373

  19. 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

  20. The role of the ubiquitin proteasome system in cerebellar development and medulloblastoma.

    PubMed

    Vriend, Jerry; Ghavami, Saeid; Marzban, Hassan

    2015-10-17

    Cerebellar granule cells precursors are derived from the upper rhombic lip and migrate tangentially independent of glia along the subpial stream pathway to form the external germinal zone. Postnatally, granule cells migrate from the external germinal zone radially through the Purkinje cell layer, guided by Bergmann glia fibers, to the internal granular cell layer.Medulloblastomas (MBs) are the most common malignant childhood brain tumor. Many of these tumors develop from precursor cells of the embryonic rhombic lips. Four main groups of MB are recognized. The WNT group of MBs arise primarily from the lower rhombic lip and embryonic brainstem. The SHH group of MBs originate from cerebellar granule cell precursors in the external germinal zone of the embryonic cerebellum. The cellular origins of type 3 and type 4 MBs are not clear.Several ubiquitin ligases are revealed to be significant factors in development of the cerebellum as well as in the initiation and maintenance of MBs. Proteasome dysfunction at a critical stage of development may be a major factor in determining whether progenitor cells which are destined to become granule cells differentiate normally or become MB cells. We propose the hypothesis that proteasomal activity is essential to regulate the critical transition between proliferating granule cells and differentiated granule cells and that proteasome dysfunction may lead to MB. Proteasome dysfunction could also account for various mutations in MBs resulting from deficiencies in DNA checkpoint and repair mechanisms prior to development of MBs.Data showing a role for the ubiquitin ligases β-TrCP, FBW7, Huwe1, and SKP2 in MBs suggest the possibility of a classification of MBs based on the expression (over expression or under expression) of specific ubiquitin ligases which function as oncogenes, tumor suppressors or cell cycle regulators.

  1. Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs.

    PubMed

    Hancock, J M; Dover, G A

    1988-07-01

    The set of "expansion segments" of any eukaryotic 26S/28S ribosomal RNA (rRNA) gene is responsible for the bulk of the difference in length between the prokaryotic 23S rRNA gene and the eukaryotic 26S/28S rRNA gene. The expansion segments are also responsible for interspecific fluctuations in length during eukaryotic evolution. They show a consistent bias in base composition in any species; for example, they are AT rich in Drosophila melanogaster and GC rich in vertebrate species. Dot-matrix comparisons of sets of expansion segments reveal high similarities between members of a set within any 28S rRNA gene of a species, in contrast to the little or spurious similarity that exists between sets of expansion segments from distantly related species. Similarities among members of a set of expansion segments within any 28S rRNA gene cannot be accounted for by their base-compositional bias alone. In contrast, no significant similarity exists within a set of "core" segments (regions between expansion segments) of any 28S rRNA gene, although core segments are conserved between species. The set of expansion segments of a 26S/28S gene is coevolving as a unit in each species, at the same time as the family of 28S rRNA genes, as a whole, is undergoing continual homogenization, making all sets of expansion segments from all ribosomal DNA (rDNA) arrays in a species similar in sequence. Analysis of DNA simplicity of 26S/28S rRNA genes shows a direct correlation between significantly high relative simplicity factors (RSFs) and sequence similarity among a set of expansion segments. A similar correlation exists between RSF values, overall rDNA lengths, and the lengths of individual expansion segments. Such correlations suggest that most length fluctuations reflect the gain and loss of simple sequence motifs by slippage-like mechanisms. We discuss the molecular coevolution of expansion segments, which takes place against a background of slippage-like and unequal crossing

  2. Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

    PubMed Central

    Uechi, Hiroyuki; Hamazaki, Jun; Murata, Shigeo

    2014-01-01

    The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals. PMID:24668818

  3. The proteasome and epigenetics: zooming in on histone modifications.

    PubMed

    Bach, Svitlana V; Hegde, Ashok N

    2016-08-01

    The proteasome is a structural complex of many proteins that degrades substrates marked by covalent linkage to ubiquitin. Many years of research has shown a role for ubiquitin-proteasome-mediated proteolysis in synaptic plasticity and memory mainly in degrading synaptic, cytoplasmic and nuclear proteins. Recent work indicates that the proteasome has wider proteolytic and non-proteolytic roles in processes such as histone modifications that affect synaptic plasticity and memory. In this review, we assess the evidence gathered from neuronal as well as non-neuronal cell types regarding the function of the proteasome in positive or negative regulation of posttranslational modifications of histones, such as acetylation, methylation and ubiquitination. We discuss the critical roles of the proteasome in clearing excess histone proteins in various cellular contexts and the possible non-proteolytic functions in regulating transcription of target genes. In addition, we summarize the current literature on diverse chromatin-remodeling machineries, such as histone acetyltransferases, deacetylates, methyltransferases and demethylases, as targets for proteasomal degradation across experimental models. Lastly, we provide a perspective on how proteasomal regulation of histone modifications may modulate synaptic plasticity in the nervous system.

