WorldWideScience

Sample records for hes6 mediates proteasomal

  1. HES6 enhances the motility of alveolar rhabdomyosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wickramasinghe, Caroline M [MRC Cancer Cell Unit, Hutchison-MRC Research centre, Addenbrooke' s Hospital Cambridge, CB2 0XZ (United Kingdom); MRC Laboratory of Molecular Biology, Addenbrooke' s Hospital Cambridge, CB2 0QH (United Kingdom); Domaschenz, Renae [MRC Cancer Cell Unit, Hutchison-MRC Research centre, Addenbrooke' s Hospital Cambridge, CB2 0XZ (United Kingdom); Gene Regulation and Chromatin Group, MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London W12 ONN (United Kingdom); Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research centre, Addenbrooke' s Hospital Cambridge, CB2 0XZ (United Kingdom); Department of Pathophysiology, Faculty of Pharmaceutical Sciences, Doshisha Women' s College of Liberal Arts, Kodo, Kyotanabe, Kyoto 610-0395 (Japan); Williamson, Daniel [Molecular Cytogenetics, The Institute of Cancer Research, Sutton SM2 5NG (United Kingdom); Northern Institute for Cancer Research, Paul O' Gorman Building, Medical School, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH (United Kingdom); Missiaglia, Edoardo; Shipley, Janet [Molecular Cytogenetics, The Institute of Cancer Research, Sutton SM2 5NG (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research centre, Addenbrooke' s Hospital Cambridge, CB2 0XZ (United Kingdom); Jones, Philip H, E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research centre, Addenbrooke' s Hospital Cambridge, CB2 0XZ (United Kingdom)

    2013-01-01

    Absract: HES6, a member of the hairy-enhancer-of-split family of transcription factors, plays multiple roles in myogenesis. It is a direct target of the myogenic transcription factor MyoD and has been shown to regulate the formation of the myotome in development, myoblast cell cycle exit and the organization of the actin cytoskeleton during terminal differentiation. Here we investigate the expression and function of HES6 in rhabdomyosarcoma, a soft tissue tumor which expresses myogenic genes but fails to differentiate into muscle. We show that HES6 is expressed at high levels in the subset of alveolar rhabdomyosarcomas expressing PAX/FOXO1 fusion genes (ARMSp). Knockdown of HES6 mRNA in the ARMSp cell line RH30 reduces proliferation and cell motility. This phenotype is rescued by expression of mouse Hes6 which is insensitive to HES6 siRNA. Furthermore, expression microarray analysis indicates that the HES6 knockdown is associated with a decrease in the levels of Transgelin, (TAGLN), a regulator of the actin cytoskeleton. Knockdown of TAGLN decreases cell motility, whilst TAGLN overexpression rescues the motility defect resulting from HES6 knockdown. These findings indicate HES6 contributes to the pathogenesis of ARMSp by enhancing both proliferation and cell motility.

  2. Reconfiguration of the proteasome during chaperone-mediated assembly

    OpenAIRE

    Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A.; Lovell, Scott; Battaile, Kevin P.; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, Daniel

    2013-01-01

    The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric ? ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the ? ring 1?4 . Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit 5?10 . We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by th...

  3. Hes6 is required for actin cytoskeletal organization in differentiating C2C12 myoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Malone, Caroline M.P.; Domaschenz, Renae; Amagase, Yoko [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Dunham, Ian [EMBL-European Bioinformatics Institute (EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD (United Kingdom); Murai, Kasumi [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom); Jones, Philip H., E-mail: phj20@cam.ac.uk [MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Addenbrooke' s Hospital, Cambridge CB2 0XZ (United Kingdom)

    2011-07-01

    Hes6 is a member of the hairy-enhancer-of-split family of transcription factors that regulate proliferating cell fate in development and is known to be expressed in developing muscle. Here we investigate its function in myogenesis in vitro. We show that Hes6 is a direct transcriptional target of the myogenic transcription factors MyoD and Myf5, indicating that it is integral to the myogenic transcriptional program. The localization of Hes6 protein changes during differentiation, becoming predominantly nuclear. Knockdown of Hes6 mRNA levels by siRNA has no effect on cell cycle exit or induction of myosin heavy chain expression in differentiating C2C12 myoblasts, but F-actin filament formation is disrupted and both cell motility and myoblast fusion are reduced. The knockdown phenotype is rescued by expression of Hes6 cDNA resistant to siRNA. These results define a novel role for Hes6 in actin cytoskeletal dynamics in post mitotic myoblasts.

  4. Amyloid-β precursor protein facilitates the regulator of calcineurin 1-mediated apoptosis by downregulating proteasome subunit α type-5 and proteasome subunit β type-7.

    Science.gov (United States)

    Wu, Yili; Deng, Yu; Zhang, Shuting; Luo, Yawen; Cai, Fang; Zhang, Zhuohua; Zhou, Weihui; Li, Tingyu; Song, Weihong

    2015-01-01

    Individuals with Down syndrome (DS), caused by trisomy of chromosome 21, inevitably develop characteristic Alzheimer's disease (AD) neuropathology, including neuritic plaques, neurofibrillary tangles, and neuronal loss. Amyloid-β protein, the major component of neuritic plaques, is the proteolytic product of amyloid-β precursor protein (APP). APP and the regulator of calcineurin 1 (RCAN1) genes on chromosome 21 play a pivotal role in promoting plaque formation and neuronal apoptosis. However, the mechanism underlying AD pathogenesis in DS is not well defined. In this study, we demonstrated that APP significantly increased RCAN1 level in both cells and transgenic mice. Overexpression of APP significantly reduced the expression of 2 proteasome subunits, proteasome subunit α type-5 and proteasome subunit β type-7, leading to the inhibition of proteasomal degradation of RCAN1. Furthermore, knockdown of RCAN1 expression attenuated APP-induced neuronal apoptosis. Taken together, the results clearly showed that APP has a previously unknown function in regulating RCAN1-mediated neuronal apoptosis through the proteasome pathway. Our study demonstrates a novel mechanism by which overexpression of APP and RCAN1 causes neurodegeneration and AD pathogenesis in DS, and it provides new insights into the potential of targeting APP-induced proteasomal impairment and RCAN1 accumulation for AD and DS treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

    Directory of Open Access Journals (Sweden)

    Sayem Miah

    Full Text Available Breast tumor kinase (BRK, also known as protein tyrosine kinase 6 (PTK6, is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

  6. Degradation of regulator of calcineurin 1 (RCAN1) is mediated by both chaperone-mediated autophagy and ubiquitin proteasome pathways.

    Science.gov (United States)

    Liu, Heng; Wang, Pin; Song, Weihong; Sun, Xiulian

    2009-10-01

    Regulator of calcineurin 1 (RCAN1), a gene identified from the critical region of Down syndrome, has been implied in pathogenesis of Alzheimer's disease (AD). RCAN1 expression was shown to be increased in AD brains; however, the mechanism of RCAN1 gene regulation is not well defined. The present study was designed to investigate the molecular mechanism of RCAN1 protein degradation. In addition to being degraded through the ubiquitin proteasome pathway, we found that lysosomal inhibition markedly increased RCAN1 protein expression in a time- and dosage-dependent manner. Inhibition of macroautophagy reduced RCAN1 expression, indicating that RCAN1 degradation is not through a macroautophagy pathway. However, disruption of chaperone-mediated autophagy (CMA) increased RCAN1 expression. Two CMA recognition motifs were identified in RCAN1 protein to mediate its degradation through a CMA-lysosome pathway. A promoter assay further demonstrated that inhibition of RCAN1 degradation in cells reduced calcineurin-NFAT activity. Dysfunctions of ubiquitin-proteasome and autophagy-lysosome pathways have been implicated in neurodegenerative diseases. Therefore, elucidation of RCAN1 degradation by a ubiquitin proteasome pathway and CMA-lysosome pathway in the present study may greatly advance our understanding of AD pathogenesis.

  7. Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels.

    Science.gov (United States)

    Hsu, Po-Hao; Ma, Yu-Ting; Fang, Ya-Ching; Huang, Jing-Jia; Gan, Yu-Ling; Chang, Pei-Tzu; Jow, Guey-Mei; Tang, Chih-Yung; Jeng, Chung-Jiuan

    2017-01-18

    Mammalian Eag1 (Kv10.1) potassium (K+) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K+ channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels.

  8. Lung perfusion in hemorrhagic shock of rats. The effects of resuscitation with whole blood, saline or hes 6%

    Energy Technology Data Exchange (ETDEWEB)

    Turhanoglu, S.; Kaya, S.; Kararmaz, A.; Turhanoglu, A.D. [Dicle Univ., Diyarbakir (Turkey). Medical School

    2001-12-01

    This study was undertaken to determine the effects of various resuscitation regimens on lung perfusion following resuscitation from hemorrhagic shock. Fourty male Sprague-Dawley rats (250-300 g) were used. The rats were divided randomly into four groups (n=10 for each) and were sedated with intramuscular ketamine (100 mg/kg). We measured blood pressure, rectal temperature and lung perfusion using radioscintigraphy with a technetium colloid indicator. The systolic blood pressure was decreased 75% by removing blood via v. jugularis in the first three groups and group 4 was accepted as the control group, and blood volume was not diminished. Then the first three groups were resuscitated with autologous blood containing 125 units heparine/ml in group 1, saline in group 2, and hydroxyethyl starch (HES) 6% in group 3. After the correction of hypovolemia, all animals were injected 100 Bg (0.1 cc) technetium 99m macroaggregated albumin ({sup 99m}Tc MAA) via penil vein. After injection of {sup 99m}Tc MAA, 3 minutes fixed images were detected by a {gamma} camera in posterior position at 15 minutes and 5 hours. {sup 99m}Tc MMA ''wash out'' rate in lung was determined quantitatively at 5 hours. Compared to a control group, lung perfusion was decreased significantly in groups resuscitated with saline, and HES 6% while perfusion was restored with autologous blood. We conclude that heparinized autologous blood saved lung capillary circulation in hemorrhagic shock in rats. (author)

  9. The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Dijk, Marianne van; Murphy, Eoin [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); Morrell, Ruth [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Knapper, Steven [Department of Haematology, School of Medicine, Cardiff University, Heath Park, CF14 4XN Cardiff (United Kingdom); O' Dwyer, Michael [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Medicine, National University of Ireland, University Road, Galway (Ireland); Samali, Afshin; Szegezdi, Eva, E-mail: eva.szegezdi@nuigalway.ie [Apoptosis Research Center, National University of Ireland, University Road, Galway (Ireland); School of Natural Sciences, National University of Ireland, University Road, Galway (Ireland)

    2011-03-15

    Acute myeloid leukemia (AML) is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification), which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

  10. The Proteasome Inhibitor Bortezomib Sensitizes AML with Myelomonocytic Differentiation to TRAIL Mediated Apoptosis

    Directory of Open Access Journals (Sweden)

    Eva Szegezdi

    2011-03-01

    Full Text Available Acute myeloid leukemia (AML is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification, which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.

  11. Urokinase receptor mediates doxorubicin-induced vascular smooth muscle cell senescence via proteasomal degradation of TRF2.

    Science.gov (United States)

    Hodjat, Mahshid; Haller, Hermann; Dumler, Inna; Kiyan, Yulia

    2013-01-01

    The anthracycline doxorubicin is a widely used effective anti-cancer drug. However, its application and dosage are severely limited due to its cardiotoxicity. The exact mechanisms of doxorubicin-induced cardiotoxic side effects remain poorly understood. Even less is known about the impact of doxorubicin treatment on vascular damage. We found that low doses of doxorubicin induced a senescent response in human primary vascular smooth muscle cells (VSMC). We observed that expression of urokinase receptor (uPAR) was upregulated in response to doxorubicin. Furthermore, the level of uPAR expression played a decisive role in developing doxorubicin-induced senescence. uPAR silencing in human VSMC by means of RNA interference as well as uPAR knockout in mouse VSMC resulted in abrogation of doxorubicin-induced cellular senescence. On the contrary, uPAR overexpression promoted VSMC senescence. We further found that proteasomal degradation of telomeric repeat binding factor 2 (TRF2) mediates doxorubicin-induced VSMC senescence. Our results demonstrate that uPAR controls the ubiquitin-proteasome system in VSMC and regulates doxorubicin-induced TRF2 ubiquitination and proteasomal degradation via this mechanism. Therefore, VSMC senescence induced by low doses of doxorubicin may contribute to vascular damage upon doxorubicin treatment. uPAR-mediated TRF2 ubiquitination and proteasomal degradation are further identified as a molecular mechanism underlying this process. Copyright © 2012 S. Karger AG, Basel.

  12. A novel role for ATM in regulating proteasome-mediated protein degradation through suppression of the ISG15 conjugation pathway.

    Directory of Open Access Journals (Sweden)

    Laurence M Wood

    2011-01-01

    Full Text Available Ataxia Telangiectasia (A-T is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.

  13. HIV protease-mediated activation of sterically capped proteasome inhibitors and substrates.

    Science.gov (United States)

    Buckley, Dennis L; Corson, Timothy W; Aberle, Nicholas; Crews, Craig M

    2011-02-02

    Strategies for selectively killing HIV-infected cells present an appealing alternative to traditional antiretroviral drugs. We show here the first example of an inactive “Trojan horse” molecule that releases a cytotoxic, small-molecule proteasome inhibitor upon cleavage by HIV-1 protease. As a proof-of-concept strategy, the protein avidin was used to block entry of the compound into the proteasome in the absence of HIV-1 protease. We demonstrate that this strategy is also feasible without requiring an exogenous protein; a polylysine dendrimer-containing molecule is unable to enter the proteasome until cleaved by HIV-1 protease. These results demonstrate that conditional proteasome inhibitors could prove useful in the development of new tools for chemical biology and future therapeutics.

  14. Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

    Science.gov (United States)

    Wike, Candice L; Graves, Hillary K; Wason, Arpit; Hawkins, Reva; Gopalakrishnan, Jay; Schumacher, Jill; Tyler, Jessica K

    2016-08-17

    The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

  15. PIAS1-mediated sumoylation promotes STUbL-dependent proteasomal degradation of the human telomeric protein TRF2.

    Science.gov (United States)

    Her, Joonyoung; Jeong, Yu Young; Chung, In Kwon

    2015-10-24

    The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex.

    Directory of Open Access Journals (Sweden)

    David S Reis

    2013-10-01

    Full Text Available The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS, has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquinated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone or a translation inhibitor (anisomycin in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning highlight the role of UPS-mediated degradation as critical for synaptic plasticity.

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

    Science.gov (United States)

    Li, Sheng; Yang, Li-Juan; Wang, Ping; He, Yu-Jiao; Huang, Jun-Mei; Liu, Han-Wei; Shen, Xiao-Fei; Wang, Fei

    2016-01-01

    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. 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. 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. 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. 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-mediated IFNAR1 degradation. This may provide a novel

  18. Proteasome nuclear import mediated by Arc3 can influence efficient DNA damage repair and mitosis in Schizosaccharomyces pombe

    DEFF Research Database (Denmark)

    Cabrera, Rodrigo; Sha, Zhe; Vadakkan, Tegy J.

    2010-01-01

    Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasome....... Proteasome nuclear import is reduced when Arc3 is inactivated, leading to hypersensitivity to DNA damage and inefficient cyclin-B degradation, two events occurring in the nucleus. These data suggest that proteasomes display Arc3-dependent mobility in the cell, and mobile proteasomes can efficiently access...

  19. Trehalose activates autophagy and decreases proteasome inhibitor-induced endoplasmic reticulum stress and oxidative stress-mediated cytotoxicity in hepatocytes.

    Science.gov (United States)

    Honma, Yuichi; Sato-Morita, Miyuki; Katsuki, Yuka; Mihara, Hitomi; Baba, Ryoko; Harada, Masaru

    2017-03-11

    Endoplasmic reticulum stress is associated with the pathophysiology of various liver diseases. Endoplasmic reticulum stress mediates the accumulation of abnormal proteins and leads to oxidative stress, cytoplasmic inclusion body formation, and apoptosis in hepatocytes. Autophagy is a bulk degradation pathway for long-lived cytoplasmic proteins or damaged organelles and is also a major degradation pathway for many aggregate-prone and disease-causing proteins. We previously reported that rapamycin, a mammalian target of rapamycin inhibitor, activated autophagy and decreased proteasome inhibitor-mediated ubiquitinated protein accumulation, cytoplasmic inclusion body formation, and apoptosis in hepatocytes. Trehalose is a non-reducing disaccharide that has been shown to activate autophagy. It has been reported to decrease aggregate-prone proteins and ameliorate cytotoxicity in neurodegenerative disease models. However, the effects of trehalose in hepatocytes are unclear. We show here that trehalose activated autophagy and reduced endoplasmic reticulum stress, cytoplasmic inclusion body formation, and apoptosis in proteasome inhibitor-treated liver-derived cultured cells. To our knowledge, this is the first report showing that trehalose activates autophagy and has cytoprotective effects in hepatocytes. Our findings suggest that trehalose can become a therapeutic agent for endoplasmic reticulum stress-related liver diseases. © 2017 The Japan Society of Hepatology.

  20. Inhibition of chaperone-mediated autophagy prevents glucotoxicity in the Caenorhabditis elegans mev-1 mutant by activation of the proteasome.

    Science.gov (United States)

    Eisermann, Dorothé Jenni; Wenzel, Uwe; Fitzenberger, Elena

    2017-02-26

    Chronic hyperglycemia is a hallmark of diabetes mellitus and the main cause of diabetes-associated complications. Increased intracellular glucose levels lead to damaged proteins and in consequence disturb cellular proteostasis. As an important contributor to the maintenance and restoration of proteostasis, autophagy mediates the lysosomal degradation of damaged proteins or entire cellular organelles. In the present study we used the stress-sensitive mev-1 mutant of the nematode Caenorhabditis elegans in order to assess the role of lmp-2, a homologue of the lysosome associated membrane protein type 2A, in the context of glucotoxicity, which was achieved by feeding glucose in a liquid medium. Knockdown of lmp-2 by RNA interference completely prevented the survival reduction caused by glucose under heat stress. Those effects were associated with the prevention of (1) increased lysosome formation and (2) reduction of proteasomal activity, which were observed under glucose feeding. Finally, the survival reduction due to knockdown of ubiquitin remained unaffected by the additional lmp-2 knockdown in the absence or presence of glucose. In conclusion, our study provides evidence that lmp-2, a key player in chaperone-mediated autophagy, is functional in C. elegans, too. Inhibition of lmp-2 prevents the reduction of proteasomal activity by glucose and thereby prevents also glucotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Prion-mediated neurodegeneration is associated with early impairment of the ubiquitin?proteasome system

    OpenAIRE

    2015-01-01

    Prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of misfolded prion protein (PrPSc) in the brain. The critical relationship between aberrant protein misfolding and neurotoxicity currently remains unclear. The accumulation of aggregation-prone proteins has been linked to impairment of the ubiquitin?proteasome system (UPS) in a variety of neurodegenerative disorders, including Alzheimer?s, Parkinson?s and Huntington?s diseases. As the principal r...

  2. BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation.

    Science.gov (United States)

    Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro

    2017-08-26

    Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Tariquidar sensitizes multiple myeloma cells to proteasome inhibitors via reduction of hypoxia-induced P-gp-mediated drug resistance.

    Science.gov (United States)

    Muz, Barbara; Kusdono, Hubert D; Azab, Feda; de la Puente, Pilar; Federico, Cinzia; Fiala, Mark; Vij, Ravi; Salama, Noha N; Azab, Abdel Kareem

    2017-12-01

    Multiple myeloma (MM) presents a poor prognosis and high lethality of patients due to development of drug resistance. P-glycoprotein (P-gp), a drug-efflux transporter, is upregulated in MM patients post-chemotherapy and is involved in the development of drug resistance since many anti-myeloma drugs (including proteasome inhibitors) are P-gp substrates. Hypoxia develops in the bone marrow niche during MM progression and has long been linked to chemoresistance. Additionally, hypoxia-inducible transcription factor (HIF-1α) was demonstrated to directly regulate P-gp expression. We found that in MM patients P-gp expression positively correlated with the hypoxic marker, HIF-1α. Hypoxia increased P-gp protein expression and its efflux capabilities in MM cells in vitro using flow cytometry. We reported herein that hypoxia-mediated resistance to carfilzomib and bortezomib in MM cells is due to P-gp activity and was reversed by tariquidar, a P-gp inhibitor. These results suggest combining proteasome inhibitors with P-gp inhibition for future clinical studies.

  4. Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells.

    Science.gov (United States)

    Qin, Jian-Zhong; Ziffra, Jeffrey; Stennett, Lawrence; Bodner, Barbara; Bonish, Brian K; Chaturvedi, Vijaya; Bennett, Frank; Pollock, Pamela M; Trent, Jeffrey M; Hendrix, Mary J C; Rizzo, Paola; Miele, Lucio; Nickoloff, Brian J

    2005-07-15

    Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

  5. Ginsenoside Rd Is Efficacious Against Acute Ischemic Stroke by Suppressing Microglial Proteasome-Mediated Inflammation.

    Science.gov (United States)

    Zhang, Guangyun; Xia, Feng; Zhang, Yunxia; Zhang, Xiao; Cao, Yuhong; Wang, Ling; Liu, Xuedong; Zhao, Gang; Shi, Ming

    2016-05-01

    A great deal of attention has been paid to neuroprotective therapies for cerebral ischemic stroke. Our two recent clinical trials showed that ginsenoside Rd (Rd), a kind of monomeric compound extracted from Chinese herbs, Panax ginseng and Panax notoginseng, was safe and efficacious for the treatment of ischemic stroke. In this study, we conducted a pooled analysis of the data from 199 patients with acute ischemic stroke in the first trial and 390 in the second to reanalyze the efficacy and safety of Rd. Moreover, animal stroke models were carried out to explore the possible molecular mechanisms underlying Rd neuroprotection. The pooled analysis showed that compared with placebo group, Rd could improve patients' disability as assessed by modified Rankin Scale (mRS) score on day 90 post-stroke and reduce neurologic deficits on day 15 or day 90 post-stroke as assessed by NIH Stroke Scale (NIHSS) and Barthel Index (BI) scores. For neuroprotective mechanisms, administration of Rd 4 h after stroke could inhibit ischemia-induced microglial activation, decrease the expression levels of various proinflammatory cytokines, and suppress nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα) phosphorylation and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation. An in vitro proteasome activity assay revealed a significant inhibitory effect of Rd on proteasome activity in microglia. Interestingly, Rd was showed to have less side effects than glucocorticoid. Therefore, our study demonstrated that Rd could safely improve the outcome of patients with ischemic stroke, and this therapeutic effect may result from its capability of suppressing microglial proteasome activity and sequential inflammation.

  6. Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation.

    Science.gov (United States)

    Gardiol, D; Kühne, C; Glaunsinger, B; Lee, S S; Javier, R; Banks, L

    1999-09-30

    Previous studies have shown that the oncogenic HPV E6 proteins form a complex with the human homologue of the Drosophila tumour suppressor protein, discs large (Dlg). This is mediated by the carboxy terminus of the E6 proteins and involves recognition of at least one PDZ domain of Dlg. This region of E6 is not conserved amongst E6 proteins from the low risk papillomavirus types and, hence, binding of HPV E6 proteins to Dlg correlates with the oncogenic potential of these viruses. We have performed studies to investigate the consequences of the interaction between E6 and Dlg. Mutational analysis of both the HPV18 E6 and Dlg proteins has further defined the regions of E6 and Dlg necessary for complex formation. Strikingly, co-expression of wild type HPV18 E6 with Dlg in vitro or in vivo results in a dramatic decrease in the amount of Dlg protein, whereas mutants of E6 which fail to complex with Dlg have minimal effect on Dlg protein levels. The oncogenic HPV16 E6 also decreased the Dlg levels, but this was not observed with the low risk HPV11 E6 protein. Moreover, a region within the first 544 amino acids of Dlg containing the three PDZ domains confers susceptibility to E6 mediated degradation. Finally, treatment of cells with a proteasome inhibitor overrides the capacity of E6 to degrade Dlg. These results demonstrate that Dlg is targeted by high risk HPV E6 proteins for proteasome mediated degradation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

    2009-08-28

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

  8. CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

    Directory of Open Access Journals (Sweden)

    Sylvain Cardinaud

    2011-05-01

    Full Text Available Cytotoxic CD8+ T cells (CTLs play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF. We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D and a majority encoding an asparagine (Q9VF/5N. We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

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

    Directory of Open Access Journals (Sweden)

    Sheng Li

    2016-06-01

    Full Text Available 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

  10. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of β-secretase

    Science.gov (United States)

    Singh, Amir Kumar; Pati, Uttam

    2015-01-01

    In patient with Alzheimer’s disease (AD), deposition of amyloid-beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β-secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin–proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates Aβ generation and AD-related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK-APP cells, BACE1 is a new substrate of E3-ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing Aβ. CHIPUbox domain physically interacts with BACE1; however, both U-box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK-APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53’s DNA-binding conformation and its binding upon 5′ UTR element (+127 to +150). We have thus discovered that CHIP regulates p53-mediated trans-repression of BACE1 at both transcriptional and post-translational level. We propose that a CHIP–BACE1–p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD. PMID:25773675

  11. The Xanthomonas campestris type III effector XopJ targets the host cell proteasome to suppress salicylic-acid mediated plant defence.

    Directory of Open Access Journals (Sweden)

    Suayib Üstün

    Full Text Available The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv requires type III effector proteins (T3Es for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

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

    Energy Technology Data Exchange (ETDEWEB)

    Honma, Yuichi; Harada, Masaru, E-mail: msrharada@med.uoeh-u.ac.jp

    2013-08-15

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

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

    Directory of Open Access Journals (Sweden)

    Ercan Cacan

    2015-12-01

    Full Text Available 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.

  14. Different Stability and Proteasome-Mediated Degradation Rate of SMN Protein Isoforms.

    Directory of Open Access Journals (Sweden)

    Denise Locatelli

    Full Text Available The key pathogenic steps leading to spinal muscular atrophy (SMA, a genetic disease characterized by selective motor neuron degeneration, are not fully clarified. The full-length SMN protein (FL-SMN, the main protein product of the disease gene SMN1, plays an established role in the cytoplasm in snRNP biogenesis ultimately leading to mRNA splicing within the nucleus. It is also involved in the mRNA axonal transport. However, to what extent the impairment of these two SMN functions contributes to SMA pathogenesis remains unknown. A shorter SMN isoform, axonal-SMN or a-SMN, with more specific axonal localization, has been discovered, but whether it might act in concert with FL-SMN in SMA pathogenesis is not known. As a first step in defining common or divergent intracellular roles of FL-SMN vs a-SMN proteins, we here characterized the turn-over of both proteins and investigated which pathway contributed to a-SMN degradation. We performed real time western blot and confocal immunofluorescence analysis in easily controllable in vitro settings. We analyzed co-transfected NSC34 and HeLa cells and cell clones stably expressing both a-SMN and FL-SMN proteins after specific blocking of transcript or protein synthesis and inhibition of known intracellular degradation pathways. Our data indicated that whereas the stability of both FL-SMN and a-SMN transcripts was comparable, the a-SMN protein was characterized by a much shorter half-life than FL-SMN. In addition, as already demonstrated for FL-SMN, the Ub/proteasome pathway played a major role in the a-SMN protein degradation. We hypothesize that the faster degradation rate of a-SMN vs FL-SMN is related to the protection provided by the protein complex in which FL-SMN is assembled. The diverse a-SMN vs FL-SMN C-terminus may dictate different protein interactions and complex formation explaining the different localization and role in the neuronal compartment, and the lower expression and stability of a-SMN.

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

    Science.gov (United States)

    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. 2009 Wiley-Liss, Inc.

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

    Science.gov (United States)

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

    2015-12-22

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

  17. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA1.

    Science.gov (United States)

    Gilkerson, Jonathan; Kelley, Dior R; Tam, Raymond; Estelle, Mark; Callis, Judy

    2015-06-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCF(TIR1/AFB) (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS(6x)-HA(3x)) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS(6x)-HA(3x)-IAA1 and Lys-less HIS(6x)-HA(3x)-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS(6x)-HA(3x)-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data

  18. Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

    Directory of Open Access Journals (Sweden)

    Verbrugge Sue Ellen

    2013-01-01

    Full Text Available Abstract Background In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI bortezomib (BTZ treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. Methods Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC. Results Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit introducing an amino acid substitution (Met45Ile in the BTZ-binding pocket, (b a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. Conclusions These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

  19. Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model

    OpenAIRE

    Zollner, Thomas M.; Podda, Maurizio; Pien, Christine; Elliott, Peter J.; Kaufmann, Roland; Boehncke, Wolf-Henning

    2002-01-01

    There is increasing evidence that bacterial superantigens contribute to inflammation and T cell responses in psoriasis. Psoriatic inflammation entails a complex series of inductive and effector processes that require the regulated expression of various proinflammatory genes, many of which require NF-κB for maximal trans-activation. PS-519 is a potent and selective proteasome inhibitor based upon the naturally occurring compound lactacystin, which inhibits NF-κB activation by blocking the degr...

  20. Bifunctional anti-huntingtin proteasome-directed intrabodies mediate efficient degradation of mutant huntingtin exon 1 protein fragments.

    Directory of Open Access Journals (Sweden)

    David C Butler

    Full Text Available Huntington's disease (HD is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG(n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt, formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs, expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1 significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q by ~80-90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation.

  1. Priming the proteasome by protein kinase G: a novel cardioprotective mechanism of sildenafil

    OpenAIRE

    Zhang, Hanming; Wang, Xuejun

    2015-01-01

    The proteasome mediates the degradation of most cellular proteins including misfolded proteins, pivotal to intracellular protein hemostasis. Proteasome functional insufficiency is implicated in a large subset of human failing hearts. Experimental studies have established proteasome functional insufficiency as a major pathogenic factor, rationalizing proteasome enhancement as a potentially new therapeutic strategy for congestive heart failure. Protein kinase G activation known to be cardioprot...

  2. Modelling Proteasome and Proteasome Regulator Activities

    Directory of Open Access Journals (Sweden)

    Juliane Liepe

    2014-06-01

    Full Text Available Proteasomes are key proteases involved in a variety of processes ranging from the clearance of damaged proteins to the presentation of antigens to CD8+ T-lymphocytes. Which cleavage sites are used within the target proteins and how fast these proteins are degraded have a profound impact on immune system function and many cellular metabolic processes. The regulation of proteasome activity involves different mechanisms, such as the substitution of the catalytic subunits, the binding of regulatory complexes to proteasome gates and the proteasome conformational modifications triggered by the target protein itself. Mathematical models are invaluable in the analysis; and potentially allow us to predict the complex interactions of proteasome regulatory mechanisms and the final outcomes of the protein degradation rate and MHC class I epitope generation. The pioneering attempts that have been made to mathematically model proteasome activity, cleavage preference variation and their modification by one of the regulatory mechanisms are reviewed here.

  3. Neurofibromatosis type 2 tumor suppressor protein, NF2, induces proteasome-mediated degradation of JC virus T-antigen in human glioblastoma.

    Directory of Open Access Journals (Sweden)

    Sarah Beltrami

    Full Text Available Neurofibromatosis type 2 protein (NF2 has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV tumor antigen (T-antigen as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation.

  4. Elevation of proteasomal substrate levels sensitizes cells to apoptosis induced by inhibition of proteasomal deubiquitinases.

    Science.gov (United States)

    Sun, Chao; Roboti, Peristera; Puumalainen, Marjo-Riitta; Fryknäs, Mårten; Wang, Xin; D'Arcy, Padraig; Hult, Malin; High, Stephen; Linder, Stig; Swanton, Eileithyia

    2014-01-01

    Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s) underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system.

  5. Elevation of proteasomal substrate levels sensitizes cells to apoptosis induced by inhibition of proteasomal deubiquitinases.

    Directory of Open Access Journals (Sweden)

    Chao Sun

    Full Text Available Inhibitors of the catalytic activity of the 20S proteasome are cytotoxic to tumor cells and are currently in clinical use for treatment of multiple myeloma, whilst the deubiquitinase activity associated with the 19S regulatory subunit of the proteasome is also a valid target for anti-cancer drugs. The mechanisms underlying the therapeutic efficacy of these drugs and their selective toxicity towards cancer cells are not known. Here, we show that increasing the cellular levels of proteasome substrates using an inhibitor of Sec61-mediated protein translocation significantly increases the extent of apoptosis that is induced by inhibition of proteasomal deubiquitinase activity in both cancer derived and non-transformed cell lines. Our results suggest that increased generation of misfolded proteasome substrates may contribute to the mechanism(s underlying the increased sensitivity of tumor cells to inhibitors of the ubiquitin-proteasome system.

  6. Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

    Directory of Open Access Journals (Sweden)

    Chaowalit Yuajit

    Full Text Available Cyst enlargement in polycystic kidney disease (PKD involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h with steviol (100 microM also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h with steviol (100 microM markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

  7. The proteasome maturation protein POMP increases proteasome assembly and activity in psoriatic lesional skin.

    Science.gov (United States)

    Zieba, Barbara A; Henry, Laurent; Lacroix, Matthieu; Jemaà, Mohamed; Lavabre-Bertrand, Thierry; Meunier, Laurent; Coux, Olivier; Stoebner, Pierre-Emmanuel

    2017-10-01

    The ubiquitin proteasome pathway is involved in the pathogenesis of psoriasis and proteasome subunits are increased in lesional psoriatic skin. Recent works have highlighted that proteasome levels can be regulated through modulation of proteasome assembly notably by the proteasome maturation protein POMP. To investigate whether proteasome assembly and POMP expression are modified in psoriatic skin. Proteasome assembly as well as expression of proteasome regulators were assessed in non-lesional and lesional psoriatic skin using native gel electrophoresis and western blots respectively. The protein and mRNA expression levels of POMP were compared by western blots, immunohistochemistry and quantitative polymerase chain reaction. The role of POMP in keratinocyte proliferation and differentiation was assessed by silencing POMP gene expression by RNA interference in human immortalized keratinocyte HaCaT cells. Both 20S and 26S proteasomes (and their respective proteolytic activities) as well as the main proteasome regulators are increased in lesional psoriatic skin. POMP binds to 20S precursor complexes and is overexpressed in lesional epidermal psoriatic skin, supporting that POMP-mediated proteasome assembly is increased in psoriatic skin. POMP silencing inhibited HaCaT cell proliferation and induced apoptosis through the inhibition of the proteasome assembly. Moreover POMP partial depletion decreased the expression of the differentiation markers keratin 10 and involucrin during the [Ca2+]-induced HaCaT cells differentiation. Altogether these results establish a potential role for POMP and proteasome assembly in psoriasis pathogenesis. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  8. The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

    Directory of Open Access Journals (Sweden)

    Adrian O'Hara

    Full Text Available The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2 and regenerating protein 1 (Reg1. Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

  9. The role of proteasome beta subunits in gastrin-mediated transcription of plasminogen activator inhibitor-2 and regenerating protein1.

    Science.gov (United States)

    O'Hara, Adrian; Howarth, Alice; Varro, Andrea; Dimaline, Rod

    2013-01-01

    The hormone gastrin physiologically regulates gastric acid secretion and also contributes to maintaining gastric epithelial architecture by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2) and regenerating protein 1 (Reg1). Here we examine the role of proteasome subunit PSMB1 in the transcriptional regulation of PAI-2 and Reg1 by gastrin, and its subcellular distribution during gastrin stimulation. We used the gastric cancer cell line AGS, permanently transfected with the CCK2 receptor (AGS-GR) to study gastrin stimulated expression of PAI-2 and Reg1 reporter constructs when PSMB1 was knocked down by siRNA. Binding of PSMB1 to the PAI-2 and Reg1 promoters was assessed by chromatin immunoprecipitation (ChIP) assay. Subcellular distribution of PSMB1 was determined by immunocytochemistry and Western Blot. Gastrin robustly increased expression of PAI-2 and Reg1 in AGS-GR cells, but when PSMB1 was knocked down the responses were dramatically reduced. In ChIP assays, following immunoprecipitation of chromatin with a PSMB1 antibody there was a substantial enrichment of DNA from the gastrin responsive regions of the PAI-2 and Reg1 promoters compared with chromatin precipitated with control IgG. In AGS-GR cells stimulated with gastrin there was a significant increase in the ratio of nuclear:cytoplasmic PSMB1 over the same timescale as recruitment of PSMB1 to the PAI-2 and Reg1 promoters seen in ChIP assays. We conclude that PSMB1 is part of the transcriptional machinery required for gastrin stimulated expression of PAI-2 and Reg1, and that its change in subcellular distribution in response to gastrin is consistent with this role.

  10. Marine Streptomyces sp. derived antimycin analogues suppress HeLa cells via depletion HPV E6/E7 mediated by ROS-dependent ubiquitin–proteasome system

    Science.gov (United States)

    Zhang, Weiyi; Che, Qian; Tan, Hongsheng; Qi, Xin; Li, Jing; Li, Dehai; Gu, Qianqun; Zhu, Tianjiao; Liu, Ming

    2017-01-01

    Four new antimycin alkaloids (1–4) and six related known analogs (5–10) were isolated from the culture of a marine derived Streptomyces sp. THS-55, and their structures were elucidated by extensive spectroscopic analysis. All of the compounds exhibited potent cytotoxicity in vitro against HPV-transformed HeLa cell line. Among them, compounds 6–7 were derived as natural products for the first time, and compound 5 (NADA) showed the highest potency. NADA inhibited the proliferation, arrested cell cycle distribution, and triggered apoptosis in HeLa cancer cells. Our molecular mechanic studies revealed NADA degraded the levels of E6/E7 oncoproteins through ROS-mediated ubiquitin-dependent proteasome system activation. This is the first report that demonstrates antimycin alkaloids analogue induces the degradation of high-risk HPV E6/E7 oncoproteins and finally induces apoptosis in cervical cancer cells. The present work suggested that these analogues could serve as lead compounds for the development of HPV-infected cervical cancer therapeutic agents, as well as research tools for the study of E6/E7 functions. PMID:28176847

  11. The Proteasome Distinguishes between Heterotypic and Homotypic Lysine-11-Linked Polyubiquitin Chains

    Directory of Open Access Journals (Sweden)

    Guinevere L. Grice

    2015-07-01

    Full Text Available Proteasome-mediated degradation occurs with proteins principally modified with lysine-48 polyubiquitin chains. Whether the proteasome also can bind atypical ubiquitin chains, including those linked by lysine-11, has not been well established. This is critically important, as lysine-11 polyubiquitination has been implicated in both proteasome-mediated degradation and non-degradative outcomes. Here we demonstrate that pure homotypic lysine-11-linked chains do not bind strongly to the mammalian proteasome. By contrast, heterotypic polyubiquitin chains, containing lysine-11 and lysine-48 linkages, not only bind to the proteasome but also stimulate the proteasomal degradation of the cell-cycle regulator cyclin B1. Thus, while heterotypic lysine-11-linked chains facilitate proteasomal degradation, homotypic lysine-11 linkages adopt conformations that prevent association with the proteasome. Our data demonstrate the capacity of the proteasome to bind ubiquitin chains of distinct topology, with implications for the recognition and diverse biological functions of mixed ubiquitin chains.

  12. Proteasomes and protein conjugation across domains of life

    OpenAIRE

    Maupin-Furlow, Julie

    2011-01-01

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pat...

  13. Genetics of Proteasome Diseases

    Directory of Open Access Journals (Sweden)

    Aldrin V. Gomes

    2013-01-01

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

  14. An historic perspective of proteasome inhibition.

    Science.gov (United States)

    Esseltine, Dixie-Lee; Mulligan, George

    2012-07-01

    The ubiquitin-proteasome system (UPS) and associated signaling pathways are regarded today as an exciting area of development for novel therapeutics. However, two decades ago, following the discovery and elucidation of ubiquitin and the 26S proteasome as key mediators of protein turnover, the concept of inhibiting the UPS was not even considered a feasible therapeutic approach due to the assumption that inhibition of this pathway would have widespread deleterious effects. Subsequent clinical developments with the first-in-class proteasome inhibitor bortezomib have radically overturned that view, with the proteasome now recognized as a validated target and proteasome inhibition demonstrated to be a highly successful treatment for a number of hematologic malignancies. Here we provide a historic perspective on the emergence of proteasome inhibition, sharing some of the lessons learned along the way. We describe the development of bortezomib and the elucidation of the effects of its novel mechanism of action, and place the cutting-edge work described elsewhere in this issue in the context of these historic developments. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Combined autophagy and proteasome inhibition

    Science.gov (United States)

    Vogl, Dan T; Stadtmauer, Edward A; Tan, Kay-See; Heitjan, Daniel F; Davis, Lisa E; Pontiggia, Laura; Rangwala, Reshma; Piao, Shengfu; Chang, Yunyoung C; Scott, Emma C; Paul, Thomas M; Nichols, Charles W; Porter, David L; Kaplan, Janeen; Mallon, Gayle; Bradner, James E; Amaravadi, Ravi K

    2014-01-01

    The efficacy of proteasome inhibition for myeloma is limited by therapeutic resistance, which may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation. Preclinical studies demonstrate that autophagy inhibition with hydroxychloroquine augments the antimyeloma efficacy of the proteasome inhibitor bortezomib. We conducted a phase I trial combining bortezomib and hydroxychloroquine for relapsed or refractory myeloma. We enrolled 25 patients, including 11 (44%) refractory to prior bortezomib. No protocol-defined dose-limiting toxicities occurred, and we identified a recommended phase 2 dose of hydroxychloroquine 600 mg twice daily with standard doses of bortezomib, at which we observed dose-related gastrointestinal toxicity and cytopenias. Of 22 patients evaluable for response, 3 (14%) had very good partial responses, 3 (14%) had minor responses, and 10 (45%) had a period of stable disease. Electron micrographs of bone marrow plasma cells collected at baseline, after a hydroxychloroquine run-in, and after combined therapy showed therapy-associated increases in autophagic vacuoles, consistent with the combined effects of increased trafficking of misfolded proteins to autophagic vacuoles and inhibition of their degradative capacity. Combined targeting of proteasomal and autophagic protein degradation using bortezomib and hydroxychloroquine is therefore feasible and a potentially useful strategy for improving outcomes in myeloma therapy. PMID:24991834

  16. Lysine Residues Are Not Required for Proteasome-Mediated Proteolysis of the Auxin/Indole Acidic Acid Protein IAA11[OPEN

    Science.gov (United States)

    Gilkerson, Jonathan; Estelle, Mark

    2015-01-01

    Although many ubiquitin-proteasome substrates have been characterized in plants, very little is known about the corresponding ubiquitin attachment(s) underlying regulated proteolysis. Current dogma asserts that ubiquitin is typically covalently attached to a substrate through an isopeptide bond between the ubiquitin carboxy terminus and a substrate lysyl amino group. However, nonlysine (non-Lys) ubiquitin attachment has been observed in other eukaryotes, including the N terminus, cysteine, and serine/threonine modification. Here, we investigate site(s) of ubiquitin attachment on indole-3-acetic acid1 (IAA1), a short-lived Arabidopsis (Arabidopsis thaliana) Auxin/indole-3-acetic acid (Aux/IAA) family member. Most Aux/IAA proteins function as negative regulators of auxin responses and are targeted for degradation after ubiquitination by the ubiquitin ligase SCFTIR1/AFB (for S-Phase Kinase-Associated Protein1, Cullin, F-box [SCF] with Transport Inhibitor Response1 [TIR1]/Auxin Signaling F-box [AFB]) by an interaction directly facilitated by auxin. Surprisingly, using a Histidine-Hemaglutinin (HIS6x-HA3x) epitope-tagged version expressed in vivo, Lys-less IAA1 was ubiquitinated and rapidly degraded in vivo. Lys-substituted versions of IAA1 localized to the nucleus as Yellow Fluorescent Protein fusions and interacted with both TIR1 and IAA7 in yeast (Saccharomyces cerevisiae) two-hybrid experiments, indicating that these proteins were functional. Ubiquitination on both HIS6x-HA3x-IAA1 and Lys-less HIS6x-HA3x-IAA1 proteins was sensitive to sodium hydroxide treatment, indicative of ubiquitin oxyester formation on serine or threonine residues. Additionally, base-resistant forms of ubiquitinated IAA1 were observed for HIS6x-HA3x-IAA1, suggesting additional lysyl-linked ubiquitin on this protein. Characterization of other Aux/IAA proteins showed that they have diverse degradation rates, adding additional complexity to auxin signaling. Altogether, these data indicate that Aux

  17. Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5) expression through both hypoxia inducible factor-1α and proteasome-mediated pathways.