  4. Proteasome activation as a novel anti-aging strategy.

    PubMed

    Gonos, Efstathios

    2014-10-01

    Aging and longevity are two multifactorial biological phenomena whose knowledge at molecular level is still limited. We have studied proteasome function in replicative senescence and cell survival (Mol Aspects Med 35, 1-71, 2014). We have observed reduced levels of proteasome content and activities in senescent cells due to the down-regulation of the catalytic subunits of the 20S complex (J Biol Chem 278, 28026-28037, 2003). In support, partial inhibition of proteasomes in young cells by specific inhibitors induces premature senescence which is p53 dependent (Aging Cell 7, 717-732, 2008). Stable over-expression of catalytic subunits or POMP resulted in enhanced proteasome assembly and activities and increased cell survival following treatments with various oxidants. Importantly, the developed "proteasome activated" human fibroblasts cell lines exhibit a delay of senescence by approximately 15% (J Biol Chem 280, 11840-11850, 2005; J Biol Chem 284, 30076-30086, 2009). Our current work proposes that proteasome activation is an evolutionary conserved mechanism, as it can delay aging in various in vivo systems. Moreover, additional findings indicate that the recorded proteasome activation by many inducers is Nrf2-dependent (J Biol Chem 285, 8171-8184, 2010). Finally, we have studied the proteolysis processes of various age-related proteins and we have identified that CHIP is a major p53 E3 ligase in senescent fibroblasts (Free Rad Biol Med 50, 157-165, 2011).

  5. Activities of proteasome and m-calpain are essential for Chikungunya virus replication.

    PubMed

    Karpe, Yogesh A; Pingale, Kunal D; Kanade, Gayatri D

    2016-10-01

    Replication of many viruses is dependent on the ubiquitin proteasome system. The present study demonstrates that Chikungunya virus replication increases proteasome activity and induces unfolded protein response (UPR) in cultured cells. Further, it was seen that the virus replication was dependent on the activities of proteasomes and m-calpain. Proteasome inhibition induced accumulation of polyubiquitinated proteins and earlier visualization of UPR.

  6. Karyotypes, heterochromatin, and physical mapping of 18S-26S rDNA in Cactaceae.

    PubMed

    Las Peñas, M L; Urdampilleta, J D; Bernardello, G; Forni-Martins, E R

    2009-01-01

    Karyotype analyses in members of the four Cactaceae subfamilies were performed. Numbers and karyotype formula obtained were: Pereskioideae = Pereskiaaculeata(2n = 22; 10 m + 1 sm), Maihuenioideae = Maihuenia patagonica (2n = 22, 9 m + 2 sm; 2n = 44, 18 m + 4 sm), Opuntioideae = Cumulopuntia recurvata(2n = 44; 20 m + 2 sm), Cactoideae = Acanthocalycium spiniflorum (2n = 22; 10 m + 1 sm),Echinopsis tubiflora (2n = 22; 10 m + 1 sm), Trichocereus candicans (2n = 22, 22 m). Chromosomes were small, the average chromosome length was 2.3 mum. Diploid species and the tetraploid C. recurvata had one terminal satellite, whereas the remaining tetraploid species showed four satellited chromosomes. Karyotypes were symmetrical. No CMA(-)/DAPI(+) bands were detected, but CMA(+)/DAPI(-) bands associated with NOR were always found. Pericentromeric heterochromatin was found in C. recurvata, A. spiniflorum, and the tetraploid cytotype of M. patagonica. The locations of the 18S-26S rDNA sites in all species coincided with CMA(+)/DAPI(-) bands; the same occurred with the sizes and numbers of signals for each species. This technique was applied for the first time in metaphase chromosomes in cacti. NOR-bearing pair no.1 may be homeologous in all species examined. In Cactaceae, the 18S-26S loci seem to be highly conserved.

  7. Development of proteasome inhibitors as research tools and cancer drugs

    PubMed Central

    2012-01-01

    The proteasome is the primary site for protein degradation in mammalian cells, and proteasome inhibitors have been invaluable tools in clarifying its cellular functions. The anticancer agent bortezomib inhibits the major peptidase sites in the proteasome’s 20S core particle. It is a “blockbuster drug” that has led to dramatic improvements in the treatment of multiple myeloma, a cancer of plasma cells. The development of proteasome inhibitors illustrates the unpredictability, frustrations, and potential rewards of drug development but also emphasizes the dependence of medical advances on basic biological research. PMID:23148232

  8. Sent to destroy: the ubiquitin proteasome system regulates cell signaling and protein quality control in cardiovascular development and disease.