    Science.gov (United States)

    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.

  18. Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin-26 S proteasome system in the living rat.

    Science.gov (United States)

    Dominy, John E; Hirschberger, Lawrence L; Coloso, Relicardo M; Stipanuk, Martha H

    2006-02-15

    Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin-26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.

  19. Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin–26 S proteasome system in the living rat

    Science.gov (United States)

    Dominy, John E.; Hirschberger, Lawrence L.; Coloso, Relicardo M.; Stipanuk, Martha H.

    2005-01-01

    Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin–26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism. PMID:16262602

  20. The ubiquitin-proteasome system

    Indian Academy of Sciences (India)

    proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.

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

    Directory of Open Access Journals (Sweden)

    David S. Pitcher

    2015-07-01

    Full Text Available The proteasome inhibitor Bortezomib is used to treat multiple myeloma (MM. Bortezomib inhibits protein degradation by inactivating proteasomes' active-sites. MM cells are exquisitely sensitive to Bortezomib – exhibiting a low-nanomolar IC50 – suggesting that minimal inhibition of degradation suffices to kill MM cells. Instead, we report, a low Bortezomib concentration, contrary to expectation, achieves severe inhibition of proteasome activity in MM cells: the degree of inhibition exceeds what one would expect from the small proportion of active-sites that Bortezomib inhibits. Our data indicate that Bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity. Comparing MM cells to other, Bortezomib-resistant, cancer cells shows that the degree of proteasome inhibition is the greatest in MM cells and only there leads to proteasome stress, providing an explanation for why Bortezomib is effective against MM but not other cancers.

  2. Tissue-nonspecific alkaline phosphatase with an Asp(289)-->Val mutation fails to reach the cell surface and undergoes proteasome-mediated degradation.

    Science.gov (United States)

    Ishida, Yoko; Komaru, Keiichi; Ito, Masahiro; Amaya, Yoshihiro; Kohno, Shoji; Oda, Kimimitsu

    2003-07-01

    A missense mutation in the gene of tissue-nonspecific alkaline phosphatase, which replaces aspartic acid at position 289 with valine [TNSALP (D289V)], was reported in a lethal hypophosphatasia patient [Taillandier, A. et al. (1999) Hum. Mut. 13, 171-172]. To define the molecular defects of TNSALP (D289V), this mutant protein in transiently transfected COS-1 cells was analyzed biochemically and morphologically. TNSALP (D289V) exhibited no alkaline phosphatase activity and mainly formed a disulfide-linked high molecular mass aggregate. Cell-surface biotinylation, digestion with phosphatidylinositol-specific phospholipase C and an immunofluorescence study showed that the mutant protein failed to appear on the cell surface and was accumulated intracellularly. In agreement with this, pulse/chase experiments demonstrated that TNSALP (D289V) remained endo-beta-N-acetyl- glucosaminidase H-sensitive throughout the chase and was eventually degraded, indicating that the mutant protein is unable to reach the medial-Golgi. Proteasome inhibitors strongly blocked the degradation of TNSALP (D289V), and furthermore the mutant protein was found to be ubiquitinated. Besides, another naturally occurring TNSALP with a Glu(218)-->Gly mutation was also found to be polyubiquitinated and degraded in the proteasome. Since the acidic amino acids at positions 218 and 289 of TNSALP are thought to be directly involved in the Ca(2+) coordination, these results suggest the critical importance of calcium binding in post-translational folding and assembly of the TNSALP molecule.

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

    Science.gov (United States)

    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; Felder, Robin A.; Jose, Pedro A.

    2008-01-01

    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. PMID:18464932

  4. Ethylene-Induced Stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 Is Mediated by Proteasomal Degradation of EIN3 Binding F-Box 1 and 2 That Requires EIN2 in Arabidopsis[C][W

    Science.gov (United States)

    An, Fengying; Zhao, Qiong; Ji, Yusi; Li, Wenyang; Jiang, Zhiqiang; Yu, Xiangchun; Zhang, Chen; Han, Ying; He, Wenrong; Liu, Yidong; Zhang, Shuqun; Ecker, Joseph R.; Guo, Hongwei

    2010-01-01

    Plant responses to ethylene are mediated by regulation of EBF1/2-dependent degradation of the ETHYLENE INSENSITIVE3 (EIN3) transcription factor. Here, we report that the level of EIL1 protein is upregulated by ethylene through an EBF1/2-dependent pathway. Genetic analysis revealed that EIL1 and EIN3 cooperatively but differentially regulate a wide array of ethylene responses, with EIL1 mainly inhibiting leaf expansion and stem elongation in adult plants and EIN3 largely regulating a multitude of ethylene responses in seedlings. When EBF1 and EBF2 are disrupted, EIL1 and EIN3 constitutively accumulate in the nucleus and remain unresponsive to exogenous ethylene application. Further study revealed that the levels of EBF1 and EBF2 proteins are downregulated by ethylene and upregulated by silver ion and MG132, suggesting that ethylene stabilizes EIN3/EIL1 by promoting EBF1 and EBF2 proteasomal degradation. Also, we found that EIN2 is indispensable for mediating ethylene-induced EIN3/EIL1 accumulation and EBF1/2 degradation, whereas MKK9 is not required for ethylene signal transduction, contrary to a previous report. Together, our studies demonstrate that ethylene similarly regulates EIN3 and EIL1, the two master transcription factors coordinating myriad ethylene responses, and clarify that EIN2 but not MKK9 is required for ethylene-induced EIN3/EIL1 stabilization. Our results also reveal that EBF1 and EBF2 act as essential ethylene signal transducers that by themselves are subject to proteasomal degradation. PMID:20647342

  5. Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system.

    Science.gov (United States)

    Cerqueira, Paula G; Passos-Silva, Danielle G; Vieira-da-Rocha, João P; Mendes, Isabela Cecilia; de Oliveira, Karla A; Oliveira, Camila F B; Vilela, Liza F F; Nagem, Ronaldo A P; Cardoso, Joseane; Nardelli, Sheila C; Krieger, Marco A; Franco, Glória R; Macedo, Andrea M; Pena, Sérgio D J; Schenkman, Sérgio; Gomes, Dawidson A; Guerra-Sá, Renata; Machado, Carlos R

    2017-03-01

    In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase.

    Science.gov (United States)

    Braun-Falco, Markus; Eisenried, Angelika; Büning, Hildegard; Ring, Johannes

    2005-05-01

    Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis. This study was undertaken to find factors that influence rAAV-2-mediated in vitro gene transfer into human keratinocytes and, consequently, ways to optimize gene delivery. Transduction experiments using rAAV-2 vectors expressing green fluorescent protein (GFP) demonstrated that impaired cellular trafficking of vector particles and high levels of autophosphorylation at epidermal growth factor receptor tyrosine kinase (EGF-R TK) have a negative influence on gene transfer into keratinocytes. Treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient augmentation of GFP expression in up to 37% of cells. Treatment with EGF-R TK inhibitors (quinazoline type) enhanced transgene expression in 10-14.5% of the cells. Gene expression was stable for more than 10 weeks and persisted until proliferative senescence occurred. This stable gene expression allows speculation that keratinocyte stem cells have initially been transduced. These findings might have relevance for the use of rAAV-2 vectors in skin gene therapy: transient enhancement of rAAV-2 transduction with proteasome inhibitors might be useful for genetic promotion of wound healing or skin-directed vaccination. Treatment with quinazolines may increase rAAV-2 transduction of keratinocyte stem cells, which is important for gene therapy approaches to inherited diseases.

  7. Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.

    Science.gov (United States)

    Murakawa, Yasuhiro; Sonoda, Eiichiro; Barber, Louise J; Zeng, Weihua; Yokomori, Kyoko; Kimura, Hiroshi; Niimi, Atsuko; Lehmann, Alan; Zhao, Guang Yu; Hochegger, Helfrid; Boulton, Simon J; Takeda, Shunichi

    2007-09-15

    Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.

  8. Inhibition of melanogenesis by jineol from Scolopendra subspinipes mutilans via MAP-Kinase mediated MITF downregulation and the proteasomal degradation of tyrosinase.

    Science.gov (United States)

    Alam, Md Badrul; Bajpai, Vivek K; Lee, JungIn; Zhao, Peijun; Byeon, Jung-Hee; Ra, Jeong-Sic; Majumder, Rajib; Lee, Jong Sung; Yoon, Jung-In; Rather, Irfan A; Park, Yong-Ha; Kim, Kangmin; Na, MinKyun; Lee, Sang-Han

    2017-04-10

    In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin.

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

    Science.gov (United States)

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

    2015-07-06

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l(-/-) mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. © 2015 Gerhardt et al.

  10. The cilia-regulated proteasome and its role in the development of ciliopathies and cancer.

    Science.gov (United States)

    Gerhardt, Christoph; Leu, Tristan; Lier, Johanna Maria; Rüther, Ulrich

    2016-01-01

    The primary cilium is an essential structure for the mediation of numerous signaling pathways involved in the coordination and regulation of cellular processes essential for the development and maintenance of health. Consequently, ciliary dysfunction results in severe human diseases called ciliopathies. Since many of the cilia-mediated signaling pathways are oncogenic pathways, cilia are linked to cancer. Recent studies demonstrate the existence of a cilia-regulated proteasome and that this proteasome is involved in cancer development via the progression of oncogenic, cilia-mediated signaling. This review article investigates the association between primary cilia and cancer with particular emphasis on the role of the cilia-regulated proteasome.

  11. Proteasome inhibitors with photocontrolled activity.

    Science.gov (United States)

    Hansen, Mickel J; Velema, Willem A; de Bruin, Gerjan; Overkleeft, Herman S; Szymanski, Wiktor; Feringa, Ben L

    2014-09-22

    Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non-specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure. Here we describe the design, synthesis, and activity of photoswitchable proteasome inhibitors. Substantial differences in proteasome inhibitory activity in cell extracts were observed before and after irradiation with light. The presented results show potential for the development of chemotherapeutic agents that can be switched on and off with light, constituting a new strategy for spatiotemporally modulating proteasomal activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Proteasome inhibitors in cancer therapy

    Directory of Open Access Journals (Sweden)

    Wioletta Romaniuk

    2015-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Achuth Padmanabhan

    2016-03-01

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

  14. Proteasome inhibitor patents (2010 - present).

    Science.gov (United States)

    Metcalf, Rainer; Scott, Latanya M; Daniel, Kenyon G; Dou, Q Ping

    2014-04-01

    Over the past 3 years, numerous patents and patent applications have been submitted and published involving compounds designed to inhibit the proteasome. Proteasome inhibition has been of great interest in cancer research since disruption of proteolysis leads to a significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells. The current standards in proteasome inhibition are the only FDA-approved inhibitors, bortezomib and carfilzomib. Although these drugs are quite effective in treating multiple myeloma and other blood tumors, there are shortcomings, including toxicities and resistance. Most of the current patents attempt to improve on existing compounds, by increasing bioavailability and selectivity, while attempting to reduce toxicity. A general categorization of similar compounds was employed to evaluate and compare drug design strategies. This review focuses on novel compounds and subsequent analogs developed for proteasome inhibition, used in preventing and treating human cancers. A comprehensive description and categorization of patents related to each type of compound and its derivatives, as well as their uses and efficacies as anticancer agents is included. A review of combination therapy patents has also been included. Although there are many diverse chemical scaffolds being published, there are few patented proteasome inhibitors whose method of inhibition is genuinely novel. Most patents utilize a destructive chemical warhead to attack the catalytic threonine residue of the proteasome active sites. Few patents try to depart from this, emphasizing the need for developing new mechanisms of action and specific targeting.

  15. CREB activates proteasomal degradation of DSCR1/RCAN1.

    Science.gov (United States)

    Seo, Su Ryeon; Chung, Kwang Chul

    2008-06-11

    The cyclic AMP response element-binding protein (CREB) is involved in the development and function of the nervous system. Here, we find that CREB decreases the protein level of Regulator of Calcineurin Activity 1 (RCAN1/DSCR1/MCIP1), which is overexpressed in the brain of Down Syndrome (DS) patients. Decrease of RCAN1 by CREB was blocked by proteasome inhibitors, indicating that this decrease is mediated by the ubiquitin-proteasome pathway. Furthermore, we found that the ability of CREB to activate the degradation of RCAN1 depends on its transcriptional activation. Consistently, CREB-enhanced the ubiquitination and turnover rate of RCAN1. Our results reveal a new regulatory role for CREB in DS pathology through the proteasomal degradation of RCAN1.

  16. Role of proteasomes in disease

    Directory of Open Access Journals (Sweden)

    Dahlmann Burkhardt

    2007-11-01

    Full Text Available Abstract 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; http://www.targetedproteinsdb.com.

  17. Proteasomal regulation of caspase-8 in cancer cell apoptosis.

    Science.gov (United States)

    Fiandalo, Michael V; Schwarze, Steven R; Kyprianou, Natasha

    2013-06-01

    Previous studies demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of caspase-8. The present study investigated the impact of proteasome inhibition on caspase-8 stability, ubiquitination, trafficking, and activation in cancer cells. Using caspase-8 deficient neuroblastoma (NB7) cells for reconstituting non-cleavable mutant forms of caspase-8, we demonstrated that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8, in response to proteasome inhibitor and GST-TRAIL. Moreover in the LNCaP human prostate cancer cells, caspase-8 polyubiquitination occurs after TRAIL stimulation and caspase-8 processing. Subcellular fractionation analysis revealed caspase-8 activity in both cytosol and plasma membrane fractions in both NB7 reconstituted caspase-8 cell lines, as well the LNCaP prostate cancer cells. The present results suggest that caspase-8 stabilization through proteasome inhibition leads to reactivation of the extrinsic pathway of apoptosis and identify E3 ligase mediating caspase-8 polyubiquitination, as a novel molecular target. Inhibition of this E3 ligase in combination with TRAIL towards restoring apoptosis signaling activation may have potential therapeutic significance in resistant tumors.

  18. Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis

    NARCIS (Netherlands)

    Hougardy, BMT; Maduro, JH; van der Zee, AGJ; de Groot, DJA; van den Heuvel, FAJ; de Vries, EGE; de Jong, S

    2006-01-01

    In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize

  19. Regulation of SUMO2 Target Proteins by the Proteasome in Human Cells Exposed to Replication Stress

    DEFF Research Database (Denmark)

    Bursomanno, Sara; McGouran, Joanna F; Kessler, Benedikt M

    2015-01-01

    In human cells, SUMO2 is predominantly conjugated to target proteins in response to cellular stress. Previous studies suggested that proteins conjugated to SUMO2, but not to SUMO1, could be regulated by the ubiquitin-mediated proteasome system. Hence, we set out to understand the role...... of the proteasome in determining the fate of proteins conjugated to SUMO2 when cells are treated with DNA replication stress conditions. We conducted a quantitative proteomic analysis in a U2OS cell line stably expressing SUMO2(Q87R) tagged with StrepHA in the presence or absence of epoxomicin (EPOX), a proteasome...

  20. The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

    DEFF Research Database (Denmark)

    Pietroni, Paola; Vasisht, Nishi; Cook, Jonathan P

    2013-01-01

    The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the endoplasmic reticulum (ER) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted...... model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show here that the proteasome has a more complex role in ricin intoxication than previously recognised, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors...

  1. ABA-dependent inhibition of the ubiquitin proteasome system during germination at high temperature in Arabidopsis.

    Science.gov (United States)

    Chiu, Rex Shun; Pan, Shiyue; Zhao, Rongmin; Gazzarrini, Sonia

    2016-12-01

    During germination, endogenous and environmental factors trigger changes in the transcriptome, translatome and proteome to break dormancy. In Arabidopsis thaliana, the ubiquitin proteasome system (UPS) degrades proteins that promote dormancy to allow germination. While research on the UPS has focused on the identification of proteasomal substrates, little information is known about the regulation of its activity. Here we characterized the activity of the UPS during dormancy release and maintenance by monitoring protein ubiquitination and degradation of two proteasomal substrates: Suc-LLVY-AMC, a well characterized synthetic substrate, and FUSCA3 (FUS3), a dormancy-promoting transcription factor degraded by the 26S proteasome. Our data indicate that proteasome activity and protein ubiquitination increase during imbibition at optimal temperature (21°C), and are required for seed germination. However, abscisic acid (ABA) and supraoptimal temperature (32°C) inhibit germination by dampening both protein ubiquitination and proteasome activity. Inhibition of UPS function by high temperature is reduced by the ABA biosynthesis inhibitor, fluridone, and in ABA biosynthetic mutants, suggesting that it is ABA dependent. Accordingly, inhibition of FUS3 degradation at 32°C is also dependent on ABA. Native gels show that inhibition of proteasome activity is caused by interference with the 26S/30S ratio as well as free 19S and 20S levels, impacting the proteasome degradation cycle. Transfer experiments show that ABA-mediated inhibition of proteasome activity at 21°C is restricted to the first 2 days of germination, a time window corresponding to seed sensitivity to environmental and ABA-mediated growth inhibition. Our data show that ABA and high temperature inhibit germination under unfavourable growth conditions by repressing the UPS. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  2. Glatiramer Acetate and Nanny Proteins Restrict Access of the Multiple Sclerosis Autoantigen Myelin Basic Protein to the 26S Proteasome

    Directory of Open Access Journals (Sweden)

    Ekaterina Kuzina

    2014-01-01

    Full Text Available We recently showed that myelin basic protein (MBP is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM, as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA, which is used to treat multiple sclerosis (MS and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.

  3. Combined inhibition of p97 and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells.

    Directory of Open Access Journals (Sweden)

    Holger W Auner

    Full Text Available Inhibition of the proteasome is a widely used strategy for treating multiple myeloma that takes advantage of the heavy secretory load that multiple myeloma cells (MMCs have to deal with. Resistance of MMCs to proteasome inhibition has been linked to incomplete disruption of proteasomal endoplasmic-reticulum (ER-associated degradation (ERAD and activation of non-proteasomal protein degradation pathways. The ATPase p97 (VCP/Cdc48 has key roles in mediating both ERAD and non-proteasomal protein degradation and can be targeted pharmacologically by small molecule inhibition. In this study, we compared the effects of p97 inhibition with Eeyarestatin 1 and DBeQ on the secretory apparatus of MMCs with the effects induced by the proteasome inhibitor bortezomib, and the effects caused by combined inhibition of p97 and the proteasome. We found that p97 inhibition elicits cellular responses that are different from those induced by proteasome inhibition, and that the responses differ considerably between MMC lines. Moreover, we found that dual inhibition of both p97 and the proteasome terminally disrupts ER configuration and intracellular protein metabolism in MMCs. Dual inhibition of p97 and the proteasome induced high levels of apoptosis in all of the MMC lines that we analysed, including bortezomib-adapted AMO-1 cells, and was also effective in killing primary MMCs. Only minor toxicity was observed in untransformed and non-secretory cells. Our observations highlight non-redundant roles of p97 and the proteasome in maintaining secretory homeostasis in MMCs and provide a preclinical conceptual framework for dual targeting of p97 and the proteasome as a potential new therapeutic strategy in multiple myeloma.

  4. Features of proteasome functioning in malignant tumors

    Science.gov (United States)

    Kondakova, I. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Slonimskaya, E. M.; Kolomiets, L. A.; Afanas'ev, S. G.; Choinzonov, Y. L.

    2017-09-01

    Proteasome ubiquitin system is the important system of intracellular proteolysis. The activity of the proteasomes may undergo changes during cancer development. We studied the chymotrypsin-like activity of proteasomes, their subunit composition, and their association with tumor stage in breast cancer, head and neck squamous cell carcinoma, endometrial cancer, renal cancer, bladder cancer, stomach cancer, ovarian cancer, and colorectal cancer. The increase in chymotrypsin-like activity of proteasomes and decrease in total proteasome pool compared with adjacent tissues were shown in all malignant tumors excluding kidney cancer. The increase in chymotrypsin-like activity of proteasomes was found in primary tumors with all types of metastasis: lymphogenous of head and neck squamous cell carcinoma, intraperitoneal metastasis of ovarian cancer, hematogenous metastasis colorectal cancer. The exception was kidney cancer, in which there was a decrease in chymotrypsin-like activity with distant metastasis.

  5. Reversible phosphorylation of the 26S proteasome

    Directory of Open Access Journals (Sweden)

    Xing Guo

    2017-03-01

    Full Text Available ABSTRACT The 26S proteasome at the center of the ubiquitin-proteasome system (UPS is essential for virtually all cellular processes of eukaryotes. A common misconception about the proteasome is that, once made, it remains as a static and uniform complex with spontaneous and constitutive activity for protein degradation. Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions. In this review, we summarize the history and current understanding of proteasome phosphorylation, and advocate the idea of targeting proteasome kinases/phosphatases as a new strategy for clinical interventions of several human diseases.

  6. 26 S proteasomes function as stable entities

    DEFF Research Database (Denmark)

    Hendil, Klavs B; Hartmann-Petersen, Rasmus; Tanaka, Keiji

    2002-01-01

    Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes...... dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700...... subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates...

  7. In Vivo Pharmacodynamic Imaging of Proteasome Inhibition

    Directory of Open Access Journals (Sweden)

    Erin A. Kimbrel

    2009-05-01

    Full Text Available Inhibiting the proteolytic activity of the 26S proteasome has been shown to have selective apoptotic effects on cancer cells and to be clinically efficacious in certain malignancies. There is an unmet medical need for additional proteasome inhibitors, and their development will be facilitated by surrogate markers of proteasome function. Toward this end, ectopic fusion of the destruction domain from ornithine decarboxylase (ODC to reporter proteins is often used for assessing proteasome function. For luciferase-based reporters, we hypothesized that the oxygen-dependent destruction domain (ODD from hypoxia-inducible factor 1α (HIF-1α may provide improved sensitivity over luciferase-ODC, owing to its extremely rapid turnover by the proteasome (HIF-1α has a half-life of less than 5 minutes. In the current study, we show that ODD-luciferase affords a greater dynamic range and faster kinetics than luciferase-ODC in sensing proteasome inhibition in vitro. Importantly, ODD-luciferase also serves as an effective in vivo marker of proteasome function in xenograft tumor models, with inhibition being detected by noninvasive imaging within 3 hours of bortezomib administration. These data establish ODD-luciferase as a surrogate marker of proteasome function that can be used both in vitro and in vivo for the development of novel proteasome inhibitors.

  8. Aging: when the ubiquitin–proteasome machinery collapses

    Directory of Open Access Journals (Sweden)

    Mohamed A. Eldeeb

    2017-05-01

    Full Text Available In mammalian cells, protein degradation is an essential and dynamic process that is crucial for survival, growth, differentiation and proliferation of cells. Tellingly, the majority of intracellular proteins are degraded via the ubiquitin–proteasome system (UPS. UPS-mediated protein degradation serves qualitative and quantitative roles within the cellular proteome. For instance, UPS specifically targets misfolded, aggregated, toxic, mutant and otherwise structurally abnormal proteins for destruction and hence prevent aggregation and accumulation of toxic proteins. Furthermore, several cellular regulatory proteins, including cell cycle regulators, transcription factors, DNA replication and DNA repair proteins are selectively targeted for degradation via UPS and thus contribute to maintaining protein homeostasis (proteostasis and proper functional proteome. Concomitantly, the deregulation of proteostasis may lead to several pathological disorders including aging-associated pathologies. Remarkably, augmenting the proteasomal activity has been linked to longevity in model organisms and protect these organisms from symptoms linked to protein homeostasis disorders. Herein I comment briefly on the recent work revealing the pivotal role of ubiquitin–proteasome-mediated protein degradation with respect to regulating aging process in model organisms.

  9. Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells.

    Science.gov (United States)

    Ramos de Carvalho, J Emanuel; Verwoert, Milan T; Vogels, Ilse M C; Schipper-Krom, Sabine; Van Noorden, Cornelis J F; Reits, Eric A; Klaassen, Ingeborg; Schlingemann, Reinier O

    2017-10-27

    Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α7, β5, and β5i were assessed. Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H2O2-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β2 and β5i/β1 and reduced activity of β5/β1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells. © 2017 S. Karger AG, Basel.

  10. Identification and Characterisation of a Proteasome -

    DEFF Research Database (Denmark)

    Andersen, Katrine Mølgaard

    is responsible for the degradation of most intracellular proteins. To sustain its function, the proteasome is supported by a still increasing number of interacting proteins or co-factors. In the work presented here, two new proteasome interacting proteins are identified and characterised in humans...

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

    Science.gov (United States)

    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.

  12. Defective Proteasome Delivery of Polyubiquitinated Proteins by Ubiquilin-2 Proteins Containing ALS Mutations.

    Directory of Open Access Journals (Sweden)

    Lydia Chang

    Full Text Available Ubiquilin proteins facilitate delivery of ubiquitinated proteins to the proteasome for degradation. Interest in the proteins has been heightened by the discovery that gene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS. However, the mechanisms by which the mutations cause ALS are not known. Here we report on the underlying defect of ubiquilin-2 proteins containing ALS-linked mutations in affecting proteasome-mediated degradation. We found that overexpression of ubiquilin-2 proteins containing any one of five different ALS mutations slow degradation of Myc, a prototypic proteasome substrate. Examination of coprecipitating proteins indicated that the mutant proteins are generally capable of binding polyubiquitinated proteins, but defective in binding the proteasome. GST-pulldown studies revealed that many of the mutants bind weaker to the S5a subunit of the proteasome, compared with wild type (WT ubiquilin-2 protein. The results suggest the mutant proteins are unable to deliver their captured cargo to the proteasome for degradation, which presumably leads to toxicity. Quantification of cell death is consistent with this idea. Measurement of protein turnover further indicated the mutant proteins have longer half-lives than WT ubiquilin-2. Our studies provide novel insight into the mechanism by which ALS-linked mutations in UBQLN2 interfere with protein degradation.

  13. Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development.

    Directory of Open Access Journals (Sweden)

    Amin Al-Shami

    Full Text Available Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/- mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/- mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/- mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.

  14. Enhancement of 26S proteasome functionality connects oxidative stress and vascular endothelial inflammatory response in diabetes mellitus.

    Science.gov (United States)

    Liu, Hongtao; Yu, Shujie; Xu, Wenjia; Xu, Jian

    2012-09-01

    Although the connection of oxidative stress and inflammation has been long recognized in diabetes mellitus, the underlying mechanisms are not fully elucidated. This study defined the role of 26S proteasomes in promoting vascular inflammatory response in early diabetes mellitus. The 26S proteasome functionality, markers of autophagy, and unfolded protein response were assessed in (1) cultured 26S proteasome reporter cells and endothelial cells challenged with high glucose, (2) transgenic reporter (Ub(G76V)-green fluorescence protein) and wild-type (C57BL/6J) mice rendered diabetic, and (3) genetically diabetic (Akita and OVE26) mice. In glucose-challenged cells, and also in aortic, renal, and retinal tissues from diabetic mice, enhanced 26S proteasome functionality was observed, evidenced by augmentation of proteasome (chymotrypsin-like) activities and reduction in 26S proteasome reporter proteins, accompanied by increased nitrotyrosine-containing proteins. Also, whereas inhibitor of the nuclear factor κ-light-chain-enhancer of activated B cells α proteins were decreased, an increase was found in nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) nucleus translocation, which enhanced the NF-κB-mediated proinflammatory response, without affecting markers of autophagy or unfolded protein response. Importantly, the alterations were abolished by MG132 administration, small interfering RNA knockdown of PA700 (proteasome activator protein complex), or superoxide scavenging in vivo. Early hyperglycemia enhances 26S proteasome functionality, not autophagy or unfolded protein response, through peroxynitrite/superoxide-mediated PA700-dependent proteasomal activation, which elevates NF- ĸB-mediated endothelial inflammatory response in early diabetes mellitus.

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

    Directory of Open Access Journals (Sweden)

    Karl Andrew Rodriguez

    2014-11-01

    Full Text Available 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 2.24mg/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.

  16. Proteasome addiction defined in Ewing’s sarcoma is effectively targeted by a novel class of 19S proteasome inhibitors

    Science.gov (United States)

    Shukla, Neerav; Somwar, Romel; Smith, Roger S.; Ambati, Sri; Munoz, Stanley; Merchant, Melinda; D’Arcy, Padraig; Wang, Xin; Kobos, Rachel; Antczak, Christophe; Bhinder, Bhavneet; Shum, David; Radu, Constantin; Yang, Guangbin; Taylor, Barry S.; Ng, Charlotte K.Y.; Weigelt, Britta; Khodos, Inna; de Stanchina, Elisa; Reis-Filho, Jorge S.; Ouerfelli, Ouathek; Linder, Stig; Djaballah, Hakim; Ladanyi, Marc

    2017-01-01

    Ewing’s sarcoma (EWS) is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of EWS cells to provide the basis for the development of novel therapies, and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds which selectively inhibit growth of EWS cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in EWS cells also identified the proteasome as a node of vulnerability in EWS cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in EWS cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of EMS with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for EWS. PMID:27256563

  17. Use of proteasome inhibitors in anticancer therapy

    Directory of Open Access Journals (Sweden)

    Sara M. Schmitt

    2011-10-01

    Full Text Available The importance of the ubiquitin-proteasome pathway to cellular function has brought it to the forefront in the search for new anticancer therapies. The ubiquitin-proteasome pathway has proven promising in targeting various human cancers. The approval of the proteasome inhibitor bortezomib for clinical treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma has validated the ubiquitin-proteasome as a rational target. Bortezomib has shown positive results in clinical use but some toxicity and side effects, as well as resistance, have been observed, indicating that further development of novel, less toxic drugs is necessary. Because less toxic drugs are necessary and drug development can be expensive and time-consuming, using existing drugs that can target the ubiquitin-proteasome pathway in new applications, such as cancer therapy, may be effective in expediting the regulatory process and bringing new drugs to the clinic. Toward this goal, previously approved drugs, such as disulfiram, as well as natural compounds found in common foods, such as green tea polyphenol (--EGCG and the flavonoid apigenin, have been investigated for their possible proteasome inhibitory and cell death inducing abilities. These compounds proved quite promising in preclinical studies and have now moved into clinical trials, with preliminary results that are encouraging. In addition to targeting the catalytic activity of the proteasome pathway, upstream regulators, such as the 19S regulatory cap, as well as E1, E2, and E3, are now being investigated as potential drug targets. This review outlines the development of novel proteasome inhibitors from preclinical to clinical studies, highlighting their abilities to inhibit the tumor proteasome and induce apoptosis in several human cancers.

  18. Proteasome impairment by α-synuclein.

    Directory of Open Access Journals (Sweden)

    Lisa Zondler

    Full Text Available Parkinson's disease (PD is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients' midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V-GFP cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.

  19. Involvement of the ubiquitin-proteasome system in the expression of extracellular matrix genes in retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    J. Emanuel Ramos de Carvalho

    2018-03-01

    Full Text Available Emerging evidence suggests that dysfunction of the ubiquitin-proteasome system is involved in the pathogenesis of numerous senile degenerative diseases including retinal disorders. The aim of this study was to assess whether there is a link between proteasome regulation and retinal pigment epithelium (RPE-mediated expression of extracellular matrix genes. For this purpose, human retinal pigment epithelial cells (ARPE-19 were treated with different concentrations of transforming growth factor-β (TGFβ, connective tissue growth factor (CTGF, interferon-γ (IFNγ and the irreversible proteasome inhibitor epoxomicin. First, cytotoxicity and proliferation assays were carried out. The expression of proteasome-related genes and proteins was assessed and proteasome activity was determined. Then, expression of fibrosis-associated factors fibronectin (FN, fibronectin EDA domain (FN EDA, metalloproteinase-2 (MMP-2, tissue inhibitor of metalloproteinases-1 (TIMP-1 and peroxisome proliferator-associated receptor-γ (PPARγ was assessed. The proteasome inhibitor epoxomicin strongly arrested cell cycle progression and down-regulated TGFβ gene expression, which in turn was shown to induce expression of pro-fibrogenic genes in ARPE-19 cells. Furthermore, epoxomicin induced a directional shift in the balance between MMP-2 and TIMP-1 and was associated with down-regulation of transcription of extracellular matrix genes FN and FN-EDA and up-regulation of the anti-fibrogenic factor PPARγ. In addition, both CTGF and TGFβ were shown to affect expression of proteasome-associated mRNA and protein levels. Our results suggest a link between proteasome activity and pro-fibrogenic mechanisms in the RPE, which could imply a role for proteasome-modulating agents in the treatment of retinal disorders characterized by RPE-mediated fibrogenic responses.

  20. Recognition of Client Proteins by the Proteasome.

    Science.gov (United States)

    Yu, Houqing; Matouschek, Andreas

    2017-05-22

    The ubiquitin proteasome system controls the concentrations of regulatory proteins and removes damaged and misfolded proteins from cells. Proteins are targeted to the protease at the center of this system, the proteasome, by ubiquitin tags, but ubiquitin is also used as a signal in other cellular processes. Specificity is conferred by the size and structure of the ubiquitin tags, which are recognized by receptors associated with the different cellular processes. However, the ubiquitin code remains ambiguous, and the same ubiquitin tag can target different proteins to different fates. After binding substrate protein at the ubiquitin tag, the proteasome initiates degradation at a disordered region in the substrate. The proteasome has pronounced preferences for the initiation site, and its recognition represents a second component of the degradation signal.

  1. The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

    Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

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

    Directory of Open Access Journals (Sweden)

    Candida Vannini

    Full Text Available 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.

  4. mAChR-dependent decrease in proteasome activity in the gustatory cortex is necessary for novel taste learning.

    Science.gov (United States)

    Rosenberg, Tali; Elkobi, Alina; Rosenblum, Kobi

    2016-11-01

    Regulation of protein degradation via the ubiquitin proteasome system is crucial for normal learning and synaptic plasticity processes. While some studies reveal that increased proteasome degradation is necessary for different types of learning, others suggest the proteasome to be a negative regulator of plasticity. We aim to understand the molecular and cellular processes taking place in the gustatory cortex (GC), which underlie appetitive and aversive forms of taste learning. Previously, we have shown that N-methyl d-aspartic acid receptor (NMDAR)-dependent upregulation of proteasome activity 4h after novel taste learning is necessary for the association of novel taste with malaise and formation of conditioned taste aversion (CTA). Here, we first identify a correlative increase in proteasome activity in the GC immediately after novel taste learning and study the upstream and downstream effectors of this modulated proteasome activity. Interestingly, proteasome-mediated degradation was reduced in the GC, 20min after novel taste consumption in a muscarinic acetylcholine receptor (mAChR)-dependent and NMDAR-independent manner. This reduction in protein degradation led to an increased amount of p70 S6 kinase (p70S6k), which was abolished in the presence of mAChR antagonist scopolamine. Infusion of lactacystin, a proteasome inhibitor, to the GC precluded the amnestic effect of scopolamine. This study shows for the first time that following novel taste learning there is a cortical, mAChR-dependent reduced proteasome activity that enables the memory of taste familiarity. Moreover, inhibition of degradation in the GC attenuates novel taste learning and of p70 S6 kinase correlative increased expression. These results shed light on the complex regulation of protein synthesis and degradation machineries in the cortex following novel taste experience. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Mammalian 26S proteasomes remain intact during protein degradation

    DEFF Research Database (Denmark)

    Kriegenburg, Franziska; Seeger, Michael; Saeki, Yasushi

    2008-01-01

    It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)...

  6. Dss1 Is a 26S Proteasome Ubiquitin Receptor

    OpenAIRE

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

    2014-01-01

    Summary The ubiquitin-proteasome system is the major pathway for protein degradation in eukaryotic cells. Proteins to be degraded are conjugated to ubiquitin chains that act as recognition signals for the 26S proteasome. The proteasome subunits Rpn10 and Rpn13 are known to bind ubiquitin, but genetic and biochemical data suggest the existence of at least one other substrate receptor. Here, we show that the phylogenetically conserved proteasome subunit Dss1 (Sem1) binds ubiquitin chains linked...

  7. Molecular characterization of 26S proteasome regulatory subunit in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-17

    Jun 17, 2009 ... a T. verrucoum gene encoding a protein that belongs to. 26S proteasome family, which is hereby referred to as. Tv26S-Proteasome. Analysis of the amino acid sequence of this gene shows a significant homology with other eukaryotic 26S proteasome family such as those of T. rubrum (Naeimi et al., 2007), ...

  8. IFNγ-inducible proteasome components in immune responses

    NARCIS (Netherlands)

    de Graaf, N.

    2011-01-01

    Protein degradation results in the production of peptides that can be presented to CD8 T cells in MHC class I molecules on the cell surface. The proteasome is the main protease responsible for protein degradation. Especially the IFNγ-inducible proteasome components (proteasome activator PA28 and the

  9. The role of 19S proteasome associated deubiquitinases in activity-dependent hippocampal synaptic plasticity.

    Science.gov (United States)

    Yun, Di; Zhuang, Yinghan; Kreutz, Michael R; Behnisch, Thomas

    2018-01-31

    Posttranslational modification and degradation of proteins by the ubiquitin-proteasome system (UPS) is crucial to synaptic transmission. It is well established that 19S proteasome associated deubiquitinases (DUBs) reverse the process of ubiquitination by removing ubiquitin from their substrates. However, their potential contribution to hippocampal synaptic plasticity has not been addressed in detail. Here, we report that inhibition of the 19S proteasome associated DUBs, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14) by b-AP15 results in an accumulation of polyubiquitinated proteins and a reduction of monomeric ubiquitin without overt effects on 26S proteasome activity. b-AP15 led to a suppression of mTOR-p70S6K signaling and an increase in levels of p-p38 MAPK, two pathways essentially involved in establishing various forms of activity-dependent plasticity. Additionally, b-AP15 impaired the induction of late-phase long-term potentiation (L-LTP), induced the transformation of mGluR-mediated protein synthesis-independent long-term depression (early-LTD) to L-LTD and promoted heterosynaptic stabilization through synaptic tagging/capture (STC) in the hippocampal CA1 region of mice. The activity of 19S proteasome associated DUBs was also required for the enhancement of short-term potentiation (STP) induced by brain-derived neurotrophic factor (BDNF). Altogether, these results indicate an essential role of 19S proteasome associated DUBs in regulating activity-dependent hippocampal synaptic plasticity. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Targeting proteasomes with natural occurring compounds in cancer treatment.

    Science.gov (United States)

    Cecarini, V; Cuccioloni, M; Mozzicafreddo, M; Bonfili, L; Angeletti, M; Eleuteri, A M

    2011-03-01

    Aberrant cellular proliferation and compromised apoptotic pathways are hallmarks of cancer aggressiveness, and in this framework, the role of protein degradation machineries has been extensively dissected. Among proteases, the proteasome is unequivocally central in the intracellular regulation of both these processes, thus several proteasome-directed therapies have been investigated, aiming at controlling its activity and possibly restoring normal cell functions. Numerous studies reported proteasome inhibitors (both synthetic and natural occurring) to potently and selectively induce apoptosis in many types of cancer cells. In this review we discuss recent advances in proteasomal modulation by some natural occurring polyphenols, globally providing evidence of the proteasome role as therapeutic target in cancer treatment.

  11. A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster.

    Science.gov (United States)

    Pickering, Andrew M; Staab, Trisha A; Tower, John; Sieburth, Derek; Davies, Kelvin J A

    2013-02-15

    In mammalian cells, hydrogen peroxide (H(2)O(2))-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 H(2)O(2) 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.

  12. Native and aspirin-triggered lipoxins control innate immunity by inducing proteasomal degradation of TRAF6.

    Science.gov (United States)

    Machado, Fabiana S; Esper, Lísia; Dias, Alexandra; Madan, Rajat; Gu, YuanYuan; Hildeman, David; Serhan, Charles N; Karp, Christopher L; Aliberti, Júlio

    2008-05-12

    Innate immune signaling is critical for the development of protective immunity. Such signaling is, perforce, tightly controlled. Lipoxins (LXs) are eicosanoid mediators that play key counterregulatory roles during infection. The molecular mechanisms underlying LX-mediated control of innate immune signaling are of interest. In this study, we show that LX and aspirin (ASA)-triggered LX (ATL) inhibit innate immune signaling by inducing suppressor of cytokine signaling (SOCS) 2-dependent ubiquitinylation and proteasome-mediated degradation of TNF receptor-associated factor (TRAF) 2 and TRAF6, which are adaptor molecules that couple TNF and interleukin-1 receptor/Toll-like receptor family members to intracellular signaling events. LX-mediated degradation of TRAF6 inhibits proinflammatory cytokine production by dendritic cells. This restraint of innate immune signaling can be ablated by inhibition of proteasome function. In vivo, this leads to dysregulated immune responses, accompanied by increased mortality during infection. Proteasomal degradation of TRAF6 is a central mechanism underlying LX-driven immune counterregulation, and a hitherto unappreciated mechanism of action of ASA. These findings suggest a new molecular target for drug development for diseases marked by dysregulated inflammatory responses.

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

    Directory of Open Access Journals (Sweden)

    Nandita Sahana

    Full Text Available 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.

  14. Inhibition of proteasome activity by the dietary flavonoid apigenin is associated with growth inhibition in cultured breast cancer cells and xenografts.

    Science.gov (United States)

    Chen, Di; Landis-Piwowar, Kristin R; Chen, Marina S; Dou, Q Ping

    2007-01-01

    Proteasome inhibition is an attractive approach to anticancer therapy and may have relevancy in breast cancer treatment. Natural products, such as dietary flavonoids, have been suggested as natural proteasome inhibitors with potential use for cancer prevention and therapeutics. We previously reported that apigenin, a flavonoid widely distributed in many fruits and vegetables, can inhibit proteasome activity and can induce apoptosis in cultured leukemia Jurkat T cells. Whether apigenin has proteasome-inhibitory activity in the highly metastatic human breast MDA-MB-231 cells and xenografts,however, is unknown. MDA-MB-231 breast cancer cell cultures and xenografts were treated with apigenin, followed by measurement of reduced cellular viability/proliferation,proteasome inhibition, and apoptosis induction. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity, by ubiquitinated proteins, and by accumulation of proteasome target proteins in extracts of the treated cells or tumors. Apoptotic cell death was measured by caspase-3/caspase-7 activation, poly(ADP-ribose) polymerase cleavage, and immunohistochemistry for terminal nucleotidyltransferase-mediated nick end labeling positivity. We report for the first time that apigenin inhibits the proteasomal chymotrypsin-like activity and induces apoptosis not only in cultured MDA-MB-231 cells but also in MDA-MB-231 xenografts. Furthermore, while apigenin has antibreast tumor activity, no apparent toxicity to the tested animals was observed. We have shown that apigenin is an effective proteasome inhibitor in cultured breast cancer cells and in breast cancer xenografts. Furthermore, apigenin induces apoptotic cell death in human breast cancer cells and exhibits anticancer activities in tumors. The results suggest its potential benefits in breast cancer prevention and treatment.

  15. Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

    Science.gov (United States)

    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. PMID:23300704

  16. Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

    Science.gov (United States)

    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. Copyright © 2013 Federation of European Biochemical Societies. All rights reserved.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. Keratin 6a reorganization for ubiquitin-proteasomal processing is a direct antimicrobial response.

    Science.gov (United States)

    Chan, Jonathan K L; Yuen, Don; Too, Priscilla Hiu-Mei; Sun, Yan; Willard, Belinda; Man, David; Tam, Connie

    2017-11-30

    Skin and mucosal epithelia deploy antimicrobial peptides (AMPs) to eliminate harmful microbes. We reported that the intermediate filament keratin 6a (K6a) is constitutively processed into antimicrobial fragments in corneal epithelial cells. In this study, we show that K6a network remodeling is a host defense response that directly up-regulates production of keratin-derived AMPs (KAMPs) by the ubiquitin-proteasome system (UPS). Bacterial ligands trigger K6a phosphorylation at S19, S22, S37, and S60, leading to network disassembly. Mutagenic analysis of K6a confirmed that the site-specific phosphorylation augmented its solubility. K6a in the cytosol is ubiquitinated by cullin-RING E3 ligases for subsequent proteasomal processing. Without an appreciable increase in K6a gene expression and proteasome activity, a higher level of cytosolic K6a results in enhanced KAMP production. Although proteasome-mediated proteolysis is known to produce antigenic peptides in adaptive immunity, our findings demonstrate its new role in producing AMPs for innate immune defense. Manipulating K6a phosphorylation or UPS activity may provide opportunities to harness the innate immunity of epithelia against infection. © 2018 Chan et al.