    PubMed

    Willis, Monte S; Townley-Tilson, W H Davin; Kang, Eunice Y; Homeister, Jonathon W; Patterson, Cam

    2010-02-19

    The ubiquitin proteasome system (UPS) plays a crucial role in biological processes integral to the development of the cardiovascular system and cardiovascular diseases. The UPS prototypically recognizes specific protein substrates and places polyubiquitin chains on them for subsequent destruction by the proteasome. This system is in place to degrade not only misfolded and damaged proteins, but is essential also in regulating a host of cell signaling pathways involved in proliferation, adaptation to stress, regulation of cell size, and cell death. During the development of the cardiovascular system, the UPS regulates cell signaling by modifying transcription factors, receptors, and structural proteins. Later, in the event of cardiovascular diseases as diverse as atherosclerosis, cardiac hypertrophy, and ischemia/reperfusion injury, ubiquitin ligases and the proteasome are implicated in protecting and exacerbating clinical outcomes. However, when misfolded and damaged proteins are ubiquitinated by the UPS, their destruction by the proteasome is not always possible because of their aggregated confirmations. Recent studies have discovered how these ubiquitinated misfolded proteins can be destroyed by alternative "specific" mechanisms. The cytosolic receptors p62, NBR, and histone deacetylase 6 recognize aggregated ubiquitinated proteins and target them for autophagy in the process of "selective autophagy." Even the ubiquitination of multiple proteins within whole organelles that drive the more general macro-autophagy may be due, in part, to similar ubiquitin-driven mechanisms. In summary, the crosstalk between the UPS and autophagy highlight the pivotal and diverse roles the UPS plays in maintaining protein quality control and regulating cardiovascular development and disease.

  9. It Is All about (U)biquitin: Role of Altered Ubiquitin-Proteasome System and UCHL1 in Alzheimer Disease.

    PubMed

    Tramutola, Antonella; Di Domenico, Fabio; Barone, Eugenio; Perluigi, Marzia; Butterfield, D Allan

    2016-01-01

    Free radical-mediated damage to macromolecules and the resulting oxidative modification of different cellular components are a common feature of aging, and this process becomes much more pronounced in age-associated pathologies, including Alzheimer disease (AD). In particular, proteins are particularly sensitive to oxidative stress-induced damage and these irreversible modifications lead to the alteration of protein structure and function. In order to maintain cell homeostasis, these oxidized/damaged proteins have to be removed in order to prevent their toxic accumulation. It is generally accepted that the age-related accumulation of "aberrant" proteins results from both the increased occurrence of damage and the decreased efficiency of degradative systems. One of the most important cellular proteolytic systems responsible for the removal of oxidized proteins in the cytosol and in the nucleus is the proteasomal system. Several studies have demonstrated the impairment of the proteasome in AD thus suggesting a direct link between accumulation of oxidized/misfolded proteins and reduction of this clearance system. In this review we discuss the impairment of the proteasome system as a consequence of oxidative stress and how this contributes to AD neuropathology. Further, we focus the attention on the oxidative modifications of a key component of the ubiquitin-proteasome pathway, UCHL1, which lead to the impairment of its activity.

  10. A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly

    PubMed Central

    Tomko, Robert J.; Taylor, David W.; Chen, Zhuo A.; Wang, Hong-Wei; Rappsilber, Juri; Hochstrasser, Mark

    2015-01-01

    Summary Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes. PMID:26451487

  11. A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly.

    PubMed

    Tomko, Robert J; Taylor, David W; Chen, Zhuo A; Wang, Hong-Wei; Rappsilber, Juri; Hochstrasser, Mark

    2015-10-08

    Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes.

  12. 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.

  13. Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome.

    PubMed

    Groll, Michael; Berkers, Celia R; Ploegh, Hidde L; Ovaa, Huib

    2006-03-01

    The dipeptide boronic acid bortezomib, also termed VELCADE, is a proteasome inhibitor now in use for the treatment of multiple myeloma, and its use for the treatment of other malignancies is being explored. We determined the crystal structure of the yeast 20S proteasome in complex with bortezomib to establish the specificity and binding mode of bortezomib to the proteasome's different catalytically active sites. This structure should enable the rational design of new boronic acid derivatives with improved affinities and specificities for individual active subunits.

  14. Proteasome Inhibition by Fellutamide B Induces Nerve Growth Factor Synthesis

    PubMed Central

    Hines, John; Groll, Michael; Fahnestock, Margaret; Crews, Craig M.

    2008-01-01

    SUMMARY Neurotrophic small molecules have the potential to aid in the treatment of neuronal injury and neurodegenerative diseases. The natural product fellutamide B, originally isolated from Penicillium fellutanum, potently induces nerve growth factor (NGF) release from fibroblasts and glial-derived cells, although the mechanism for this neurotrophic activity has not been elucidated. Here, we report that fellutamide B potently inhibits proteasome catalytic activity. High resolution structural information obtained from co-crystallization of the 20S proteasome reveals novel aspects regarding β-subunit binding and adduct formation by fellutamide B to inhibit their hydrolytic activity. We demonstrate that fellutamide B and other proteasome inhibitors increased NGF gene transcription via a cis-acting element (or elements) in the promoter. These results demonstrate an unrecognized connection between proteasome inhibition and NGF production, suggesting a possible new strategy in the development of neurotrophic agents. PMID:18482702

  15. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  16. Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation*

    PubMed Central

    Goitea, Victor E.; Hallak, Marta E.

    2015-01-01

    Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm. PMID:25969538

  17. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    SciTech Connect

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin–proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

  18. 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.