  20. Uch2/Uch37 is the major deubiquitinating enzyme associated with the 26S proteasome in fission yeast

    DEFF Research Database (Denmark)

    Stone, Miranda; Hartmann-Petersen, Rasmus; Seeger, Michael

    2004-01-01

    Conjugation of proteins to ubiquitin plays a central role for a number of cellular processes including endocytosis, DNA repair and degradation by the 26S proteasome. However, ubiquitination is reversible as a number of deubiquitinating enzymes mediate the disassembly of ubiquitin-protein conjugat...

  1. Primary proteasome inhibition results in cardiac dysfunction

    Science.gov (United States)

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

    2013-01-01

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

  2. Proteasome inhibitors therapeutic strategies for cancer.

    Science.gov (United States)

    D'Alessandro, Annamaria; Pieroni, Luisa; Ronci, Maurizio; D'Aguanno, Simona; Federici, Giorgio; Urbani, Andrea

    2009-01-01

    Aberrations in the Ubiquitin-Proteasome System (UPS) have been recently connected to the pathogenesis of several human protein degradation disorders (e.g., cancer and neurodegenerative diseases), so that proteasome is now considered an important target for drug discovery. Small molecules able to inhibit and modulate UPS have been, in fact, described as novel tools for a new approach in anti-cancer therapy. In particular Proteasome Inhibitors (PIs), blocking activation of nuclear factor-kappa B (NF-kB), trigger a decreased cellular proliferation and angiogenic cytokine production, induce cell death and inhibit tumor cell adhesion to stroma. Furthermore, several studies have demonstrated that PIs potentiate the activity of other anti-cancer treatment, in part by down-regulating chemoresistance pathways. Therefore pharmacologic, preclinical, and clinical data suggested the use of PIs in anticancer strategies, for their potential therapeutic relevance in the treatment of cancer and inflammatory-related diseases. This review focuses on recent advances in the development of PIs anticancer agents highlighting both novel patented compounds and novel therapeutic protocol of intervention.

  3. The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

    Science.gov (United States)

    Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G

    2011-01-01

    Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.

  4. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization....... From the perspective of mediatization research, the most important effect of the media stems from their embeddedness in culture and society....

  5. RNAi Screening Reveals Proteasome- and Cullin3-Dependent Stages in Vaccinia Virus Infection

    Directory of Open Access Journals (Sweden)

    Jason Mercer

    2012-10-01

    Full Text Available A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection. Validation and analysis of clustered hits revealed previously unknown processes during virus entry, including a mechanism for genome uncoating. Viral core proteins were found to be already ubiquitinated during virus assembly. After entering the cytosol of an uninfected cell, the viral DNA was released from the core through the activity of the cell’s proteasomes. Next, a Cullin3-based ubiquitin ligase mediated a further round of ubiquitination and proteasome action. This was needed in order to initiate viral DNA replication. The results accentuate the value of large-scale RNAi screens in providing directions for detailed cell biological investigation of complex pathways. The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sanghwan; Hur, Eu-gene [Yeungnam University, College of Pharmacy, Gyeongsan-si, Gyeongsangbuk-do 712‐749 (Korea, Republic of); Ryoo, In-geun; Jung, Kyeong-Ah [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420‐743 (Korea, Republic of); Kwak, Jiyeon [Inha University, College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon 402‐751 (Korea, Republic of); Kwak, Mi-Kyoung, E-mail: mkwak@catholic.ac.kr [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420‐743 (Korea, Republic of)

    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

  7. Activating Cell Death Ligand Signaling Through Proteasome Inhibition

    Science.gov (United States)

    2009-05-01

    the proteasome inhibitor bort - ezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer. J Clin Oncol...Effects of the proteasome inhibitor bort - ezomib on osteolytic human prostate cancer cell metastases. Prostate cancer and prostatic diseases 2005; 8:327

  8. Proteasomes from structure to function: perspectives from Archaea.

    Science.gov (United States)

    Maupin-Furlow, Julie A; Humbard, Matthew A; Kirkland, P Aaron; Li, Wei; Reuter, Christopher J; Wright, Amy J; Zhou, G

    2006-01-01

    Insight into the world of proteolysis has expanded considerably over the past decade. Energy-dependent proteases, such as the proteasome, are no longer viewed as nonspecific degradative enzymes associated solely with protein catabolism but are intimately involved in controlling biological processes that span life to death. The proteasome maintains this exquisite control by catalyzing the precisely timed and rapid turnover of key regulatory proteins. Proteasomes also interplay with chaperones to ensure protein quality and to readjust the composition of the proteome following stress. Archaea encode proteasomes that are highly related to those of eukaryotes in basic structure and function. Investigations of archaeal proteasomes coupled with those of eukaryotes has greatly facilitated our understanding of the molecular mechanisms that govern regulated protein degradation by this elaborate nanocompartmentalized machine.

  9. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Li, E-mail: luli7300@126.com [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao [Department of Anatomy, Shanxi Medical University, Taiyuan 030001 (China); Li, Ang [Department of Anatomy, University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Xiao, Zhi-Cheng, E-mail: zhicheng.xiao@monash.edu [Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical College, Kunming 650031 (China); Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Melbourne 3800 (Australia)

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  10. Alterations of the intracellular peptidome in response to the proteasome inhibitor bortezomib.

    Directory of Open Access Journals (Sweden)

    Julia S Gelman

    Full Text Available Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours, and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.

  11. Bortezomib Warhead-Switch Confers Dual Activity against Mycobacterial Caseinolytic Protease and Proteasome and Selectivity against Human Proteasome

    Directory of Open Access Journals (Sweden)

    Thomas Dick

    2017-04-01

    Full Text Available Mycobacteria harbor two main degradative proteolytic machineries, the caseinolytic protease ClpP1P2 and a proteasome. We recently showed that Bortezomib inhibits ClpP1P2 and exhibits whole cell activity against Mycobacterium tuberculosis. Bortezomib, a dipeptide with a boronic acid warhead, is a human proteasome inhibitor approved for cancer therapy. The boronic acid warhead of the compound has been shown to drive potency against both the human proteasome and ClpP1P2 protease. Selectivity for the bacterial ClpP1P2 protease over the human proteasome is lacking but needs to be achieved to move this new anti-tuberculosis lead forward. In this study we explored whether an alternative warhead could influence Bortezomib's selectivity. We synthesized an analog containing a chloromethyl ketone instead of the boronic acid warhead and determined potencies against the bacterial and human enzymes. Surprisingly, the analog retained activity against mycobacterial ClpP1P2 and was active against the mycobacterial proteasome, but was devoid of activity against the human proteasome. Interrogation of a set of chloromethyl ketone peptides identified three additional compounds similarly inhibiting both ClpP1P2 and the proteasome in the bacteria while leaving the human proteasome untouched. Finally, we showed that these compounds display bactericidal activity against M. tuberculosis with cytotoxicity ranging from acceptable to undetectable. These results suggest that selectivity over the human proteasome is achievable. Selectivity, together with dual-targeting of mycobacterial ClpP1P2 and proteasome makes this new scaffold an attractive starting point for optimization.

  12. Concentration-dependent Effects of Proteasomal Inhibition on tau Processing in a Cellular Model of Tauopathy

    Science.gov (United States)

    Hamano, Tadanori; Gendron, Tania F.; Ko, Li-wen; Yen, Shu-Hui

    2009-01-01

    Tauopathies are characterized by accumulation of filamentous tau aggregates. These aggregates can be recapitulated in transfectant M1C overproducing wild-type human brain tau 4R0N via the tetracycline off (TetOff) inducible expression mechanism. To determine the contribution of proteasomes to tau degradation and aggregation, we exposed M1C cells to epoxomicin (Epx; 2-50 nM) or MG132 (0.5 μM) on the 3rd or 4th day of a 5-day TetOff induction and demonstrated a reduction of proteasomal activity. Cultures treated with 2 nM Exp showed accumulation of full-length tau without affecting ubiquitin and β-catenin immunoblotting profiles. In contrast, cells treated with 10, 50 nM Epx or MG132 displayed changes in ubiquitin or β-catenin immunoblotting profiles and extensive tau degradation/truncation. The increase of tau degradation/truncation was accompanied with accumulation of oligomers and sarkosyl-insoluble aggregates of tau, augmented thioflavin-binding and activation of caspases and calpains. Truncated, oligomeric and sarkosyl-insoluble tau derivatives appeared with caspase-specific cleavage and their production was diminished when pretreated with a pan-caspase inhibitor. The results demonstrate (i) a dose-dependent, opposite effect of proteasome inhibition on tau processing, (ii) the participation of proteasome-dependent, ubiquitination-independent mechanisms in tau degradation and aggregation, and (iii) the promotion of tau aggregation by caspase-mediated tau degradation/truncation. PMID:19636403

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

    Directory of Open Access Journals (Sweden)

    Anice Moumen

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

  14. An extract of Artemisia dracunculus L. inhibits ubiquitin-proteasome activity and preserves skeletal muscle mass in a murine model of diabetes.

    Directory of Open Access Journals (Sweden)

    Heather Kirk-Ballard

    Full Text Available Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasome-dependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011 improves insulin action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-A(y murine model of type 2 diabetes were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces Atrogin-1 and MuRF-1 protein levels. In the KK-A(y murine model of type 2 diabetes, skeletal muscle ubiquitylation and proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in the preservation of muscle mass in type 2 diabetes.

  15. Proteasome expression and activity in cancer and cancer stem cells.

    Science.gov (United States)

    Voutsadakis, Ioannis A

    2017-03-01

    Proteasome is a multi-protein organelle that participates in cellular proteostasis by destroying damaged or short-lived proteins in an organized manner guided by the ubiquitination signal. By being in a central place in the cellular protein complement homeostasis, proteasome is involved in virtually all cell processes including decisions on cell survival or death, cell cycle, and differentiation. These processes are important also in cancer, and thus, the proteasome is an important regulator of carcinogenesis. Cancers include a variety of cells which, according to the cancer stem cell theory, descend from a small percentage of cancer stem cells, alternatively termed tumor-initiating cells. These cells constitute the subsets that have the ability to propagate the whole variety of cancer and repopulate tumors after cytostatic therapies. Proteasome plays a role in cellular processes in cancer stem cells, but it has been found to have a decreased function in them compared to the rest of cancer cells. This article will discuss the transcriptional regulation of proteasome sub-unit proteins in cancer and in particular cancer stem cells and the relationship of the proteasome with the pluripotency that is the defining characteristic of stem cells. Therapeutic opportunities that present from the understanding of the proteasome role will also be discussed.

  16. Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease.

    Science.gov (United States)

    Liu, Yanying; Hettinger, Casey L; Zhang, Dong; Rezvani, Khosrow; Wang, Xuejun; Wang, Hongmin

    2014-05-01

    The ubiquitin proteasome system (UPS) is impaired in Huntington's disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntington's disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane-mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntington's disease and other intractable disorders. Accumulation of mutant huntingtin (mHtt) protein causes Huntington's disease (HD). Sulforaphane (SFN), a naturally occurring compound, increased proteasome and autophagy activities in vivo and enhanced mHtt turnover and cell survival in HD cell models. SFN-mediated mHtt degradation is mainly through the proteasome pathway. These data suggest that SFN can be a therapeutic reagent for treating HD and other intractable disorders. © 2014 International Society for Neurochemistry.

  17. IFNγ-inducible proteasome components in immune responses

    OpenAIRE

    de Graaf, N.

    2011-01-01

    Protein degradation results in the production of peptides that can be presented to CD8 T cells in MHC class I molecules on the cell surface. The proteasome is the main protease responsible for protein degradation. Especially the IFNγ-inducible proteasome components (proteasome activator PA28 and the immunoproteasome subunits LMP2, LMP7 and MECL-1) play a central role in MHC I antigen processing, as well as in other aspects of immune responses. We studied the function of IFNγ-inducible proteas...

  18. Patented small molecule inhibitors in the ubiquitin proteasome system

    Directory of Open Access Journals (Sweden)

    Colland Frédéric

    2007-11-01

    Full Text Available Abstract Deregulation of the ubiquitin proteasome system (UPS has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. The recent approval of the proteasome inhibitor Velcade® (bortezomib for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. We review here new patented proteasome inhibitors and patented small molecule inhibitors targeting more specific UPS components, such as E3 ubiquitin ligases and deubiquitylating enzymes. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com.

  19. Detection of antibodies to the 20s proteasome by ELISA

    DEFF Research Database (Denmark)

    Jørgensen, Karin Meinike; Frederiksen, Jette Lautrup; Nielsen, Christoffer Tandrup

    2013-01-01

    The presence of antibodies against the 20S proteasome has been correlated with diseases like multiple sclerosis (MS) and systemic lupus erythematosus (SLE) but no definite association has been established. In order to investigate this further, we optimized an ELISA for proteasome antibodies...... and applied it to test a total of 324 serum and plasma samples from MS patients, SLE patients, and healthy controls. Our results yield a functional and reliable assay but no correlation between the amount of proteasome antibodies present and the development of MS or SLE could be established....

  20. It Is All about (Ubiquitin: Role of Altered Ubiquitin-Proteasome System and UCHL1 in Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Antonella Tramutola

    2016-01-01

    Full Text Available 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.

  1. Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway

    Directory of Open Access Journals (Sweden)

    Raúl Sánchez-Lanzas

    2017-12-01

    Full Text Available Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7 by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag or C-terminus (V5 of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag or C-terminus (V5 were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging. While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell.

  2. Proteasome inhibitors as experimental therapeutics of autoimmune diseases

    NARCIS (Netherlands)

    Verbrugge, C.S.E.; Scheper, R.J.; Lems, W.F.; de Gruijl, T.D.; Jansen, G.

    2015-01-01

    Current treatment strategies for rheumatoid arthritis (RA) consisting of disease-modifying anti-rheumatic drugs or biological agents are not always effective, hence driving the demand for new experimental therapeutics. The antiproliferative capacity of proteasome inhibitors (PIs) has received

  3. Reversible 26S Proteasome Disassembly upon Mitochondrial Stress

    Directory of Open Access Journals (Sweden)

    Nurit Livnat-Levanon

    2014-06-01

    Full Text Available In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mitochondrial networks fragment, and cellular reactive oxygen species (ROS levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent substrate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S proteasome activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging.

  4. Changes in proteasome structure and function caused by HAMLET in tumor cells.

    Science.gov (United States)

    Gustafsson, Lotta; Aits, Sonja; Onnerfjord, Patrik; Trulsson, Maria; Storm, Petter; Svanborg, Catharina

    2009-01-01

    Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

    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.

  6. Proteasome inhibitors: recent advances and new perspectives in medicinal chemistry.

    Science.gov (United States)

    Genin, E; Reboud-Ravaux, M; Vidal, J

    2010-01-01

    The search for proteasome inhibitors began fifteen years ago. These inhibitors proved to be powerful tools for investigating many important cellular processes regulated by the ubiquitin-proteasome pathway. Targeting the proteasome pathway can also lead to new treatments for disorders like cancer, muscular dystrophies, inflammation and immune diseases. This is already true for cancer; the FDA approved bortezomib, a potent proteasome inhibitor, for treating multiple myeloma in 2003, and mantle cell lymphoma in 2006. The chemical structures identified in some of the early proteasome inhibitors have led to the development of new anti-cancer drugs (CEP-18770, Carfilzomib, NPI-0052). All these molecules are covalent bonding inhibitors that react with the catalytic Thr1-O(gamma) of the three types of active site. This review covers recent developments in medicinal chemistry of natural and synthetic proteasome inhibitors. Advances in non-covalent inhibitors that have no reactive group will be highlighted as they should minimize side-effects. New structures and new modes of action have been recently identified that open the door to new drug candidates for treating a range of diseases.

  7. A mammalian nervous system-specific plasma membrane proteasome complex that modulates neuronal function

    Science.gov (United States)

    Ramachandran, Kapil V.; Margolis, Seth S.

    2017-01-01

    In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It remains unclear how proteasomes can acutely regulate such processes, as this is inconsistent with their canonical role in proteostasis. Here, we made the discovery of a mammalian nervous system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is tightly associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of this membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked extracellular peptide production and attenuated neuronal activity-induced calcium signaling. Moreover, membrane proteasome-derived peptides are sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes primarily function to maintain proteostasis, and highlight a form of neuronal communication through a membrane proteasome complex. PMID:28287632

  8. The homeodomain transcription factor Hoxa2 interacts with and promotes the proteasomal degradation of the E3 ubiquitin protein ligase RCHY1.

    Directory of Open Access Journals (Sweden)

    Isabelle Bergiers

    Full Text Available Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2, an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53.

  9. Pale body-like inclusion formation and neurodegeneration following depletion of 26S proteasomes in mouse brain neurones are independent of α-synuclein.

    Directory of Open Access Journals (Sweden)

    Simon M L Paine

    Full Text Available Parkinson's disease (PD is characterized by the progressive degeneration of substantia nigra pars compacta (SNpc dopaminergic neurones and the formation of Lewy bodies (LB in a proportion of the remaining neurones. α-synuclein is the main component of LB, but the pathological mechanisms that lead to neurodegeneration associated with LB formation remain unclear. Three pivotal elements have emerged in the development of PD: α-synuclein, mitochondria and protein degradation systems. We previously reported a unique model, created by conditional genetic depletion of 26S proteasomes in the SNpc of mice, which mechanistically links these three elements with the neuropathology of PD: progressive neurodegeneration and intraneuronal inclusion formation. Using this model, we tested the hypothesis that α-synuclein was essential for the formation of inclusions and neurodegeneration caused by 26S proteasomal depletion. We found that both of these processes were independent of α-synuclein. This provides an important insight into the relationship between the proteasome, α-synuclein, inclusion formation and neurodegeneration. We also show that the autophagy-lysosomal pathway is not activated in 26S proteasome-depleted neurones. This leads us to suggest that the paranuclear accumulation of mitochondria in inclusions in our model may reflect a role for the ubiquitin proteasome system in mitochondrial homeostasis and that neurodegeneration may be mediated through mitochondrial factors linked to inclusion biogenesis.

  10. Proteasome Activators, PA28α and PA28β, Govern Development of Microvascular Injury in Diabetic Nephropathy and Retinopathy

    Directory of Open Access Journals (Sweden)

    Saeed Yadranji Aghdam

    2016-01-01

    Full Text Available Diabetic nephropathy (DN and diabetic retinopathy (DR are major complications of type 1 and type 2 diabetes. DN and DR are mainly caused by injury to the perivascular supporting cells, the mesangial cells within the glomerulus, and the pericytes in the retina. The genes and molecular mechanisms predisposing retinal and glomerular pericytes to diabetic injury are poorly characterized. In this study, the genetic deletion of proteasome activator genes, PA28α and PA28β genes, protected the diabetic mice in the experimental STZ-induced diabetes model against renal injury and retinal microvascular injury and prolonged their survival compared with wild type STZ diabetic mice. The improved wellbeing and reduced renal damage was associated with diminished expression of Osteopontin (OPN and Monocyte Chemoattractant Protein-1 (MCP-1 in the glomeruli of STZ-injected PA28α/PA28β double knockout (Pa28αβDKO mice and also in cultured mesangial cells and retinal pericytes isolated from Pa28αβDKO mice that were grown in high glucose. The mesangial PA28-mediated expression of OPN under high glucose conditions was suppressed by peptides capable of inhibiting the binding of PA28 to the 20S proteasome. Collectively, our findings demonstrate that diabetic hyperglycemia promotes PA28-mediated alteration of proteasome activity in vulnerable perivascular cells resulting in microvascular injury and development of DN and DR.

  11. Changes of the proteasomal system during the aging process.

    Science.gov (United States)

    Baraibar, Martin A; Friguet, Bertrand

    2012-01-01

    Accumulation of oxidized and damaged proteins is a hallmark of the aging process in different organs and tissues. Intracellular protein degradation is normally the most efficient mechanism to prevent toxicity associated with the accumulation of altered proteins without affecting the cellular reserves of amino acids. Protein degradation by the proteasomal system is a key process for the maintenance of cellular protein homeostasis and has come into the focus of aging research during the last decade. During the last few years, several lines of evidence have indicated that proteasome function is impaired during aging, suggesting that this decreased activity might be causally related to the aging process and the occurrence of age-associated diseases. This chapter reviews the proteasome status in organs, tissues, cells, and model organisms during aging as well as the molecular mechanisms involved in the age-related decline of proteasome function. Finally, interventions aimed at rejuvenating proteasome function as a potential antiaging strategy are discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Milly M Choy

    2015-11-01

    Full Text Available 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.

  13. Association of proteasomal activity with metastasis in luminal breast cancer

    Science.gov (United States)

    Shashova, E. E.; Fesik, E. A.; Doroshenko, A. V.

    2017-09-01

    Chimotrypsin-like (ChTL) and caspase-like (CL) proteasomal activities were investigated in different variants of the tumor progression of luminal breast cancer. Patients with primary luminal breast cancer (n = 123) in stage T1-3N0-2M0 who had not received neoadjuvant treatment were included in this study. Proteasome ChTL and CL activities were determined in the samples of tumor and adjacent tissues. The coefficients of chymotrypsin-like (kChTL) and caspase-like (kCL) proteasome activity were also calculated as the ratio of the corresponding activity in the tumor tissue to activity in the adjacent tissue. ChTL, CL, kChTL and kCL in the tissues of luminal A and B breast cancer with lymphogenic metastasis were compared, and their association with hematogenous metastasis was evaluated. On the one hand, CL activity of proteasomes increased in luminal A breast cancer with extensive lymphogenic metastasis (N2), on the other hand it decreased in the luminal B subtype of cancer. The ratio of proteasomal activity in the tumor and adjacent tissues plays a significant role in the hematogenic pathway of breast cancer progression and is associated with poor metastatic-free survival.

  14. Proteomic remodeling of proteasome in right heart failure.

    Science.gov (United States)

    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. © 2013.

  15. Degradation of pro-insulin-receptor proteins by proteasomes.

    Science.gov (United States)

    Cruz, Miguel; Velasco, Eduardo; Kumate, Jesús

    2004-01-01

    Type-2 diabetes is characterized by hyperinsulinemia, peripheral insulin resistance, and diminished tyrosine phosphorylation activity. It has been recently shown that proteasomes are implicated in the degradation of the insulin receptor substrate-1 (IRS-1) but not in that of the insulin receptor (IR). However, it is unknown whether proteasomes are involved in pro-IR degradation. We used CHO-IR and the 3T3-L1 cells treated with insulin at different concentrations and compared the proteasome activity of IRS-1, IR, and pro-IR degradation either in presence or in absence of lactacystin. A total of 100 nM of insulin allowed degradation of IRS-1 after 6 h of incubation. At 1,000 nM of insulin, pro-IR degradation began at 1 h of incubation, similar to IRS-1 degradation. Surprisingly, at a higher concentration (10 microM) of insulin, a drastic decrease of proteins was observed from the first minute of incubation. This activity was blocked by lactacystin, a specific proteasome inhibitor. According to these results, we propose that pro-IR is degraded by proteasomes.

  16. Cardiac protection by preconditioning is generated via an iron-signal created by proteasomal degradation of iron proteins.

    Directory of Open Access Journals (Sweden)

    Baruch E Bulvik

    Full Text Available Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC was shown to be mediated by a transient 'iron-signal' that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this 'iron signal' and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min ('delay' prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the 'delay'. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the 'iron-signal' by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the 'delay'. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

  17. A novel link between the proteasome pathway and the signal transduction pathway of the Bone Morphogenetic Proteins (BMPs

    Directory of Open Access Journals (Sweden)

    Kim Richard H

    2002-06-01

    Full Text Available Abstract Background The intracellular signaling events of the Bone Morphogenetic Proteins (BMPs involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood. Results Here we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome β subunit HsN3 and the ornithine decarboxylase antizyme (Az. The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses. Conclusions Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

  18. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kee K.; Adelstein, Robert S.; Kawamoto, Sachiyo, E-mail: kawamots@mail.nih.gov

    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.

  19. Regulation of serum response factor-dependent gene expression by proteasome inhibitors.

    Science.gov (United States)

    Sandbo, Nathan; Qin, Yimin; Taurin, Sebastien; Hogarth, D Kyle; Kreutz, Barry; Dulin, Nickolai O

    2005-03-01

    Serum response factor (SRF) is activated by contractile and hypertrophic agonists, such as endothelin-1 (ET1) to stimulate expression of cytoskeletal proteins in vascular smooth muscle cells (VSMCs). While studying the regulation of smooth muscle alpha-actin (SMA) expression at the level of protein stability, we discovered that inhibition of proteasome-dependent protein degradation by N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) or lactacystin (LC) did not enhance the levels of SMA, but, unexpectedly, attenuated SMA expression in response to ET1, without affecting the viability of VSMCs. Down-regulation of SMA protein by MG132 or LC occurred at the level of SMA transcription and via the inhibition of SRF activity. By contrast, MG132 and LC potentiated the activity of activator protein-1 transcription factor. Regulation of SRF by MG132 was not related to inhibition of nuclear factor-kappaB, an established target of proteasome inhibitors, and was not mediated by protein kinase A, a powerful regulator of SRF activity. Signaling studies indicate that inhibition of ET1-induced SRF activity by MG132 occurs at the level downstream of heterotrimeric G proteins Gq/11 and G13, of small GTPase RhoA, and of actin dynamics but at the level of SRF-DNA binding. MG132 treatment did not result in ubiquitination or accumulation of SRF. By contrast, the levels of c-Jun were rapidly increased upon incubation of cells with MG132, and ectopic overexpression of c-Jun mimicked the effect of MG132 on SRF activity. Together, these data suggest that inhibition of proteasome results in down-regulation of SMA expression via up-regulation of c-Jun and repression of SRF activity at the level of DNA binding.

  20. Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS).

    Science.gov (United States)

    Keil, Jeffrey M; Shen, Zhouxin; Briggs, Steven P; Patrick, Gentry N

    2010-10-18

    The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca(2+) entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3's), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca(2+) homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

  1. Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS.

    Directory of Open Access Journals (Sweden)

    Jeffrey M Keil

    2010-10-01

    Full Text Available The ubiquitin proteasome system (UPS mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1. STIM1 is as an endoplasmic reticulum (ER calcium sensor that has been shown to regulate store-operated Ca(2+ entry (SOCE. We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3's, an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca(2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

  2. Bioinformatic analysis of functional differences between the immunoproteasome and the constitutive proteasome

    NARCIS (Netherlands)

    Kesmir, C.; Noort, V. van; Boer, R.J. de; Hogeweg, P.

    2003-01-01

    Intracellular proteins are degraded largely by proteasomes. In cells stimulated with gamma interferon, the active proteasome subunits are replaced by "immuno" subunits that form immunoproteasomes. Phylogenetic analysis of the immunosubunits has revealed that they evolve faster than their

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

    Directory of Open Access Journals (Sweden)

    Benxu Cheng

    2016-01-01

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

  4. The possible role of the ubiquitin proteasome system in the development of atherosclerosis in diabetes

    Directory of Open Access Journals (Sweden)

    sasso Ferdinando

    2007-10-01

    -resistance, post-prandial hyperglycemia and chronic hyperglycemia play a role in the atherosclerotic process and may require intervention 67. Moreover, it is important to recognize that these risk factors frequently "cluster" inindividual patients and possibly interact with each other, favouring the atherosclerosis progression toward plaque instability. Thus, a fundamental question is, "which is the common soil hypothesis that may unifying the burden of all these factors on atherosclerosis of diabetic patients? Because evidences suggest that insulin-resistance, diabetes and CHD share in common a deregulation of ubiquitin-proteasome system (UPS, the major pathway for nonlysosomal intracellular protein degradation in eucaryotic cells 89, in this review ubiquitin-proteasome deregulation is proposed as the common persistent pathogenic factor mediating the initial stage of the atherosclerosis as well as the progression to complicated plaque in diabetic patients.

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

    Science.gov (United States)

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

    2013-07-05

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. A heterodimeric complex that promotes the assembly of mammalian 20S proteasomes

    DEFF Research Database (Denmark)

    Hirano, Yoko; Hendil, Klavs B.; Yashiroda, Hideki

    2005-01-01

    The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells 1, 2 . It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes 3 . The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four...

  7. Stressing the ubiquitin-proteasome system without 20S proteolytic inhibition selectively kills cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Ravi K Anchoori

    Full Text Available Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

  8. Hijacking of the Ubiquitin/Proteasome Pathway by the HIV Auxiliary Proteins

    Directory of Open Access Journals (Sweden)

    Tanja Seissler

    2017-10-01

    Full Text Available The ubiquitin-proteasome system (UPS ensures regulation of the protein pool in the cell by ubiquitination of proteins followed by their degradation by the proteasome. It plays a central role in the cell under normal physiological conditions as well as during viral infections. On the one hand, the UPS can be used by the cell to degrade viral proteins, thereby restricting the viral infection. On the other hand, it can also be subverted by the virus to its own advantage, notably to induce degradation of cellular restriction factors. This makes the UPS a central player in viral restriction and counter-restriction. In this respect, the human immunodeficiency viruses (HIV-1 and 2 represent excellent examples. Indeed, many steps of the HIV life cycle are restricted by cellular proteins, some of which are themselves components of the UPS. However, HIV itself hijacks the UPS to mediate defense against several cellular restriction factors. For example, the HIV auxiliary proteins Vif, Vpx and Vpu counteract specific restriction factors by the recruitment of cellular UPS components. In this review, we describe the interplay between HIV and the UPS to illustrate its role in the restriction of viral infections and its hijacking by viral proteins for counter-restriction.

  9. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development.

    Science.gov (United States)

    Kim, Kee K; Adelstein, Robert S; Kawamoto, Sachiyo

    2014-08-08

    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. Published by Elsevier Inc.

  10. Ubiquitin-Proteasome System in ABA Signaling: From Perception to Action.

    Science.gov (United States)

    Yu, Feifei; Wu, Yaorong; Xie, Qi

    2016-01-04

    Protein post-translational modification (PTM) by ubiquitination has been observed during many aspects of plant growth, development, and stress responses. The ubiquitin-proteasome system precisely regulates phytohormone signaling by affecting protein activity, localization, assembly, and interaction ability. Abscisic acid (ABA) is a major phytohormone, and plays important roles in plants under normal or stressed growth conditions. The ABA signaling pathway is composed of phosphatases, kinases, transcription factors, and membrane ion channels. It has been reported that multiple ABA signaling transducers are subjected to the regulations by ubiquitination. In particular, recent studies have identified different types of E3 ligases that mediate ubiquitination of ABA receptors in different cell compartments. This review focuses on modulation of these components by monoubiquitination or polyubiquitination that occurs in the plasma membrane, endomembranes, and from the cytosol to the nucleus; this implies the existence of retrograde and trafficking processes that are regulated by ubiquitination in ABA signaling. A number of single-unit E3 ligases, components of multi-subunit E3 ligases, E2s, and specific subunits of the 26S proteasome involved in ABA signal regulation are discussed. Dissecting the precise functions of ubiquitination in the ABA pathway may help us understand key factors in the signaling of other phytohormones regulated by ubiquitination and other types of PTMs. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  11. Dynamic Behavior of the RNA Polymerase II and the Ubiquitin Proteasome System During the Neuronal DNA Damage Response to Ionizing Radiation.

    Science.gov (United States)

    Casafont, Iñigo; Palanca, Ana; Lafarga, Vanesa; Mata-Garrido, Jorge; Berciano, Maria T; Lafarga, Miguel

    2016-12-01

    Neurons are highly vulnerable to genotoxic agents. To restore genome integrity upon DNA lesions, neurons trigger a DNA damage response (DDR) that requires chromatin modifications and transcriptional silencing at DNA damage sites. To study the reorganization of the active RNA polymerase II (Pol II), which transcribes all mRNA-encoding genes, and the participation of the ubiquitin-proteasome system (UPS) in the neuronal DDR, we have used rat sensory ganglion neurons exposed to X-rays (4 Gy) ionizing radiation (IR). In control neurons, Pol II appears concentrated in numerous chromatin microfoci identified as transcription factories by the incorporation of 5'-fluorouridine into nascent RNA. Upon IR treatment, numerous IR-induced foci (IRIF), which were immunoreactive for γH2AX and 53BP1, were observed as early as 30 min post-IR; their number progressively reduced at 3 h, 1 day, and 3 days post-IR. The formation of IRIF was associated with a decrease in Pol II levels by both immunofluorescence and Western blotting. Treatment with the proteasome inhibitor bortezomib strongly increased Pol II levels in both control and irradiated neurons, suggesting that proteasome plays a proteolytic role by clearing stalled Pol II complexes at DNA damage sites, as a prelude to DNA repair. Neuronal IRIF recruited ubiquitylated proteins, including ubiquitylated histone H2A (Ub-H2A), and the catalytic proteasome 20S. Ub-H2A has been associated with transcriptional silencing at DNA damage sites. On the other hand, the participation of UPS in neuronal DDR may be essential for the ubiquitylation of Pol II and other proteasome substrates of the DNA repair machinery and their subsequent proteasome-mediated degradation.

  12. Regulation of TRAIL-Receptor Expression by the Ubiquitin-Proteasome System

    Science.gov (United States)

    Sarhan, Dhifaf; D’Arcy, Padraig; Lundqvist, Andreas

    2014-01-01

    The tumor necrosis factor (TNF)-related apoptosis-inducing ligand- receptor (TRAIL-R) family has emerged as a key mediator of cell fate and survival. Ligation of TRAIL ligand to TRAIL-R1 or TRAIL-R2 initiates the extrinsic apoptotic pathway characterized by the recruitment of death domains, assembly of the death-inducing signaling complex (DISC), caspase activation and ultimately apoptosis. Conversely the decoy receptors TRAIL-R3 and TRAIL-R4, which lack the pro-apoptotic death domain, function to dampen the apoptotic response by competing for TRAIL ligand. The tissue restricted expression of the decoy receptors on normal but not cancer cells provides a therapeutic rational for the development of selective TRAIL-mediated anti-tumor therapies. Recent clinical trials using agonistic antibodies against the apoptosis-inducing TRAIL receptors or recombinant TRAIL have been promising; however the number of patients in complete remission remains stubbornly low. The mechanisms of TRAIL resistance are relatively unexplored but may in part be due to TRAIL-R down-regulation or shedding of TRAIL-R by tumor cells. Therefore a better understanding of the mechanisms underlying TRAIL resistance is required. The ubiquitin-proteasome system (UPS) has been shown to regulate TRAIL-R members suggesting that pharmacological inhibition of the UPS may be a novel strategy to augment TRAIL-based therapies and increase efficacies. We recently identified b-AP15 as an inhibitor of proteasome deubiquitinase (DUB) activity. Interestingly, exposure of tumor cell lines to b-AP15 resulted in increased TRAIL-R2 expression and enhanced sensitivity to TRAIL-mediated apoptosis and cell death in vitro and in vivo. In conclusion, targeting the UPS may represent a novel strategy to increase the cell surface expression of pro-apoptotic TRAIL-R on cancer cells and should be considered in clinical trials targeting TRAIL-receptors in cancer patients. PMID:25318057

  13. Regulation of TRAIL-Receptor Expression by the Ubiquitin-Proteasome System

    Directory of Open Access Journals (Sweden)

    Dhifaf Sarhan

    2014-10-01

    Full Text Available The tumor necrosis factor (TNF-related apoptosis-inducing ligand- receptor (TRAIL-R family has emerged as a key mediator of cell fate and survival. Ligation of TRAIL ligand to TRAIL-R1 or TRAIL-R2 initiates the extrinsic apoptotic pathway characterized by the recruitment of death domains, assembly of the death-inducing signaling complex (DISC, caspase activation and ultimately apoptosis. Conversely the decoy receptors TRAIL-R3 and TRAIL-R4, which lack the pro-apoptotic death domain, function to dampen the apoptotic response by competing for TRAIL ligand. The tissue restricted expression of the decoy receptors on normal but not cancer cells provides a therapeutic rational for the development of selective TRAIL-mediated anti-tumor therapies. Recent clinical trials using agonistic antibodies against the apoptosis-inducing TRAIL receptors or recombinant TRAIL have been promising; however the number of patients in complete remission remains stubbornly low. The mechanisms of TRAIL resistance are relatively unexplored but may in part be due to TRAIL-R down-regulation or shedding of TRAIL-R by tumor cells. Therefore a better understanding of the mechanisms underlying TRAIL resistance is required. The ubiquitin-proteasome system (UPS has been shown to regulate TRAIL-R members suggesting that pharmacological inhibition of the UPS may be a novel strategy to augment TRAIL-based therapies and increase efficacies. We recently identified b-AP15 as an inhibitor of proteasome deubiquitinase (DUB activity. Interestingly, exposure of tumor cell lines to b-AP15 resulted in increased TRAIL-R2 expression and enhanced sensitivity to TRAIL-mediated apoptosis and cell death in vitro and in vivo. In conclusion, targeting the UPS may represent a novel strategy to increase the cell surface expression of pro-apoptotic TRAIL-R on cancer cells and should be considered in clinical trials targeting TRAIL-receptors in cancer patients.

  14. Polymerase eta is a short-lived, proteasomally degraded protein that is temporarily stabilized following UV irradiation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Skoneczna, Adrianna; McIntyre, Justyna; Skoneczny, Marek; Policinska, Zofia; Sledziewska-Gojska, Ewa

    2007-03-02

    Saccharomyces cerevisiae Rad30 is the homolog of human DNA polymerase eta whose inactivation leads to the cancer-prone syndrome xeroderma pigmentosum variant. Both human and yeast polymerase eta are responsible for error-free bypass of UV-induced cis-syn pyrimidine dimers and several other DNA lesions. Here we show, using yeast strains expressing TAP-tagged Rad30, that the level of this protein is post-translationally regulated via ubiquitination and proteasome-mediated degradation. The half-life of Rad30 is 20 min and it increases due to proteasomal defects. Mutations inactivating components of the Skp1/cullin/ F-box (SCF) ubiquitin ligase complex: Skp1 and the F-box protein Ufo1 stabilize Rad30. Our results indicate also that ultraviolet irradiation causes transient stabilization of Rad30, which leads, in turn, to temporary accumulation of this polymerase in the cell. We conclude that proteolysis plays an important role in regulating the cellular abundance of Rad30. These results are the first indication of a role for controlled proteasomal degradation in modulating cellular level of translesion DNA polymerase in eukaryotes.

  15. A mathematical model of protein degradation by the proteasome

    NARCIS (Netherlands)

    Luciani, F.; Kesmir, C.; Mishto, M.; Or-Guil, 'M.; Boer, R.J. de

    2005-01-01

    The proteasome is the major protease for intracellular protein degradation. The influx rate of protein substrates and the exit rate of the fragments/products are regulated by the size of the axial channels. Opening the channels is known to increase the overall degradation rate and to change the

  16. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    Science.gov (United States)

    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…

  17. 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.

    Science.gov (United States)

    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

  18. 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.

    Directory of Open Access Journals (Sweden)

    Scott A Keith

    2016-02-01

    Full Text Available 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

  19. An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

    Science.gov (United States)

    Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

    2014-01-01

    Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc

  20. 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

    Science.gov (United States)

    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

  1. The co-chaperone p23 is degraded by caspases and the proteasome during apoptosis

    DEFF Research Database (Denmark)

    Mollerup, Jens; Berchtold, Martin Werner

    2005-01-01

    The heat shock protein 90 co-chaperone p23 has recently been shown to be up-regulated in cancer cells and down-regulated in atheroschlerotic plaques. We found that p23 is degraded during apoptosis induced by several stimuli, including Fas and TNFa-receptor activation as well as staurosporine...... treatment. Caspase inhibition protected p23 from degradation in several cell lines. In addition, recombinant caspase-3 and 8 cleaved p23 at Asp 142 generating a degradation product of 18 kDa as seen in apoptotic cells. Truncated p23 is further degraded in a proteasome dependent process during apoptosis....... Furthermore, we found that the anti-aggregating activity of truncated p23 was reduced compared to full length p23 indicating that caspase mediated p23 degradation contributes to protein destabilisation in apoptosis....

  2. Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

    Science.gov (United States)

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

    2014-12-01

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

  3. Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

    Directory of Open Access Journals (Sweden)

    Britta Stadelmann

    2014-12-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Christian Muñoz

    2015-01-01

    Full Text Available 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.

  6. Role of the Ubiquitin Proteasome System in Regulating Skin Pigmentation

    Directory of Open Access Journals (Sweden)

    Hideya Ando

    2009-10-01

    Full Text Available Pigmentation of the skin, hair and eyes is regulated by tyrosinase, the critical rate-limiting enzyme in melanin synthesis by melanocytes. Tyrosinase is degraded endogenously, at least in part, by the ubiquitin proteasome system (UPS. Several types of inherited hypopigmentary diseases, such as oculocutaneous albinism and Hermansky-Pudlak syndrome, involve the aberrant processing and/or trafficking of tyrosinase and its subsequent degradation which can occur due to the quality-control machinery. Studies on carbohydrate modifications have revealed that tyrosinase in the endoplasmic reticulum (ER is proteolyzed via ER-associated protein degradation and that tyrosinase degradation can also occur following its complete maturation in the Golgi. Among intrinsic factors that regulate the UPS, fatty acids have been shown to modulate tyrosinase degradation in contrasting manners through increased or decreased amounts of ubiquitinated tyrosinase that leads to its accelerated or decelerated degradation by proteasomes.

  7. Cylindrocyclophanes with Proteasome Inhibitory Activity from the Cyanobacterium Nostoc sp

    Science.gov (United States)

    Chlipala, George E.; Sturdy, Megan; Krunic, Aleksej; Lantvit, Daniel D.; Shen, Qi; Porter, Kyle; Swanson, Steven M.; Orjala, Jimmy

    2010-01-01

    Material collected from a parkway in the city of Chicago afforded the isolation of a Nostoc species (UIC 10022A). The extract of this strain displayed significant inhibition of the 20S proteasome as well as antiproliferative activity against HT29, MCF7, NCI-H460, and SF268 cancer cell lines. A standardized dereplication protocol allowed for the rapid identification of three known (11-13) and nine new (1-9) chlorinated cylindrocyclophanes from less than 100 mg of organic extract. Scale-up isolation of 1-9 and 11-13 from a larger extract was guided by LC-UV-MS data. In addition, KBr enrichment of the culture media afforded the isolation of a brominated cylindrocyclophane (10). Biological evaluation of 1-5, 9, and 10-13 revealed a large range of activity against the 20S proteasome and allowed the determination of preliminary structure-activity relationships (SAR) of the cylindrocyclophane pharmacophore. PMID:20825206

  8. The Role of Ubiquitine Proteasome Pathway in Carcinogenesis

    Directory of Open Access Journals (Sweden)

    N.Ceren Sumer Turanligil

    2010-02-01

    Full Text Available Ubiquitin works as a marker protein which targets misfolded or injured proteins to cellular degradation. It brings the abnormal proteins to a subcellular organelle named proteasome and it maintains the degradation of proteins in limited lenghts of peptides by leaving the process withuout being changed. Mistakes in ubiquitin-dependent proteolysis in various steps of carcinogenesis is known. In this review, we dealed with the effects of ubiquitin-proteasome pathway (UPP on carcinogenesis via intercellular signaling molecules like Ras, transcription factors like NF-kB, cytokines like TNF-alfa Tumor necrosis factor, protooncogenes like p53 and MDM2(murine double minute 2, components of cell cycle and DNA repair proteins like BRCA1. We also focused on the relationship of UPP on antigen presentation which is active in immune response and its place in the aetiology of colon cancer to provide a specific example. [Archives Medical Review Journal 2010; 19(1.000: 36-55

  9. Prediction of proteasome cleavage motifs by neural networks

    DEFF Research Database (Denmark)

    Kesimir, C.; Nussbaum, A.K.; Schild, H.

    2002-01-01

    physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...... Class I molecules. Here we demonstrate that such an approach produces an accurate prediction of the CTL the epitopes in HIV Nef. The method is available at www.cbs.dtu.dk/services/NetChop/....

  10. Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

    Directory of Open Access Journals (Sweden)

    Qureshi Asaf A

    2012-07-01

    Full Text Available Abstract Background Altered immune function during ageing results in increased production of nitric oxide (NO and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin in lipopolysaccharide (LPS-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1β, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. Results The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P 40%; P 60%; P 40%; P P  Conclusions The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1β, IL-6, and NO levels, in response to inflammatory stimuli

  11. Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Matsumoto, Hotaru [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan)

    2016-07-29

    We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. -- Highlights: •Puromycin exhibits the O-propargyl-puromycin effect. •Puromycin induces SUMO redistribution upon proteasome inhibition. •Ubiquitin and RNF4 accumulate at PML-nuclear bodies with SUMO-2/3. •The ubiquitin family may function in nuclear sequestration of immature proteins.

  12. Proteasomal degradation of TRIM5alpha during retrovirus restriction.

    Directory of Open Access Journals (Sweden)

    Christopher James Rold

    2008-05-01

    Full Text Available The host protein TRIM5alpha inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5alpha. Here, we show that TRIM5alpha is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5alpha-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5alpha protein but not the nonrestrictive human TRIM5alpha protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5alpha was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5alpha and TRIMCyp proteins. We also detected degradation of endogenous TRIM5alpha in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5alpha degradation by a proteasome-dependent mechanism.

  13. Degradation of nicastrin involves both proteasome and lysosome.

    Science.gov (United States)

    He, Guiqiong; Qing, Hong; Tong, Yigang; Cai, Fang; Ishiura, Shoichi; Song, Weihong

    2007-05-01

    The glycoprotein nicastrin (NCT) is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the gamma-secretase complex, NCT plays a crucial role in its activation. Considerable information exists on the distribution, structure and function of NCT; however, little is known of its proteolysis. The present study is aimed at exploring the molecular mechanism of NCT degradation. We found that either proteasomal or lysosomal inhibition can significantly increase the levels of both endogenous and exogenous NCT in various cell lines, and the effect of these inhibitions on NCT was time- and dose-dependent. Immunofluorescent microscopic analysis revealed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in the lysosomal apparatus. Co-immunoprecipitation can pull down both NCT and ubiquitin. Taken together, our results demonstrate that NCT degradation involves both the proteasome and the lysosome.

  14. The role of the proteasome in the generation of MHC class I ligands and immune responses.

    Science.gov (United States)

    Sijts, E J A M; Kloetzel, P M

    2011-05-01

    The ubiquitin-proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses.

  15. Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide

    Science.gov (United States)

    Samanovic, Marie I.; Tu, Shengjiang; Novák, Ondřej; Iyer, Lakshminarayan M.; McAllister, Fiona E.; Aravind, L.; Gygi, Steven P.; Hubbard, Stevan R.; Strnad, Miroslav; Darwin, K. Heran

    2015-01-01

    Summary One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO-resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homologue of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report for the first time that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO. PMID:25728768

  16. Role of proteasomes in transcription and their regulation by covalent modifications.

    Science.gov (United States)

    Mittenberg, Alexey G; Moiseeva, Tatyana N; Barlev, Nickolai A

    2008-05-01

    The 26S proteasome is an abundant multi-subunit complex, which, in addition to lysosomes, represents a major cellular "protein degradation factory". The proteasome complex possesses protease, ATPase/helicase, and RNAse enzymatic activities, which are used by the latter to regulate various physiological processes. Recent findings have revealed an important role of proteasomes in transcriptional regulation. Although proteasomes are well documented to undergo various post-translational modifications, little is known about their functional significance, in particular in the process of gene regulation in response to various forms of stress. Here, we review the data on the role of proteasomes in gene regulation and their post-translational modifications as well as discuss potential mechanisms by which proteasomal activity may be regulated by genotoxic stress.

  17. Clinical Use of Proteasome Inhibitors in the Treatment of Multiple Myeloma

    Directory of Open Access Journals (Sweden)

    Noah M. Merin

    2014-12-01

    Full Text Available Multiple myeloma (MM is an incurable hematological malignancy characterized by the clonal proliferation of neoplastic plasma cells. The use of proteasome inhibitors in the treatment of MM has led to significant improvements in outcomes. This article reviews data on the use of the two approved proteasome inhibitors (bortezomib and carlfilzomib, as well as newer agents under development. Emphasis is placed on the clinical use of proteasome inhibitors, including management of side effects and combination with other agents.

  18. Preclinical activity of the oral proteasome inhibitor MLN9708 in Myeloma bone disease.

    Science.gov (United States)

    Garcia-Gomez, Antonio; Quwaider, Dalia; Canavese, Miriam; Ocio, Enrique M; Tian, Ze; Blanco, Juan F; Berger, Allison J; Ortiz-de-Solorzano, Carlos; Hernández-Iglesias, Teresa; Martens, Anton C M; Groen, Richard W J; Mateo-Urdiales, Joaquín; Fraile, Susana; Galarraga, Miguel; Chauhan, Dharminder; San Miguel, Jesús F; Raje, Noopur; Garayoa, Mercedes

    2014-03-15

    MLN9708 (ixazomib citrate), which hydrolyzes to pharmacologically active MLN2238 (ixazomib), is a next-generation proteasome inhibitor with demonstrated preclinical and clinical antimyeloma activity, but yet with an unknown effect on myeloma bone disease. Here, we investigated its bone anabolic and antiresorptive effects in the myeloma setting and in comparison with bortezomib in preclinical models. The in vitro effect of MLN2238 was tested on osteoclasts and osteoclast precursors from healthy donors and patients with myeloma, and on osteoprogenitors derived from bone marrow mesenchymal stem cells also from both origins. We used an in vivo model of bone marrow-disseminated human myeloma to evaluate MLN2238 antimyeloma and bone activities. Clinically achievable concentrations of MLN2238 markedly inhibited in vitro osteoclastogenesis and osteoclast resorption; these effects involved blockade of RANKL (receptor activator of NF-κB ligand)-induced NF-κB activation, F-actin ring disruption, and diminished expression of αVβ3 integrin. A similar range of MLN2238 concentrations promoted in vitro osteoblastogenesis and osteoblast activity (even in osteoprogenitors from patients with myeloma), partly mediated by activation of TCF/β-catenin signaling and upregulation of the IRE1 component of the unfolded protein response. In a mouse model of bone marrow-disseminated human multiple myeloma, orally administered MLN2238 was equally effective as bortezomib to control tumor burden and also provided a marked benefit in associated bone disease (sustained by both bone anabolic and anticatabolic activities). Given favorable data on pharmacologic properties and emerging clinical safety profile of MLN9708, it is conceivable that this proteasome inhibitor may achieve bone beneficial effects in addition to its antimyeloma activity in patients with myeloma. ©2014 AACR.

  19. A mammalian nervous-system-specific plasma membrane proteasome complex that modulates neuronal function.

    Science.gov (United States)

    Ramachandran, Kapil V; Margolis, Seth S

    2017-04-01

    In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It is unclear how proteasomes are able to acutely regulate such processes, as this action is inconsistent with their canonical role in proteostasis. Here we describe a mammalian nervous-system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is closely associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of the membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked the production of extracellular peptides and attenuated neuronal-activity-induced calcium signaling. Moreover, we observed that membrane-proteasome-derived peptides were sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes function primarily to maintain proteostasis, and highlight a form of neuronal communication that takes place through a membrane proteasome complex.

  20. Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication.

    Directory of Open Access Journals (Sweden)

    Nickolay Neznanov

    2008-04-01

    Full Text Available Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

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

    Science.gov (United States)

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

    2015-10-01

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

  2. Proteasome Activation by Hepatitis C Core Protein is Reversed by Ethanol-Induced Oxidative Stress

    Science.gov (United States)

    Osna, Natalia A.; White, Ronda L.; Krutik, Viatcheslav M.; Wang, Ting; Weinman, Steven A.; Donohue, Terrence M.

    2008-01-01

    Background and Aims The proteasome is a major cellular proteinase. Its activity is modulated by cellular oxidants. Hepatitis C core protein and ethanol exposure both cause enhanced oxidant generation. The aim was to investigate whether core protein, by its ability to generate oxidants, alters proteasome activity and whether these alterations are further affected byethanol exposure. Methods These interactions were examined in Huh-7 cell lines that expressed inducible HCV core protein and/or constitutive cytochrome P450 2E1 (CYP2E1) and as purified components in a cell free system. Chymotrypsin-like proteasome activity was measured fluorometrically. Results Proteasome activity in core-positive 191-20 cells was 20% higher than that in core-negative cells and was enhanced three-fold in CYP2E1-expressing L14 cells. Exposure of core-positive cells to glutathione ethyl ester, catalase, or the CYP2E1 inhibitor, DAS, partially reversed the elevation of proteasome activity in core-positive cells, while ethanol exposure suppressed proteasome activity. The results indicate that proteasome activity was up-regulated by low levels of core-induced oxidative stress and down-regulated by high levels of ethanol-elicited stress. These findings were partially mimicked in cell free system. Addition of core protein enhanced the peptidase activity of purified 20S proteasome containing the proteasome activator, PA28 and was further potentiated by addition of liver mitochondrial and/or microsome fractions. However, proteasome activation was significantly attenuated when fractions were obtained from ethanol-fed animals. Conclusions HCV core protein interacts with PA28, mitochondrial and ER proteins to cause low levels of oxidant stress and proteasome activation, which is dampened during ethanol metabolism when oxidant generation is higher. PMID:18549882

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

    Science.gov (United States)

    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

  4. Targeting neuroblastoma stem cells with retinoic acid and proteasome inhibitor.

    Directory of Open Access Journals (Sweden)

    Barbara Hämmerle

    Full Text Available Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2 neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

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

    Directory of Open Access Journals (Sweden)

    Jonathan Wills

    Full Text Available 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.

  6. BDNF Induces Striatal-Enriched Protein Tyrosine Phosphatase 61 Degradation Through the Proteasome.

    Science.gov (United States)

    Saavedra, Ana; Puigdellívol, Mar; Tyebji, Shiraz; Kurup, Pradeep; Xu, Jian; Ginés, Silvia; Alberch, Jordi; Lombroso, Paul J; Pérez-Navarro, Esther

    2016-08-01

    Brain-derived neurotrophic factor (BDNF) promotes synaptic strengthening through the regulation of kinase and phosphatase activity. Conversely, striatal-enriched protein tyrosine phosphatase (STEP) opposes synaptic strengthening through inactivation or internalization of signaling molecules. Here, we investigated whether BDNF regulates STEP levels/activity. BDNF induced a reduction of STEP61 levels in primary cortical neurons, an effect that was prevented by inhibition of tyrosine kinases, phospholipase C gamma, or the ubiquitin-proteasome system (UPS). The levels of pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204), two STEP substrates, increased in BDNF-treated cultures, and blockade of the UPS prevented STEP61 degradation and reduced BDNF-induced GluN2B and ERK1/2 phosphorylation. Moreover, brief or sustained cell depolarization reduced STEP61 levels in cortical neurons by different mechanisms. BDNF also promoted UPS-mediated STEP61 degradation in cultured striatal and hippocampal neurons. In contrast, nerve growth factor and neurotrophin-3 had no effect on STEP61 levels. Our results thus indicate that STEP61 degradation is an important event in BDNF-mediated effects.

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

    Directory of Open Access Journals (Sweden)

    Sachiko Tsukamoto

    2005-06-01

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

  8. Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells

    Directory of Open Access Journals (Sweden)

    Amanda C. Stacer

    2015-08-01

    Full Text Available Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.

  9. Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells.

    Science.gov (United States)

    Stacer, Amanda C; Wang, Hanxiao; Fenner, Joseph; Dosch, Joseph S; Salomonnson, Anna; Luker, Kathryn E; Luker, Gary D; Rehemtulla, Alnawaz; Ross, Brian D

    2015-08-01

    Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed the activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient-derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor- and metastasis-initiating cancer cells and a potential biomarker for outcomes in patients with several different cancers.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-01-01

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

  11. Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells

    NARCIS (Netherlands)

    Ramos de Carvalho, J. Emanuel; Verwoert, Milan T.; Vogels, Ilse M. C.; Schipper-Krom, Sabine; van Noorden, Cornelis J. F.; Reits, Eric A.; Klaassen, Ingeborg; Schlingemann, Reinier O.

    2018-01-01

    Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome

  12. Exploring dual electrophiles in peptide-based proteasome inhibitors: carbonyls and epoxides

    NARCIS (Netherlands)

    Xin, B.T.; Bruin, G. de; Verdoes, M.; Filippov, D.V.; Marel, G.A. van der; Overkleeft, H.S.

    2014-01-01

    Peptide epoxyketones are potent and selective proteasome inhibitors. Selectivity is governed by the epoxyketone dual electrophilic warhead, which reacts with the N-terminal threonine 1,2-amino alcohol uniquely present in proteasome active sites. We studied a series of C-terminally modified

  13. Characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui

    Directory of Open Access Journals (Sweden)

    B. Franzetti

    2002-01-01

    Full Text Available A 20S proteasome, comprising two subunits α and β, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome, obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

  14. Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis

    Science.gov (United States)

    Palombella, Vito J.; Conner, Elaine M.; Fuseler, John W.; Destree, Antonia; Davis, Jonathan M.; Laroux, F. Stephen; Wolf, Robert E.; Huang, Jianqing; Brand, Stephen; Elliott, Peter J.; Lazarus, Douglas; McCormack, Teresa; Parent, Lana; Stein, Ross; Adams, Julian; Grisham, Matthew B.

    1998-01-01

    The transcription factor NF-κB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-κB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IκB is degraded by the ubiquitin–proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-κB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin–proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-κB and the subsequent transcription of genes that are regulated by NF-κB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IκBα degradation and NF-κB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin–proteasome pathway and NF-κB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect. PMID:9861028

  15. The ubiquitin proteasome system in Caenorhabditis elegans and its regulation☆

    Science.gov (United States)

    Papaevgeniou, Nikoletta; Chondrogianni, Niki

    2014-01-01

    Protein degradation constitutes a major cellular function that is responsible for maintenance of the normal cellular physiology either through the degradation of normal proteins or through the elimination of damaged proteins. The Ubiquitin–Proteasome System (UPS)1 is one of the main proteolytic systems that orchestrate protein degradation. Given that up- and down- regulation of the UPS system has been shown to occur in various normal (such as ageing) and pathological (such as neurodegenerative diseases) processes, the exogenous modulation of the UPS function and activity holds promise of (a) developing new therapeutic interventions against various diseases and (b) establishing strategies to maintain cellular homeostasis. Since the proteasome genes are evolutionarily conserved, their role can be dissected in simple model organisms, such as the nematode, Caenorhabditis elegans. In this review, we survey findings on the redox regulation of the UPS in C. elegans showing that the nematode is an instrumental tool in the identification of major players in the UPS pathway. Moreover, we specifically discuss UPS-related genes that have been modulated in the nematode and in human cells and have resulted in similar effects thus further exhibiting the value of this model in the study of the UPS. PMID:24563851

  16. Comparative study of the biochemical properties of proteasomes in domestic animals.

    Science.gov (United States)

    Raule, Mary; Cerruti, Fulvia; Cascio, Paolo

    2015-07-15

    Information on the biochemical properties of proteasomes is lacking or, at best, only fragmentary for most species of veterinary interest. Moreover, direct comparison of the limited data available on the enzymatic features of proteasomes in domestic animals is rendered difficult due to the heterogeneity of the experimental settings used. This represents a clear drawback in veterinary research, given the crucial involvement of proteasomes in control of several physiological and pathological processes. We performed the first comparative analysis of key biochemical properties of proteasomes obtained from 8 different domestic mammals. Specifically, we investigated the three main peptidase activities of constitutive and immunoproteasomes in parallel and systematically checked the sensitivity of the chymotryptic site to three of the most potent and selective inhibitors available. Overall, there was substantial similarity in the enzymatic features of proteasomes among the species examined, although some interesting species-specific features were observed. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Distinct temporal requirements for autophagy and the proteasome in yeast meiosis.

    Science.gov (United States)

    Wen, Fu-ping; Guo, Yue-shuai; Hu, Yang; Liu, Wei-xiao; Wang, Qian; Wang, Yuan-ting; Yu, Hai-Yan; Tang, Chao-ming; Yang, Jun; Zhou, Tao; Xie, Zhi-ping; Sha, Jia-hao; Guo, Xuejiang; Li, Wei

    2016-01-01

    Meiosis is a special type of cellular renovation that involves 2 successive cell divisions and a single round of DNA replication. Two major degradation systems, the autophagy-lysosome and the ubiquitin-proteasome, are involved in meiosis, but their roles have yet to be elucidated. Here we show that autophagy mainly affects the initiation of meiosis but not the nuclear division. Autophagy works not only by serving as a dynamic recycling system but also by eliminating some negative meiotic regulators such as Ego4 (Ynr034w-a). In a quantitative proteomics study, the proteasome was found to be significantly upregulated during meiotic divisions. We found that proteasomal activity is essential to the 2 successive meiotic nuclear divisions but not for the initiation of meiosis. Our study defines the roles of autophagy and the proteasome in meiosis: Autophagy mainly affects the initiation of meiosis, whereas the proteasome mainly affects the 2 successive meiotic divisions.

  18. Txl1 and Txc1 are co-factors of the 26S proteasome in fission yeast

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Jensen, Camilla; Kriegenburg, Franziska

    2011-01-01

    The 26S proteasome is a large proteolytic particle present in the cytosol and nucleus of eukaryotic cells. Most intracellular proteins, including those affected by oxidative damage, are degraded by the proteasome. The human thioredoxin, Txnl1, is known to associate with the 26S proteasome and the...

  19. Proteasome Inhibitor Up Regulates Liver Antioxidative Enzymes in Rat Model of Alcoholic Liver Disease

    Science.gov (United States)

    Bardag-Gorce, Fawzia; Oliva, Joan; Lin, Andrew; Li, Jun; French, Barbara A.; French, Samuel W.

    2010-01-01

    Oxidative stress occurs in the liver of rats fed alcohol chronically due to ethanol metabolism by CYP2E1, causing liver injury. The proteasome is considered as an antioxidant defense in the cell because of its activity in removing damaged and oxidized proteins, but a growing body of evidence shows that proteasome inhibitor treatment, at a non toxic low dose, provides protection against oxidative stress. In the present study, rats were fed ethanol for 4 weeks and were treated with the proteasome inhibitor PS-341 (Bortezomib, Velcade®). Exposure to proteasome inhibitor elicited the elevation of antioxidative defense by enhancing the levels of mRNA and protein expression transcripts of glutathione reductase (GSR), glutathione synthetase (GSS), glutathione peroxidase 2 (GPX2), and superoxide dismutase 2 (SOD2) in the liver of rats fed ethanol chronically, while ethanol alone did not increase these genes mRNA. Our results also showed that glutamate cysteine ligase catalytic subunit (GCLC), a rate-limiting enzyme in glutathione biosynthesis, was also up regulated in the liver of rats fed ethanol and injected with PS-431. Nrf2 mRNA level was significantly decreased in the liver of ethanol fed rats, as well as in the livers of animal fed ethanol and treated with proteasome inhibitor, indicating that the mechanism by which proteasome inhibitor up regulates the antioxidant response element is not due to regulation of Nrf2. However, ATF4, a major regulator of antioxidant response elements, was significantly up regulated by proteasome inhibitor treatment. The beneficial effects of proteasome inhibitor treatment also reside in the reversibility of the drug because the proteasome activity was significantly increased 72h post treatment. In conclusion, proteasome inhibitor treatment used at a non toxic low dose has potential protective effects against oxidative stress due to chronic ethanol feeding. PMID:21036165

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

    Directory of Open Access Journals (Sweden)

    Anna Pagotto

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

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

    Science.gov (United States)

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

    2013-01-01

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

  2. Proteomic Analysis of the E3 Ubiquitin-Ligase Hakai Highlights a Role in Plasticity of the Cytoskeleton Dynamics and in the Proteasome System.

    Science.gov (United States)

    Díaz-Díaz, Andrea; Casas-Pais, Alba; Calamia, Valentina; Castosa, Raquel; Martinez-Iglesias, Olaia; Roca-Lema, Daniel; Santamarina, Isabel; Valladares-Ayerbes, Manuel; Calvo, Lourdes; Chantada, Venancio; Figueroa, Angélica

    2017-08-04

    Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.

  3. Quercetin improves the activity of the ubiquitin-proteasomal system in 150Q mutated huntingtin-expressing cells but exerts detrimental effects on neuronal survivability.

    Science.gov (United States)

    Chakraborty, J; Rajamma, U; Jana, N; Mohanakumar, K P

    2015-10-01

    Quercetin, a strong free radical scavenger, is investigated for neuroprotective effects in a Neuro 2a cell line conditionally transfected with 16Q huntingtin (Htt) and 150Q Htt, which express the protein upon stimulation. Cells were protected from death by a 20-µM dose of quercetin on the second day of Htt induction, but 30-100-µM doses of the drug caused further toxicity in both 16Q and 150Q cells, as indicated by MTT assay and by significant reductions in the number of cells bearing neurites on the second day. A significant decrease in the number of cells containing aggregate was seen in induced 150Q cells treated with 20 µM but not for those treated with 40 or 50 µM quercetin up to 4 days of induction. Mutated Htt (mHtt)-induced reduction in proteasomal activity of the ubiquitin-proteasomal system (UPS) was significantly attenuated by 20 µM quercetin. However, neither mitochondrial membrane potential loss nor colocalization of 20S proteasome with mHtt aggregate was corrected by quercetin treatment. Our results imply that the neuroprotective effect of quercetin arises out of the upregulation of UPS activity, which causes a decrease in the number of mHtt aggregate-harboring cells. The increased neurotoxicity could result from the continued association of mHtt with 20S proteasome and the failure of quercetin to correct mitochondrial membrane potential loss. These results suggest that, although quercetin at a low dose protects against mHtt-mediated cell death, higher doses are toxic to the cells, clearly demarcating a narrow therapeutic window for this dietary flavonoid. © 2015 Wiley Periodicals, Inc.

  4. Peripheral proteasome and caspase activity in Parkinson disease and Alzheimer disease.

    Science.gov (United States)

    Blandini, F; Sinforiani, E; Pacchetti, C; Samuele, A; Bazzini, E; Zangaglia, R; Nappi, G; Martignoni, E

    2006-02-28

    Defects of the ubiquitin-proteasome (UP) system, a multicatalytic complex degrading polyubiquitinated proteins, may intervene in the pathogenesis of neurodegenerative disorders characterized by intracellular formation of protein aggregates such as Parkinson disease (PD) and Alzheimer disease (AD) by inducing proapoptotic conditions. The authors measured the activity of proteolytic UP core, proteasome 20S, and of proapoptotic caspase-3 and -9 in peripheral blood lymphocytes (PBLs) of PD and AD patients to establish whether changes in these systems are detectable peripherally. Proteasome 20S activity was reduced in PBLs of treated PD patients vs healthy controls (mean +/- SEM: 1.0 +/- 0.1 vs 2.3 +/- 0.2 nmol 7-amino-4-methylcoumarin (AMC)/10(6) cells, p Parkinson's Disease Rating Scale score) were inversely correlated with proteasome 20S activity and directly correlated with caspase-3 activity. An inverse correlation was also observed in PD patients between caspase-3 activity and proteasome 20S activity. No significant changes in proteasome 20S or caspase activity or correlations between biochemical and clinical variables were found in patients with AD. A decrease in proteasome activity, possibly related to caspase activation, is detectable in peripheral blood lymphocytes of patients with Parkinson disease but not patients with Alzheimer disease, suggesting that these variables may be considered for the development of peripheral biomarkers of Parkinson disease.

  5. Proteasome- and Ethanol-Dependent Regulation of HCV-Infection Pathogenesis

    Directory of Open Access Journals (Sweden)

    Natalia A. Osna

    2014-09-01

    Full Text Available This paper reviews the role of the catabolism of HCV and signaling proteins in HCV protection and the involvement of ethanol in HCV-proteasome interactions. HCV specifically infects hepatocytes, and intracellularly expressed HCV proteins generate oxidative stress, which is further exacerbated by heavy drinking. The proteasome is the principal proteolytic system in cells, and its activity is sensitive to the level of cellular oxidative stress. Not only host proteins, but some HCV proteins are degraded by the proteasome, which, in turn, controls HCV propagation and is crucial for the elimination of the virus. Ubiquitylation of HCV proteins usually leads to the prevention of HCV propagation, while accumulation of undegraded viral proteins in the nuclear compartment exacerbates infection pathogenesis. Proteasome activity also regulates both innate and adaptive immunity in HCV-infected cells. In addition, the proteasome/immunoproteasome is activated by interferons, which also induce “early” and “late” interferon-sensitive genes (ISGs with anti-viral properties. Cleaving viral proteins to peptides in professional immune antigen presenting cells and infected (“target” hepatocytes that express the MHC class I-antigenic peptide complex, the proteasome regulates the clearance of infected hepatocytes by the immune system. Alcohol exposure prevents peptide cleavage by generating metabolites that impair proteasome activity, thereby providing escape mechanisms that interfere with efficient viral clearance to promote the persistence of HCV-infection.

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

    Science.gov (United States)

    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.

  7. Circulating 20S Proteasome Is Independently Associated with Abdominal Muscle Mass in Hemodialysis Patients

    Science.gov (United States)

    Fukasawa, Hirotaka; Kaneko, Mai; Niwa, Hiroki; Matsuyama, Takashi; Yasuda, Hideo; Kumagai, Hiromichi; Furuya, Ryuichi

    2015-01-01

    Protein-energy wasting is highly prevalent in hemodialysis patients, and it contributes to patient morbidity and mortality. The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and it is involved in the regulation of basic cellular processes. However, the role of this system in the determination of nutritional status is largely unknown. To examine a relationship between protein-energy wasting and the ubiquitin-proteasome system, a cross-sectional study of 76 hemodialysis patients was performed. Plasma concentrations of 20S proteasome were studied to evaluate its association with muscle and fat mass, which were investigated by abdominal muscle and fat areas measured using computed tomography and by creatinine production estimated using the creatinine kinetic model. Plasma 20S proteasome concentrations significantly and negatively correlated with abdominal muscle areas and creatinine production (rho = -0.263, P < 0.05 and rho = -0.241, P < 0.05, respectively), but not abdominal subcutaneous and visceral fat areas. Multiple regression analyses showed that 20S proteasome was a significant independent predictor of abdominal muscle area (P < 0.05). In conclusion, plasma 20S proteasome concentrations were independently associated with abdominal muscle mass in hemodialysis patients. Our findings indicate a relationship between circulating 20S proteasomes and muscle metabolism in these patients. Trial Registration UMIN Clinical Trials Registry UMIN000012341 PMID:25803510

  8. Biological significance of co- and post-translational modifications of the yeast 26S proteasome.

    Science.gov (United States)

    Hirano, Hisashi; Kimura, Yayoi; Kimura, Ayuko

    2016-02-16

    In yeast (Saccharomyces cerevisiae), co- and post-translational modifications of the 26S proteasome, a large protein complex, were comprehensively detected by proteomic techniques, and their functions were investigated. The presence, number, site, and state of co- and post-translational modifications of the 26S proteasome differ considerably among yeast, human, and mouse. The roles of phosphorylation, N(α)-acetylation, N(α)-myristoylation, N(α)-methylation, and N-terminal truncation in the yeast 26S proteasome were investigated. Although there is only one modification site for either N(α)-acetylation, N(α)-myristoylation, or N(α)-methylation, these modifications play an important role in the functions of the yeast proteasome. In contrast, there are many phosphorylation sites in the yeast 26S proteasome. However, the phosphorylation patterns might be a few, suggesting that tiny modifications exert considerable effects on the function of the proteasome. Protein co- and post-translational modifications produce different protein species which often have different functions. The yeast 26S proteasome, a large protein complex, consisting of many subunits has a number of co- and post-translational modification sites. This review describes the effects of the modifications on the function of the protein complex. This article is part of a Special Issue entitled: Protein species. Guest Editors: Peter Jungblut, Hartmut Schlüter and Bernd Thiede. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Circulating 20S proteasome is independently associated with abdominal muscle mass in hemodialysis patients.

    Directory of Open Access Journals (Sweden)

    Hirotaka Fukasawa

    Full Text Available Protein-energy wasting is highly prevalent in hemodialysis patients, and it contributes to patient morbidity and mortality. The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and it is involved in the regulation of basic cellular processes. However, the role of this system in the determination of nutritional status is largely unknown. To examine a relationship between protein-energy wasting and the ubiquitin-proteasome system, a cross-sectional study of 76 hemodialysis patients was performed. Plasma concentrations of 20S proteasome were studied to evaluate its association with muscle and fat mass, which were investigated by abdominal muscle and fat areas measured using computed tomography and by creatinine production estimated using the creatinine kinetic model. Plasma 20S proteasome concentrations significantly and negatively correlated with abdominal muscle areas and creatinine production (rho = -0.263, P < 0.05 and rho = -0.241, P < 0.05, respectively, but not abdominal subcutaneous and visceral fat areas. Multiple regression analyses showed that 20S proteasome was a significant independent predictor of abdominal muscle area (P < 0.05. In conclusion, plasma 20S proteasome concentrations were independently associated with abdominal muscle mass in hemodialysis patients. Our findings indicate a relationship between circulating 20S proteasomes and muscle metabolism in these patients.UMIN Clinical Trials Registry UMIN000012341.

  10. Reduction of RPT6/S8 (a Proteasome Component) and Proteasome Activity in the Cortex is Associated with Cognitive Impairment in Lewy Body Dementia.

    Science.gov (United States)

    Alghamdi, Amani; Vallortigara, Julie; Howlett, David R; Broadstock, Martin; Hortobágyi, Tibor; Ballard, Clive; Thomas, Alan J; O'Brien, John T; Aarsland, Dag; Attems, Johannes; Francis, Paul T; Whitfield, David R

    2017-01-01

    Lewy body dementia is the second most common neurodegenerative dementia and is pathologically characterized by α-synuclein positive cytoplasmic inclusions, with varying amounts of amyloid-β (Aβ) and hyperphosphorylated tau (tau) aggregates in addition to synaptic loss. A dysfunctional ubiquitin proteasome system (UPS), the major proteolytic pathway responsible for the clearance of short lived proteins, may be a mediating factor of disease progression and of the development of α-synuclein aggregates. In the present study, protein expression of a key component of the UPS, the RPT6 subunit of the 19S regulatory complex was determined. Furthermore, the main proteolytic-like (chymotrypsin- and PGPH-) activities have also been analyzed. The middle frontal (Brodmann, BA9), inferior parietal (BA40), and anterior cingulate (BA24) gyrus' cortex were selected as regions of interest from Parkinson's disease dementia (PDD, n = 31), dementia with Lewy bodies (DLB, n = 44), Alzheimer's disease (AD, n = 16), and control (n = 24) brains. Clinical and pathological data available included the MMSE score. DLB, PDD, and AD were characterized by significant reductions of RPT6 (one-way ANOVA, p < 0.001; Bonferroni post hoc test) in prefrontal cortex and parietal cortex compared with controls. Strong associations were observed between RPT6 levels in prefrontal, parietal cortex, and anterior cingulate gyrus and cognitive impairment (p = 0.001, p = 0.001, and p = 0.008, respectively). These findings highlight the involvement of the UPS in Lewy body dementia and indicate that targeting the UPS may have the potential to slow down or reduce the progression of cognitive impairment in DLB and PDD.

  11. The role of proteasome inhibition in nonsmall cell lung cancer.

    Science.gov (United States)

    Escobar, Mauricio; Velez, Michel; Belalcazar, Astrid; Santos, Edgardo S; Raez, Luis E

    2011-01-01

    Lung cancer therapy with current available chemotherapeutic agents is mainly palliative. For these and other reasons there is now a great interest to find targeted therapies that can be effective not only palliating lung cancer or decreasing treatment-related toxicity, but also giving hope to cure these patients. It is already well known that the ubiquitin-proteasome system like other cellular pathways is critical for the proliferation and survival of cancer cells; thus, proteosome inhibition has become a very attractive anticancer therapy. There are several phase I and phase II clinical trials now in non-small cell lung cancer and small cell lung cancer using this potential target. Most of the trials use bortezomib in combination with chemotherapeutic agents. This paper tends to make a state-of-the-art review based on the available literature regarding the use of bortezomib as a single agent or in combination with chemotherapy in patients with lung cancer.

  12. The Role of Proteasome Inhibition in Nonsmall Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Mauricio Escobar

    2011-01-01

    Full Text Available Lung cancer therapy with current available chemotherapeutic agents is mainly palliative. For these and other reasons there is now a great interest to find targeted therapies that can be effective not only palliating lung cancer or decreasing treatment-related toxicity, but also giving hope to cure these patients. It is already well known that the ubiquitin-proteasome system like other cellular pathways is critical for the proliferation and survival of cancer cells; thus, proteosome inhibition has become a very attractive anticancer therapy. There are several phase I and phase II clinical trials now in non-small cell lung cancer and small cell lung cancer using this potential target. Most of the trials use bortezomib in combination with chemotherapeutic agents. This paper tends to make a state-of-the-art review based on the available literature regarding the use of bortezomib as a single agent or in combination with chemotherapy in patients with lung cancer.

  13. Proteasomal diseases are a new branch of autoinflammatory pathology

    Directory of Open Access Journals (Sweden)

    Evgeny Stanislavovich Fedorov

    2013-12-01

    Full Text Available The paper deals with a new autoinflammatory disease entity that is proteasomal diseases. The latter include three nosological entities: Nakajo–Nishimura syndrome (NNS, Japanese autoinflammatory syndrome with lipodystrophy; chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE syndrome; joint contractures, muscular atrophy, microcytic anemia, and panniculitisinduced lipodystrophy (JMP syndrome. All the three conditions are caused by mutations in one PSMB8 gene encoding the immunoproteasome subunit β5i. Unlike other autoinflammatory syndromes that are mainly IL-1-dependent, the leading component of the pathogenesis of these diseases is IL-6/γ−interferonі system hyperactivation. These diseases are characterized by childhoodonset, retarded physical development, different skin and muscular lesions, lipodystrophy, joint contractures, and hypochromic anemia, as well as elevated levels of acutephase markers; autoimmune disorders may joint in time.

  14. VSV virotherapy improves chemotherapy by triggering apoptosis due to proteasomal degradation of Mcl-1.

    Science.gov (United States)

    Schache, P; Gürlevik, E; Strüver, N; Woller, N; Malek, N; Zender, L; Manns, M; Wirth, T; Kühnel, F; Kubicka, S

    2009-07-01

    Overexpression of myeloid cell leukemia 1 protein (Mcl-1), an anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member, contributes to chemotherapy resistance of tumors. The short half-life of Mcl-1 makes it an interesting target for therapeutic agents that negatively interfere with cellular protein biosynthesis, such as oncolytic viruses. Vesicular Stomatitis Virus (VSV) has been established as the oncolytic virus that efficiently disrupts de novo protein biosynthesis of infected cells. Here, we show that after VSV infection, Mcl-1 protein levels rapidly declined, whereas the expression of other members of the Bcl-2 family remained unchanged. Mcl-1 elimination was a consequence of proteasomal degradation, as overexpression of a degradation-resistant Mcl-1 mutant restored Mcl-1 levels. Mcl-1 rescue inhibited apoptosis and thereby confirmed that Mcl-1 downregulation contributes to VSV-induced apoptosis. In vitro, VSV virotherapy in combination with chemotherapy revealed an enhanced therapeutic effect compared with the single treatments, which could be reverted by Mcl-1 rescue or RNA interference (RNAi)-mediated knockdown of pro-apoptotic Bax and Bak proteins. Finally, in a tumor mouse model, combinations of doxorubicin and VSV showed a superior therapeutic efficacy compared with VSV or doxorubicin alone. In summary, our data indicate that VSV virotherapy is an attractive strategy to overcome tumor resistance against conventional chemotherapy by elimination of Mcl-1.

  15. Sequence analysis of β-subunit genes of the 20S proteasome in patients with relapsed multiple myeloma treated with bortezomib or dexamethasone

    NARCIS (Netherlands)

    D.I. Lichter (David); H. Danaee (Hadi); M.D. Pickard (Michael); O. Tayber (Olga); M. Sintchak (Michael); H. Shi (Hongliang); P.G. Richardson (Paul Gerard); J. Cavenagh (Jamie); J. Bladé (Joan); T. Facon (Thierry); R. Niesvizky; M. Alsina (Melissa); W. Dalton (William); P. Sonneveld (Pieter); S. Lonial (Sagar); H. van de Velde (Helgi); D. Ricci (Deborah); D.-L. Esseltine (Dixie-Lee); W.L. Trepicchio (William); G. Mulligan (George); K.C. Anderson (Kenneth)

    2012-01-01

    textabstractVariations within proteasome β (PSMB) genes, which encode the β subunits of the 20S proteasome, may affect proteasome function, assembly, and/or binding of proteasome inhibitors. To investigate the potential association between PSMB gene variants and treatment-emergent resistance to

  16. Preclinical evaluation of the proteasome inhibitor bortezomib in cancer therapy

    Directory of Open Access Journals (Sweden)

    Morgan Gareth

    2005-06-01

    Full Text Available Abstract Bortezomib is a highly selective, reversible inhibitor of the 26S proteasome that is indicated for single-agent use in the treatment of patients with multiple myeloma who have received at least 2 prior therapies and are progressing on their most recent therapy. Clinical investigations have been completed or are under way to evaluate the safety and efficacy of bortezomib alone or in combination with chemotherapy in multiple myeloma, both at relapse and presentation, as well as in other cancer types. The antiproliferative, proapoptotic, antiangiogenic, and antitumor activities of bortezomib result from proteasome inhibition and depend on the altered degradation of a host of regulatory proteins. Exposure to bortezomib has been shown to stabilize p21, p27, and p53, as well as the proapoptotic Bid and Bax proteins, caveolin-1, and inhibitor κB-α, which prevents activation of nuclear factor κB-induced cell survival pathways. Bortezomib also promoted the activation of the proapoptotic c-Jun-NH2 terminal kinase, as well as the endoplasmic reticulum stress response. The anticancer effects of bortezomib as a single agent have been demonstrated in xenograft models of multiple myeloma, adult T-cell leukemia, lung, breast, prostate, pancreatic, head and neck, and colon cancer, and in melanoma. In these preclinical in vivo studies, bortezomib treatment resulted in decreased tumor growth, angiogenesis, and metastasis, as well as increased survival and tumor apoptosis. In several in vitro and/or in vivo cancer models, bortezomib has also been shown to enhance the antitumor properties of several antineoplastic treatments. Importantly, bortezomib was generally well tolerated and did not appear to produce additive toxicities when combined with other therapies in the dosing regimens used in these preclinical in vivo investigations. These findings provide a rationale for further clinical trials using bortezomib alone or in combination regimens with

  17. Ubiquitin-proteasome system involvement in Huntington’s disease

    Directory of Open Access Journals (Sweden)

    Zaira eOrtega

    2014-09-01

    Full Text Available Huntington’s disease (HD is a genetic autosomal dominant neurodegenerative disease caused by the expansion of a CAG repeat in the huntingtin (htt gene. This triplet expansion encodes a polyglutamine stretch (polyQ in the N-terminus of the high molecular weight (348-kDa and ubiquitously expressed protein huntingtin (htt. Normal individuals have between 6 and 35 CAG triplets, while expansions longer than 40 repeats lead to HD. The onset and severity of the disease depend on the length of the polyQ tract: the longer the polyQ is, the earlier the disease begins and the more severe the symptoms are. One of the main histopathological hallmarks of HD is the presence of intraneuronal proteinaceous inclusion bodies (IBs, whose prominent and invariant feature is the presence of Ubiquitin (Ub; therefore, they can be detected with anti-ubiquitin and anti-proteasome antibodies. This, together with the observation that mutations in components of the Ubiquitin Proteasome system (UPS give rise to some neurodegenerative diseases, suggests that UPS impairment may be causative of HD. Even though the link between disrupted Ub homeostasis and protein aggregation to HD is undisputed, the functional significance of these correlations and their mechanistic implications remains unresolved. Moreover, there is no consistent evidence documenting an accompanying decrease in levels of free Ub or disruption of Ub pool dynamics in neurodegenerative disease or models thus suggesting that the Ub-conjugate accumulation may be benign and just underlie lesion in 26S function. In this chapter we will elaborate on the different studies that have been performed using different experimental approaches, in order to shed light to this matter.

  18. Lead discovery and chemical biology approaches targeting the ubiquitin proteasome system.

    Science.gov (United States)

    Akinjiyan, Favour A; Carbonneau, Seth; Ross, Nathan T

    2017-10-15

    Protein degradation is critical for proteostasis, and the addition of polyubiquitin chains to a substrate is necessary for its recognition by the 26S proteasome. Therapeutic intervention in the ubiquitin proteasome system has implications ranging from cancer to neurodegeneration. Novel screening methods and chemical biology tools for targeting E1-activating, E2-conjugating and deubiquitinating enzymes will be discussed in this review. Approaches for targeting E3 ligase-substrate interactions as well as the proteasome will also be covered, with a focus on recently described approaches. Copyright © 2017. Published by Elsevier Ltd.

  19. Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

    DEFF Research Database (Denmark)

    Andersen, Katrine M; Klausen, Louise Kjær; Prag, Søren

    2009-01-01

    The 26S proteasome is a large proteolytic machine, which degrades most intracellular proteins. We found that thioredoxin, Txnl1/TRP32, binds to Rpn11, a subunit of the regulatory complex of the human 26S proteasome. Txnl1 is abundant, metabolically stable and widely expressed and is present...... in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about -250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26S proteasome. eEF1A1 is therefore a likely physiological substrate...

  20. Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

    Science.gov (United States)

    Sundaresan, Sinju; Meininger, Cameron A; Kang, Anthony J; Photenhauer, Amanda L; Hayes, Michael M; Sahoo, Nirakar; Grembecka, Jolanta; Cierpicki, Tomasz; Ding, Lin; Giordano, Thomas J; Else, Tobias; Madrigal, David J; Low, Malcolm J; Campbell, Fiona; Baker, Ann-Marie; Xu, Haoxing; Wright, Nicholas A; Merchant, Juanita L

    2017-12-01

    The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. MEN1-associated

  1. Proteasome inhibition improves diaphragm function in an animal model for COPD.

    NARCIS (Netherlands)

    Hees, H.W.H. van; Ottenheijm, C.A.C.; Ennen, L.; Linkels, M.; Dekhuijzen, R.; Heunks, L.M.A.

    2011-01-01

    Diaphragm muscle weakness in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. Recent studies indicate that increased contractile protein degradation by the proteasome contributes to diaphragm weakness in patients with COPD. The aim of

  2. Structural characterization of the interaction of Ubp6 with the 26S proteasome

    NARCIS (Netherlands)

    Aufderheide, Antje; Beck, Florian; Stengel, Florian; Hartwig, Michaela; Schweitzer, Andreas; Pfeifer, Günter; Goldberg, Alfred L; Sakata, Eri; Baumeister, Wolfgang; Förster, Friedrich|info:eu-repo/dai/nl/412516438

    2015-01-01

    In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6;

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

    Science.gov (United States)

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

    2012-01-31

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

  4. Nigrostriatal proteasome inhibition impairs dopamine neurotransmission and motor function in minipigs

    DEFF Research Database (Denmark)

    Lillethorup, Thea Pinholt; Glud, Andreas Nørgaard; Alstrup, Aage Kristian Olsen

    2018-01-01

    Parkinson's disease (PD) is characterized by degeneration of dopaminergic neurons in the substantia nigra leading to slowness and stiffness of limb movement with rest tremor. Using ubiquitin proteasome system inhibitors, rodent models have shown nigrostriatal degeneration and motor impairment. We...

  5. Secondary Metabolites Produced by an Endophytic Fungus Pestalotiopsis sydowiana and Their 20S Proteasome Inhibitory Activities.

    Science.gov (United States)

    Xia, Xuekui; Kim, Soonok; Liu, Changheng; Shim, Sang Hee

    2016-07-20

    Fungal endophytes have attracted attention due to their functional diversity. Secondary metabolites produced by Pestalotiopsis sydowiana from a halophyte, Phragmites communis Trinus, were investigated. Eleven compounds, including four penicillide derivatives (1-4) and seven α-pyrone analogues (5-10) were isolated from cultures of P. sydowiana. The compounds were identified based on spectroscopic data. The inhibitory activities against the 20S proteasome were evaluated. Compounds 1-3, 5, and 9-10 showed modest proteasome inhibition activities, while compound 8 showed strong activity with an IC50 of 1.2 ± 0.3 μM. This is the first study on the secondary metabolites produced by P. sydowiana and their proteasome inhibitory activities. The endophytic fungus P. sydowiana might be a good resource for proteasome inhibitors.