  19. Copper modulates the degradation of copper chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome.

    PubMed

    Bertinato, Jesse; L'Abbé, Mary R

    2003-09-12

    Copper chaperones are copper-binding proteins that directly insert copper into specific targets, preventing the accumulation of free copper ions that can be toxic to the cell. Despite considerable advances in the understanding of copper transfer from copper chaperones to their target, to date, there is no information regarding how the activity of these proteins is regulated in higher eukaryotes. The insertion of copper into the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1) depends on the copper chaperone for SOD1 (CCS). We have recently reported that CCS protein is increased in tissues of rats fed copper-deficient diets suggesting that copper may regulate CCS expression. Here we show that whereas copper deficiency increased CCS protein in rats, mRNA level was unaffected. Rodent and human cell lines cultured in the presence of the specific copper chelator 2,3,2-tetraamine displayed a dose-dependent increase in CCS protein that could be reversed with the addition of copper but not iron or zinc to the cells. Switching cells from copper-deficient to copper-rich medium promoted the rapid degradation of CCS, which could be blocked by the proteosome inhibitors MG132 and lactacystin but not a cysteine protease inhibitor or inhibitors of the lysosomal degradation pathway. In addition, CCS degradation was slower in copper-deficient cells than in cells cultured in copper-rich medium. Together, these data show that copper regulates CCS expression by modulating its degradation by the 26 S proteosome and suggest a novel role for CCS in prioritizing the utilization of copper when it is scarce.

  20. Reconfiguration of the proteasome during chaperone-mediated assembly.

    PubMed

    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

    2013-05-23

    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 carboxy-terminal tails inserting into pockets of the α-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae 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 non-hydrolysable ATP analogue, 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 proteasomes, 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.

  1. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    PubMed Central

    Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

    2012-01-01

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

  2. 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

  3. On the linkage between the ubiquitin-proteasome system and the mitochondria.

    PubMed

    Lehmann, Gilad; Udasin, Ronald G; Ciechanover, Aaron

    2016-04-22

    Several metabolic pathways critical for cellular homeostasis occur in the mitochondria. Because of the evolution of mitochondria and their physical separation, these pathways have traditionally been thought to be free from regulation by the ubiquitin-proteasome system. This perception has recently been challenged by evidence for the presence of ubiquitin system components in the mitochondria. Furthermore, it has been shown that certain mitochondrial proteins are conjugated by ubiquitin, and some of them are degraded by the proteasome. Of particular interest is the finding that some of these proteins are localized to the inner membrane and matrix, which rules out that their targeting is mediated by the cytosolic ubiquitin system. However, the extent of the involvement of the ubiquitin system in mitochondrial regulation is not known. The present study addresses this surprising finding, employing several independent approaches. First, we identified reported ubiquitin conjugates in human and yeast mitochondria and found that a large fraction of the mitochondrial proteome (62% in human) is ubiquitinated, with most proteins localized to the inner membrane and matrix. Next, we searched the literature and found that numerous ubiquitin system components localize to the mitochondria and/or contain mitochondrial targeting sequences. Finally, we identified reported protein-protein interactions between ubiquitin system components and mitochondrial proteins. These unexpected findings suggest that mitochondrial regulation by the ubiquitin system is fundamental and may have broad biomedical implications.

  4. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  5. Skeletal muscle myotubes of the severely obese exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux

    PubMed Central

    Bollinger, Lance M.; Powell, Jonathan J. S.; Houmard, Joseph A.; Witczak, Carol A.; Brault, Jeffrey J.

    2015-01-01

    Objective Whole-body protein metabolism is dysregulated with obesity. Our goal was to determine if activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. Methods We utilized primary Human Skeletal Muscle cell (HSkM) cultures since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean (BMI ≤ 26.0 kg/m2) and 8 severely obese (BMI ≥ 39.0) women were examined basally and when stimulated to atrophy (serum and amino acid starvation). Results HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in the lean and an increase in HSkM from obese subjects. HSkMC from the obese also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from lean and obese individuals under basal and starved conditions. Conclusions Our data indicate that muscle cells of the lean and severely obese have innate differences in management of protein degradation, which may explain their metabolic differences. PMID:26010327

  6. 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.

  7. Drug Synergism of Proteasome Inhibitors and Mitotane by Complementary Activation of ER Stress in Adrenocortical Carcinoma Cells.

    PubMed

    Kroiss, Matthias; Sbiera, Silviu; Kendl, Sabine; Kurlbaum, Max; Fassnacht, Martin