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

    OpenAIRE

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

    2012-01-01

    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 sig...

  7. Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

    Directory of Open Access Journals (Sweden)

    Nora Semren

    Full Text Available Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ, provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ. OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

  8. Purification and characterization of a proteasome from the hyperthermophilic archaeon pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, M.W.; Bauer, S.H.; Kelly, R.M. [North Carolina State Univ., Raleigh, NC (United States)

    1997-03-01

    A 640-kDa proteasome consisting of {alpha} (25-kDa) and {beta} (22-kDa) subunits, and with a temperature optimum of 95{degrees}C, was purified from crude cell extracts of a hyperthermophilic archaeon, Pyrococcus furiosus. Although this is the fourth member of the kingdom Euryarchaeota (and the first hyperthermophile) found to contain a proteasome, none has been identified among the members of the kingdom Crenarchaeota. 38 refs., 5 figs., 2 tabs.

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

    Science.gov (United States)

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

    2015-06-01

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

  10. Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes

    DEFF Research Database (Denmark)

    Hirano, Y.; Hayashi, H.; Iemura, S.

    2006-01-01

    The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, a1-7ß1-7ß1-7a1-7. It is reported that the biogenesis of mammalian 20...

  11. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

    Directory of Open Access Journals (Sweden)

    Antonio Pereira-Neves

    Full Text Available Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF, also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin

  12. Dp71 is regulated by phosphorylation and ubiquitin-proteasome system in neuronal cells.

    Science.gov (United States)

    Fujimoto, Takahiro; Yaoi, Takeshi; Fushiki, Shinji; Itoh, Kyoko

    2017-10-21

    The Dystrophin (Dp) gene is responsible for Duchenne muscular dystrophy (DMD), which is characterized by progressive muscular degeneration and variable degrees of cognitive impairment. Although Dp71 is the most abundant among the Dp isoforms in the brain, the regulatory mechanisms of the related expression levels have not been elucidated. In this study, we found that the constitutive expression levels of Dp71 in PC12 cells were sensitive to proteasomal inhibition. The ectopic expression of FLAG-tagged ubiquitin revealed that Dp71 was ubiquitinated intracellularly. Interestingly, proteasomal inhibition was accompanied by a posttranslational accumulation of modified Dp71, which was restored by protein phosphatase treatment in vitro, indicating that phosphorylation is responsible for the modification and affects the proteasome-dependent degradation of Dp71. Proteasomal activity-sensitive phosphorylated Dp71 is closely associated with syntrophin, a well-known binding partner of Dp71, and syntrophin is also regulated by proteasomal activity in a similar way to Dp71, suggesting that the posttranslational regulatory machinery for Dp71 level is coupled with Dp71-syntrophin molecular complex. Taken together, our results indicated that the expression levels of Dp71 are posttranslationally regulated by the phosphorylation-ubiquitin-proteasomal pathway, which may indicate the presence of regulatory mechanisms underlying the proteostasis of both Dp and its molecular complex, which may lead to better therapeutic approaches for the treatment of Dp-related diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The Effect of Proteasome Inhibition on the Generation of the Human Leukocyte Antigen (HLA) Peptidome*

    Science.gov (United States)

    Milner, Elena; Gutter-Kapon, Lilach; Bassani-Strenberg, Michal; Barnea, Eilon; Beer, Ilan; Admon, Arie

    2013-01-01

    The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides. PMID:23538226

  14. Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Matthew A. Humbard

    2010-01-01

    Full Text Available Proteasomes are composed of 20S core particles (CPs of α- and β-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α1 and α2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS. Using this approach, several phosphosites were mapped including α1 Thr147, α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α1, thus, revealing a new type of proteasomal modification. Probing the biological role of α1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α1. The α1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.

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

    Directory of Open Access Journals (Sweden)

    Romina A. Guedes

    2016-07-01

    Full Text Available 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.

  16. Adrm1, a putative cell adhesion regulating protein, is a novel proteasome-associated factor

    DEFF Research Database (Denmark)

    Jørgensen, Jakob Ploug; Lauridsen, Anne-Marie; Kristensen, Poul

    2006-01-01

    . Adrm1 was present almost exclusively in soluble 26 S proteasomes, albeit a small fraction was membrane-associated, like proteasomes. Adrm1 was found in cells in amounts equimolar with S6a, a 26 S proteasome subunit. HeLa cells contain no pool of free Adrm1 but recombinant Adrm1 could bind to pre......We have identified Adrm1 as a novel component of the regulatory ATPase complex of the 26 S proteasome: Adrm1 was precipitated with an antibody to proteasomes and vice versa. Adrm1 co-migrated with proteasomes on gel-filtration chromatography and non-denaturing polyacrylamide gel electrophoresis....... Adrm1 has been described as an interferon-gamma-inducible, heavily glycosylated membrane protein of 110 kDa. However, we found Adrm1 in mouse tissues only as a 42 kDa peptide, corresponding to the mass of the non-glycosylated peptide chain, and it could not be induced in HeLa cells with interferon...

  17. Proteasome (Prosome Subunit Variations during the Differentiation of Myeloid U937 Cells

    Directory of Open Access Journals (Sweden)

    Laurent Henry

    1997-01-01

    Full Text Available 20S proteasomes (prosomes/multicatalytic proteinase are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol‐myristate‐acetate or retinoic acid plus 1,25‐dihydroxy‐cholecalciferol by western blot, flow cytometry and immuno‐fluorescence. p25K (C3, p27K (IOTA and p30/33K (C2 subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA+VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno‐fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA+VD‐induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA‐induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non‐induced cells; these membrane proteins disappeared when treated with RA+VD, whereas some increased on PMA‐induced cells. The differential changes in the distribution and type of proteasomes in RA+VD and PMA‐induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

  18. plantsUPS: a database of plants' Ubiquitin Proteasome System

    Directory of Open Access Journals (Sweden)

    Su Zhen

    2009-05-01

    Full Text Available Abstract Background The ubiquitin 26S/proteasome system (UPS, a serial cascade process of protein ubiquitination and degradation, is the last step for most cellular proteins. There are many genes involved in this system, but are not identified in many species. The accumulating availability of genomic sequence data is generating more demands in data management and analysis. Genomics data of plants such as Populus trichocarpa, Medicago truncatula, Glycine max and others are now publicly accessible. It is time to integrate information on classes of genes for complex protein systems such as UPS. Results We developed a database of higher plants' UPS, named 'plantsUPS'. Both automated search and manual curation were performed in identifying candidate genes. Extensive annotations referring to each gene were generated, including basic gene characterization, protein features, GO (gene ontology assignment, microarray probe set annotation and expression data, as well as cross-links among different organisms. A chromosome distribution map, multi-sequence alignment, and phylogenetic trees for each species or gene family were also created. A user-friendly web interface and regular updates make plantsUPS valuable to researchers in related fields. Conclusion The plantsUPS enables the exploration and comparative analysis of UPS in higher plants. It now archives > 8000 genes from seven plant species distributed in 11 UPS-involved gene families. The plantsUPS is freely available now to all users at http://bioinformatics.cau.edu.cn/plantsUPS.

  19. Inhibitor-binding mode of homobelactosin C to proteasomes: New insights into class I MHC ligand generation

    Science.gov (United States)

    Groll, Michael; Larionov, Oleg V.; Huber, Robert; de Meijere, Armin

    2006-01-01

    Most class I MHC ligands are generated from the vast majority of cellular proteins by proteolysis within the ubiquitin–proteasome pathway and are presented on the cell surface by MHC class I molecules. Here, we present the crystallographic analysis of yeast 20S proteasome in complex with the inhibitor homobelactosin C. The structure reveals a unique inhibitor-binding mode and provides information about the composition of proteasomal primed substrate-binding sites. IFN-γ inducible substitution of proteasomal constitutive subunits by immunosubunits modulates characteristics of generated peptides, thus producing fragments with higher preference for binding to MHC class I molecules. The structural data for the proteasome:homobelactosin C complex provide an explanation for involvement of immunosubunits in antigen generation and open perspectives for rational design of ligands, inhibiting exclusively constitutive proteasomes or immunoproteasomes. PMID:16537370

  20. The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM-1 and FEM-3 for degradation by the proteasome.

    Science.gov (United States)

    Jäger, Sibylle; Schwartz, Hillel T; Horvitz, H Robert; Conradt, Barbara

    2004-08-24

    The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and downstream of her-1 and probably tra-2. When expressed in mammalian cells, SEL-10 protein coimmunoprecipitates with FEM-1, FEM-2, and FEM-3, which are required for masculinization, and FEM-1 and FEM-3 are targeted by SEL-10 for proteasomal degradation. We propose that SEL-10-mediated proteolysis of FEM-1 and FEM-3 is required for normal hermaphrodite development.

  1. The Caenorhabditis elegans F-box protein SEL-10 promotes female development and may target FEM-1 and FEM-3 for degradation by the proteasome

    Science.gov (United States)

    Jäger, Sibylle; Schwartz, Hillel T.; Horvitz, H. Robert; Conradt, Barbara

    2004-01-01

    The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and downstream of her-1 and probably tra-2. When expressed in mammalian cells, SEL-10 protein coimmunoprecipitates with FEM-1, FEM-2, and FEM-3, which are required for masculinization, and FEM-1 and FEM-3 are targeted by SEL-10 for proteasomal degradation. We propose that SEL-10-mediated proteolysis of FEM-1 and FEM-3 is required for normal hermaphrodite development. PMID:15306688

  2. N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases.

    Science.gov (United States)

    Schauer, S; Burster, T; Spindler-Barth, M

    2012-06-01

    Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action. © 2012 The Authors. Insect Molecular Biology © 2012 The Royal Entomological Society.

  3. Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Aleksandra Glogowska

    2012-05-01

    Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

  4. Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

    Science.gov (United States)

    Sánchez-Martín, David; Martínez-Torrecuadrada, Jorge; Teesalu, Tambet; Sugahara, Kazuki N.; Alvarez-Cienfuegos, Ana; Ximénez-Embún, Pilar; Fernández-Periáñez, Rodrigo; Martín, M. Teresa; Molina-Privado, Irene; Ruppen-Cañás, Isabel; Blanco-Toribio, Ana; Cañamero, Marta; Cuesta, Ángel M.; Compte, Marta; Kremer, Leonor; Bellas, Carmen; Alonso-Camino, Vanesa; Guijarro-Muñoz, Irene; Sanz, Laura; Ruoslahti, Erkki; Alvarez-Vallina, Luis

    2013-01-01

    Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer. PMID:23918357

  5. ROS inhibitor N-acetyl-l-cysteine antagonizes the activity of proteasome inhibitors

    Science.gov (United States)

    Halasi, Marianna; Wang, Ming; Chavan, Tanmay S.; Gaponenko, Vadim; Hay, Nissim; Gartel, Andrei L.

    2015-01-01

    NAC (N-acetyl-l-cysteine) is commonly used to identify and test ROS (reactive oxygen species) inducers, and to inhibit ROS. In the present study, we identified inhibition of proteasome inhibitors as a novel activity of NAC. Both NAC and catalase, another known scavenger of ROS, similarly inhibited ROS levels and apoptosis associated with H2O2. However, only NAC, and not catalase or another ROS scavenger Trolox, was able to prevent effects linked to proteasome inhibition, such as protein stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors. Recently, NAC was used as a ROS inhibitor to functionally characterize a novel anticancer compound, piperlongumine, leading to its description as a ROS inducer. In contrast, our own experiments showed that this compound depicts features of proteasome inhibitors including suppression of FOXM1 (Forkhead box protein M1), stabilization of cellular proteins, induction of ROS-independent apoptosis and enhanced accumulation of ubiquitin conjugates. In addition, NAC, but not catalase or Trolox, interfered with the activity of piperlongumine, further supporting that piperlongumine is a proteasome inhibitor. Most importantly, we showed that NAC, but not other ROS scavengers, directly binds to proteasome inhibitors. To our knowledge, NAC is the first known compound that directly interacts with and antagonizes the activity of proteasome inhibitors. Taken together, the findings of the present study suggest that, as a result of the dual nature of NAC, data interpretation might not be straightforward when NAC is utilized as an antioxidant to demonstrate ROS involvement in drug-induced apoptosis. PMID:23772801

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

    Science.gov (United States)

    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. © 2015 The Author(s).

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation.

    Science.gov (United States)

    Schwartzkopff, Benjamin; Platta, Harald W; Hasan, Sohel; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-05-14

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p. © 2015 Authors.

  9. The Ubiquitin-Proteasome System and Microvascular Complications of Diabetes

    Directory of Open Access Journals (Sweden)

    Saeed Yadranji Aghdam

    2013-01-01

    Full Text Available The ubiquitin-proteasome system (UPS is the mainstay of protein quality control which regulates cell cycle, differentiation and various signal transduction pathways in eukaryotic cells. The timely and selective degradation of surplus and/or aberrant proteins by the UPS is essential for normal cellular physiology. Any disturbance, delay or exaggeration in the process of selection, sequestration, labeling for degradation and degradation of target proteins by the UPS will compromise cellular and tissue homeostasis. High blood glucose or hyperglycemia caused by diabetes disrupts normal vascular function in several target organs including the retina and kidney resulting in the development of diabetic retinopathy (DR and diabetic nephropathy (DN. We and others have shown that hyperglycemia and oxidative stress modulate UPS activity in the retina and kidney. The majority of studies have focused on the kidney and provided insights into the contribution of dysregulated UPS to microvascular damage in DN. The eye is a unique organ in which a semi-fluid medium, the vitreous humor, separates the neural retina and its anastomosed blood vessels from the semi-solid lens tissue. The complexity of the cellular and molecular components of the eye may require a normal functioning and well tuned UPS for healthy vision. Altered UPS activity may contribute to the development of retinal microvascular complications of diabetes. A better understanding of the molecular nature of the ocular UPS function under normal and diabetic conditions is essential for development of novel strategies targeting its activity. This review will discuss the association of retinal vascular cell UPS activity with microvascular damage in DR with emphasis on alterations of the PA28 subunits of the UPS.

  10. Regulation of dimethyl-fumarate toxicity by proteasome inhibitors.

    Science.gov (United States)

    Booth, Laurence; Cruickshanks, Nichola; Tavallai, Seyedmehrad; Roberts, Jane L; Peery, Matthew; Poklepovic, Andrew; Dent, Paul

    2014-01-01

    The present studies examined the biology of the multiple sclerosis drug dimethyl-fumarate (DMF) or its in vivo breakdown product and active metabolite mono-methyl-fumarate (MMF), alone or in combination with proteasome inhibitors, in primary human glioblastoma (GBM) cells. MMF enhanced velcade and carfilzomib toxicity in multiple primary GBM isolates. Similar data were obtained in breast and colon cancer cells. MMF reduced the invasiveness of GBM cells, and enhanced the toxicity of ionizing radiation and temozolomide. MMF killed freshly isolated activated microglia which was associated with reduced IL-6, TGFβ and TNFα production. The combination of MMF and the multiple sclerosis drug Gilenya further reduced both GBM and activated microglia viability and cytokine production. Over-expression of c-FLIP-s or BCL(-)XL protected GBM cells from MMF and velcade toxicity. MMF and velcade increased plasma membrane localization of CD95, and knock down of CD95 or FADD blocked the drug interaction. The drug combination inactivated AKT, ERK1/2 and mTOR. Molecular inhibition of AKT/ERK/mTOR signaling enhanced drug combination toxicity whereas molecular activation of these pathways suppressed killing. MMF and velcade increased the levels of autophagosomes and autolysosomes and knock down of ATG5 or Beclin1 protected cells. Inhibition of the eIF2α/ATF4 arm or the IRE1α/XBP1 arm of the ER stress response enhanced drug combination lethality. This was associated with greater production of reactive oxygen species and quenching of ROS suppressed cell killing.

  11. Pregnancy is associated with decreased cardiac proteasome activity and oxidative stress in mice.

    Directory of Open Access Journals (Sweden)

    Andrea Iorga

    Full Text Available During pregnancy, the heart develops physiological hypertrophy. Proteasomal degradation has been shown to be altered in various models of pathological cardiac hypertrophy. Since the molecular signature of pregnancy-induced heart hypertrophy differs significantly from that of pathological heart hypertrophy, we investigated whether the cardiac proteasomal proteolytic pathway is affected by pregnancy in mice. We measured the proteasome activity, expression of proteasome subunits, ubiquitination levels and reactive oxygen production in the hearts of four groups of female mice: i non pregnant (NP at diestrus stage, ii late pregnant (LP, iii one day post-partum (PP1 and iv 7 days post-partum (PP7. The activities of the 26 S proteasome subunits β1 (caspase-like, and β2 (trypsin-like were significantly decreased in LP (β1∶83.26 ± 1.96%; β2∶74.74 ± 1.7%, normalized to NP whereas β5 (chymotrypsin-like activity was not altered by pregnancy but significantly decreased 1 day post-partum. Interestingly, all three proteolytic activities of the proteasome were restored to normal levels 7 days post-partum. The decrease in proteasome activity in LP was not due to the surge of estrogen as estrogen treatment of ovariectomized mice did not alter the 26 S proteasome activity. The transcript and protein levels of RPN2 and RPT4 (subunits of 19 S, β2 and α7 (subunits of 20 S as well as PA28α and β5i (protein only were not significantly different among the four groups. High resolution confocal microscopy revealed that nuclear localization of both core (20S and RPT4 in LP is increased ∼2-fold and is fully reversed in PP7. Pregnancy was also associated with decreased production of reactive oxygen species and ubiquitinated protein levels, while the de-ubiquitination activity was not altered by pregnancy or parturition. These results indicate that late pregnancy is associated with decreased ubiquitin-proteasome proteolytic activity and oxidative stress.

  12. Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase*

    Science.gov (United States)

    Medalia, Noa; Beer, Avital; Zwickl, Peter; Mihalache, Oana; Beck, Martin; Medalia, Ohad; Navon, Ami

    2009-01-01

    In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide. PMID:19363223

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

    Directory of Open Access Journals (Sweden)

    Ilaria Postiglione

    2015-08-01

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

  14. Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast.

    Science.gov (United States)

    Velichutina, Irina; Connerly, Pamela L; Arendt, Cassandra S; Li, Xia; Hochstrasser, Mark

    2004-02-11

    The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/alpha3 subunit is deleted. Purified pre9Delta proteasomes show a two-fold enrichment for the Pre6/alpha4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/alpha4 is expressed in yeast, it complements not only pre6Delta but also pre6Delta pre9Delta mutants; therefore, the plant alpha4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9Delta cells, suggesting an advantage for Pre9 over Pre6 incorporation at the alpha3 position that facilitates correct assembly.

  15. Role of ubiquitin-proteasome system (UPS) in left ventricular hypertrophy (LVH).

    Science.gov (United States)

    Cacciapuoti, Federico

    2014-01-01

    Cardiac hypertrophy is a key compensatory mechanism acting in response to pressure or volume overload, involving some alterations in signaling transduction pathways and transcription factors-regulation. These changes result in enhanced proteins' synthesis leading to Left Ventricular Hypertrophy (LVH). It is known that the main function of Ubiquitin-Proteasome System (UPS) is to prevent accumulation of damaged, misfolded and mutant proteins by proteolysis. But emerging evidences suggest that UPS also attends to the cells' growth, favoring proteins' synthesis, subsequently evolving in LVH. The role of the proteasome in to favor cellular hypertrophy consists in upregulation of the catalytic proteasome subunit, with prevalence of proteins-synthesis on proteins degradation. It is also evident that UPS inhibition may prevent cells' growth opposing to the hypertrophy. In fact in several experimental models, UPS inhibition demonstrated to be able to prevent or reverse cardiac hypertrophy induced by abdominal aortic banding (AAB). That can happen with several proteasome inhibitors acting by multifactorial mechanisms. These evidences induce to hypothesize that, in the future, in patients with the increased volume overload by systemic hypertension, some proteasome-inhibitors could be used to antagonize or prevent LVH without reducing peripheral high blood pressure levels too.

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

    Science.gov (United States)

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

    2011-02-01

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

  17. Mmi1, the yeast homologue of mammalian TCTP, associates with stress granules in heat-shocked cells and modulates proteasome activity.

    Directory of Open Access Journals (Sweden)

    Mark Rinnerthaler

    Full Text Available As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a. We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.

  18. Rotavirus NSP1 inhibits NFkappaB activation by inducing proteasome-dependent degradation of beta-TrCP: a novel mechanism of IFN antagonism.

    Directory of Open Access Journals (Sweden)

    Joel W Graff

    2009-01-01

    Full Text Available Mechanisms by which viruses counter innate host defense responses generally involve inhibition of one or more components of the interferon (IFN system. Multiple steps in the induction and amplification of IFN signaling are targeted for inhibition by viral proteins, and many of the IFN antagonists have direct or indirect effects on activation of latent cytoplasmic transcription factors. Rotavirus nonstructural protein NSP1 blocks transcription of type I IFNalpha/beta by inducing proteasome-dependent degradation of IFN-regulatory factors 3 (IRF3, IRF5, and IRF7. In this study, we show that rotavirus NSP1 also inhibits activation of NFkappaB and does so by a novel mechanism. Proteasome-mediated degradation of inhibitor of kappaB (IkappaBalpha is required for NFkappaB activation. Phosphorylated IkappaBalpha is a substrate for polyubiquitination by a multisubunit E3 ubiquitin ligase complex, Skp1/Cul1/F-box, in which the F-box substrate recognition protein is beta-transducin repeat containing protein (beta-TrCP. The data presented show that phosphorylated IkappaBalpha is stable in rotavirus-infected cells because infection induces proteasome-dependent degradation of beta-TrCP. NSP1 expressed in isolation in transiently transfected cells is sufficient to induce this effect. Targeted degradation of an F-box protein of an E3 ligase complex with a prominent role in modulation of innate immune signaling and cell proliferation pathways is a unique mechanism of IFN antagonism and defines a second strategy of immune evasion used by rotaviruses.

  19. Proteasome regulation of the chromodomain protein MRG-1 controls the balance between proliferative fate and differentiation in the C. elegans germ line.

    Science.gov (United States)

    Gupta, Pratyush; Leahul, Lindsay; Wang, Xin; Wang, Chris; Bakos, Brendan; Jasper, Katie; Hansen, Dave

    2015-01-15

    The level of stem cell proliferation must be tightly controlled for proper development and tissue homeostasis. Multiple levels of gene regulation are often employed to regulate stem cell proliferation to ensure that the amount of proliferation is aligned with the needs of the tissue. Here we focus on proteasome-mediated protein degradation as a means of regulating the activities of proteins involved in controlling the stem cell proliferative fate in the C. elegans germ line. We identify five potential E3 ubiquitin ligases, including the RFP-1 RING finger protein, as being involved in regulating proliferative fate. RFP-1 binds to MRG-1, a homologue of the mammalian chromodomain-containing protein MRG15 (MORF4L1), which has been implicated in promoting the proliferation of neural precursor cells. We find that C. elegans with reduced proteasome activity, or that lack RFP-1 expression, have increased levels of MRG-1 and a shift towards increased proliferation in sensitized genetic backgrounds. Likewise, reduction of MRG-1 partially suppresses stem cell overproliferation. MRG-1 levels are controlled independently of the spatially regulated GLP-1/Notch signalling pathway, which is the primary signal controlling the extent of stem cell proliferation in the C. elegans germ line. We propose a model in which MRG-1 levels are controlled, at least in part, by the proteasome, and that the levels of MRG-1 set a threshold upon which other spatially regulated factors act in order to control the balance between the proliferative fate and differentiation in the C. elegans germ line. © 2015. Published by The Company of Biologists Ltd.

  20. Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors

    Directory of Open Access Journals (Sweden)

    Kwai Fung Hui

    2017-11-01

    Full Text Available Epstein-Barr virus (EBV is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma. To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers.

  1. Trypanocidal activity of the proteasome inhibitor and anti-cancer drug bortezomib

    Directory of Open Access Journals (Sweden)

    Wang Xia

    2009-07-01

    Full Text Available Abstract The proteasome inhibitor and anti-cancer drug bortezomib was tested for in vitro activity against bloodstream forms of Trypanosoma brucei. The concentrations of bortezomib required to reduce the growth rate by 50% and to kill all trypanosomes were 3.3 nM and 10 nM, respectively. In addition, bortezomib was 10 times more toxic to trypanosomes than to human HL-60 cells. Moreover, exposure of trypanosomes to 10 nM bortezomib for 16 h was enough to kill 90% of the parasites following incubation in fresh medium. However, proteasomal peptidase activities of trypanosomes exposed to bortezomib were only inhibited by 10% and 30% indicating that the proteasome is not the main target of the drug. The results suggest that bortezomib may be useful as drug for the treatment of human African trypanosomiasis.

  2. The influence of proteasome inhibitor on the expression of cardiomyocytes damage markers after incubation with doxorubicin

    Directory of Open Access Journals (Sweden)

    Tereszkiewicz Sylwia

    2014-06-01

    Full Text Available The aim of the study was to verify the thesis that the cardiotoxic effects of doxorubicin are connected with activation of the ubiquitin - proteasome pathway followed by protein degradation. The expression of myocardial damage markers - fatty acid binding protein (H-FABP and brain natriuretic peptide (BNP was evaluated in rat fetal cardiomyocytes simultaneously treated with doxorubicin and the proteasome inhibitor - bortezomib. The level of H-FABP and BNP protein under the influence of doxorubicin was decreased below the detection threshold with unchanged (H-FABP or elevated (BNP mRNA expression level. Against the expectations, the inhibitor of proteasome did not abolish this effect. The observed abnormal expression of BNP and H-FABP protein after doxorubicin treatment makes their diagnostic significance in anthracycline cardiotoxicity questionable.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-10

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

  4. The putative roles of the ubiquitin/proteasome pathway in resistance to anticancer therapy.

    Science.gov (United States)

    Smith, Laura; Lind, Michael J; Drew, Philip J; Cawkwell, Lynn

    2007-11-01

    The ubiquitin/proteasome (UP) pathway plays a significant role in many important biological functions and alterations in this pathway have been shown to contribute to the pathology of many human diseases, including cancer. Proteasome inhibition has been well established as a rational strategy for the treatment of multiple myeloma and is currently under investigation for the treatment of other haematological malignancies and solid tumours. Recent evidence suggests that proteasome inhibition may also sensitise tumour cells to the actions of both conventional chemotherapy and radiotherapy, suggesting that this pathway may modify clinical response to anticancer therapy. However, conflicting evidence exists as to the roles of the UP pathway in resistance to treatment. This review endeavours to discuss such roles.

  5. Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

    Science.gov (United States)

    Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However,...

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

    Directory of Open Access Journals (Sweden)

    Cory W Baumann

    Full Text Available 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.

  7. Interplay between Molecular Chaperones and the Ubiquitin-Proteasome System in Targeting of Misfolded Proteins for Degradation

    DEFF Research Database (Denmark)

    Poulsen, Esben Guldahl

    interacting with purified 26S proteasomes, and the subsequent characterization of two novel proteasome interacting proteins. The third study was aimed at analyzing the chaperone-assisted pathway leading to degradation of misfolded kinetochore proteins in S. pombe. In this study chaperones, E2s, E3s and DUBs...

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

    Directory of Open Access Journals (Sweden)

    Yasuhiko Izumi

    2015-11-01

    Full Text Available 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.

  9. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also foun...

  10. Proteasome-based mechanisms of intrinsic and acquired bortezomib resistance in non-small cell lung cancer

    NARCIS (Netherlands)

    de Wilt, Leonie H. A. M.; Jansen, Gerrit; Assaraf, Yehuda G.; van Meerloo, Johan; Cloos, Jacqueline; Schimmer, Aaron D.; Chan, Elena T.; Kirk, Christopher J.; Peters, Godefridus J.; Kruyt, Frank A. E.

    2012-01-01

    The proteasome inhibitor bortezomib, registered for Multiple Myeloma treatment, is currently explored for activity in solid tumors including non-small cell lung cancer (NSCLC). Here we studied the proteasome-based mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells.

  11. Postnatal proteasome inhibition induces neurodegeneration and cognitive deficiencies in adult mice: a new model of neurodevelopment syndrome.

    Directory of Open Access Journals (Sweden)

    Rocío Romero-Granados

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

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

    Directory of Open Access Journals (Sweden)

    Noolu Bindu

    2013-01-01

    Full Text Available Abstract 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

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

    Science.gov (United States)

    Noolu, Bindu; Ajumeera, Rajanna; Chauhan, Anitha; Nagalla, Balakrishna; Manchala, Raghunath; Ismail, Ayesha

    2013-01-09

    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. 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. 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. Our study suggests M. koenigii leaves to be a potent source of proteasome inhibitors that lead to cancer cell death. Therefore, identification of active component(s) from the leaf

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

    Directory of Open Access Journals (Sweden)

    Shawn W Zimmerman

    Full Text Available 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

  15. Characterization of the Proteasome Accessory Factor (paf) Operon in Mycobacterium tuberculosis▿

    OpenAIRE

    Festa, Richard A.; Pearce, Michael J.; Darwin, K. Heran

    2007-01-01

    In a previous screen for Mycobacterium tuberculosis mutants that are hypersusceptible to reactive nitrogen intermediates (RNI), two genes associated with the M. tuberculosis proteasome were identified. One of these genes, pafA (proteasome accessory factor A), encodes a protein of unknown function. In this work, we determined that pafA is in an operon with two additional genes, pafB and pafC. In order to assess the contribution of these genes to RNI resistance, we isolated mutants with transpo...

  16. Proteasome activity related with the daily physical activity of COPD patients

    Directory of Open Access Journals (Sweden)

    Lee KY

    2017-05-01

    Full Text Available Kang-Yun Lee,1,2,* Tzu-Tao Chen,1,* Ling-Ling Chiang,1,3 Hsiao-Chi Chuang,1,3 Po-Hao Feng,1,2 Wen-Te Liu,1–3 Kuan-Yuan Chen,1 Shu-Chuan Ho1,3 1Division of Pulmonary Medicine, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, 2Division of Pulmonary Medicine, Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, 3School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan *These authors contributed equally to this work Background: COPD is a debilitating disease that affects patients’ daily lives. One’s daily physical activity (DPA decreases due to multifactorial causes, and this decrease is correlated with a poor prognosis in COPD patients. Muscle wasting may at least be partly due to increased activity of the ubiquitin proteasome pathway and apoptosis.Methods: This study investigated the relationships among DPA, circulating proteasome activity, and protein carbonyl in COPD patients and healthy subjects (HSs. This study included 57 participants (42 patients and 15 healthy subjects. Ambulatory DPA was measured using actigraphy, and oxygen saturation was measured with a pulse oximeter.Results: COPD patients had lower DPA, lower 6 min walking distance (6MWD, lower delta saturation pulse oxygenation (SpO2 during the 6MWT, and lower delta SpO2 during DPA than HSs. COPD patients had higher proteasome activity and protein carbonyl than HSs. Circulating proteasome activity was significantly negatively correlated with DPA (r=−0.568, P<0.05 in COPD patients, whereas delta SpO2 during the 6MWT was significantly positively correlated with proteasome activity (r=0.685, P<0.05 in HSs. Protein carbonyl was significantly negatively correlated with the body mass index (r=−0.318, P<0.05, mid-arm circumference (r=0.350, P<0.05, calf circumference (r=0.322, P<0.05, forced expiratory volume in the first second (r=−0.441, P<0

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

    Directory of Open Access Journals (Sweden)

    Lafuente Esther M

    2010-09-01

    Full Text Available Abstract 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

  18. Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity.

    Science.gov (United States)

    Li, Hao; Ponder, Elizabeth L; Verdoes, Martijn; Asbjornsdottir, Kristijana H; Deu, Edgar; Edgington, Laura E; Lee, Jeong Tae; Kirk, Christopher J; Demo, Susan D; Williamson, Kim C; Bogyo, Matthew

    2012-12-21

    The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Proteasomes raise the microtubule dynamics in influenza A (H1N1) virus-infected LLC-MK2 cells.

    Science.gov (United States)

    De Conto, Flora; Chezzi, Carlo; Fazzi, Alessandra; Razin, Sergey V; Arcangeletti, Maria Cristina; Medici, Maria Cristina; Gatti, Rita; Calderaro, Adriana

    2015-12-01

    The dynamics of microtubule networks are known to have an impact on replication of influenza A virus in some cellular models. Here we present evidence suggesting that at late stages of LLC-MK2 cell infection by influenza A (H1N1) virus the ubiquitin-proteasome protein degradation system participates in destabilization of microtubules, and favours virus replication. Chemical inhibition of proteasome activity partially suppresses influenza A virus replication, while stimulation of proteasome activity favours influenza A virus replication. Conversely, in another cellular model, A549 cells, inhibitors and activators of proteasomes have a small effect on influenza A virus replication. These data suggest that influenza A virus might take selective advantage of proteasome functions in order to set up a favourable cytoskeletal "environment" for its replication and spread. Furthermore, the relationship between influenza virus and the host cell is likely to depend on both the cellular model and the virus strain.

  20. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Di, E-mail: DiWu@mail.nankai.edu.cn; Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  1. Parkin promotes proteasomal degradation of p62: implication of selective vulnerability of neuronal cells in the pathogenesis of Parkinson’s disease

    Directory of Open Access Journals (Sweden)

    Pingping Song

    2016-01-01

    Full Text Available Abstract Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson’s disease (PD, respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN and striatum (STR regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.

  2. Synergistic antitumor effects of radiation and proteasome inhibitor treatment in pancreatic cancer through the induction of autophagy and the downregulation of TRAF6.

    Science.gov (United States)

    Chiu, Hui-Wen; Lin, Shu-Wen; Lin, Li-Ching; Hsu, Yung-Ho; Lin, Yuh-Feng; Ho, Sheng-Yow; Wu, Yuan-Hua; Wang, Ying-Jan

    2015-09-01

    Ninety percent of human pancreatic cancer is characterized by activating K-RAS mutations. TRAF6 is an oncogene that plays a vital role in K-RAS-mediated oncogenesis. We investigated the synergistic effect of combining ionizing radiation (IR) and proteasome inhibitor (MG132). Furthermore, following combined treatment with IR and MG132, we analyzed the expression of TRAF6 and the mechanism of human pancreatic cancer cell death in vitro and in an orthotopic pancreatic cancer mouse model. The combined treatment groups displayed synergistic cell killing effects and induced endoplasmic reticulum stress in human pancreatic cancer cells. The combined treatment groups were characterized by enhanced cytotoxicity, which resulted from increased autophagy induction through the inhibition of TRAF6. Significantly reduced cytotoxicity was observed following MG132 and IR treatment of MIA PaCa-2 cells pre-treated with 3-MA (an autophagy inhibitor). Down-regulation of TRAF6 led to a significant increase in apoptosis and autophagy. In an orthotopic xenograft model of SCID mice, combination MG132 and IR therapy resulted in a significant increase in the tumor growth delay time and a decreased tumor tissue expression of TRAF6. IR combined with a proteasome inhibitor or TRAF6 inhibition could represent a new therapeutic strategy for human pancreatic cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. An Unexpected Major Role for Proteasome-Catalyzed Peptide Splicing in Generation of T Cell Epitopes: Is There Relevance for Vaccine Development?

    Directory of Open Access Journals (Sweden)

    Anouk C. M. Platteel

    2017-11-01

    Full Text Available Efficient and safe induction of CD8+ T cell responses is a desired characteristic of vaccines against intracellular pathogens. To achieve this, a new generation of safe vaccines is being developed accommodating single, dominant antigens of pathogens of interest. In particular, the selection of such antigens is challenging, since due to HLA polymorphism the ligand specificities and immunodominance hierarchies of pathogen-specific CD8+ T cell responses differ throughout the human population. A recently discovered mechanism of proteasome-mediated CD8+ T cell epitope generation, i.e., by proteasome-catalyzed peptide splicing (PCPS, expands the pool of peptides and antigens, presented by MHC class I HLA molecules. On the cell surface, one-third of the presented self-peptides are generated by PCPS, which coincides with one-fourth in terms of abundance. Spliced epitopes are targeted by CD8+ T cell responses during infection and, like non-spliced epitopes, can be identified within antigen sequences using a novel in silico strategy. The existence of spliced epitopes, by enlarging the pool of peptides available for presentation by different HLA variants, opens new opportunities for immunotherapies and vaccine design.

  4. Sestrin 2 protein regulates platelet-derived growth factor receptor β (Pdgfrβ) expression by modulating proteasomal and Nrf2 transcription factor functions.

    Science.gov (United States)

    Tomasovic, Ana; Kurrle, Nina; Sürün, Duran; Heidler, Juliana; Husnjak, Koraljka; Poser, Ina; Schnütgen, Frank; Scheibe, Susan; Seimetz, Michael; Jaksch, Peter; Hyman, Anthony; Weissmann, Norbert; von Melchner, Harald

    2015-04-10

    We recently identified the antioxidant protein Sestrin 2 (Sesn2) as a suppressor of platelet-derived growth factor receptor β (Pdgfrβ) signaling and Pdgfrβ signaling as an inducer of lung regeneration and injury repair. Here, we identified Sesn2 and the antioxidant gene inducer nuclear factor erythroid 2-related factor 2 (Nrf2) as positive regulators of proteasomal function. Inactivation of Sesn2 or Nrf2 induced reactive oxygen species-mediated proteasomal inhibition and Pdgfrβ accumulation. Using bacterial artificial chromosome (BAC) transgenic HeLa and mouse embryonic stem cells stably expressing enhanced green fluorescent protein-tagged Sesn2 at nearly endogenous levels, we also showed that Sesn2 physically interacts with 2-Cys peroxiredoxins and Nrf2 albeit under different reductive conditions. Overall, we characterized a novel, redox-sensitive Sesn2/Pdgfrβ suppressor pathway that negatively interferes with lung regeneration and is up-regulated in the emphysematous lungs of patients with chronic obstructive pulmonary disease (COPD). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Proteasome-dependent and -independent mechanisms for FosB destabilization: identification of FosB degron domains and implications for DeltaFosB stability.

    Science.gov (United States)

    Carle, Tiffany L; Ohnishi, Yoshinori N; Ohnishi, Yoko H; Alibhai, Imran N; Wilkinson, Matthew B; Kumar, Arvind; Nestler, Eric J

    2007-05-01

    The transcription factor DeltaFosB (Delta FosB) accumulates in a region-specific manner in the brain during chronic exposure to stress, drugs of abuse or other chronic stimuli. Once induced, DeltaFosB persists in the brain for at least several weeks following cessation of the chronic stimulus. The biochemical basis of the persistent expression of DeltaFosB has remained unknown. Here, we show that the FosB C-terminus, absent in DeltaFosB as a result of alternative splicing, contains two degron domains. Pulse-chase experiments of C-terminal truncation mutants of full-length FosB indicate that removal of its most C-terminal degron increases its half-life approximately fourfold, and prevents its proteasome-mediated degradation and ubiquitylation, properties similar to DeltaFosB. In addition, removal of a second degron domain, which generates DeltaFosB, further stabilizes FosB approximately twofold, but in a proteasome-independent manner. These data indicate that alternative splicing specifically removes two destabilizing elements from FosB in order to generate a longer-lived transcription factor, DeltaFosB, in response to chronic perturbations to the brain.

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

    Science.gov (United States)

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

    2014-01-03

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

  7. Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation*

    Science.gov (United States)

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

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8+ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation. PMID:24247241

  8. Increased expression of (immuno)proteasome subunits during epileptogenesis is attenuated by inhibition of the mammalian target of rapamycin pathway.

    Science.gov (United States)

    Broekaart, Diede W M; van Scheppingen, Jackelien; Geijtenbeek, Karlijne W; Zuidberg, Mark R J; Anink, Jasper J; Baayen, Johannes C; Mühlebner, Angelika; Aronica, Eleonora; Gorter, Jan A; van Vliet, Erwin A

    2017-08-01

    Inhibition of the mammalian target of rapamycin (mTOR) pathway reduces epileptogenesis in various epilepsy models, possibly by inhibition of inflammatory processes, which may include the proteasome system. To study the role of mTOR inhibition in the regulation of the proteasome system, we investigated (immuno)proteasome expression during epileptogenesis, as well as the effects of the mTOR inhibitor rapamycin. The expression of constitutive (β1, β5) and immunoproteasome (β1i, β5i) subunits was investigated during epileptogenesis using immunohistochemistry in the electrical post-status epilepticus (SE) rat model for temporal lobe epilepsy (TLE). The effect of rapamycin was studied on (immuno)proteasome subunit expression in post-SE rats that were treated for 6 weeks. (Immuno)proteasome expression was validated in the brain tissue of patients who had SE or drug-resistant TLE and the effect of rapamycin was studied in primary human astrocyte cultures. In post-SE rats, increased (immuno)proteasome expression was detected throughout epileptogenesis in neurons and astrocytes within the hippocampus and piriform cortex and was most evident in rats that developed a progressive form of epilepsy. Rapamycin-treated post-SE rats had reduced (immuno)proteasome protein expression and a lower number of spontaneous seizures compared to vehicle-treated rats. (Immuno)proteasome expression was also increased in neurons and astrocytes within the human hippocampus after SE and in patients with drug-resistant TLE. In vitro studies using cultured human astrocytes showed that interleukin (IL)-1β-induced (immuno)proteasome gene expression could be attenuated by rapamycin. Because dysregulation of the (immuno)proteasome system is observed before the occurrence of spontaneous seizures in rats, is associated with progression of epilepsy, and can be modulated via the mTOR pathway, it may represent an interesting novel target for drug treatment in epilepsy. Wiley Periodicals, Inc. © 2017

  9. Involvement of the proteasome in IL-1beta induced suppression of islets of Langerhans in the rat.

    Science.gov (United States)

    Sternesjö, Johnny; Karlsen, Allan E; Sandler, Stellan

    2003-01-01

    The cytokine IL-1beta suppresses rodent islets of Langerhans in vitro. Presently we used inhibitors of the proteasome to investigate if these compounds could counteract the suppressive effects of the cytokine. Thus, isolated rat islets were cultured and pre-treated with proteasome inhibitors and subsequently exposed for 48 h to 25 U/ml human IL-1beta. After this period functional tests were carried out. The rate of glucose oxidation (pmol/10 islets x 90 min) was suppressed by IL-1beta (115 +/- 17 vs. control 380 +/- 57). Pre-treatment with 10 microM of the proteasome inhibitor MG115 (N-carbobenzoxyl-leu-leu-norvalinal) and 100 microM of the calpain inhibitor norLEU (N-acetyl-leu-leu-norleucinal; known to affect proteasome activity) counteracted the suppressive effects (253 +/- 17 and 262 +/- 10 respectively). The calpain inhibitor alIMET (N-acetyl-leu-leu-methional) had no effect. MG115 (10 microM) and norLEU (100 microM) blocked nitric oxide formation induced by IL-1beta, while alIMET was without effect. We also investigated if IL-1beta could influence the expression of two inducible proteasome subunits, namely LMP2 and LMP7, and found that the cytokine increased the mRNA expression of the proteasome subunit LMP2 in islets, and that the proteasome inhibitor MG115 prevented this increase. In conclusion our study shows that IL-1beta increases the transcription of the proteasome subunit LMP2, and that the proteasome is involved in IL-1beta induced suppression of islet function. Moreover, the observation that inhibitors of the proteasome protect islets against IL-1beta induced inhibition of glucose metabolism, suggests that these compounds might be worthwile to explore in future therapies against the development of type 1 diabetes.

  10. Proteolytic processing of ovalbumin and beta-galactosidase by the proteasome to a yield antigenic peptides

    National Research Council Canada - National Science Library

    Dick, LR; Aldrich, C; Jameson, SC; Moomaw, CR; Pramanik, BC; Doyle, CK; DeMartino, GN; Bevan, MJ; Forman, JM; Slaughter, CA

    1994-01-01

    ... in the proteolytic processing of polypeptide Ags. We have tested the ability of the 20S proteasome to produce peptides that can be presented by class I molecules as targets for killing by OVA-specific and beta-galactosidase- specific CTL clones...

  11. Suppression of the macrophage proteasome by ethanol impairs MHC class I antigen processing and presentation.

    Directory of Open Access Journals (Sweden)

    Alain J D'Souza

    Full Text Available Alcohol binge-drinking (acute ethanol consumption is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.

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

    Science.gov (United States)

    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. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  13. Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells

    NARCIS (Netherlands)

    Somborac-Bacura, Anita; van der Toorn, Marco; Franciosi, Lorenza; Slebos, Dirk-Jan; Zanic-Grubisic, Tihana; Bischoff, Rainer; van Oosterhout, Antoon J. M.

    Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease. Cigarette smoke (CS) causes oxidative stress and severe damage to proteins in the lungs. One of the main systems to protect cells from the accumulation of damaged proteins is the ubiquitin-proteasome pathway. In

  14. Suppression of the Oncogenic Transcription Factor FOXM1 by Proteasome Inhibitors

    Directory of Open Access Journals (Sweden)

    Andrei L. Gartel

    2014-01-01

    Full Text Available The oncogenic transcription factor FOXM1 is one of the key regulators of tumorigenesis. We found that FOXM1 upregulates its own transcription and its protein stability depends on its interaction with the chaperone nucleophosmin. We also determined that FOXM1 is negatively regulated by the tumor suppressor p53. We identified the thiazole antibiotics Siomycin A and thiostrepton as inhibitors of transcriptional activity and FOXM1 expression via proteasome inhibition. In addition, we found that all tested proteasome inhibitors target FOXM1. We showed synergy between thiostrepton and bortezomib in different human cancer cell lines and in vivo. We generated isogenic human cancer cell lines of different origin with wild-type p53 or p53 knockdown and we demonstrated that proteasome inhibitors induce p53-independent apoptosis in these cells. Using RNA-interference or proteasome inhibitors to inhibit FOXM1 we found that suppression of FOXM1 sensitized human cancer cells to apoptosis induced by DNA-damaging agents or oxidative stress. We encapsulated thiostrepton into micelle-nanoparticles and after injection we detected accumulation of nanoparticles in tumors and in the livers of treated mice. This treatment led to inhibition of human xenograft tumor growth in nude mice. Our data indicate that targeting FOXM1 increases apoptosis and inhibits tumor growth.

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

    Science.gov (United States)

    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...

  16. Bioinformatic analysis of functional differences between the immunoproteasome and the constitutive proteasome

    DEFF Research Database (Denmark)

    Kesmir, Can; van Noort, V.; de Boer, R.J.

    2003-01-01

    not yet been quantified how different the specificity of two forms of the proteasome are. The main question, which still lacks direct evidence, is whether the immunoproteasome generates more MHC ligands. Here we use bioinformatics tools to quantify these differences and show that the immunoproteasome...

  17. High Levels of Serum Ubiquitin and Proteasome in a Case of HLA-B27 Uveitis

    Directory of Open Access Journals (Sweden)

    Settimio Rossi

    2017-02-01

    Full Text Available In this paper, the authors describe a case of high serum levels of ubiquitin and proteasome in a woman under an acute attack of autoimmune uveitis. The woman was 52 years old, diagnosed as positive for the Human leukocyte antigen-B27 gene, and came to our observation in January 2013 claiming a severe uveitis attack that involved the right eye. During the acute attack of uveitis, this woman had normal serum biochemical parameters but higher levels of serum ubiquitin and proteasome 20S subunit, with respect to a healthy volunteer matched for age and sex. These levels correlated well with the clinical score attributed to uveitis. After the patient was admitted to therapy, she received oral prednisone in a de-escalation protocol (doses from 50 to 5 mg/day for four weeks. Following this therapy, she had an expected reduction of clinical signs and score for uveitis, but concomitantly she had a reduction of the serum levels of ubiquitin, poliubiquitinated proteins (MAb-FK1 and proteasome 20S activity. Therefore, a role for ubiquitin and proteasome in the development of human autoimmune uveitis has been hypothesized.

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

    Science.gov (United States)

    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...

  19. Optimization of peptidomimetic boronates bearing a P3 bicyclic scaffold as proteasome inhibitors.

    Science.gov (United States)

    Troiano, Valeria; Scarbaci, Kety; Ettari, Roberta; Micale, Nicola; Cerchia, Carmen; Pinto, Andrea; Schirmeister, Tanja; Novellino, Ettore; Grasso, Silvana; Lavecchia, Antonio; Zappalà, Maria

    2014-08-18

    A new series of pseudopeptide boronate proteasome inhibitors (2-3) was developed, through optimization of our previously described analogs of bortezomib, bearing a bicyclic 1,6-naphthyridin-5(6H)-one scaffold as P3 fragment (1). The biological evaluation on human 20S proteasome displayed a promising inhibition profile, especially for compounds bearing a P2 ethylene fragment, which exhibited Ki values in the nanomolar range for the ChT-L activity (e.g. 2a, Ki = 0.057 μM) and considerable selectivity for proteasome over bovine pancreatic α-chymotrypsin. Docking experiments into the yeast 20S proteasome revealed that the ligands are accommodated predominantly into the ChT-L site and that they covalently bind to the active site threonine residue via boron atom. Within the cellular assays performed against a 60 cancer cell line panel, compounds 3e and 3f demonstrated also good antiproliferative activity and compound 3f emerged as promising lead compound for the development of anticancer agents targeting melanoma and non-small cell lung cancer. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

    Science.gov (United States)

    2003-04-01

    two independent experiments. rapamycin on mammalian cells is its inhibitory effect Caspase-3 activity in normal ( MRC5 ) and AT- on 26s proteasome...Exponentially growing normal ( MRC5 ) cin could account for the observed G, arrest in wild- fibroblast and fibroblast from a patient with ataxia telang

  1. High Levels of Serum Ubiquitin and Proteasome in a Case of HLA-B27 Uveitis.

    Science.gov (United States)

    Rossi, Settimio; Gesualdo, Carlo; Maisto, Rosa; Trotta, Maria Consiglia; Di Carluccio, Nadia; Brigida, Annalisa; Di Iorio, Valentina; Testa, Francesco; Simonelli, Francesca; D'Amico, Michele; Di Filippo, Clara

    2017-02-26

    In this paper, the authors describe a case of high serum levels of ubiquitin and proteasome in a woman under an acute attack of autoimmune uveitis. The woman was 52 years old, diagnosed as positive for the Human leukocyte antigen-B27 gene, and came to our observation in January 2013 claiming a severe uveitis attack that involved the right eye. During the acute attack of uveitis, this woman had normal serum biochemical parameters but higher levels of serum ubiquitin and proteasome 20S subunit, with respect to a healthy volunteer matched for age and sex. These levels correlated well with the clinical score attributed to uveitis. After the patient was admitted to therapy, she received oral prednisone in a de-escalation protocol (doses from 50 to 5 mg/day) for four weeks. Following this therapy, she had an expected reduction of clinical signs and score for uveitis, but concomitantly she had a reduction of the serum levels of ubiquitin, poliubiquitinated proteins (MAb-FK1) and proteasome 20S activity. Therefore, a role for ubiquitin and proteasome in the development of human autoimmune uveitis has been hypothesized.

  2. Combination of Proteasomal Inhibitors Lactacystin and MG132 Induced Synergistic Apoptosis in Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Robert B. Shirley

    2005-12-01

    Full Text Available The proteasome inhibitor Velcade (bortezomib/PS-341 has been shown to block the targeted proteolytic degradation of short-lived proteins that are involved in cell maintenance, growth, division, and death, advocating the use of proteasomal inhibitors as therapeutic agents. Although many studies focused on the use of one proteasomal inhibitor for therapy, we hypothesized that the combination of proteasome inhibitors Lactacystin (AG Scientific, Inc., San Diego, CA and MG132 (Biomol International, Plymouth Meeting, PA may be more effective in inducing apoptosis. Additionally, this regimen would enable the use of sublethal doses of individual drugs, thus reducing adverse effects. Results indicate a significant increase in apoptosis when LNCaP prostate cancer cells were treated with increasing levels of Lactacystin, MG132, or a combination of sublethal doses of these two inhibitors. Furthermore, induction in apoptosis coincided with a significant loss of IKKα, IKKβ, and IKKγ proteins and NFκB activity. In addition to describing effective therapeutic agents, we provide a model system to facilitate the investigation of the mechanism of action of these drugs and their effects on the IKK-NFκB axis.

  3. THE PROTEASOME IS A TARGET OF OXIDATIVE DAMAGE IN HUMAN RETINA PIGMENT EPITHELIAL CELLS

    Science.gov (United States)

    Purpose: Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study is to investigate the effect of oxidative stress on the UPP in retina pigment epithelial cells. Methods: To mimic physiological oxidative stress...

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

    NARCIS (Netherlands)

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

    2016-01-01

    The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long

  5. POSSIBLE ROLE OF LIVER PROTEASOMES IN THE REALIZATION OF MECHANISMS OF TRANSPLANTATION TOLERANCE

    Directory of Open Access Journals (Sweden)

    G. A. Bozhok

    2011-01-01

    Full Text Available In contrast to the majority of organs in liver non-specific immunity predominates over adaptive one, and in response to the antigen presentation develops preferably not immune reaction but immunological tolerance. Tolerance is considered to provide some processes, such as apoptosis of reactive T-cells, immune deviation and active suppression of immune reactions. At the same time there are the grounds for believing that an important role in regulation of liver immune response is played by proteasomes, intracellular multiprotease protein complexes. This is confirmed by the fact of application of proteasome inhibitor bortezomib as immune suppressor in transplantology. Immune 26S- and 20S-proteoasomes participate in the formation of antigen oligopeptides and play a key role in T-cell immune response. It has been shown that the pool of proteasomes is subjected to significant changes during ontogenesis of immune competent organs. The changes in the pool of proteasosmes occur likely during the development of specific tolerance in transplantation too. The knowledge of the peculiarities of proteasome functioning and regularities of alterations of their shapes will enable the revealing of the mechanisms responsible for either graft rejection or acceptance. 

  6. Role of Proteasome-Dependent Protein Degradation in Long-Term Operant Memory in "Aplysia"

    Science.gov (United States)

    Lyons, Lisa C.; Gardner, Jacob S.; Gandour, Catherine E.; Krishnan, Harini C.

    2017-01-01

    We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in "Aplysia" using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through…

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

    Science.gov (United States)

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

    2016-08-15

    The success of inhibition of the proteasome by formation of covalent bonds is a major victory over the long held-view that this would lead to binding the wrong targets and undoubtedly lead to toxicity. Great challenges are now found in uncovering ensembles of new moieties capable of forming long lasting ties. We have introduced peptido sulfonyl fluorides for this purpose. Tuning the reactivity of this electrophilic trap may be crucial for modulating the biological action. Here we describe incorporation of a vinyl moiety into a peptido sulfonyl fluoride backbone, which should lead to a combined attack of the proteasome active site threonine on the double bond and the sulfonyl fluoride. Although this led to strong proteasome inhibitors, in vitro studies did not unambiguously demonstrate the formation of the proposed seven-membered ring structure. Possibly, formation of a seven-membered covalent adduct with the proteosomal active site threonine can only be achieved within the context of the enzyme. Nevertheless, this dual warhead concept may provide exclusive possibilities for duration and selectivity of proteasome inhibition. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Differential Regulation of the Autophagy and Proteasome Pathways in Skeletal Muscles in Sepsis.

    Science.gov (United States)

    Stana, Flavia; Vujovic, Marija; Mayaki, Dominique; Leduc-Gaudet, Jean-Philippe; Leblanc, Philippe; Huck, Laurent; Hussain, Sabah N A

    2017-09-01

    Skeletal muscle fiber atrophy develops in response to severe sepsis, but it is unclear as to how the proteolytic pathways that are involved in its development are differentially regulated. We investigated the link between sepsis-induced fiber atrophy and activation of the proteasome and autophagy pathways and whether the degree of activation is more severe and sustained in limb muscles than it is in the diaphragm. Randomized controlled experiment. Animal research laboratory. Adult male C57/BL6 mice. Two groups of animals were studied. The sepsis group was subjected to a cecal ligation and perforation technique, whereas the control (sham) group was subjected to abdominal surgery without cecal ligation and perforation. Measurements for both groups were performed 24, 48, and 96 hours after the surgical procedure. Atrophy was quantified in the diaphragm and tibialis anterior by measuring fiber diameter. Autophagy was evaluated using electron microscopic detection of autophagosomes and by measuring LC3B protein lipidation and autophagy-related protein expressions. Proteasomal degradation was quantified by measuring chymotrypsin-like activity of the 26S proteasome and messenger RNA expressions of muscle-specific E3 ligases. Sepsis triggered transient fiber atrophy in the diaphragm that lasted for 24 hours and prolonged atrophy in the tibialis anterior that persisted for 96 hours. The autophagy and proteasome pathways were activated in both muscles at varying intensities over the time course of sepsis. Activation was more pronounced in the tibialis anterior than in the diaphragm. Sepsis inhibited the V-Akt thymoma viral oncogene homolog 1 and complex 1 of the mammalian target of rapamycin pathways and stimulated the AMP-activated protein kinase pathway in both muscles. Sepsis triggers more severe and sustained muscle fiber atrophy in limb muscles when compared with respiratory muscle. This response is associated with enhanced proteasomal and autophagic proteolytic pathway

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

    Directory of Open Access Journals (Sweden)

    Xian Fu

    2016-05-01

    Full Text Available 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.

  10. High-resolution cryo-EM proteasome structures in drug development.

    Science.gov (United States)

    Morris, Edward P; da Fonseca, Paula C A

    2017-06-01

    With the recent advances in biological structural electron microscopy (EM), protein structures can now be obtained by cryo-EM and single-particle analysis at resolutions that used to be achievable only by crystallographic or NMR methods. We have explored their application to study protein-ligand interactions using the human 20S proteasome, a well established target for cancer therapy that is also being investigated as a target for an increasing range of other medical conditions. The map of a ligand-bound human 20S proteasome served as a proof of principle that cryo-EM is emerging as a realistic approach for more general structural studies of protein-ligand interactions, with the potential benefits of extending such studies to complexes that are unfavourable to other methods and allowing structure determination under conditions that are closer to physiological, preserving ligand specificity towards closely related binding sites. Subsequently, the cryo-EM structure of the Plasmodium falciparum 20S proteasome, with a new prototype specific inhibitor bound, revealed the molecular basis for the ligand specificity towards the parasite complex, which provides a framework to guide the development of highly needed new-generation antimalarials. Here, the cryo-EM analysis of the ligand-bound human and P. falciparum 20S proteasomes is reviewed, and a complete description of the methods used for structure determination is provided, including the strategy to overcome the bias orientation of the human 20S proteasome on electron-microscope grids and details of the icr3d software used for three-dimensional reconstruction.

  11. Inhibition of proteasomal degradation of rpn4 impairs nonhomologous end-joining repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Donghong Ju

    Full Text Available BACKGROUND: The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the transcription factor Rpn4 induces the proteasome genes and is rapidly degraded by the assembled proteasome. The integrity of the Rpn4-proteasome feedback loop is critical for cell viability under stressed conditions. We have demonstrated that inhibition of Rpn4 degradation sensitizes cells to DNA damage, particularly in response to high doses of DNA damaging agents. The underlying mechanism, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast genetics and biochemical approach we show that inhibition of Rpn4 degradation displays a synthetic growth defect with deletion of the MEC1 checkpoint gene and sensitizes several checkpoint mutants to DNA damage. In addition, inhibition of Rpn4 degradation leads to a defect in repair of double-strand breaks (DSBs by nonhomologous end-joining (NHEJ. The expression levels of several key NHEJ genes are downregulated and the recruitment of Yku70 to a DSB is reduced by inhibition of Rpn4 degradation. We find that Rpn4 and the proteasome are recruited to a DSB, suggesting their direct participation in NHEJ. Inhibition of Rpn4 degradation may result in a concomitant delay of release of Rpn4 and the proteasome from a DSB. CONCLUSION/SIGNIFICANCE: This study provides the first evidence for the role of proteasomal degradation of Rpn4 in NHEJ.

  12. Nutrigenomics of high fat diet induced obesity in mice suggests relationships between susceptibility to fatty liver disease and the proteasome.

    Directory of Open Access Journals (Sweden)

    Helen Waller-Evans

    Full Text Available Nutritional factors play important roles in the etiology of obesity, type 2 diabetes mellitus and their complications through genotype x environment interactions. We have characterised molecular adaptation to high fat diet (HFD feeding in inbred mouse strains widely used in genetic and physiological studies. We carried out physiological tests, plasma lipid assays, obesity measures, liver histology, hepatic lipid measurements and liver genome-wide gene transcription profiling in C57BL/6J and BALB/c mice fed either a control or a high fat diet. The two strains showed marked susceptibility (C57BL/6J and relative resistance (BALB/c to HFD-induced insulin resistance and non alcoholic fatty liver disease (NAFLD. Global gene set enrichment analysis (GSEA of transcriptome data identified consistent patterns of expression of key genes (Srebf1, Stard4, Pnpla2, Ccnd1 and molecular pathways in the two strains, which may underlie homeostatic adaptations to dietary fat. Differential regulation of pathways, including the proteasome, the ubiquitin mediated proteolysis and PPAR signalling in fat fed C57BL/6J and BALB/c suggests that altered expression of underlying diet-responsive genes may be involved in contrasting nutrigenomic predisposition and resistance to insulin resistance and NAFLD in these models. Collectively, these data, which further demonstrate the impact of gene x environment interactions on gene expression regulations, contribute to improved knowledge of natural and pathogenic adaptive genomic regulations and molecular mechanisms associated with genetically determined susceptibility and resistance to metabolic diseases.

  13. Nutrigenomics of high fat diet induced obesity in mice suggests relationships between susceptibility to fatty liver disease and the proteasome.

    Science.gov (United States)

    Waller-Evans, Helen; Hue, Christophe; Fearnside, Jane; Rothwell, Alice R; Lockstone, Helen E; Caldérari, Sophie; Wilder, Steven P; Cazier, Jean-Baptiste; Scott, James; Gauguier, Dominique

    2013-01-01

    Nutritional factors play important roles in the etiology of obesity, type 2 diabetes mellitus and their complications through genotype x environment interactions. We have characterised molecular adaptation to high fat diet (HFD) feeding in inbred mouse strains widely used in genetic and physiological studies. We carried out physiological tests, plasma lipid assays, obesity measures, liver histology, hepatic lipid measurements and liver genome-wide gene transcription profiling in C57BL/6J and BALB/c mice fed either a control or a high fat diet. The two strains showed marked susceptibility (C57BL/6J) and relative resistance (BALB/c) to HFD-induced insulin resistance and non alcoholic fatty liver disease (NAFLD). Global gene set enrichment analysis (GSEA) of transcriptome data identified consistent patterns of expression of key genes (Srebf1, Stard4, Pnpla2, Ccnd1) and molecular pathways in the two strains, which may underlie homeostatic adaptations to dietary fat. Differential regulation of pathways, including the proteasome, the ubiquitin mediated proteolysis and PPAR signalling in fat fed C57BL/6J and BALB/c suggests that altered expression of underlying diet-responsive genes may be involved in contrasting nutrigenomic predisposition and resistance to insulin resistance and NAFLD in these models. Collectively, these data, which further demonstrate the impact of gene x environment interactions on gene expression regulations, contribute to improved knowledge of natural and pathogenic adaptive genomic regulations and molecular mechanisms associated with genetically determined susceptibility and resistance to metabolic diseases.

  14. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

    Energy Technology Data Exchange (ETDEWEB)

    Maeda, Tomoji, E-mail: t-maeda@nichiyaku.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan); Nakajima, Toshihiro [Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku, Shinjyuku, Tokyo, Tokyo, 160-8402 (Japan); Komano, Hiroto, E-mail: hkomano@iwate-med.ac.jp [Department of Neuroscience, School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-Cho, Shiwagun, Iwate, 028-3603 (Japan)

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

  15. Age-related and sex-specific differences in proteasome activity in individual Drosophila flies from wild type, longevity-selected and stress resistant strains

    DEFF Research Database (Denmark)

    Hansen, Tina Østergaard; Sarup, Pernille Merete; Loeschcke, Volker

    2012-01-01

    In this study we have measured the caspase-like proteasome activity in individual male and female Drosophila flies by using a non-denaturing lysing technique that consistently extracts total protein. The mean proteasome activity in control C1 females was more than 2 times higher as compared...... with that in C1 males. However, in longevity-selected LS1 flies the proteasome activity was significantly lower compared to C1 flies, but the sex differences were maintained to some extent. Five other stress resistant lines also had significantly reduced proteasome activity in both sexes. During ageing......, there was a progressive decrease in proteasome activity in C1 females, but not in C1 males. This age-related decline in proteasome activity observed in C1 females was not observed in LS1 flies. We conclude that the proteasome activity in control male and female flies is significantly different from each other...

  16. Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon

    Directory of Open Access Journals (Sweden)

    Kremsner Peter G

    2008-09-01

    Full Text Available Abstract Background The emergence and spread of Plasmodium falciparum resistance to almost all available antimalarial drugs necessitates the search for new chemotherapeutic compounds. The ubiquitin/proteasome system plays a major role in overall protein turnover, especially in fast dividing eukaryotic cells including plasmodia. Previous studies show that the 20S proteasome is expressed and catalytically active in plasmodia and treatment with proteasome inhibitors arrests parasite growth. This is the first comprehensive screening of proteasome inhibitors with different chemical modes of action against laboratory strains of P. falciparum. Subsequently, a selection of inhibitors was tested in field isolates from Lambaréné, Gabon. Methods Epoxomicin, YU101, YU102, MG132, MG115, Z-L3-VS, Ada-Ahx3-L3-VS, lactacystin, bortezomib (Velcade®, gliotoxin, PR11 and PR39 were tested and compared to chloroquine- and artesunate-activities in a standardized in vitro drug susceptibility assay against P. falciparum laboratory strains 3D7, D10 and Dd2. Freshly obtained field isolates from Lambaréné, Gabon, were used to measure the activity of chloroquine, artesunate, epoxomicin, MG132, lactacystin and bortezomib. Parasite growth was detected through histidine-rich protein 2 (HRP2 production. Raw data were fitted by a four-parameter logistic model and individual inhibitory concentrations (50%, 90%, and 99% were calculated. Results Amongst all proteasome inhibitors tested, epoxomicin showed the highest activity in chloroquine-susceptible (IC50: 6.8 nM [3D7], 1.7 nM [D10] and in chloroquine-resistant laboratory strains (IC50: 10.4 nM [Dd2] as well as in field isolates (IC50: 8.5 nM. The comparator drug artesunate was even more active (IC50: 1.0 nM, whereas all strains were chloroquine-resistant (IC50: 113 nM. Conclusion The peptide α',β'-epoxyketone epoxomicin is highly active against P. falciparum regardless the grade of the parasite's chloroquine

  17. Enzymatice Activation of Proteasome Inhibitor Prodrugs by Prostate-Specific Antigen as Targeted Therapy for Prostate Cancer

    National Research Council Canada - National Science Library

    Denmeade, Samuel

    2001-01-01

    .... In the original proposal, proteasome inhibitors were selected as the cytotoxic agent. Initial studies revealed that this approach was not tenable due to the inherent instability of these compounds...

  18. Enzymatic Activation of Proteasome Inhibitor Prodrugs by Prostate-Specific Antigen as Targeted Therapy for Prostate Cancer

    National Research Council Canada - National Science Library

    Denmeade, Samuel

    2000-01-01

    .... In the original proposal, proteasome inhibitors were selected as the cytotoxic agent. Initial studies revealed that this approach was not tenable due to the inherent instability of these compounds...

  19. Gambogic Acid Is a Tissue-Specific Proteasome Inhibitor In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Xiaofen Li

    2013-01-01

    Full Text Available Gambogic acid (GA is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

  20. Salinosporamides A and B Inhibit Proteasome Activity and Delay the Degradation of N-end Rule Model Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Seungkyun; Bang, Daein; Choi, Wonhoon; Lee, Minjae [Kyung Hee Univ., Yongin (Korea, Republic of); Kim, Seonghwan; Oh, Dongchan [Seoul National Univ., Seoul (Korea, Republic of)

    2013-05-15

    The proteasome, which is highly evolutionarily conserved, is responsible for the degradation of most short-lived proteins in cells. Small-molecule inhibitors targeting the proteasome's degradative activity have been extensively developed as lead compounds for various human diseases. An exemplified molecule is bortezomib, which was approved by FDA in 2003 for the treatment of multiple myeloma. Here, using transiently and stably expressed N-end rule model substrates in mammalian cells, we evaluated and identified that salinosporamide A and salinosporamide B effectively inhibited the proteasomal degradation. Considering that a variety of proteasome substrates are implicated in the pathogenesis of many diseases, they have the potential to be clinically applicable as therapeutic agents.

  1. The 11S Proteasomal Activator REGγ Impacts Polyglutamine-Expanded Androgen Receptor Aggregation and Motor Neuron Viability through Distinct Mechanisms

    Directory of Open Access Journals (Sweden)

    Jill M. Yersak

    2017-05-01

    Full Text Available Spinal and bulbar muscular atrophy (SBMA is caused by expression of a polyglutamine (polyQ-expanded androgen receptor (AR. The inefficient nuclear proteasomal degradation of the mutant AR results in the formation of nuclear inclusions containing amino-terminal fragments of the mutant AR. PA28γ (also referred to as REGγ is a nuclear 11S-proteasomal activator with limited proteasome activation capabilities compared to its cytoplasmic 11S (PA28α, PA28β counterparts. To clarify the role of REGγ in polyQ-expanded AR metabolism, we carried out genetic and biochemical studies in cell models of SBMA. Overexpression of REGγ in a PC12 cell model of SBMA increased polyQ-expanded AR aggregation and contributed to polyQ-expanded AR toxicity in the presence of dihydrotestosterone (DHT. These effects of REGγ were independent of its association with the proteasome and may be due, in part, to the decreased binding of polyQ-expanded AR by the E3 ubiquitin-ligase MDM2. Unlike its effects in PC12 cells, REGγ overexpression rescued transgenic SBMA motor neurons from DHT-induced toxicity in a proteasome binding-dependent manner, suggesting that the degradation of a specific 11S proteasome substrate or substrates promotes motor neuron viability. One potential substrate that we found to play a role in mutant AR toxicity is the splicing factor SC35. These studies reveal that, depending on the cellular context, two biological roles for REGγ impact cell viability in the face of polyQ-expanded AR; a proteasome binding-independent mechanism directly promotes mutant AR aggregation while a proteasome binding-dependent mechanism promotes cell viability. The balance between these functions likely determines REGγ effects on polyQ-expanded AR-expressing cells.

  2. Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis†

    OpenAIRE

    Cheroni, Cristina; Marino, Marianna; Tortarolo, Massimo; Veglianese, Pietro; De Biasi, Silvia; Fontana, Elena; Zuccarello, Laura Vitellaro; Maynard, Christa J.; Dantuma, Nico P; Bendotti, Caterina

    2008-01-01

    In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by re...

  3. Small interfering RNA targeting mcl-1 enhances proteasome inhibitor-induced apoptosis in various solid malignant tumors

    Directory of Open Access Journals (Sweden)

    Zhou Wei

    2011-11-01

    Full Text Available Abstract Background Targeting the ubiquitin-proteasome pathway is a promising approach for anticancer strategies. Recently, we found Bik accumulation in cancer cell lines after they were treated with bortezomib. However, recent evidence indicates that proteasome inhibitors may also induce the accumulation of anti-apoptotic Bcl-2 family members. The current study was designed to analyze the levels of several anti-apoptotic members of Bcl-2 family in different human cancer cell lines after they were treated with proteasome inhibitors. Methods Different human cancer cell lines were treated with proteasome inhibitors. Western blot were used to investigate the expression of Mcl-1 and activation of mitochondrial apoptotic signaling. Cell viability was investigated using SRB assay, and induction of apoptosis was measured using flow cytometry. Results We found elevated Mcl-1 level in human colon cancer cell lines DLD1, LOVO, SW620, and HCT116; human ovarian cancer cell line SKOV3; and human lung cancer cell line H1299, but not in human breast cancer cell line MCF7 after they were treated with bortezomib. This dramatic Mcl-1 accumulation was also observed when cells were treated with other two proteasome inhibitors, MG132 and calpain inhibitor I (ALLN. Moreover, our results showed Mcl-1 accumulation was caused by stabilization of the protein against degradation. Reducing Mcl-1 accumulation by Mcl-1 siRNA reduced Mcl-1 accumulation and enhanced proteasome inhibitor-induced cell death and apoptosis, as evidenced by the increased cleavage of caspase-9, caspase-3, and poly (ADP-ribose polymerase. Conclusions Our results showed that it was not only Bik but also Mcl-1 accumulation during the treatment of proteasome inhibitors, and combining proteasome inhibitors with Mcl-1 siRNA would enhance the ultimate anticancer effect suggesting this combination might be a more effective strategy for cancer therapy.

  4. Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda).

    Science.gov (United States)

    Götze, Sandra; Saborowski, Reinhard; Martínez-Cruz, Oliviert; Muhlia-Almazán, Adriana; Sánchez-Paz, Arturo

    2017-11-01

    Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsin-like (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Cas-like to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L-1. The K M values ranged from 0.072 mmol L-1 for the Try-like activity of P. vannamei to 0.309 mmol L-1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing ~80-90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

  5. Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System

    Directory of Open Access Journals (Sweden)

    Florian Full

    2017-10-01

    Full Text Available Gammaherpesviruses like Epstein-Barr virus (EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS, an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML protein-associated nuclear bodies (PML-NBs by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.

  6. Mitochondrial and Ubiquitin Proteasome System Dysfunction in Ageing and Disease: Two Sides of the Same Coin?

    Directory of Open Access Journals (Sweden)

    Jaime M. Ross

    2015-08-01

    Full Text Available Mitochondrial dysfunction and impairment of the ubiquitin proteasome system have been described as two hallmarks of the ageing process. Additionally, both systems have been implicated in the etiopathogenesis of many age-related diseases, particularly neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease. Interestingly, these two systems are closely interconnected, with the ubiquitin proteasome system maintaining mitochondrial homeostasis by regulating organelle dynamics, the proteome, and mitophagy, and mitochondrial dysfunction impairing cellular protein homeostasis by oxidative damage. Here, we review the current literature and argue that the interplay of the two systems should be considered in order to better understand the cellular dysfunction observed in ageing and age-related diseases. Such an approach may provide valuable insights into molecular mechanisms underlying the ageing process, and further discovery of treatments to counteract ageing and its associated diseases. Furthermore, we provide a hypothetical model for the heterogeneity described among individuals during ageing.

  7. Pulse treatment with the proteasome inhibitor bortezomib inhibits osteoclast resorptive activity in clinically relevant conditions

    DEFF Research Database (Denmark)

    Boissy, P; Levin Andersen, Thomas; Lund, T

    2008-01-01

    Myeloma bone disease is due to bone degradation by osteoclasts, and absence of repair by bone forming osteoblasts. Recent observations suggest that the anti-myeloma drug bortezomib, a proteasome inhibitor, stimulates bone formation and may inhibit bone resorption. Here, we tested bortezomib...... on cultured osteoclasts in conditions mimicking the pulse treatment used in the clinic, thereby avoiding continuous proteasome inhibition and unselective toxicity. A 3h pulse with 25nM bortezomib followed by a 3-day culture in its absence markedly inhibited osteoclast activity as evaluated through bone...... cells drastically reduced their survival. We measured next the levels of two bone resorption markers in patients during the 3 days following five and seven therapeutic bortezomib administrations, respectively. These levels decreased significantly already 1-2 days after injection, and then increased...

  8. Identification of 2-thioxoimidazolidin-4-one derivatives as novel noncovalent proteasome and immunoproteasome inhibitors.

    Science.gov (United States)

    Maccari, Rosanna; Ettari, Roberta; Adornato, Ilenia; Naß, Alexandra; Wolber, Gerhard; Bitto, Alessandra; Mannino, Federica; Aliquò, Federica; Bruno, Giuseppe; Nicolò, Francesco; Previti, Santo; Grasso, Silvana; Zappalà, Maria; Ottanà, Rosaria

    2017-12-26

    This paper describes the design, synthesis, and biological evaluation of 2-thioxoimidazolidin-4-one derivatives as inhibitors of proteasome and immunoproteasome, potential targets for the treatment of hematological malignancies. In particular, we focused our efforts on the design of noncovalent inhibitors, which might be a promising therapeutic option potentially devoid of drawbacks and side-effects related to irreversible inhibition. Among all the synthesized compounds, we identified a panel of active inhibitors with Ki values towards one or two chymotrypsin-like activities of proteasome (β5c) and immunoproteasome (β5i and β1i subunits) in the low micromolar range. Docking studies suggested a unique binding mode of the molecules in the catalytic site of immunoproteasome proteolytic subunits. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    Energy Technology Data Exchange (ETDEWEB)

    Radulescu, Razvan T., E-mail: ratura@gmx.net [Molecular Concepts Research (MCR), Muenster (Germany); Duckworth, William C. [Department of Medicine, Phoenix VA Health Care System, Phoenix, AZ (United States); Levy, Jennifer L. [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States); Fawcett, Janet, E-mail: janet.fawcett@va.gov [Research Service, Phoenix VA Health Care System, Phoenix, AZ (United States)

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  10. Oxidative stress regulates the ubiquitin-proteasome system and immunoproteasome functioning in a mouse model of X-adrenoleukodystrophy.

    Science.gov (United States)

    Launay, Nathalie; Ruiz, Montserrat; Fourcade, Stéphane; Schlüter, Agatha; Guilera, Cristina; Ferrer, Isidre; Knecht, Erwin; Pujol, Aurora

    2013-03-01

    Oxidative damage is a pivotal aetiopathogenic factor in X-linked adrenoleukodystrophy. This is a neurometabolic disease characterized by the accumulation of very-long-chain fatty acids owing to the loss of function of the peroxisomal transporter Abcd1. Here, we used the X-linked adrenoleukodystrophy mouse model and patient's fibroblasts to detect malfunctioning of the ubiquitin-proteasome system resulting from the accumulation of oxidatively modified proteins, some involved in bioenergetic metabolism. Furthermore, the immunoproteasome machinery appears upregulated in response to oxidative stress, in the absence of overt inflammation. i-Proteasomes are recruited to mitochondria when fibroblasts are exposed to an excess of very-long-chain fatty acids in response to oxidative stress. Antioxidant treatment regulates proteasome expression, prevents i-proteasome induction and translocation of i-proteasomes to mitochondria. Our findings support a key role of i-proteasomes in quality control in mitochondria during oxidative damage in X-linked adrenoleukodystrophy, and perhaps in other neurodegenerative conditions with similar pathogeneses.

  11. Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis.

    Science.gov (United States)

    Chandra, Abhishek; Wang, Luqiang; Young, Tiffany; Zhong, Leilei; Tseng, Wei-Ju; Levine, Michael A; Cengel, Keith; Liu, X Sherry; Zhang, Yejia; Pignolo, Robert J; Qin, Ling

    2018-01-01

    Bone atrophy and its related fragility fractures are frequent, late side effects of radiotherapy in cancer survivors and have a detrimental impact on their quality of life. In another study, we showed that parathyroid hormone 1-34 and anti-sclerostin antibody attenuates radiation-induced bone damage by accelerating DNA repair in osteoblasts. DNA damage responses are partially regulated by the ubiquitin proteasome pathway. In the current study, we examined whether proteasome inhibitors have similar bone-protective effects against radiation damage. MG132 treatment greatly reduced radiation-induced apoptosis in cultured osteoblastic cells. This survival effect was owing to accelerated DNA repair as revealed by γH2AX foci and comet assays and to the up-regulation of Ku70 and DNA-dependent protein kinase, catalytic subunit, essential DNA repair proteins in the nonhomologous end-joining pathway. Administration of bortezomib (Bzb) reversed the loss of trabecular bone structure and strength in mice at 4 wk after focal radiation. Histomorphometry revealed that Bzb significantly increased the number of osteoblasts and activity in the irradiated area and suppressed the number and activity of osteoclasts, regardless of irradiation. Two weeks of Bzb treatment accelerated DNA repair in bone-lining osteoblasts and thus promoted their survival. Meanwhile, it also inhibited bone marrow adiposity. Taken together, we demonstrate a novel role of proteasome inhibitors in treating radiation-induced osteoporosis.-Chandra, A., Wang, L., Young, T., Zhong, L., Tseng, W.-J., Levine, M. A., Cengel, K., Liu, X. S., Zhang, Y., Pignolo, R. J., Qin, L. Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis. © FASEB.

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

    Science.gov (United States)

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

    2006-01-01

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

  13. Synergy between proteasome inhibitors and imatinib mesylate in chronic myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Zheng Hu

    Full Text Available BACKGROUND: Resistance developed by leukemic cells, unsatisfactory efficacy on patients with chronic myeloid leukemia (CML at accelerated and blastic phases, and potential cardiotoxity, have been limitations for imatinib mesylate (IM in treating CML. Whether low dose IM in combination with agents of distinct but related mechanisms could be one of the strategies to overcome these concerns warrants careful investigation. METHODS AND FINDINGS: We tested the therapeutic efficacies as well as adverse effects of low dose IM in combination with proteasome inhibitor, Bortezomib (BOR or proteasome inhibitor I (PSI, in two CML murine models, and investigated possible mechanisms of action on CML cells. Our results demonstrated that low dose IM in combination with BOR exerted satisfactory efficacy in prolongation of life span and inhibition of tumor growth in mice, and did not cause cardiotoxicity or body weight loss. Consistently, BOR and PSI enhanced IM-induced inhibition of long-term clonogenic activity and short-term cell growth of CML stem/progenitor cells, and potentiated IM-caused inhibition of proliferation and induction of apoptosis of BCR-ABL+ cells. IM/BOR and IM/PSI inhibited Bcl-2, increased cytoplasmic cytochrome C, and activated caspases. While exerting suppressive effects on BCR-ABL, E2F1, and beta-catenin, IM/BOR and IM/PSI inhibited proteasomal degradation of protein phosphatase 2A (PP2A, leading to a re-activation of this important negative regulator of BCR-ABL. In addition, both combination therapties inhibited Bruton's tyrosine kinase via suppression of NFkappaB. CONCLUSION: These data suggest that combined use of tyrosine kinase inhibitor and proteasome inhibitor might be helpful for optimizing CML treatment.

  14. Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

    Directory of Open Access Journals (Sweden)

    Barré-Sinoussi Françoise

    2009-05-01

    Full Text Available Abstract Background The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. Results Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+ and BeWo-CD4+ (CD4+, CCR5+, CXCR4+ cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. Conclusion Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

  15. Tat-binding protein-1 (TBP-1), an ATPase of 19S regulatory particles of the 26S proteasome, enhances androgen receptor function in cooperation with TBP-1-interacting protein/Hop2.

    Science.gov (United States)

    Satoh, Tetsurou; Ishizuka, Takahiro; Tomaru, Takuya; Yoshino, Satoshi; Nakajima, Yasuyo; Hashimoto, Koshi; Shibusawa, Nobuyuki; Monden, Tsuyoshi; Yamada, Masanobu; Mori, Masatomo

    2009-07-01

    The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

  16. The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation

    Directory of Open Access Journals (Sweden)

    Marcel Stark

    2014-01-01

    Full Text Available The standardized Ginkgo biloba extract EGb 761 has well-described antioxidative activities and effects on different cytoprotective signaling pathways. Consequently, a potential use of EGb 761 in neurodegenerative diseases has been proposed. A common characteristic feature of a variety of such disorders is the pathologic formation of protein aggregates, suggesting a crucial role for protein homeostasis. In this study, we show that EGb 761 increased the catalytic activity of the proteasome and enhanced protein degradation in cultured cells. We further investigated this effect in a cellular model of Huntington’s disease (HD by employing cells expressing pathologic variants of a polyglutamine protein (polyQ protein. We show that EGb 761 affected these cells by (i increasing proteasome activity and (ii inducing a more efficient degradation of aggregation-prone proteins. These results demonstrate a novel activity of EGb 761 on protein aggregates by enhancing proteasomal protein degradation, suggesting a therapeutic use in neurodegenerative disorders with a disturbed protein homeostasis.

  17. Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions.

    Science.gov (United States)

    Concannon, Claire; Lahue, Robert S

    2014-01-01

    Trinucleotide repeat (TNR) expansion underpins a number of inheritable neurological human disorders. Multiple mechanisms are thought to contribute to the expansion process. The incorrect processing of the repeat tract by DNA repair proteins can drive this mutation process forward, as expansions are suppressed following ablation of certain repair factors in mouse models and cell models of disease. Nucleotide excision repair (NER) is one repair pathway implicated in TNR instability, although most previous work focussed on TNR contractions, not expansions. Here we investigated the role of NER in modulating expansions of threshold-length (CTG·CAG) repeats in yeast. We show that both the global genome and transcription-coupled repair subpathways promote expansions of threshold-length TNRs. Furthermore, NER works with the 26S proteasome to drive expansions, based on analysis of double mutants defective in both pathways, and of Rad23, a protein involved in both NER and the shuttling of ubiquitinated proteins to the proteasome. This work provides the first evidence that both subpathways of NER can promote threshold-length TNR expansions and that NER interacts with the proteasome to drive expansions. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Analyzing proteasomal subunit expression reveals Rpt4 as a prognostic marker in stage II colorectal cancer.

    LENUS (Irish Health Repository)

    2012-02-01

    Colorectal cancer is a leading cause of cancer-related deaths worldwide. Early diagnosis and treatment of colorectal cancer is the key to improving survival rates and as such a need exists to identify patients who may benefit from adjuvant chemotherapy. The dysregulation of the ubiquitin-proteasome system (UPS) has been implicated in oncogenesis and cancer cell survival, and proteasome inhibitors are in clinical use for a number of malignancies including multiple myeloma. In our study, we examined the protein expression of several key components of the UPS in colorectal cancer using immunohistochemistry to determine expression levels of ubiquitinylated proteins and the proteasomal subunits, 20S core and Rpt4 in a cohort of 228 patients with colon cancer. Multivariate Cox analysis revealed that neither the intensity of either ubiquitinylated proteins or the 20S core was predictive in either Stage II or III colon cancer for disease free survival or overall survival. In contrast, in Stage II patients increased Rpt4 staining was significantly associated with disease free survival (Cox proportional hazard ratio 0.605; p = 0.0217). Our data suggest that Rpt4 is an independent prognostic variable for Stage II colorectal cancer and may aid in the decision of which patients undergo adjuvant chemotherapy.

  19. Interleukin-15 enhances proteasomal degradation of bid in normal lymphocytes: implications for large granular lymphocyte leukemias.

    Science.gov (United States)

    Hodge, Deborah L; Yang, Jun; Buschman, Matthew D; Schaughency, Paul M; Dang, Hong; Bere, William; Yang, Yili; Savan, Ram; Subleski, Jeff J; Yin, Xiao-Ming; Loughran, Thomas P; Young, Howard A

    2009-05-01

    Large granular lymphocyte (LGL) leukemia is a clonal proliferative disease of T and natural killer (NK) cells. Interleukin (IL)-15 is important for the development and progression of LGL leukemia and is a survival factor for normal NK and T memory cells. IL-15 alters expression of Bcl-2 family members, Bcl-2, Bcl-XL, Bim, Noxa, and Mcl-1; however, effects on Bid have not been shown. Using an adoptive transfer model, we show that NK cells from Bid-deficient mice survive longer than cells from wild-type control mice when transferred into IL-15-null mice. In normal human NK cells, IL-15 significantly reduces Bid accumulation. Decreases in Bid are not due to alterations in RNA accumulation but result from increased proteasomal degradation. IL-15 up-regulates the E3 ligase HDM2 and we find that HDM2 directly interacts with Bid. HDM2 suppression by short hairpin RNA increases Bid accumulation lending further support for HDM2 involvement in Bid degradation. In primary leukemic LGLs, Bid levels are low but are reversed with bortezomib treatment with subsequent increases in LGL apoptosis. Overall, these data provide a novel molecular mechanism for IL-15 control of Bid that potentially links this cytokine to leukemogenesis through targeted proteasome degradation of Bid and offers the possibility that proteasome inhibitors may aid in the treatment of LGL leukemia.

  20. Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp transcription factors

    Directory of Open Access Journals (Sweden)

    Pathi Satya

    2011-08-01

    Full Text Available Abstract Background Betulinic acid (BA inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells. Methods The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a and ZBTB10 mRNA expression. Results BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS, ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10. Conclusions These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.