    2016-12-01

    Mitotane is the only drug approved for treatment of the orphan disease adrenocortical carcinoma (ACC) and was recently shown to be the first clinically used drug acting through endoplasmic reticulum (ER)-stress induced by toxic lipids. Since mitotane has limited clinical activity as monotherapy, we here study the potential of activating ER-stress through alternative pathways. The single reliable NCI-H295 cell culture model for ACC was used to study the impact MG132, bortezomib (BTZ) and carfilzomib (CFZ) on mRNA and protein expression of ER-stress markers, cell viability and steroid hormone secretion. We found all proteasome inhibitors alone to trigger expression of mRNA (spliced X-box protein 1, XBP1) and protein markers indicative of the inositol-requiring enzyme 1 (IRE1) dependent pathway of ER-stress but not phosphorylation of eukaryotic initiation factor 2α (eIF2α), a marker of the PRKR-like endoplasmic reticulum kinase (PERK)-dependent pathway. Whereas mitotane alone activated both pathways, combination of BTZ and CFZ with low-dose mitotane blocked mitotane-induced eIF2α phosphorylation but increased XBP1-mRNA splicing indicating that proteasome inhibitors can commit signalling towards a single ER-stress pathway in ACC cells. By applying the median effect model of drug combinations using cell viability as a read out, we determined significant drug synergism between mitotane and both BTZ and CFZ. In conclusion, combination of mitotane with activators of ER-stress through the unfolded protein response is synergistic in an ACC cell culture model. Since proteasome inhibitors are readily available clinically, they are attractive candidates to study for ACC treatment in clinical trials in combination with mitotane.

  8. Proteomic remodeling of proteasome in right heart failure.

    PubMed

    Fessart, Delphine; Martin-Negrier, Marie-Laure; Claverol, Stéphane; Thiolat, Marie-Laure; Crevel, Huguette; Toussaint, Christian; Bonneu, Marc; Muller, Bernard; Savineau, Jean-Pierre; Delom, Frederic

    2014-01-01

    The development of right heart failure (RHF) is characterized by alterations of right ventricle (RV) structure and function, but the mechanisms of RHF remain still unknown. Thus, understanding the RHF is essential for improved therapies. Therefore, identification by quantitative proteomics of targets specific to RHF may have therapeutic benefits to identify novel potential therapeutic targets. The objective of this study was to analyze the molecular mechanisms changing RV function in the diseased RHF and thus, to identify novel potential therapeutic targets. For this, we have performed differential proteomic analysis of whole RV proteins using two experimental rat models of RHF. Differential protein expression was observed for hundred twenty six RV proteins including proteins involved in structural constituent of cytoskeleton, motor activity, structural molecule activity, cytoskeleton protein binding and microtubule binding. Interestingly, further analysis of down-regulated proteins, reveals that both protein and gene expressions of proteasome subunits were drastically decreased in RHF, which was accompanied by an increase of ubiquitinated proteins. Interestingly, the proteasomal activities chymotrypsin and caspase-like were decreased whereas trypsin-like activity was maintained. In conclusion, this study revealed the involvement of ubiquitin-proteasome system (UPS) in RHF. Three deregulated mechanisms were discovered: (1) decreased gene and protein expressions of proteasome subunits, (2) decreased specific activity of proteasome; and (3) a specific accumulation of ubiquitinated proteins. This modulation of UPS of RV may provide a novel therapeutic avenue for restoration of cardiac function in the diseased RHF.

  9. Targeting proteasome ubiquitin receptor Rpn13 in multiple myeloma.

    PubMed

    Song, Y; Ray, A; Li, S; Das, D S; Tai, Y T; Carrasco, R D; Chauhan, D; Anderson, K C

    2016-09-01

    Proteasome inhibitor bortezomib is an effective therapy for relapsed and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance can limit its long-term utility. Recent research has focused on targeting ubiquitin receptors upstream of 20S proteasome, with the aim of generating less toxic therapies. Here we show that 19S proteasome-associated ubiquitin receptor Rpn13 is more highly expressed in MM cells than in normal plasma cells. Rpn13-siRNA (small interfering RNA) decreases MM cell viability. A novel agent RA190 targets Rpn13 and inhibits proteasome function, without blocking the proteasome activity or the 19S deubiquitylating activity. CRISPR/Cas9 Rpn13-knockout demonstrates that RA190-induced activity is dependent on Rpn13. RA190 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma and overcomes bortezomib resistance. Anti-MM activity of RA190 is associated with induction of caspase-dependent apoptosis and unfolded protein response-related apoptosis. MM xenograft model studies show that RA190 is well tolerated, inhibits tumor growth and prolongs survival. Combining RA190 with bortezomib, lenalidomide or pomalidomide induces synergistic anti-MM activity. Our preclinical data validates targeting Rpn13 to overcome bortezomib resistance, and provides the framework for clinical evaluation of Rpn13 inhibitors, alone or in combination, to improve patient outcome in MM.

  10. Clioquinol - a novel copper-dependent and independent proteasome inhibitor.

    PubMed

    Schimmer, A D

    2011-03-01

    Clioquinol (5-chloro-7-iodo-quinolin-8-ol) was used in the 1950's-1970's as an oral anti-parasitic agent. More recently, studies have demonstrated that Clioquinol displays preclinical efficacy in the treatment of malignancy. Its anti-cancer activity relates, at least in part, to its ability to inhibit the proteasome through mechanisms dependent and independent of its ability to bind heavy metals such as copper. By acting as a metal ionophore Clioquinol transports metal ions from the extracellular environment into the cell and mobilizes weakly bound intracellular stores. It then directs the metal to the proteasome resulting in disruption of this enzymatic complex. In addition, Clioquinol is capable of directly inhibiting the proteasome at higher concentrations. Thus, Clioquinol represents a novel therapeutic strategy to inhibit the proteasome. Given the prior toxicology and pharmacology studies, Clioquinol could be rapidly repositioned for a new anti-cancer indication. This review highlights the mechanism of action of Clioquinol as a proteasome inhibitor. In addition, it discusses the human pharmacology and toxicology studies and how this information would guide a phase I clinical trial of this agent for patients with malignancy.