  1. Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni

    Science.gov (United States)

    de Paula, Renato G.; Ornelas, Alice M. M.; Moreira, Érika B. C.; Badoco, Fernanda Rafacho; Magalhães, Lizandra G.; Verjovski-Almeida, Sergio; Rodrigues, Vanderlei

    2017-01-01

    Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites’ tegument. We identified 1,919 genes with a statistically significant (q-value ≤ 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG-132 caused important changes in the worm tegument; peeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis

  2. Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Enyara R Morais

    Full Text Available Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA. Scanning electron microscopy was used to document changes in the parasites' tegument. We identified 1,919 genes with a statistically significant (q-value ≤ 0.025 differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG-132 caused important changes in the worm tegument; peeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two

  3. Proteomic analysis reveals that proteasome subunit beta 6 is involved in hypoxia-induced pulmonary vascular remodeling in rats.

    Directory of Open Access Journals (Sweden)

    Jian Wang

    Full Text Available Chronic hypoxia (CH is known to be one of the major causes of pulmonary hypertension (PH, which is characterized by sustained elevation of pulmonary vascular resistance resulting from vascular remodeling. In this study, we investigated whether the ubiquitin proteasome system (UPS was involved in the mechanism of hypoxia-induced pulmonary vascular remodeling. We isolated the distal pulmonary artery (PA from a previously defined chronic hypoxic pulmonary hypertension (CHPH rat model, performed proteomic analyses in search of differentially expressed proteins belonging to the UPS, and subsequently identified their roles in arterial remodeling.Twenty-two proteins were differently expressed between the CH and normoxic group. Among them, the expression of proteasome subunit beta (PSMB 1 and PSMB6 increased after CH exposure. Given that PSMB1 is a well-known structural subunit and PSMB6 is a functional subunit, we sought to assess whether PSMB6 could be related to the multiple functional changes during the CHPH process. We confirmed the proteomic results by real-time PCR and Western blot. With the increase in quantity of the active subunit, proteasome activity in both cultured pulmonary artery smooth muscle cells (PASMCs and isolated PA from the hypoxic group increased. An MTT assay revealed that the proteasome inhibitor MG132 was able to attenuate the hypoxia-induced proliferation of PASMC in a dose-dependent manner. Knockdown of PSMB6 using siRNA also prevented hypoxia-induced proliferation.The present study revealed the association between increased PSMB6 and CHPH. CH up-regulated proteasome activity and the proliferation of PASMCs, which may have been related to increased PSMB6 expression and the subsequently enhanced functional catalytic sites of the proteasome. These results suggested an essential role of the proteasome during CHPH development, a novel finding requiring further study.

  4. Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

    Science.gov (United States)

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-01-01

    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. PMID:25341660

  5. Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

    Directory of Open Access Journals (Sweden)

    Alfonso Lavorgna

    2014-10-01

    Full Text Available 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.

  6. Expression of the 26S proteasome subunit RPN10 is upregulated by salt stress in Dunaliella viridis.

    Science.gov (United States)

    Sun, Xiaobin; Meng, Xiangzong; Xu, Zhengkai; Song, Rentao

    2010-08-15

    Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472 bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Deltarpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5-3 M NaCl, but decreased significantly at extremely high salinities (4-5 M NaCl). When a salinity shock of 1-3 M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36 h, and then gradually elevated to the level observed at 3 M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis. Copyright 2010 Elsevier GmbH. All rights reserved.

  7. Role of the proteasome in the regulation of estrogen receptor alpha turnover and function in MCF-7 breast carcinoma cells.

    Science.gov (United States)

    Laïos, Ioanna; Journé, Fabrice; Nonclercq, Denis; Vidal, Doris Salazar; Toillon, Robert-Alain; Laurent, Guy; Leclercq, Guy

    2005-03-01

    Estrogen receptor alpha (ER) turnover in MCF-7 cells was assessed by pulse chase analysis and measurement of ER steady-state level. In untreated cells, degradation of (35)S-labeled ER was characterized by a slow phase followed by a more rapid decline. Without ligand, ER elimination was totally compensated by synthesis which maintained receptor homeostasis. Estradiol (E(2)) and the pure antiestrogen RU 58,668 abolished the slow phase of ER breakdown and enhanced the degradation of neosynthesized ER, producing a low ER steady-state level. By contrast, the partial antiestrogen OH-Tam was ineffective in this respect and caused ER accumulation. Regardless of the conditions, ER breakdown was abolished by proteasome inhibition (MG-132). ER ligands decreased cell capacity to bind [(3)H]E(2), even in the presence of MG-132, indicating that the regulation of ER level and E(2) binding capacity occurs through distinct mechanisms. MG-132 partially blocked the basal transcription of an ERE-dependent reporter gene and modified the ability of E(2) to induce the expression of the latter: the hormone was unable to restore the transactivation activity measured without MG-132. RU 58,668 and OH-Tam failed to enhance the inhibitory action of MG-132, suggesting that a loss of basal ER-mediated transactivation mainly affects the stimulatory effect of estrogens. Overall, our findings reveal that ER steady state level, ligand binding capacity and transactivation potency fit in a complex regulatory scheme involving distinct mechanisms, which may be dissociated from each other under various treatments.

  8. L-glutamine Schiff base copper complex as a proteasome inhibitor and an apoptosis inducer in human cancer cells.

    Science.gov (United States)

    Xiao, Yan; Bi, Caifeng; Fan, Yuhua; Cui, Cindy; Zhang, Xia; Dou, Q Ping

    2008-11-01

    Interest in the use of metallic compounds for cancer treatment has been increasing since the discovery of cisplatin. Clinical studies suggest the use of proteasome inhibitors as potential novel anticancer agents. L-glutamine is the most abundant free amino acid in the body, and has been shown to play a regulatory role in several cellular processes, including metabolism, degradation, redox potential and cellular integrity. Although glutamine is reported to play a role in the regulation of apoptosis, the effect of glutamine copper complex on tumor cells and the involved molecular mechanism have not been investigated. Here, for the first time, we report that a newly synthesized L-glutamine-containing copper complex has proteasome-inhibitory activity in human breast cancer and leukemia cells. The inhibition of the tumor proteasomal activity results in the accumulation of ubiquitinated proteins and ubiquitinated form of IkappaB-alpha, a natural proteasome substrate, followed by induction of apoptosis. Furthermore, this glutamine Schiff base copper complex selectively inhibits the proteasomal activity and induces cell death in cultured breast cancer cells, but not normal, immortalized breast cells. Our data suggest that glutamine Schiff base copper complexes have a potential use for to be used in cancer treatment and prevention.

  9. Discovery of PI-1840, a novel noncovalent and rapidly reversible proteasome inhibitor with anti-tumor activity.

    Science.gov (United States)

    Kazi, Aslamuzzaman; Ozcan, Sevil; Tecleab, Awet; Sun, Ying; Lawrence, Harshani R; Sebti, Saïd M

    2014-04-25

    The proteasome inhibitor bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, acts covalently, and is associated with undesired side effects. Therefore, noncovalent inhibitors that are less toxic and more effective against solid tumors are desirable. Structure activity relationship studies led to the discovery of PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nm) over trypsin-like and peptidylglutamyl peptide hydrolyzing (IC50 values >100 μm) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Mass spectrometry and dialysis studies demonstrate that PI-1840 is a noncovalent and rapidly reversible CT-L inhibitor. In intact cancer cells, PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax, and IκB-α, inhibits survival pathways and viability, and induces apoptosis. Furthermore, PI-1840 sensitizes human cancer cells to the mdm2/p53 disruptor, nutlin, and to the pan-Bcl-2 antagonist BH3-M6. Finally, in vivo, PI-1840 but not bortezomib suppresses the growth in nude mice of human breast tumor xenografts. These results warrant further evaluation of a noncovalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors.

  10. Cytotoxicity and Proteasome Inhibition by Alkaloid Extract from Murraya koenigii Leaves in Breast Cancer Cells-Molecular Docking Studies.

    Science.gov (United States)

    Ismail, Ayesha; Noolu, Bindu; Gogulothu, Ramesh; Perugu, Shyam; Rajanna, Ajumeera; Babu, Suresh K

    2016-12-01

    Murraya koenigii (curry tree) leaves are rich in bioactive compounds such as flavonoids, alkaloids, and coumarins. Alkaloids from M. koenigii leaves have antianalgesic, antiulcerogenic, antiobesity, and antitumor activities. In this study, we tested the cytotoxic and proteasome-inhibitory potential of a total alkaloid extract (TAE) from M. koenigii leaves in the breast cancer cell line MDA-MB-231. The TAE decreased cell viability with an IC 50 of 14.4 μg/mL and altered growth kinetics of breast cancer cells. TAE (32 μg/mL) arrested cells (35%) in the "S" phase of the cell cycle and induced apoptosis. The 26S proteasome, a multicatalytic protease complex, promotes tumor cell proliferation and protects tumor cells from apoptosis. The TAE and mahanine, a carbazole alkaloid present in M. koenigii leaves, preferentially inhibited the trypsin-like, but not the chymotrypsin-like proteolytic activity of the proteasome with an IC 50 of 162 μg/mL and 287 μM, respectively. In silico analysis of 26 compounds from M. koenigii leaves revealed significant docking scores for mahanine and two other carbazole alkaloids with the β2 and β5 subunits of the catalytic 20S proteasome. Taken together, this study demonstrates that inhibition of the proteasome is an important biological activity of M. koenigii alkaloids, which may lead to cancer cell death.

  11. Loss of FBXO7 (PARK15) results in reduced proteasome activity and models a parkinsonism-like phenotype in mice.

    Science.gov (United States)

    Vingill, Siv; Brockelt, David; Lancelin, Camille; Tatenhorst, Lars; Dontcheva, Guergana; Preisinger, Christian; Schwedhelm-Domeyer, Nicola; Joseph, Sabitha; Mitkovski, Miso; Goebbels, Sandra; Nave, Klaus-Armin; Schulz, Jörg B; Marquardt, Till; Lingor, Paul; Stegmüller, Judith

    2016-09-15

    Mutations in the FBXO7 (PARK15) gene have been implicated in a juvenile form of parkinsonism termed parkinsonian pyramidal syndrome (PPS), characterized by Parkinsonian symptoms and pyramidal tract signs. FBXO7 (F-box protein only 7) is a subunit of the SCF (SKP1/cullin-1/F-box protein) E3 ubiquitin ligase complex, but its relevance and function in neurons remain to be elucidated. Here, we report that the E3 ligase FBXO7-SCF binds to and ubiquitinates the proteasomal subunit PSMA2. In addition, we show that FBXO7 is a proteasome-associated protein involved in proteasome assembly. In FBXO7 knockout mice, we find reduced proteasome activity and early-onset motor deficits together with premature death. In addition, we demonstrate that NEX (neuronal helix-loop-helix protein-1)-Cre-induced deletion of the FBXO7 gene in forebrain neurons or the loss of FBXO7 in tyrosine hydroxylase (TH)-positive neurons results in motor defects, reminiscent of the phenotype in PARK15 patients. Taken together, our study establishes a vital role for FBXO7 in neurons, which is required for proper motor control and accentuates the importance of FBXO7 in proteasome function. © 2016 The Authors.

  12. Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction

    Science.gov (United States)

    Ros, Carlos; Burckhardt, Christoph J.; Kempf, Christoph

    2002-01-01

    The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation. PMID:12438589

  13. Targeting PP2A and proteasome activity ameliorates features of allergic airway disease in mice.

    Science.gov (United States)

    Nair, P M; Starkey, M R; Haw, T J; Liu, G; Horvat, J C; Morris, J C; Verrills, N M; Clark, A R; Ammit, A J; Hansbro, P M

    2017-12-01

    Asthma is an allergic airway disease (AAD) caused by aberrant immune responses to allergens. Protein phosphatase-2A (PP2A) is an abundant serine/threonine phosphatase with anti-inflammatory activity. The ubiquitin proteasome system (UPS) controls many cellular processes, including the initiation of inflammatory responses by protein degradation. We assessed whether enhancing PP2A activity with fingolimod (FTY720) or 2-amino-4-(4-(heptyloxy) phenyl)-2-methylbutan-1-ol (AAL(S) ), or inhibiting proteasome activity with bortezomib (BORT), could suppress experimental AAD. Acute AAD was induced in C57BL/6 mice by intraperitoneal sensitization with ovalbumin (OVA) in combination with intranasal (i.n) exposure to OVA. Chronic AAD was induced in mice with prolonged i.n exposure to crude house dust mite (HDM) extract. Mice were treated with vehicle, FTY720, AAL(S) , BORT or AAL(S) +BORT and hallmark features of AAD assessed. AAL(S) reduced the severity of acute AAD by suppressing tissue eosinophils and inflammation, mucus-secreting cell (MSC) numbers, type 2-associated cytokines (interleukin (IL)-33, thymic stromal lymphopoietin, IL-5 and IL-13), serum immunoglobulin (Ig)E and airway hyper-responsiveness (AHR). FTY720 only suppressed tissue inflammation and IgE. BORT reduced bronchoalveolar lavage fluid (BALF) and tissue eosinophils and inflammation, IL-5, IL-13 and AHR. Combined treatment with AAL(S) +BORT had complementary effects and suppressed BALF and tissue eosinophils and inflammation, MSC numbers, reduced the production of type 2 cytokines and AHR. AAL(S) , BORT and AAL(S) +BORT also reduced airway remodelling in chronic AAD. These findings highlight the potential of combination therapies that enhance PP2A and inhibit proteasome activity as novel therapeutic strategies for asthma. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

  14. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

    Science.gov (United States)

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-15

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. © 2016 Khmelinskii et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. A heart that beats for 500 years: age-related changes in cardiac proteasome activity, oxidative protein damage and expression of heat shock proteins, inflammatory factors, and mitochondrial complexes in Arctica islandica, the longest-living noncolonial animal.

    Science.gov (United States)

    Sosnowska, Danuta; Richardson, Chris; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan; Ridgway, Iain

    2014-12-01

    Study of negligibly senescent animals may provide clues that lead to better understanding of the cardiac aging process. To elucidate mechanisms of successful cardiac aging, we investigated age-related changes in proteasome activity, oxidative protein damage and expression of heat shock proteins, inflammatory factors, and mitochondrial complexes in the heart of the ocean quahog Arctica islandica, the longest-lived noncolonial animal (maximum life span potential: 508 years). We found that in the heart of A. islandica the level of oxidatively damaged proteins did not change significantly up to 120 years of age. No significant aging-induced changes were observed in caspase-like and trypsin-like proteasome activity. Chymotrypsin-like proteasome activity showed a significant early-life decline, then it remained stable for up to 182 years. No significant relationship was observed between the extent of protein ubiquitination and age. In the heart of A. islandica, an early-life decline in expression of HSP90 and five mitochondrial electron transport chain complexes was observed. We found significant age-related increases in the expression of three cytokine-like mediators (interleukin-6, interleukin-1β, and tumor necrosis factor-α) in the heart of A. islandica. Collectively, in extremely long-lived molluscs, maintenance of protein homeostasis likely contributes to the preservation of cardiac function. Our data also support the concept that low-grade chronic inflammation in the cardiovascular system is a universal feature of the aging process, which is also manifest in invertebrates. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Proteomic Profiling of Radiation-Induced Skin Fibrosis in Rats: Targeting the Ubiquitin-Proteasome System

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wenjie [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Cyrus Tang Hematology Center, Soochow University, Suzhou (China); Luo, Judong [Department of Radiotherapy, Changzhou Tumor Hospital, Soochow University, Changzhou (China); Sheng, Wenjiong; Xue, Jiao; Li, Ming [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Ji, Jiang [Department of Dermatology, the Second Affiliated Hospital of Soochow University, Suzhou (China); Liu, Pengfei [Department of Gastroenterology, the Affiliated Jiangyin Hospital of Southeast University, Jiangyin (China); Zhang, Xueguang [Institute of Medical Biotechnology and Jiangsu Stem Cell Key Laboratory, Medical College of Soochow University, Suzhou (China); Cao, Jianping [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Zhang, Shuyu, E-mail: zhang.shuyu@hotmail.com [School of Radiation Medicine and Protection and Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou (China); Cyrus Tang Hematology Center, Soochow University, Suzhou (China)

    2016-06-01

    Purpose: To investigate the molecular changes underlying the pathogenesis of radiation-induced skin fibrosis. Methods and Materials: Rat skin was irradiated to 30 or 45 Gy with an electron beam. Protein expression in fibrotic rat skin and adjacent normal tissues was quantified by label-free protein quantitation. Human skin cells HaCaT and WS-1 were treated by x-ray irradiation, and the proteasome activity was determined with a fluorescent probe. The effect of proteasome inhibitors on Transforming growth factor Beta (TGF-B) signaling was measured by Western blot and immunofluorescence. The efficacy of bortezomib in wound healing of rat skin was assessed by the skin injury scale. Results: We found that irradiation induced epidermal and dermal hyperplasia in rat and human skin. One hundred ninety-six preferentially expressed and 80 unique proteins in the irradiated fibrotic skin were identified. Through bioinformatic analysis, the ubiquitin-proteasome pathway showed a significant fold change and was investigated in greater detail. In vitro experiments demonstrated that irradiation resulted in a decline in the activity of the proteasome in human skin cells. The proteasome inhibitor bortezomib suppressed profibrotic TGF-β downstream signaling but not TGF-β secretion stimulated by irradiation in HaCaT and WS-1 cells. Moreover, bortezomib ameliorated radiation-induced skin injury and attenuated epidermal hyperplasia. Conclusion: Our findings illustrate the molecular changes during radiation-induced skin fibrosis and suggest that targeting the ubiquitin-proteasome system would be an effective countermeasure.

  17. The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

    Directory of Open Access Journals (Sweden)

    McBride William H

    2005-07-01

    Full Text Available Abstract Background By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. Methods Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. Results Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. Conclusion We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132.

  18. Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients

    DEFF Research Database (Denmark)

    Anvar, Seyed Yahya; hoen, Peter Ac; Venema, Andrea

    2011-01-01

    molecular pathways that are consistently associated with OPMD, we performed an integrated high-throughput transcriptome study in affected muscles of OPMD animal models and patients. The ubiquitin-proteasome system (UPS) was found to be the most consistently and significantly OPMD-deregulated pathway across......-progression. We demonstrate a correlation between expression trends and entrapment into PABPN1 insoluble aggregates of OPMD-deregulated E3 ligases. We also show that manipulations of proteasome and immunoproteasome activity specifically affect the accumulation and aggregation of mutant PABPN1. We suggest...

  19. The ubiquitin proteasome system in glia and its role in neurodegenerative disease

    Directory of Open Access Journals (Sweden)

    Anne H.P. Jansen

    2014-08-01

    Full Text Available The ubiquitin proteasome system (UPS is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including Amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s and Huntington’s disease, leading to the hypothesis that proteasomal impairment is contributing to these diseases. So far, most research related to the UPS in neurodegenerative diseases has been focused on neurons, while glial cells have been largely disregarded in this respect. However, glial cells are essential for proper neuronal functioning and adopt a reactive phenotype in neurodegenerative diseases, thereby contributing to an inflammatory response. This process is called reactive gliosis, which in turn affects UPS functioning in glial cells. In many neurodegenerative diseases, mostly neurons show accumulation and aggregation of ubiquitinated proteins, suggesting that glial cells may be better equipped to maintain proper protein homeostasis. During an inflammatory reaction, the immunoproteasome is induced in glia, which may contribute to a more efficient degradation of disease-related proteins. Here we review the role of the UPS in glial cells in various neurodegenerative diseases, and we discuss how studying glial cell functioning might provide essential information in unraveling mechanisms of neurodegenerative diseases.

  20. Aging is not associated with proteasome impairment in UPS reporter mice.

    Directory of Open Access Journals (Sweden)

    Casey Cook

    2009-06-01

    Full Text Available Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS. Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function.We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo.Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s.

  1. Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

    Science.gov (United States)

    Collins, Ian; Wang, Hannah; Caldwell, John J; Chopra, Raj

    2017-03-15

    Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology. © 2017 The Author(s).

  2. Aging is not associated with proteasome impairment in UPS reporter mice.

    Science.gov (United States)

    Cook, Casey; Gass, Jennifer; Dunmore, Judith; Tong, Jimei; Taylor, Julie; Eriksen, Jason; McGowan, Eileen; Lewis, Jada; Johnston, Jennifer; Petrucelli, Leonard

    2009-06-11

    Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS). Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function. We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo. Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s).

  3. Ubiquitin-Proteasome-Collagen (CUP Pathway in Preterm Premature Rupture of Fetal Membranes

    Directory of Open Access Journals (Sweden)

    Xinliang Zhao

    2017-06-01

    Full Text Available Spontaneous preterm birth (sPTB occurs before 37 gestational weeks, with preterm premature rupture of the membranes (PPROM and spontaneous preterm labor (sPTL as the predominant adverse outcomes. Previously, we identified altered expression of long non-coding RNAs (lncRNAs and message RNAs (mRNAs related to the ubiquitin proteasome system (UPS in human placentas following pregnancy loss and PTB. We therefore hypothesized that similar mechanisms might underlie PPROM. In the current study, nine pairs of ubiquitin-proteasome-collagen (CUP pathway–related mRNAs and associated lncRNAs were found to be differentially expressed in PPROM and sPTL. Pathway analysis showed that the functions of their protein products were inter-connected by ring finger protein. Twenty variants including five mutations were identified in CUP-related genes in sPTL samples. Copy number variations were found in COL19A1, COL28A1, COL5A1, and UBAP2 of sPTL samples. The results reinforced our previous findings and indicated the association of the CUP pathway with the development of sPTL and PPROM. This association was due not only to the genetic variation, but also to the epigenetic regulatory function of lncRNAs. Furthermore, the findings suggested that the loss of collagen content in PPROM could result from degradation and/or suppressed expression of collagens.

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

    Directory of Open Access Journals (Sweden)

    Gonos Efstathios

    2011-06-01

    Full Text Available Abstract 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chai, Fan; Liang, Yan [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Bi, Jiong [Laboratory of General Surgery, First Affiliated Hospital, Sun Yet-sen University, Guangzhou 510080 (China); Chen, Li; Zhang, Fan [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Cui, Youhong [Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Jiang, Jun, E-mail: jcbd@medmail.com.cn [Breast Disease Center, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    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.

  6. Proteomic Identification of Putative Biomarkers of Radiotherapy Resistance: A Possible Role for the 26S Proteasome?

    Directory of Open Access Journals (Sweden)

    Laura Smith

    2009-11-01

    Full Text Available PURPOSE: We aimed to identify putative predictive protein biomarkers of radioresistance. EXPERIMENTAL DESIGN: Three breast cancer cell lines (MCF7, MDA-MB-231, and T47D were used as in vitro models to study radioresistance. Inherent radiosensitivities were examined using a clonogenic survival assay. It was revealed that each cell line differed in their response to radiotherapy. These parental breast cancer cell lines were used to establish novel derivatives (MCF7RR, MDA-MB-231RR, and T47DRR displaying significant resistance to ionizing radiation. Derivative cells were compared with parental cells to identify putative biomarkers associated with the radioresistant phenotype. To identify these biomarkers, complementary proteomic screening approaches were exploited encompassing two-dimensional gel electrophoresis in combination with mass spectrometry, liquid chromatography coupled with tandem mass spectrometry and quantitative proteomics using iTRAQ technology. RESULTS: A large number of potential biomarkers were identified, and several of these were confirmed using Western blot analysis. In particular, a decrease in the expression of the 26S proteasome was found in all radioresistant derivatives when compared with the respective parent cells. Decreased expression of this target was also found to be associated with radioresistant laryngeal tumors (P = .05 in a small pilot immunohistochemical study. CONCLUSIONS: These findings suggest that the 26S proteasome may provide a general predictive biomarker for radiotherapy outcome.

  7. Procyanidins from Cinnamomi Cortex promote proteasome-independent degradation of nuclear Nrf2 through phosphorylation of insulin-like growth factor-1 receptor in A549 cells.

    Science.gov (United States)

    Ohnuma, Tomokazu; Sakamoto, Kazuya; Shinoda, Asumi; Takagi, Chiaki; Ohno, Shoko; Nishiyama, Takahito; Ogura, Kenichiro; Hiratsuka, Akira

    2017-12-01

    Many lines of evidence demonstrate that transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays essential roles in cancer cell proliferation and resistance to chemotherapy, thereby indicating that suppression of abnormal Nrf2 activation is needed for a new therapeutic approach. Our previous studies reported that procyanidins prepared from Cinnamomi Cortex extract (CCE) have an ability to suppress cytoprotective enzymes and cell proliferation in human cancer cells with activated Nrf2. In the present study, we investigated the mechanism of CCE procyanidin-mediated antagonization of Nrf2. CCE procyanidin treatment rapidly reduced nuclear Nrf2 expression and phosphorylated insulin-like growth factor-1 receptor (IGF-1R) in A549 cells. Nrf2 protein expression in A549 cells with reduced IGF-1R expression and function was not affected by treatment with CCE procyanidins, which suggested that CCE procyanidins decreased Nrf2 through IGF-1R. Nrf2 suppression by CCE procyanidins was mitigated in the presence of protease inhibitors, not proteasome inhibitors. In addition, CCE procyanidin treatment led to enhancement of nuclear cysteine protease activity in A549 cells. Our findings suggest a novel mechanism by which CCE procyanidins can promote proteasome-independent degradation of nuclear Nrf2 through IGF-1R phosphorylation and cysteine protease activation. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Human Papilloma Virus 16 E6 RNA Interference Enhances Cisplatin and Death Receptor-Mediated Apoptosis in Human Cervical Carcinoma Cells

    NARCIS (Netherlands)

    Tan, Shinta; Hougardy, Brigitte M. T.; Meersma, Gert J.; Schaap, Bessel; de Vries, Elisabeth G. E.; van der Zee, Ate G. J.; de Jong, Steven

    In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small

  9. Proteasomal degradation of p53 by human papillomavirus E6 oncoprotein relies on the structural integrity of p53 core domain.

    Directory of Open Access Journals (Sweden)

    Xavier Bernard

    Full Text Available The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR, circular dichroism (CD, and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.

  10. SOCS2-induced proteasome-dependent TRAF6 degradation: a common anti-inflammatory pathway for control of innate immune responses.

    Directory of Open Access Journals (Sweden)

    Cortez McBerry

    Full Text Available Pattern recognition receptors and receptors for pro-inflammatory cytokines provide critical signals to drive the development of protective immunity to infection. Therefore, counter-regulatory pathways are required to ensure that overwhelming inflammation harm host tissues. Previously, we showed that lipoxins modulate immune response during infection, restraining inflammation during infectious diseases in an Aryl hydrocarbon receptor (AhR/suppressors of cytokine signaling (SOCS2-dependent-manner. Recently, Indoleamine-pyrrole 2,3- dioxygenase (IDO-derived tryptophan metabolites, including L-kynurenine, were also shown to be involved in several counter-regulatory mechanisms. Herein, we addressed whether the intracellular molecular events induced by lipoxins mediating control of innate immune signaling are part of a common regulatory pathway also shared by L-kynurenine exposure. We demonstrate that Tumor necrosis factor receptor-associated factor (TRAF6--member of a family of adapter molecules that couple the TNF receptor and interleukin-1 receptor/Toll-like receptor families to intracellular signaling events essential for the development of immune responses--is targeted by both lipoxins and L-kynurenine via an AhR/SOCS2-dependent pathway. Furthermore, we show that LXA₄- and L-kynurenine-induced AhR activation, its subsequent nuclear translocation, leading SOCS2 expression and TRAF6 Lys47-linked poly-ubiquitination and proteosome-mediated degradation of the adapter proteins. The in vitro consequences of such molecular interactions included inhibition of TLR- and cytokine receptor-driven signal transduction and cytokine production. Subsequently, in vivo proteosome inhibition led to unresponsiveness to lipoxins, as well as to uncontrolled pro-inflammatory reactions and elevated mortality during toxoplasmosis. In summary, our results establish proteasome degradation of TRAF6 as a key molecular target for the anti-inflammatory pathway triggered by

  11. HDAC inhibitor L-carnitine and proteasome inhibitor bortezomib synergistically exert anti-tumor activity in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Hongbiao Huang

    Full Text Available Combinations of proteasome inhibitors and histone deacetylases (HDAC inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21(cip1 gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like activity assay. Here we report that (i the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii the combination also synergistically inhibits tumor growth in vivo; (iii two major pathways are involved in the synergistical effects of the combinational treatment: increased p21(cip1 expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

  12. Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

    DEFF Research Database (Denmark)

    Dutaud, Dominique; Aubry, Laurent; Henry, Laurent

    2002-01-01

    /ml with a slope of 1.004 and a coefficient of determination r2 of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients...

  13. Ubiquitin-proteasome-dependent degradation of TBP-like protein is prevented by direct binding of TFIIA.

    Science.gov (United States)

    Isogai, Momoko; Suzuki, Hidefumi; Maeda, Ryo; Tamura, Taka-Aki

    2016-11-01

    Although the majority of gene expression is driven by TATA-binding protein (TBP)-based transcription machinery, it has been reported that TBP-related factors (TRFs) are also involved in the regulation of gene expression. TBP-like protein (TLP), which is one of the TRFs and exhibits the highest affinity to TFIIA among known proteins, has recently been showed to have significant roles in gene regulation. However, how the level of TLP is maintained in vivo has remained unknown. In this study, we explored the mechanism by which TLP protein is turned over in vivo and the factor that maintains the amount of TLP. We showed that TLP is rapidly degraded by the ubiquitin-proteasome system and that tight interaction with TFIIA results in protection of TLP from ubiquitin-proteasome-dependent degradation. The half-life of TLP was shown to be less than a few hours, and the proteasome inhibitor MG132 specifically suppressed TLP degradation. Moreover, knockdown and over-expression experiments showed that TFIIA is engaged in stabilization of TLPin vivo. Thus, we showed a novel characteristic of TLP, that is, interaction with TFIIA is essential to suppress proteasome-dependent turnover of TLP, providing a further insight into TLP-governed gene regulation. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  14. Subnormothermic Perfusion in the Isolated Rat Liver Preserves the Antioxidant Glutathione and Enhances the Function of the Ubiquitin Proteasome System

    Directory of Open Access Journals (Sweden)

    Teresa Carbonell

    2016-01-01

    Full Text Available 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.

  15. 1.5-NM PROJECTION STRUCTURE OF HELA-CELL PROSOME-MCP (PROTEASOME) PROVIDED BY 2-DIMENSIONAL CRYSTALS

    NARCIS (Netherlands)

    PERKINS, GA; BERGSMASCHUTTER, W; KEEGSTRA, W; ARNBERG, AC; COUX, O; SCHERRER, K

    1994-01-01

    We grew two-dimensional crystals of HeLa cell prosomes, also called multicatalytic proteinases (MCP) and proteasomes, for a structure determination by electron microscopy. The molecules were arranged in side views in these crystals. The crystals have p21 plane group symmetry with one particle per

  16. Autophagy Regulates Proteasome Inhibitor-Induced Pigmentation in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

    Science.gov (United States)

    Juuti-Uusitalo, Kati; Koskela, Ali; Kivinen, Niko; Viiri, Johanna; Hyttinen, Juha M T; Reinisalo, Mika; Koistinen, Arto; Uusitalo, Hannu; Sinha, Debasish; Skottman, Heli; Kaarniranta, Kai

    2017-05-19

    The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.

  17. Proteasome inhibition induces DNA damage and reorganizes nuclear architecture and protein synthesis machinery in sensory ganglion neurons.

    Science.gov (United States)

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

    2014-05-01

    Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.

  18. The proteasome inhibitor bortezomib induces an inhibitory chromatin environment at a distal enhancer of the estrogen receptor-α gene.

    Directory of Open Access Journals (Sweden)

    Ginny L Powers

    Full Text Available Expression of the estrogen receptor-α (ERα gene, ESR1, is a clinical biomarker used to predict therapeutic outcome of breast cancer. Hence, there is significant interest in understanding the mechanisms regulating ESR1 gene expression. Proteasome activity is increased in cancer and we previously showed that proteasome inhibition leads to loss of ESR1 gene expression in breast cancer cells. Expression of ESR1 mRNA in breast cancer cells is controlled predominantly through a proximal promoter within ∼400 base pair (bp of the transcription start site (TSS. Here, we show that loss of ESR1 gene expression induced by the proteasome inhibitor bortezomib is associated with inactivation of a distal enhancer located 150 kilobases (kb from the TSS. Chromatin immunoprecipitation assays reveal several bortezomib-induced changes at the distal site including decreased occupancy of three critical transcription factors, GATA3, FOXA1, and AP2γ. Bortezomib treatment also resulted in decreased histone H3 and H4 acetylation and decreased occupancy of histone acetyltransferase, p300. These data suggest a mechanism to explain proteasome inhibitor-induced loss of ESR1 mRNA expression that highlights the importance of the chromatin environment at the -150 kb distal enhancer in regulation of basal expression of ESR1 in breast cancer cells.

  19. Protein repertoire impact of Ubiquitin-Proteasome System impairment: insight into the protective role of beta-estradiol.

    Science.gov (United States)

    D'Alessandro, Annamaria; D'Aguanno, Simona; Cencioni, Maria Teresa; Pieroni, Luisa; Diamantini, Adamo; Battistini, Luca; Longone, Patrizia; Spalloni, Alida; De Laurenzi, Vincenzo; Bernardini, Sergio; Federici, Giorgio; Urbani, Andrea

    2012-02-02

    The Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathways (ALP) are key mechanisms for cellular homeostasis sustenance and protein clearance. A wide number of Neurodegenerative Diseases (NDs) are tied with UPS impairment and have been also described as proteinopathies caused by aggregate-prone proteins, not efficiently removed by proteasome. Despite the large knowledge on proteasome biological role, molecular mechanisms associated with its impairment are still blur. We have pursued a comprehensive proteomic investigation to evaluate the phenotypic rearrangements in protein repertoires associated with a UPS blockage. Different functional proteomic approaches have been employed to tackle UPS impairment impact on human NeuroBlastoma (NB) cell lines responsive to proteasome inhibition by Epoxomicin. 2-Dimensional Electrophoresis (2-DE) separation combined with Mass Spectrometry and Shotgun Proteomics experiments have been employed to design a thorough picture of protein profile. Unsupervised meta-analysis of the collected proteomic data revealed that all the identified proteins relate each other in a functional network centered on beta-estradiol. Moreover we showed that treatment of cells with beta-estradiol resulted in aggregate removal and increased cell survival due to activation of the autophagic pathway. Our data may provide the molecular basis for the use of beta-estradiol in neurodegenerative disorders by induction of protein aggregate removal. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Nie XH

    2015-10-01

    Full Text Available Xiao-hu Nie,1,* Jia Ou-yang,2,* Ying Xing,3 Dan-yan Li,4 Xing-yu Dong,1 Ru-en Liu,5 Ru-xiang Xu6 1Affiliated Bayi Brain Hospital, Southern Medical University, Beijing, People’s Republic of China; 2Nanchang University Medical College, Jiangxi, People’s Republic of China; 3Department of Gastroenterology, The 98th Hospital of Nanjing Military Command, Huzhou, Zhejiang, People’s Republic of China; 4Spleen & Stomach Institution, Guangzhou University of Traditional Chinese Medicine, Guangdong, People’s Republic of China; 5Department of Neurosurgery, China–Japan Friendship Hospital, Beijing, People’s Republic of China; 6Bayi Brain Hospital, The Military General Hospital of Beijing PLA, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: 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. Keywords: paeoniflorin, glioma, apoptosis

  1. Proteasome subunit expression analysis and chemosensitivity in relapsed paediatric acute leukaemia patients receiving bortezomib-containing chemotherapy

    Directory of Open Access Journals (Sweden)

    Denise Niewerth

    2016-09-01

    Full Text Available Abstract Background Drug combinations of the proteasome inhibitor bortezomib with cytotoxic chemotherapy are currently evaluated in phase 2 and 3 trials for the treatment of paediatric acute myeloid leukaemia (AML and acute lymphocytic leukaemia (ALL. Methods We investigated whether expression ratios of immunoproteasome to constitutive proteasome in leukaemic cells correlated with response to bortezomib-containing re-induction chemotherapy in patients with relapsed and refractory acute leukaemia, enrolled in two Children’s Oncology Group phase 2 trials of bortezomib for ALL (COG-AALL07P1 and AML (COG-AAML07P1. Expression of proteasome subunits was examined in 72 patient samples (ALL n = 60, AML n = 12 obtained before start of therapy. Statistical significance between groups was determined by Mann-Whitney U test. Results Ratios of immunoproteasome to constitutive proteasome subunit expression were significantly higher in pre-B ALL cells than in AML cells for both β5i/β5 and β1i/β1 subunits (p = 0.004 and p < 0.001. These ratios correlated with therapy response in AML patients; β1i/β1 ratios were significantly higher (p = 0.028 between patients who did (n = 4 and did not reach complete remission (CR (n = 8, although for β5i/β5 ratios, this did not reach significance. For ALL patients, the subunit ratios were also higher for patients who showed a good early response to therapy but this relation was not statistically significant. Overall, for this study, the patients were treated with combination therapy, so response was not only attributed to proteasome inhibition. Moreover, the leukaemic blast cells were not purified for these samples. Conclusions These first ex vivo results encourage further studies into relative proteasome subunit expression to improve proteasome inhibition-containing therapy and as a potential indicator of bortezomib response in acute leukaemia.

  2. Nrf2- and ATF4-Dependent Upregulation of xCT Modulates the Sensitivity of T24 Bladder Carcinoma Cells to Proteasome Inhibition

    Science.gov (United States)

    Ye, Peng; Okada, Tomomi; Sato, Hideyo; Liu, Tao; Maruyama, Atsushi; Ohyama, Chikara; Itoh, Ken

    2014-01-01

    The ubiquitin-proteasome pathway degrades ubiquitinated proteins to remove damaged or misfolded protein and thus plays an important role in the maintenance of many important cellular processes. Because the pathway is also crucial for tumor cell growth and survival, proteasome inhibition by specific inhibitors exhibits potent antitumor effects in many cancer cells. xCT, a subunit of the cystine antiporter system xc−, plays an important role in cellular cysteine and glutathione homeostasis. Several recent reports have revealed that xCT is involved in cancer cell survival; however, it was unknown whether xCT affects the cytotoxic effects of proteasome inhibitors. In this study, we found that two stress-inducible transcription factors, Nrf2 and ATF4, were upregulated by proteasome inhibition and cooperatively enhance human xCT gene expression upon proteasome inhibition. In addition, we demonstrated that the knockdown of xCT by small interfering RNA (siRNA) or pharmacological inhibition of xCT by sulfasalazine (SASP) or (S)-4-carboxyphenylglycine (CPG) significantly increased the sensitivity of T24 cells to proteasome inhibition. These results suggest that the simultaneous inhibition of both the proteasome and xCT could have therapeutic benefits in the treatment of bladder tumors. PMID:25002527

  3. Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice.

    Science.gov (United States)

    Wang, Jianjun; Wang, Chuan-En; Orr, Adam; Tydlacka, Suzanne; Li, Shi-Hua; Li, Xiao-Jiang

    2008-03-24

    Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

  4. Impaired ubiquitin–proteasome system activity in the synapses of Huntington's disease mice

    Science.gov (United States)

    Wang, Jianjun; Wang, Chuan-En; Orr, Adam; Tydlacka, Suzanne; Li, Shi-Hua; Li, Xiao-Jiang

    2008-01-01

    Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin–proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models. PMID:18362179

  5. The 11S Proteasome Subunit PSME3 Is a Positive Feedforward Regulator of NF-κB and Important for Host Defense against Bacterial Pathogens

    Directory of Open Access Journals (Sweden)

    Jinxia Sun

    2016-02-01

    Full Text Available The NF-κB pathway plays important roles in immune responses. Although its regulation has been extensively studied, here, we report an unknown feedforward mechanism for the regulation of this pathway by Toll-like receptor (TLR ligands in macrophages. During bacterial infections, TLR ligands upregulate the expression of the 11S proteasome subunit PSME3 via NF-κB-mediated transcription in macrophages. PSME3, in turn, enhances the transcriptional activity of NF-κB by directly binding to and destabilizing KLF2, a negative regulator of NF-κB transcriptional activity. Consistent with this positive role of PSME3 in NF-κB regulation and importance of the NF-κB pathway in host defense against bacterial infections, the lack of PSME3 in hematopoietic cells renders the hosts more susceptible to bacterial infections, accompanied by increased bacterial burdens in host tissues. Thus, this study identifies a substrate for PSME3 and elucidates a proteolysis-dependent, but ubiquitin-independent, mechanism for NF-κB regulation that is important for host defense and innate immunity.

  6. Protein ingestion preserves proteasome activity during intense aseptic inflammation and facilitates skeletal muscle recovery in humans.

    Science.gov (United States)

    Draganidis, Dimitrios; Chondrogianni, Niki; Chatzinikolaou, Athanasios; Terzis, Gerasimos; Karagounis, Leonidas G; Sovatzidis, Apostolos; Avloniti, Alexandra; Lefaki, Maria; Protopapa, Maria; Deli, Chariklia K; Papanikolaou, Konstantinos; Jamurtas, Athanasios Z; Fatouros, Ioannis G

    2017-08-01

    The ubiquitin-proteasome system (UPS) is the main cellular proteolytic system responsible for the degradation of normal and abnormal (e.g. oxidised) proteins. Under catabolic conditions characterised by chronic inflammation, the UPS is activated resulting in proteolysis, muscle wasting and impaired muscle function. Milk proteins provide sulphur-containing amino acid and have been proposed to affect muscle inflammation. However, the response of the UPS to aseptic inflammation and protein supplementation is largely unknown. The aim of this study was to investigate how milk protein supplementation affects UPS activity and skeletal muscle function under conditions of aseptic injury induced by intense, eccentric exercise. In a double-blind, cross-over, repeated measures design, eleven men received either placebo (PLA) or milk protein concentrate (PRO, 4×20 g on exercise day and 20 g/d for the following 8 days), following an acute bout of eccentric exercise (twenty sets of fifteen eccentric contractions at 30°/s) on an isokinetic dynamometer. In each trial, muscle biopsies were obtained from the vastus lateralis muscle at baseline, as well as at 2 and 8 d post exercise, whereas blood samples were collected before exercise and at 6 h, 1 d, 2 d and 8 d post exercise. Muscle strength and soreness were assessed before exercise, 6 h post exercise and then daily for 8 consecutive days. PRO preserved chymotrypsin-like activity and attenuated the decrease of strength, facilitating its recovery. PRO also prevented the increase of NF-κB phosphorylation and HSP70 expression throughout recovery. We conclude that milk PRO supplementation following exercise-induced muscle trauma preserves proteasome activity and attenuates strength decline during the pro-inflammatory phase.