  11. Proteasome inhibition alleviates prolonged moderate compression-induced muscle pathology

    PubMed Central

    2011-01-01

    Background The molecular mechanism initiating deep pressure ulcer remains to be elucidated. The present study tested the hypothesis that the ubiquitin proteasome system is involved in the signalling mechanism in pressure-induced deep tissue injury. Methods Adult Sprague Dawley rats were subjected to an experimental compression model to induce deep tissue injury. The tibialis region of the right hind limb was subjected to 100 mmHg of static pressure for six hours on each of two consecutive days. The compression pressure was continuously monitored by a three-axial force transducer within the compression indentor. The left hind limb served as the intra-animal control. Muscle tissues underneath the compressed region were collected and used for analyses. Results Our results demonstrated that the activity of 20S proteasome and the protein abundance of ubiquitin and MAFbx/atrogin-1 were elevated in conjunction with pathohistological changes in the compressed muscle, as compared to control muscle. The administration of the proteasome inhibitor MG132 was found to be effective in ameliorating the development of pathological histology in compressed muscle. Furthermore, 20S proteasome activity and protein content of ubiquitin and MAFbx/atrogin-1 showed no apparent increase in the MG132-treated muscle following compression. Conclusion Our data suggest that the ubiquitin proteasome system may play a role in the pathogenesis of pressure-induced deep tissue injury. PMID:21385343

  12. Inhibition of histone deacetylases by chlamydocin induces apoptosis and proteasome-mediated degradation of survivin.

    PubMed

    De Schepper, Stefanie; Bruwiere, Hélène; Verhulst, Tinne; Steller, Ulf; Andries, Luc; Wouters, Walter; Janicot, Michel; Arts, Janine; Van Heusden, Jim

    2003-02-01

    The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC(50) of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase-dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

  13. Proteasomal Processing of Albumin by Renal Dendritic Cells Generates Antigenic Peptides

    PubMed Central

    Macconi, Daniela; Chiabrando, Chiara; Schiarea, Silvia; Aiello, Sistiana; Cassis, Linda; Gagliardini, Elena; Noris, Marina; Buelli, Simona; Zoja, Carla; Corna, Daniela; Mele, Caterina; Fanelli, Roberto; Remuzzi, Giuseppe; Benigni, Ariela

    2009-01-01

    The role of dendritic cells (DC) that accumulate in the renal parenchyma of non–immune-mediated proteinuric nephropathies is not well understood. Under certain circumstances, DC capture immunologically ignored antigens, including self-antigens, and present them within MHC class I, initiating an autoimmune response. We studied whether DC could generate antigenic peptides from the self-protein albumin. Exposure of rat proximal tubular cells to autologous albumin resulted in its proteolytic cleavage to form an N-terminal 24–amino acid peptide (ALB1-24). This peptide was further processed by the DC proteasome into antigenic peptides that had binding motifs for MHC class I and were capable of activating syngeneic CD8+ T cells. In vivo, the rat five-sixths nephrectomy model allowed the localization and activation of renal DC. Accumulation of DC in the renal parenchyma peaked 1 wk after surgery and decreased at 4 wk, concomitant with their appearance in the renal draining lymph nodes. DC from renal lymph nodes, loaded with ALB1-24, activated syngeneic CD8+ T cells in primary culture. The response of CD8+ T cells of five-sixths nephrectomized rats was amplified with secondary stimulation. In contrast, DC from renal lymph nodes of five-sixths nephrectomized rats treated with the proteasomal inhibitor bortezomib lost their capacity to stimulate CD8+ T cells in primary and secondary cultures. These data suggest that albumin can be a source of potentially antigenic peptides upon renal injury and that renal DC play a role in processing self-proteins through a proteasome-dependent pathway. PMID:19092126

  14. Gene therapy by proteasome activator, PA28γ, improves motor coordination and proteasome function in Huntington's disease YAC128 mice.

    PubMed

    Jeon, J; Kim, W; Jang, J; Isacson, O; Seo, H

    2016-06-02

    Huntington's disease (HD) is neurologically characterized by involuntary movements, associated with degeneration of the medium-sized spiny neurons (MSNs) and ubiquitin-positive neuronal intranuclear inclusions (NIIs). It has been reported that the proteolytic activities of the ubiquitin-proteasome system (UPS) are generally inhibited in HD patient's brain. We previously discovered that a proteasome activator (PA), PA28γ enhances proteasome activities and cell survival in in vitro HD model. In this study, we aimed to find whether PA28γ gene transfer improves the proteasome activities and pathological symptoms in in vivo HD model. We stereotaxically injected lenti-PA28γ virus into the striatum of mutant (MT) YAC128 HD mice and littermate (LM) controls at 14-18months of age, and validated their behavioral and biochemical changes at 12weeks after the injection. YAC128 mice showed a significant increase in their peptidyl-glutamyl preferring hydrolytic (PGPH) proteasome activity and the mRNA or protein levels of brain-derived neurotrophic factor (BDNF) and pro-BDNF after lenti-PA28γ injection. The number of ubiquitin-positive inclusion bodies was reduced in the striatum of YAC128 mice after lenti-PA28γ injection. YAC128 mice showed significant improvement of latency to fall on the rota-rod test after lenti-PA28γ injection. These data demonstrate that the gene therapy with PA, PA28γ can improve UPS function as well as behavioral abnormalities in HD model mice.

  15. Bacterial self-resistance to the natural proteasome inhibitor salinosporamide A

    PubMed Central

    Kale, Andrew J.; McGlinchey, Ryan P.; Lechner, Anna; Moore, Bradley S.

    2011-01-01

    Proteasome inhibitors have recently emerged as a therapeutic strategy in cancer chemotherapy but susceptibility to drug resistance limits their efficacy. The marine actinobacterium Salinispora tropica produces salinosporamide A (NPI-0052, marizomib), a potent proteasome inhibitor and promising clinical agent in the treatment of multiple myeloma. Actinobacteria also possess 20S proteasome machinery, raising the question of self-resistance. We identified a redundant proteasome β-subunit, SalI, encoded within the salinosporamide biosynthetic gene cluster and biochemically characterized the SalI proteasome complex. The SalI β-subunit has an altered substrate specificity profile, 30-fold resistance to salinosporamide A, and cross-resistance to the FDA-approved proteasome inhibitor bortezomib. An A49V mutation in SalI correlates to clinical bortezomib resistance from a human proteasome β 5-subunit A49T mutation, suggesting that intrinsic resistance to natural proteasome inhibitors may predict clinical outcomes. PMID:21882868

  16. Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

    PubMed

    Gastel, J A; Roseboom, P H; Rinaldi, P A; Weller, J L; Klein, D C

    1998-02-27

    The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

  17. 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.

  18. Progressively impaired proteasomal capacity during terminal plasma cell differentiation

    PubMed Central

    Cenci, Simone; Mezghrani, Alexandre; Cascio, Paolo; Bianchi, Giada; Cerruti, Fulvia; Fra, Anna; Lelouard, Hugues; Masciarelli, Silvia; Mattioli, Laura; Oliva, Laura; Orsi, Andrea; Pasqualetto, Elena; Pierre, Philippe; Ruffato, Elena; Tagliavacca, Luigina; Sitia, Roberto

    2006-01-01

    After few days of intense immunoglobulin (Ig) secretion, most plasma cells undergo apoptosis, thus ending the humoral immune response. We asked whether intrinsic factors link plasma cell lifespan to Ig secretion. Here we show that in the late phases of plasmacytic differentiation, when antibody production becomes maximal, proteasomal activity decreases. The excessive load for the reduced proteolytic capacity correlates with accumulation of polyubiquitinated proteins, stabilization of endogenous proteasomal substrates (including Xbp1s, IκBα, and Bax), onset of apoptosis, and sensitization to proteasome inhibitors (PI). These events can be reproduced by expressing Ig-μ chain in nonlymphoid cells. Our results suggest that a developmental program links plasma cell death to protein production, and help explaining the peculiar sensitivity of normal and malignant plasma cells to PI. PMID:16498407

  19. Proteasomal degradation of beta-carotene metabolite--modified proteins.

    PubMed

    Sommerburg, Olaf; Karius, Nicole; Siems, Werner; Langhans, Claus-Dieter; Leichsenring, Michael; Breusing, Nicolle; Grune, Tilman

    2009-01-01

    Free radical attack on beta-carotene results in the formation of high amounts of carotene breakdown products (CBPs) having biological activities. As several of the CBPs are reactive aldehydes, it has to be considered that these compounds are able to modify proteins. Therefore, the aim of the study was to investigate whether CBP-modification of proteins is leading to damaged proteins recognized and degraded by the proteasomal system. We used the model proteins tau and ferritin to test whether CBPs will modify them and whether such modifications lead to enhanced proteasomal degradation. To modify proteins, we used crude CBPs as a mixture obtained after hypochloric acid derived BC degradation, as well as several single compounds, as apo8'-carotenal, retinal, or beta-ionone. The majority of the CBPs found in our reaction mixture are well known metabolites as described earlier after BC degradation using different oxidants. CBPs are able to modify proteins, and in in vitro studies, we were able to demonstrate that the 20S proteasome is able to recognize and degrade CBP-modified proteins preferentially. In testing the proteolytic response of HT22 cells toward CBPs, we could demonstrate an enhanced protein turnover, which is sensitive to lactacystin. Interestingly, the proteasomal activity is resistant to treatment with CBP. On the other hand, we were able to demonstrate that supraphysiological levels of CBPs might lead to the formation of protein-CBP-adducts that are able to inhibit the proteasome. Therefore, the removal of CBP-modified proteins seems to be catalyzed by the proteasomal system and is effective, if the formation of CBPs is not overwhelming and leading to protein aggregates.

  20. Proteasome-mediated degradation of the C-terminus of the Alzheimer's disease beta-amyloid protein precursor: effect of C-terminal truncation on production of beta-amyloid protein.

    PubMed

    Nunan, Janelle; Williamson, Nicholas A; Hill, Andrew F; Sernee, M Fleur; Masters, Colin L; Small, David H

    2003-11-01

    The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. Our previous studies have shown that the proteasome can cleave the C-terminal cytoplasmic domain of APP. To identify proteasome cleavage sites in APP, two peptides homologous to the C-terminus of APP were incubated with recombinant 20S proteasome. Cleavage of the peptides was monitored by reversed phase high-performance liquid chromatography and mass spectrometry. Proteasome cleaved the APP C-terminal peptides at several sites, including a region around the sequence YENPTY that interacts with several APP-binding proteins. To examine the effect of this cleavage on Abeta production, APP-CTFbeta and mutant forms of APP-CTFbeta terminating on either side of the YENPTY sequence were expressed in CHO cells. Truncation of APP-CTFbeta on the N-terminal side of the YENPTY sequence at residue 677 significantly decreased the amount of Abeta produced, whereas truncation on the C-terminal side of residue 690 had little effect. The results suggest that proteasomal cleavage of the cytosolic domain of APP at the YENPTY sequence decreases gamma-secretase processing, and consequently inhibits Abeta production. Therefore, the proteasome-dependent trafficking pathway of APP may be a valid therapeutic target for altering Abeta production in the Alzheimer's disease brain.

  1. Proteasome Activity Profiling Uncovers Alteration of Catalytic β2 and β5 Subunits of the Stress-Induced Proteasome during Salinity Stress in Tomato Roots

    PubMed Central

    Kovács, Judit; Poór, Péter; Kaschani, Farnusch; Chandrasekar, Balakumaran; Hong, Tram N.; Misas-Villamil, Johana C.; Xin, Bo T.; Kaiser, Markus; Overkleeft, Herman S.; Tari, Irma; van der Hoorn, Renier A. L.

    2017-01-01

    The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic β subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic β subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each β subunit except for two genes encoding β2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled β2 and β5 subunits, and an additional acidic pI shift for labeled β5, whilst labeled β1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress. PMID:28217134

  2. Proteasome Activity Profiling Uncovers Alteration of Catalytic β2 and β5 Subunits of the Stress-Induced Proteasome during Salinity Stress in Tomato Roots.

    PubMed

    Kovács, Judit; Poór, Péter; Kaschani, Farnusch; Chandrasekar, Balakumaran; Hong, Tram N; Misas-Villamil, Johana C; Xin, Bo T; Kaiser, Markus; Overkleeft, Herman S; Tari, Irma; van der Hoorn, Renier A L

    2017-01-01

    The stress proteasome in the animal kingdom facilitates faster conversion of oxidized proteins during stress conditions by incorporating different catalytic β subunits. Plants deal with similar kind of stresses and also carry multiple paralogous genes encoding for each of the three catalytic β subunits. Here, we investigated the existence of stress proteasomes upon abiotic stress (salt stress) in tomato roots. In contrast to Arabidopsis thaliana, tomato has a simplified proteasome gene set with single genes encoding each β subunit except for two genes encoding β2. Using proteasome activity profiling on tomato roots during salt stress, we discovered a transient modification of the catalytic subunits of the proteasome coinciding with a loss of cell viability. This stress-induced active proteasome disappears at later time points and coincides with the need to degrade oxidized proteins during salt stress. Subunit-selective proteasome probes and MS analysis of fluorescent 2D gels demonstrated that the detected stress-induced proteasome is not caused by an altered composition of subunits in active proteasomes, but involves an increased molecular weight of both labeled β2 and β5 subunits, and an additional acidic pI shift for labeled β5, whilst labeled β1 remains mostly unchanged. Treatment with phosphatase or glycosidases did not affect the migration pattern. This stress-induced proteasome may play an important role in PCD during abiotic stress.

  3. Poxvirus Exploitation of the Ubiquitin-Proteasome System

    PubMed Central

    Barry, Michele; van Buuren, Nicholas; Burles, Kristin; Mottet, Kelly; Wang, Qian; Teale, Alastair

    2010-01-01

    Ubiquitination plays a critical role in many cellular processes. A growing number of viruses have evolved strategies to exploit the ubiquitin-proteasome system, including members of the Poxviridae family. Members of the poxvirus family have recently been shown to encode BTB/kelch and ankyrin/F-box proteins that interact with cullin-3 and cullin-1 based ubiquitin ligases, respectively. Multiple members of the poxvirus family also encode ubiquitin ligases with intrinsic activity. This review describes the numerous mechanisms that poxviruses employ to manipulate the ubiquitin-proteasome system. PMID:21994622

  4. 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

  5. Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

    PubMed

    Owen, Jeremy G; Charlop-Powers, Zachary; Smith, Alexandra G; Ternei, Melinda A; Calle, Paula Y; Reddy, Boojala Vijay B; Montiel, Daniel; Brady, Sean F

    2015-04-07

    In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not