  7. Targeted proteasome inhibition by Velcade induces apoptosis in human mesothelioma and breast cancer cell lines

    Science.gov (United States)

    Wang, Ying; Puliyappadamba, Vineshkumar T.; Sharma, Sunita; Yang, Huanjie; Tarca, Adi; Dou, Q. Ping; Lonardo, Fulvio; Ruckdeschel, John C.; Pass, Harvey I.; Wali, Anil

    2013-01-01

    Introduction Thoracic malignancies and human breast cancer (HBC) continue to be aggressive solid tumors that are poor responders to the existing conventional standard chemotherapeutic approaches. Malignant pleural mesothelioma (MPM) is an asbestos-related tumor of the thoracic pleura that lacks effective treatment options. Altered ubiquitin proteasome pathway is frequently encountered in many malignancies including HBC and MPM and thus serves as an important target for therapeutic intervention strategies. Although proteasome inhibitor Velcade (Bort-ezomib) has been under clinical investigation for a number of cancers, limited preclinical studies with this agent have thus far been conducted in HBC and MPM malignancies. Purpose To study the biological and molecular responses of MPM and HBC cells to Velcade treatments, and to identify mechanisms involved in transducing growth inhibitory effects of this agent. Methods Flow-cytometric analyses coupled with western immunoblotting and gene-array methodologies were utilized to determine mechanisms of Velcade-dependent growth suppression of five MPM (H2595, H2373, H2452, H2461, and H2714) and two breast cancer (MDA MB-468, SKBR-3) cell lines. Results Our data revealed significant reduction in cell growth properties that were dose and time dependent. Velcade treatment resulted in G2M phase arrest, increased expression of cyclin-dependent kinase inhibitor p21 and pro-apoptotic protein Bax. Pretreatment of mesothelioma cells with Velcade showed synergistic effect with cisplatin combination regimens. High-throughput gene expression profiling among Velcade treated and untreated mesothelioma cell lines resulted in identification of novel transducers of apoptosis such as CARP-1, XAF1, and Troy proteins. Conclusions Velcade targets cell cycle and apoptosis signaling to suppress MPM and HBC growth in part by activating novel transducers of apoptosis. This pilot study has paved way for further in-depth analysis of the downstream

  8. Proteasome LMP2/β1i subunit as biomarker for human uterine leiomyosarcoma

    Directory of Open Access Journals (Sweden)

    Takuma Hayashi

    2014-02-01

    Full Text Available Uterine leiomyosarcoma (Ut-LMS develops more frequently in the myometrium of the uterine body than in the uterine cervix. Although the development of gynecological tumors is often correlated with the secretion of female hormones that of Ut-LMS does not, and its risk factor(s remain unknown. Importantly, a diagnostic biomarker that can distinguish malignant tumor Ut-LMS from benign tumor leiomyoma (LMA, has yet to be established. Therefore, the risk factor(s associated with Ut-LMS need to be examined in order to establish a diagnosis and clinical treatment method. Mice with a homozygous deficiency for the proteasome b-ring subunit, low-molecular mass polypeptide (LMP2/b1i spontaneously develop Ut-LMS, with a disease prevalence of ~40% by 14 months of age. In recent studies, we showed that LMP2/b1i expression was absent in human Ut-LMS, but present in other human uterine mesenchymal tumors including uterine LMA. Moreover, LMP2/b1i is also known to negatively regulate human Ut-LMS tumorigenesis. Additional experiments furthermore revealed the differential expression of cyclin E and calponin h1 in human uterine mesenchymal tumors. Therefore, LMP2/b1i is a potential diagnostic biomarker when combined with the candidate molecules, cyclin E and calponin h1 for human Ut-LMS, and may be a targeted molecule for a new therapeutic approach.---------------------------------------------Cite this article as: Hayashi T, Horiuchi A Aburatani H, Ishiko O, Yaegashi N, Kanai Y, Zharhary D, Tonegawa S, Konishi I. Proteasome LMP2/ß1i subunit as biomarker for human uterine leiomyosarcoma. Int J Cancer Ther Oncol 2014; 2(1:02018.DOI: http://dx.doi.org/10.14319/ijcto.0201.8

  9. The ubiquitin proteasome system plays a role in venezuelan equine encephalitis virus infection.

    Directory of Open Access Journals (Sweden)

    Moushimi Amaya

    Full Text Available Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV and Western equine encephalitis virus (WEEV demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.

  10. Critical cysteines in Akt1 regulate its activity and proteasomal degradation: implications for neurodegenerative diseases.

    Science.gov (United States)

    Ahmad, Faraz; Nidadavolu, Prakash; Durgadoss, Lalitha; Ravindranath, Vijayalakshmi

    2014-09-01

    Impaired Akt1 signaling is observed in neurodegenerative diseases, including Parkinson׳s disease (PD). In PD models oxidative modification of Akt1 leads to its dephosphorylation and consequent loss of its kinase activity. To explore the underlying mechanism we exposed Neuro2A cells to cadmium, a pan inhibitor of protein thiol disulfide oxidoreductases, including glutaredoxin 1 (Grx1), or downregulated Grx1, which led to dephosphorylation of Akt1, loss of its kinase activity, and also decreased Akt1 protein levels. Mutation of cysteines to serines at 296 and 310 in Akt1 did not affect its basal kinase activity but abolished cadmium- and Grx1 downregulation-induced reduction in Akt1 kinase activity, indicating their critical role in redox modulation of Akt1 function and turnover. Cadmium-induced decrease in phosphorylated Akt1 correlated with increased association of wild-type (WT) Akt1 with PP2A, which was absent in the C296-310S Akt1 mutant and was also abolished by N-acetylcysteine treatment. Further, increased proteasomal degradation of Akt1 by cadmium was not seen in the C296-310S Akt1 mutant, indicating that oxidation of cysteine residues facilitates degradation of WT Akt1. Moreover, preventing oxidative modification of Akt1 cysteines 296 and 310 by mutating them to serines increased the cell survival effects of Akt1. Thus, in neurodegenerative states such as PD, maintaining the thiol status of cysteines 296 and 310 in Akt1 would be critical for Akt1 kinase activity and for preventing its degradation by proteasomes. Preventing downregulation of Akt signaling not only has long-range consequences for cell survival but could also affect the multiple roles that Akt plays, including in the Akt-mTOR signaling cascade. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants

    OpenAIRE

    Jürgen Brojatsch; Heriberto Lima; Alak K Kar; Jacobson, Lee S.; Muehlbauer, Stefan M.; Kartik Chandran; Felipe Diaz-Griffero

    2014-01-01

    Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the p...

  12. APC/C (Cdh1) controls the proteasome-mediated degradation of E2F3 during cell cycle exit

    NARCIS (Netherlands)

    Ping, Z.; Lim, R.; Bashir, T.; Pagano, M.; Guardavaccaro, D.

    2012-01-01

    E2F transcription factors regulate gene expression in concert with the retinoblastoma tumor suppressor family. These transcriptional complexes are master regulators of cell cycle progression and, in addition, control the expression of genes involved in DNA repair, G 2/M checkpoint and

  13. Prefoldins Negatively Regulate Cold Acclimation in Arabidopsis thaliana by Promoting Nuclear Proteasome-Mediated HY5 Degradation.

    Science.gov (United States)

    Perea-Resa, Carlos; Rodríguez-Milla, Miguel A; Iniesto, Elisa; Rubio, Vicente; Salinas, Julio

    2017-06-05

    The process of cold acclimation is an important adaptive response whereby many plants from temperate regions increase their freezing tolerance after being exposed to low non-freezing temperatures. The correct development of this response relies on proper accumulation of a number of transcription factors that regulate expression patterns of cold-responsive genes. Multiple studies have revealed a variety of molecular mechanisms involved in promoting the accumulation of these transcription factors. Interestingly, however, the mechanisms implicated in controlling such accumulation to ensure their adequate levels remain largely unknown. In this work, we demonstrate that prefoldins (PFDs) control the levels of HY5, an Arabidopsis transcription factor with a key role in cold acclimation by activating anthocyanin biosynthesis, in response to low temperature. Our results show that, under cold conditions, PFDs accumulate into the nucleus through a DELLA-dependent mechanism, where they interact with HY5, triggering its ubiquitination and subsequent degradation. The degradation of HY5 would result, in turn, in anthocyanin biosynthesis attenuation, ensuring the accurate development of cold acclimation. These findings uncover an unanticipated nuclear function for PFDs in plant responses to abiotic stresses. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  14. Cysteine string protein promotes proteasomal degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) by increasing its interaction with the C terminus of Hsp70-interacting protein and promoting CFTR ubiquitylation.

    Science.gov (United States)

    Schmidt, Béla Z; Watts, Rebecca J; Aridor, Meir; Frizzell, Raymond A

    2009-02-13

    Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and CHIP-mediated CFTR degradation.

  15. Validation of microarray data in human lymphoblasts shows a role of the ubiquitin-proteasome system and NF-kB in the pathogenesis of Down syndrome.

    Science.gov (United States)

    Granese, Barbara; Scala, Iris; Spatuzza, Carmen; Valentino, Anna; Coletta, Marcella; Vacca, Rosa Anna; De Luca, Pasquale; Andria, Generoso

    2013-07-05

    Down syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown. To better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade. We show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes. The present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS.

  16. PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12 and ZNF198 during Hepatitis B Virus-Induced Liver Carcinogenesis.

    Science.gov (United States)

    Zhang, Hao; Diab, Ahmed; Fan, Huitao; Mani, Saravana Kumar Kailasam; Hullinger, Ronald; Merle, Philippe; Andrisani, Ourania

    2015-06-01

    Elucidating mechanisms of hepatitis B virus (HBV)-mediated hepatocarcinogenesis is needed to gain insights into the etiology and treatment of liver cancer. Cells where HBV is replicating exhibit increased expression of Plk1 kinase and reduced levels of two transcription repression factors, SUZ12 and ZNF198. SUZ12 is an essential subunit of the transcription repressive complex PRC2. ZNF198 stabilizes the transcription repressive complex composed of LSD1, Co-REST, and HDAC1. These two transcription repressive complexes are held together by binding the long noncoding RNA HOTAIR. In this study, we linked these regulatory events mechanistically by showing that Plk1 induces proteasomal degradation of SUZ12 and ZNF198 by site-specific phosphorylation. Plk1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression of HOTAIR, significantly reducing SUZ12 and ZNF198 stability. In cells expressing the HBV X protein (HBx), downregulation of SUZ12 and ZNF198 mediated global changes in histone modifications. In turn, HBx-expressing cells propagated an altered chromatin landscape after cell division, as exemplified by changes in histone modifications of the EpCAM promoter, a target of PRC2 and LSD1/Co-REST/HDAC1 complexes. Notably, liver tumors from X/c-myc bitransgenic mice exhibited downregulation of SUZ12 and ZNF198 along with elevated expression of Plk1, HOTAIR, and EpCAM. Clinically, similar effects were documented in a set of HBV-related liver tumors consistent with the likelihood that downregulation of SUZ12 and ZNF198 leads to epigenetic reprogramming of infected hepatocytes. Because both Plk1 and HOTAIR are elevated in many human cancers, we propose that their combined effects are involved in epigenetic reprogramming associated broadly with oncogenic transformation. ©2015 American Association for Cancer Research.

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

    Science.gov (United States)

    Tomaić, V; Banks, L

    2015-01-29

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

  18. Myxoma virus attenuates expression of activating transcription factor 4 (ATF4) which has implications for the treatment of proteasome inhibitor-resistant multiple myeloma.

    Science.gov (United States)

    Dunlap, Katherine M; Bartee, Mee Y; Bartee, Eric

    2015-01-01

    The recent development of chemotherapeutic proteasome inhibitors, such as bortezomib, has improved the outcomes of patients suffering from the plasma cell malignancy multiple myeloma. Unfortunately, many patients treated with these drugs still suffer relapsing disease due to treatment-induced upregulation of the antiapoptotic protein Mcl1. We have recently demonstrated that an oncolytic poxvirus, known as myxoma, can rapidly eliminate primary myeloma cells by inducing cellular apoptosis. The efficacy of myxoma treatment on proteasome inhibitor-relapsed or -refractory myeloma, however, remains unknown. We now demonstrate that myxoma-based elimination of myeloma is not affected by cellular resistance to proteasome inhibitors. Additionally, myxoma virus infection specifically prevents expression of Mcl1 following induction of the unfolded protein response, by blocking translation of the unfolded protein response activating transcription factor (ATF)4. These results suggest that myxoma-based oncolytic therapy represents an attractive option for myeloma patients whose disease is refractory to chemotherapeutic proteasome inhibitors due to upregulation of Mcl1.

  19. PINK1 defect causes mitochondrial dysfunction, proteasomal deficit and alpha-synuclein aggregation in cell culture models of Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Wencheng Liu

    Full Text Available Mutations in PTEN induced kinase 1 (PINK1, a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD, PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased alpha-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and alpha-synclein aggregation.

  20. Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

    DEFF Research Database (Denmark)

    Hartmann-Petersen, R; Tanaka, K; Hendil, K B

    2001-01-01

    and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking......, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7...

  1. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    Directory of Open Access Journals (Sweden)

    Anita Collavoli

    2011-01-01

    Full Text Available By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB. This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

  2. In Vivo Inhibition of Proteasome Activity and Tumour Growth by Murraya koenigii Leaf Extract in Breast Cancer Xenografts and by Its Active Flavonoids in Breast Cancer Cells.

    Science.gov (United States)

    Noolu, Bindu; Gogulothu, Ramesh; Bhat, Mehrajuddin; Qadri, Syed S Y H; Reddy, V Sudhakar; Reddy, G Bhanuprakash; Ismail, Ayesha

    2016-01-01

    Inhibition of the 26S proteasome is an attractive approach for anticancer therapy. Proteasome inhibitors are known to selectively target cancer cells and make them more sensitive to chemotherapeutic agents. Murraya koenigii is a medicinally important herb of Asian origin and a rich source of bioactive compounds such as flavonoids and alkaloids. In the present study, we investigated the proteasome inhibitory and apoptotic effect of M. koenigii leaf extract in vivo in a xenograft tumor mouse model, and also assessed the toxicity if any in normal mice. M. koenigii extract did not lead to any toxicity in mice. Analysis of extract revealed the presence of flavonoid compounds which act as proteasome inhibitors. Quercetin treatment led to the decrease in the cell viability and arrest of cells in G2/M phase. Quercetin, Apigenin, Kaempferol and Rutin; flavonoids present in the leaf extract, dose-dependently inhibited the endogenous 26S proteasome activity in MDA-MB-231 cells. Reduction in tumor growth was associated with a decrease in proteasomal enzyme activities in the treated groups. Increased caspase-3 activity and TUNEL-positive cells indicated enhanced apoptosis with Murraya leaf extract treatment. Decreased expression of angiogenic and anti-apoptotic gene markers is indicative of inhibition of angiogenesis and promotion of apoptosis in the leaf extract treated tumors.

  3. Phosphorylated alpha-synuclein at Ser-129 is targeted to the proteasome pathway in a ubiquitin-independent manner.

    Science.gov (United States)

    Machiya, Youhei; Hara, Susumu; Arawaka, Shigeki; Fukushima, Shingo; Sato, Hiroyasu; Sakamoto, Masahiro; Koyama, Shingo; Kato, Takeo

    2010-12-24

    α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(½) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.

  4. Phosphorylated α-Synuclein at Ser-129 Is Targeted to the Proteasome Pathway in a Ubiquitin-independent Manner*

    Science.gov (United States)

    Machiya, Youhei; Hara, Susumu; Arawaka, Shigeki; Fukushima, Shingo; Sato, Hiroyasu; Sakamoto, Masahiro; Koyama, Shingo; Kato, Takeo

    2010-01-01

    α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t½ = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs. PMID:20959456

  5. Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients

    DEFF Research Database (Denmark)

    Anvar, Seyed Yahya; hoen, Peter Ac; Venema, Andrea

    2011-01-01

    species. We could correlate the association of the UPS OPMD-deregulated genes with stages of disease progression. The expression trend of a subset of these genes is age-associated and therefore, marks the late onset of the disease, and a second group with expression trends relating to disease...... that the natural decrease in proteasome expression and its activity during muscle aging contributes to the onset of the disease....

  6. Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro.

    Science.gov (United States)

    Yi, Young-Joo; Manandhar, Gaurishankar; Sutovsky, Miriam; Jonáková, Vera; Park, Chang-Sik; Sutovsky, Peter

    2010-03-01

    The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization. Copyright 2009. Published by Elsevier Ireland Ltd.

  7. Characterization of a Proteasome and TAP-independent Presentation of Intracellular Epitopes by HLA-B27 Molecules

    KAUST Repository

    Magnacca, A.

    2012-07-17

    Nascent HLA-class I molecules are stabilized by proteasome-derived peptides in the ER and the new complexes proceed to the cell surface through the post-ER vesicles. It has been shown, however, that less stable complexes can exchange peptides in the Trans Golgi Network (TGN). HLA-B27 are the most studied HLA-class I molecules due to their association with Ankylosing Spondylitis (AS). Chimeric proteins driven by TAT of HIV have been exploited by us to deliver viral epitopes, whose cross-presentation by the HLA-B27 molecules was proteasome and TAP-independent and not restricted to Antigen-Presenting Cells (APC). Here, using these chimeric proteins as epitope suppliers, we compared with each other and with the HLA-A2 molecules, the two HLA-B*2705 and B*2709 alleles differing at residue 116 (D116H) and differentially associated with AS. We found that the antigen presentation by the two HLA-B27 molecules was proteasome-, TAP-, and APC-independent whereas the presentation by the HLA-A2 molecules required proteasome, TAP and professional APC. Assuming that such difference could be due to the unpaired, highly reactive Cys-67 distinguishing the HLA-B27 molecules, C67S mutants in HLA-B*2705 and B*2709 and V67C mutant in HLA-A*0201 were also analyzed. The results showed that this mutation did not influence the HLA-A2-restricted antigen presentation while it drastically affected the HLA-B27-restricted presentation with, however, remarkable differences between B*2705 and B*2709. The data, together with the occurrence on the cell surface of unfolded molecules in the case of C67S-B*2705 mutant but not in that of C67S-B*2709 mutant, indicates that Cys-67 has a more critical role in stabilizing the B*2705 rather than the B*2709 complexes.

  8. Aging and SKN-1-dependent Loss of 20S Proteasome Adaptation to Oxidative Stress in C. elegans.

    Science.gov (United States)

    Raynes, Rachel; Juarez, Crystal; Pomatto, Laura C D; Sieburth, Derek; Davies, Kelvin J A

    2017-02-01

    Aging is marked by a collapse of protein homeostasis and deterioration of adaptive stress responses that often lead to disease. During aging, the induction of stress responses decline along with protein quality control. Here, we have shown that the ability to mount an adaptive response by pretreatment with minor oxidative stress is abrogated in aged Caenorhabditis elegans We have identified a defect in SKN-1 signaling sensitivity during aging and have also found an aging-related increase in basal proteasome expression and in vitro activity, however, adaptation of the 20S proteasome in response to stress is lost in old animals. Interestingly, increased activation of SKN-1 promotes stress resistance, but is unable to rescue declining adaptation during aging. Our data demonstrate that the aging-dependent decline in SKN-1 signaling negatively impacts adaptation of the 20S proteasome in response to acute oxidative stress. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Age-related modifications of egr1 expression and ubiquitin-proteasome components in pet dog hippocampus.

    Science.gov (United States)

    Ghi, Piera; Di Brisco, Fabio; Dallorto, Dario; Osella, Maria Cristina; Orsetti, Marco

    2009-05-01

    In this paper we examined the impact of age on cognitive functions and the age-related modifications of egr1 expression, an inducible transcription factor with a confirmed role in synaptic plasticity and regulation of the proteasome activity, on pet dogs. Additionally, we examined the age-related changes of some elements of the ubiquitin-proteasome system, which is the apparatus that prevents the intracellular accumulation of abnormal proteins. The results of behavioral analysis revealed that old/senior dogs (9-16-year-old) had impaired cognitive performance compared to young/middle-aged dogs (2-8-year-old) in the Reversal Learning task. Taken togheter, the results (age-related decline of Psmd4, Psmb8, CHIP, and egr1 expression; increase of Psmb9 and Hsp90 expression) suggest that the activity of the ubiquitin-proteasome system in the dog hippocampus is a multi-step process, in which abnormal proteins destined for degradation are recognized and destroyed, and shows an age-related decline. The consequent failure of the "protein quality control system" might have detrimental effects on cell physiology and lead to a progressive impairment of cognitive functions.

  10. Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

    Directory of Open Access Journals (Sweden)

    Masami Ichikawa

    Full Text Available Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.

  11. Skeletal muscle myotubes in severe obesity exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux.

    Science.gov (United States)

    Bollinger, Lance M; Powell, Jonathan J S; Houmard, Joseph A; Witczak, Carol A; Brault, Jeffrey J

    2015-06-01

    Whole-body protein metabolism is dysregulated with obesity. The goal of the study was to determine whether activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. Primary human skeletal muscle (HSkM) cell cultures were utilized since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean women (BMI ≤ 26.0 kg/m(2) ) and 8 women with severe obesity (BMI ≥ 39.0) were examined basally and when stimulated to atrophy (serum and amino acid starvation). 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 lean subjects and an increase in HSkM from subjects with obesity. HSkM in obesity also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from individuals with and without obesity under basal and starved conditions. Our data indicate that muscle cells in lean individuals and in those with severe obesity have innate differences in management of protein degradation, which may explain their metabolic differences. © 2015 The Obesity Society.

  12. 50Hz Extremely Low Frequency Electromagnetic Fields Enhance Protein Carbonyl Groups Content in Cancer Cells: Effects on Proteasomal Systems

    Directory of Open Access Journals (Sweden)

    A. M. Eleuteri

    2009-01-01

    Full Text Available Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24–72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals.

  13. PA28αβ: The Enigmatic Magic Ring of the Proteasome?

    Directory of Open Access Journals (Sweden)

    Paolo Cascio

    2014-06-01

    Full Text Available PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. In cells, PA28 also exists in larger complexes along with the 19S particle, which allows ATP-dependent degradation of proteins; although in vivo a large fraction of PA28 is present as PA28αβ-20S particles whose exact biological functions are largely unknown. Although several lines of evidence strongly indicate that PA28αβ plays a role in MHC class I antigen presentation, the exact molecular mechanisms of this activity are still poorly understood. Herein, we review current knowledge about the biochemical and biological properties of PA28αβ and discuss recent findings concerning its role in modifying the spectrum of proteasome’s peptide products, which are important to better understand the molecular mechanisms and biological consequences of PA28αβ activity.

  14. Peptidyl-prolyl isomerization targets rice Aux/IAAs for proteasomal degradation during auxin signalling.

    Science.gov (United States)

    Jing, Hongwei; Yang, Xiaolu; Zhang, Jian; Liu, Xuehui; Zheng, Huakun; Dong, Guojun; Nian, Jinqiang; Feng, Jian; Xia, Bin; Qian, Qian; Li, Jiayang; Zuo, Jianru

    2015-06-22

    In plants, auxin signalling is initiated by the auxin-promoted interaction between the auxin receptor TIR1, an E3 ubiquitin ligase, and the Aux/IAA transcriptional repressors, which are subsequently degraded by the proteasome. Gain-of-function mutations in the highly conserved domain II of Aux/IAAs abolish the TIR1-Aux/IAA interaction and thus cause an auxin-resistant phenotype. Here we show that peptidyl-prolyl isomerization of rice OsIAA11 catalysed by LATERAL ROOTLESS2 (LRT2), a cyclophilin-type peptidyl-prolyl cis/trans isomerase, directly regulates the stability of OsIAA11. NMR spectroscopy reveals that LRT2 efficiently catalyses the cis/trans isomerization of OsIAA11. The lrt2 mutation reduces OsTIR1-OsIAA11 interaction and consequently causes the accumulation of a higher level of OsIAA11 protein. Moreover, knockdown of the OsIAA11 expression partially rescues the lrt2 mutant phenotype in lateral root development. Together, these results illustrate cyclophilin-catalysed peptidyl-prolyl isomerization promotes Aux/IAA degradation, as a mechanism regulating auxin signalling.

  15. The Role of Ubiquitin and the 26S Proteasome in Plant Abiotic Stress Signaling

    Directory of Open Access Journals (Sweden)

    Stone L Sophia

    2014-04-01

    Full Text Available 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 facilitate 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.

  16. Pseudomonas syringae pv. syringae uses proteasome inhibitor syringolin A to colonize from wound infection sites.

    Directory of Open Access Journals (Sweden)

    Johana C Misas-Villamil

    2013-03-01

    Full Text Available Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA, a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA. Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.

  17. Regulation of primary cilia formation by the ubiquitin-proteasome system.

    Science.gov (United States)

    Shearer, Robert F; Saunders, Darren N

    2016-10-15

    Primary cilia form at the surface of most vertebrate cell types, where they are essential signalling antennae for signal transduction pathways important for development and cancer, including Hedgehog. The importance of primary cilia in development is clearly demonstrated by numerous disorders (known as ciliopathies) associated with disrupted cilia formation (ciliogenesis). Recent advances describing functional regulators of the primary cilium highlight an emerging role for the ubiquitin-proteasome system (UPS) as a key regulator of ciliogenesis. Although there are well-documented examples of E3 ubiquitin ligases and deubiquitases in the regulation of cilia proteins, many putative components remain unvalidated. This review explores current understanding of how the UPS influences primary cilia formation, and also how recent screen data have identified more putative regulators of the UPS. Emerging research has identified many promising leads in the search for regulators of this important organelle and may identify potential novel therapeutic targets for intervention in cancer and other disease contexts. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  18. Natural history of relapsed myeloma, refractory to immunomodulatory drugs and proteasome inhibitors: a multicenter IMWG study.

    Science.gov (United States)

    Kumar, S K; Dimopoulos, M A; Kastritis, E; Terpos, E; Nahi, H; Goldschmidt, H; Hillengass, J; Leleu, X; Beksac, M; Alsina, M; Oriol, A; Cavo, M; Ocio, E M; Mateos, M V; O'Donnell, E K; Vij, R; Lokhorst, H M; van de Donk, N W C J; Min, C; Mark, T; Turesson, I; Hansson, M; Ludwig, H; Jagannath, S; Delforge, M; Kyriakou, C; Hari, P; Mellqvist, U; Usmani, S Z; Dytfeld, D; Badros, A Z; Moreau, P; Kim, K; Otero, P R; Lee, J H; Shustik, C; Waller, D; Chng, W J; Ozaki, S; Lee, J-J; de la Rubia, J; Eom, H S; Rosinol, L; Lahuerta, J J; Sureda, A; Kim, J S; Durie, B G M

    2017-11-01

    Introduction of new myeloma therapies offers new options for patients refractory to immunomodulatory drugs (IMiDs) and proteasome inhibitors (PIs). In this multicenter study, patients with relapsed multiple myeloma, who have received at least three prior lines of therapy, are refractory to both an IMiD (lenalidomide or pomalidomide) and a PI (bortezomib or carfilzomib), and have been exposed to an alkylating agent were identified. The time patients met the above criteria was defined as time zero (T0). Five hundred and forty-three patients diagnosed between 2006 and 2014 were enrolled in this study. Median age at T0 was 62 years (range 31-87); 61% were males. The median duration between diagnosis and T0 was 3.1 years. The median number of lines of therapy before T0 was 4 (range 3-13). The median overall survival (OS) from T0 for the entire cohort was 13 (95% confidence interval (CI) 11, 15) months. At least one regimen recorded after T0 in 462 (85%) patients, with a median (95% CI) progression-free survival and OS from T0 of 5 (4, 6), and 15.2 (13, 17) months, respectively. The study provides the expected outcome of relapsed multiple myeloma that is refractory to a PI and an IMiD, a benchmark for comparison of new therapies being evaluated.

  19. COLLABORATIVE ACTION OF CELL CYCLE, MOLECULAR CHAPERONES, AND UBIQUITIN PROTEASOME SYSTEM IN NEUROONCOLOGY

    Directory of Open Access Journals (Sweden)

    Pravir Kumar

    2013-06-01

    Full Text Available he striking feature in tumor biology is uncontrolled cell proliferation and growth. Any alteration in the genetic make up may cause cell cycle deregulation that leads to aberrant cell cycle re-entry. These cascades ultimately cause cancerous situation with unwanted cell growth and division. There are several factors in cell cycle events that can lead to cancerous situations, for instance, checkpoint breach, extracellular signals, malfunctioned protein kinases, re-expression of cyclins and cyclin-CDKs complex. A crucial function of cyclinCDK complex is phosphorylation of retinoblastoma tumor suppressor gene that inhibits its ability to regulate the action of E2F transcription factor, which induces the gene expression and thus cause cell proliferation. To maintain the cellular homeostasis under tumorous condition, a line of protective mechanism is switched on such as availability of molecular chaperones; and if repair work fails, ubiquitin proteasome system comes in action. These regulatory mechanisms are highly conserved and play a critical role in maintaining several molecular events in the brain tumor or any stress situation. Misfolded proteins in tumor tissues are either rectified by chaperone activity upto a certain threshold or follow a degradation pathway by proteolytic activity of ubiquitinproteasome system. In this review, we have highlighted an extensive explorative potential of molecular chaperones in combination with ubiquitin E3 ligase enzymes activities in brain tumors.

  20. Resveratrol fuels HER2 and ERα-positive breast cancer behaving as proteasome inhibitor.

    Science.gov (United States)

    Andreani, Cristina; Bartolacci, Caterina; Wijnant, Kathleen; Crinelli, Rita; Bianchi, Marzia; Magnani, Mauro; Hysi, Albana; Iezzi, Manuela; Amici, Augusto; Marchini, Cristina

    2017-02-26

    The phytoestrogen resveratrol has been reported to possess cancer chemo-preventive activity on the basis of its effects on tumor cell lines and xenograft or carcinogen-inducible in vivo models. Here we investigated the effects of resveratrol on spontaneous mammary carcinogenesis using Δ16HER2 mice as HER2+/ERα+ breast cancer model. Instead of inhibiting tumor growth, resveratrol treatment (0.0001% in drinking water; daily intake of 4μg/mouse) shortened tumor latency and enhanced tumor multiplicity in Δ16HER2 mice. This in vivo tumor-promoting effect of resveratrol was associated with up-regulation of Δ16HER2 and down-regulation of ERα protein levels and was recapitulated in vitro by murine (CAM6) and human (BT474) tumor cell lines. Our results demonstrate that resveratrol, acting as a proteasome inhibitor, leads to Δ16HER2 accumulation which favors the formation of Δ16HER2/HER3 heterodimers. The consequential activation of downstream mTORC1/p70S6K/4EBP1 pathway triggers cancer growth and proliferation. This study provides evidence that resveratrol mechanism of action (and hence its effects) depends on the intrinsic molecular properties of the cancer model under investigation, exerting a tumor-promoting effect in luminal B breast cancer subtype models.

  1. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    Science.gov (United States)

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

    Energy Technology Data Exchange (ETDEWEB)

    Yim, Ji-Hye [Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul 01812 (Korea, Republic of); Yun, Hong Shik [Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul 01812 (Korea, Republic of); Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Lee, Su-Jae [Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791 (Korea, Republic of); Baek, Jeong-Hwa [Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul 01812 (Korea, Republic of); Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Lee, Chang-Woo [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Song, Ji-Young; Um, Hong-Duck; Park, Jong Kuk; Kim, Jae-Sung; Park, In-Chul [Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul 01812 (Korea, Republic of); Hwang, Sang-Gu, E-mail: sgh63@kcch.re.kr [Division of Radiation Cancer Biology, Korea Institute of Radiological & Medical Sciences, Seoul 01812 (Korea, Republic of)

    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. - Highlights: • PSMC5 is a radiation-sensitive biomarker in H460 cells. • PSMC5 depletion inhibits radiation-induced apoptosis in H460 cells. • PSMC5 knockdown blocks ROS generation through inhibition of the p53-p21 pathway. • PSMC5 knockdown enhances p21 degradation via AKT-dependent MDM2 stabilization.

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

    Science.gov (United States)

    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. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity.

    Science.gov (United States)

    Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

    2012-08-03

    Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity☆

    Science.gov (United States)

    Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

    2012-01-01

    Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22641158

  6. ROS and ROS-Mediated Cellular Signaling

    Directory of Open Access Journals (Sweden)

    Jixiang Zhang

    2016-01-01

    Full Text Available It has long been recognized that an increase of reactive oxygen species (ROS can modify the cell-signaling proteins and have functional consequences, which successively mediate pathological processes such as atherosclerosis, diabetes, unchecked growth, neurodegeneration, inflammation, and aging. While numerous articles have demonstrated the impacts of ROS on various signaling pathways and clarify the mechanism of action of cell-signaling proteins, their influence on the level of intracellular ROS, and their complex interactions among multiple ROS associated signaling pathways, the systemic summary is necessary. In this review paper, we particularly focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins (NF-κB, MAPKs, Keap1-Nrf2-ARE, and PI3K-Akt, ion channels and transporters (Ca2+ and mPTP, and modifying protein kinase and Ubiquitination/Proteasome System.

  7. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

    Directory of Open Access Journals (Sweden)

    Nadia A Congo Carbajosa

    Full Text Available Aberrations in the ubiquitin-proteasome system (UPS are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

  8. AAV-6 mediated efficient transduction of mouse lower airways.

    Science.gov (United States)

    Li, Wuping; Zhang, Liqun; Wu, Zhijian; Pickles, Raymond J; Samulski, R Jude

    2011-09-01

    AAV1 and AAV6 are two closely related AAV serotypes. In the present study, we found AAV6 was more efficient in transducing mouse lower airway epithelia in vitro and in vivo than AAV1. To further explore the mechanism of this difference, we found that significantly more AAV1 bound to mouse airway epithelia than AAV6, yet transduction by AAV6 was far superior. Lectin competition assays demonstrated that both AAV1 and AAV6 similarly utilize α-2, 3-, and to a lesser extend α-2, 6- linked sialic acids as the receptors for transduction. Furthermore, the rates of AAV endocytosis could not account for the transduction differences of AAV1 and AAV6. Finally, it was revealed that AAV6 was less susceptible to ubiquitin/proteasome-mediated blocks than AAV1 when transducing mouse airway epithelia. Thus compared with AAV1, AAV6 has a unique ability to escape proteasome-mediated degradation, which is likely responsible for its higher transduction efficiency in mouse airway epithelium. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory

    Science.gov (United States)

    Sol Fustiñana, María; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro

    2014-01-01

    The ubiquitin–proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  10. Systemic analysis of heat shock response induced by heat shock and a proteasome inhibitor MG132.

    Directory of Open Access Journals (Sweden)

    Hee-Jung Kim

    Full Text Available The molecular basis of heat shock response (HSR, a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor, both of which are known to induce heat shock proteins (Hsps, we compared the responses of normal mouse fibrosarcoma cell line, RIF-1, and its thermotolerant variant cell line, TR-RIF-1 (TR, to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1 genes regulated by both heat shock and MG132, (e.g. chaperones; 2 those regulated only by heat shock (e.g. DNA binding proteins including histones; and 3 those regulated only by MG132 (e.g. innate immunity and defense related molecules. This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins.

  11. Proteasome Modulator 9 SNPs are linked to hypertension in type 2 diabetes families

    Directory of Open Access Journals (Sweden)

    Gragnoli Claudia

    2011-08-01

    Full Text Available Abstract Background Chromosome 12q24 was recently associated with hypertension. Proteasome Modulator 9 (PSMD9 lies in the 12q24 locus and is in linkage with MODY3, type 2 diabetes (T2D, microvascular and macrovascular pathology, carpal tunnel syndrome, and hypercholesterolemia in Italian families. Aims Our goal was to determine whether PSMD9 is linked to elevated blood pressure/hypertension in T2D families. Methods We characterized the Italian T2D families' members for presence and/or absence of elevated blood pressure (≥ 130/80 and/or hypertension. The phenotypes were described as unknown in all cases in which the diagnosis was either unclear or the data were not available for the subject studied. We tested in the 200 Italians families for the presence of the linkage of the PSMD9 T2D risk single nucleotide polymorphisms (SNPs IVS3+nt460 A > G, IVS3+nt437 T > C and E197G A > G with elevated blood pressure/hypertension. The non-parametric linkage analysis was performed for this qualitative phenotype by using the Merlin software; the Lod score and correspondent P-value were calculated. Parametric linkage analysis was also performed. For the significant linkage score, 1000 replicates were run to calculate the empirical P-value. Results The PSMD9 gene SNPs studied are in linkage with elevated blood pressure/hypertension in our Italian families. Conclusions We conclude that the PSMD9 gene and/or any variant in linkage disequilibrium with the SNPs studied contribute to the linkage to hypertension within our family dataset. This is the first report of PSMD9 linkage to hypertension within the 12q24 locus.

  12. The Proteasome Inhibitor MG-132 Protects Hypoxic SiHa Cervical Carcinoma Cells after Cyclic Hypoxia/Reoxygenation from Ionizing Radiation

    Directory of Open Access Journals (Sweden)

    Frank Pajonk

    2006-12-01

    Full Text Available INTRODUCTION: Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of radiation therapy. This study was designed to determine how varying cycles of hypoxia/reoxygenation affect the response of cervical carcinoma cells irradiated under oxic and hypoxic conditions and whether this could be modulated by proteasome inhibition. MATERIALS AND METHODS: Plateau-phase SiHa cervical carcinoma cells in culture were exposed to varying numbers of 30-minute cycles of hypoxia/reoxygenation directly before irradiation under oxic or hypoxic conditions. 26S Proteasome activity was blocked by addition of MG-132. Clonogenic survival was measured by a colonyforming assay. RESULTS: Under oxic conditions, repeated cycles of hypoxia/reoxygenation decreased the clonogenic survival of SiHa cells. This effect was even more pronounced after the inhibition of 26S proteasome complex. In contrast, under hypoxic conditions, SiHa cells were radioresistant, as expected, but this was increased by proteasome inhibition. CONCLUSIONS: Proteasome inhibition radiosensitizes oxygenated tumor cells but may also protect tumor cells from ionizing radiation under certain hypoxic conditions.

  13. Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Cheroni, Cristina; Marino, Marianna; Tortarolo, Massimo; Veglianese, Pietro; De Biasi, Silvia; Fontana, Elena; Zuccarello, Laura Vitellaro; Maynard, Christa J; Dantuma, Nico P; Bendotti, Caterina

    2009-01-01

    In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic Ub(G76V)-GFP /SOD1G93A mice an increase in Ub(G76V)-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of Ub(G76V)-GFP was due to deficient reporter degradation. In some motor neurons the increase of Ub(G76V)-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.

  14. Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment.

    Science.gov (United States)

    Maynard, Christa J; Böttcher, Claudia; Ortega, Zaira; Smith, Ruben; Florea, Bogdan I; Díaz-Hernández, Miguel; Brundin, Patrik; Overkleeft, Hermen S; Li, Jia-Yi; Lucas, Jose J; Dantuma, Nico P

    2009-08-18

    Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.

  15. Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton.

    Science.gov (United States)

    Arcangeletti, C; Sütterlin, R; Aebi, U; De Conto, F; Missorini, S; Chezzi, C; Scherrer, K

    1997-06-01

    A new "instantaneous" fixation/extraction procedure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37 degrees C, has been explored to reexamine the relationships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called "multicatalytic proteinase (MCP) complexes" or "20S-Proteasomes." In Triton X-100-extracted epithelial, fibroblastic, and muscle cells, prosome particles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of "instantaneous fixation" has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distributed in variable proportions between the IFs and the microfilament/ stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localization on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resistance of the prosome-IF association, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymerization of prosomes with G-actin made it possible to observe "ladder-like" filamentous structures in the electron microscope, in which the prosome particles, like the "rungs of a ladder," laterally crosslink two or more actin filaments in a regular pattern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M(r) complexes (possibly mRNPs and/or 26S proteasomes), but also directly, as individual prosome particles.

  16. Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment

    OpenAIRE

    Maynard, Christa J.; Böttcher, Claudia; Ortega, Zaira; Smith, Ruben; Florea, Bogdan I.; Díaz-Hernández, Miguel; Brundin, Patrik; Overkleeft, Hermen S.; Li, Jia-Yi; Jose J Lucas; Dantuma, Nico P

    2009-01-01

    Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe...

  17. Mediatized Humanitarianism

    DEFF Research Database (Denmark)

    Vestergaard, Anne

    2014-01-01

    The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts to legiti......The article investigates the implications of mediatization for the legitimation strategies of humanitarian organizations. Based on a (full population) corpus of ~400 pages of brochure material from 1970 to 2007, the micro-textual processes involved in humanitarian organizations' efforts...... legitimation by accountancy, legitimation by institutionalization, and legitimation by compensation. The analysis relates these changes to a problem of trust associated with mediatization through processes of mediation....

  18. Syringolin A selectively labels the 20 S proteasome in murine EL4 and wild-type and bortezomib-adapted leukaemic cell lines.

    Science.gov (United States)

    Clerc, Jérôme; Florea, Bogdan I; Kraus, Marianne; Groll, Michael; Huber, Robert; Bachmann, André S; Dudler, Robert; Driessen, Christoph; Overkleeft, Herman S; Kaiser, Markus

    2009-11-02

    The natural product syringolin A (SylA) is a potent proteasome inhibitor with promising anticancer activities. To further investigate its potential as a lead structure, selectivity profiling with cell lysates was performed. At therapeutic concentrations, a rhodamine-tagged SylA derivative selectively bound to the 20 S proteasome active sites without detectable off-target labelling. Additional profiling with lysates of wild-type and bortezomib-adapted leukaemic cell lines demonstrated the retention of this proteasome target and subsite selectivity as well as potency even in clinically relevant cell lines. Our studies, therefore, propose that further development of SylA might indeed result in an improved small molecule for the treatment of leukaemia.

  19. Mammalian target of rapamycin signaling and ubiquitin proteasome-related gene expression in 3 different skeletal muscles of colostrum- versus formula-fed calves.

    Science.gov (United States)

    Sadri, H; Steinhoff-Wagner, J; Hammon, Harald M; Bruckmaier, R M; Görs, S; Sauerwein, H

    2017-11-01

    The rates of protein turnover are higher during the neonatal period than at any other time in postnatal life. The mammalian target of rapamycin (mTOR) and the ubiquitin-proteasome system are key pathways regulating cellular protein turnover. The objectives of this study were (1) to elucidate the effect of feeding colostrum versus milk-based formula on the mRNA abundance of key components of the mTOR pathway and of the ubiquitin-proteasome system in skeletal muscle of neonatal calves and (2) to compare different muscles. German Holstein calves were fed either colostrum (COL; n = 7) or milk-based formula (FOR; n = 7) up to 4 d of life. The nutrient content in formula and colostrum was similar, but formula had lower concentrations of free branched-chain AA (BCAA) and free total AA, insulin, and insulin-like growth factor (IGF)-I than colostrum. Blood samples were taken from d 1 to 4 before morning feeding and before and 2 h after the last feeding on d 4. Muscle samples from M. longissimus dorsi (MLD), M. semitendinosus (MST), and M. masseter (MM) were collected after slaughter on d 4 at 2 h after feeding. The preprandial concentrations of free total AA and BCAA, insulin, and IGF-I in plasma changed over time but did not differ between groups. Plasma free total AA and BCAA concentrations decreased in COL, whereas they increased in FOR after feeding, resulting in higher postprandial plasma total AA and BCAA concentrations in FOR than in COL. Plasma insulin concentrations increased after feeding in both groups but were higher in COL than in FOR. Plasma IGF-I concentrations decreased in COL, whereas they remained unchanged in FOR after feeding. The mRNA abundance of mTOR and ribosomal protein S6 kinase 1 (S6K1) in 3 different skeletal muscles was greater in COL than in FOR, whereas that of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) was unaffected by diet. The mRNA abundance of ubiquitin activating enzyme (UBA1) and ubiquitin conjugating enzyme 1

  20. Native and aspirin-triggered lipoxins control innate immunity by inducing proteasomal degradation of TRAF6

    OpenAIRE

    Machado, Fabiana S.; Esper, L?sia; Dias, Alexandra; Madan, Rajat; Gu, YuanYuan; Hildeman, David; Serhan, Charles N.; Karp, Christopher L.; Aliberti, J?lio

    2008-01-01

    Innate immune signaling is critical for the development of protective immunity. Such signaling is, perforce, tightly controlled. Lipoxins (LXs) are eicosanoid mediators that play key counterregulatory roles during infection. The molecular mechanisms underlying LX-mediated control of innate immune signaling are of interest. In this study, we show that LX and aspirin (ASA)-triggered LX (ATL) inhibit innate immune signaling by inducing suppressor of cytokine signaling (SOCS) 2–dependent ubiquiti...

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

    Science.gov (United States)

    Han, Jinbin; Liu, Luming; Yue, Xiaoqiang; Chang, Jinjia; Shi, Weidong; Hua, Yongqiang

    2013-12-15

    It is a therapeutic strategy for cancers including pancreatic to inhibit proteasome activity. Disulfiram (DSF) may bind copper (Cu) to form a DSF-Cu complex. DSF-Cu is capable of inducing apoptosis in cancer cells by inhibiting proteasome activity. DSF is rapidly converted to diethyldithiocarbamate (DDTC) within bodies. Copper(II) absorbed by bodies is reduced to copper(I) when it enters cells. We found that DDTC and copper(I) could form a binuclear complex which might be entitled DDTC-Cu(I), and it had been synthesized by us in the laboratory. This study is to investigate the anticancer potential of this complex on pancreatic cancer and the possible mechanism. Pancreatic cancer cell lines, SW1990, PANC-1 and BXPC-3 were used for in vitro assays. Female athymic nude mice grown SW1990 xenografts were used as animal models. Cell counting kit-8 (cck-8) assay and flow cytometry were used for analyzing apoptosis in cells. A 20S proteasome assay kit was used in proteasome activity analysis. Western blot (WB) and immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used in tumor sample analysis. The results suggest that DDTC-Cu(I) inhibit pancreatic cancer cell proliferation and proteasome activity in vitro and in vivo. Accumulation of ubiquitinated proteins, and increased p27 as well as decreased NF-κB expression were detected in tumor tissues of DDTC-Cu(I)-treated group. Our data indicates that DDTC-Cu(I) is an effective proteasome activity inhibitor with the potential to be explored as a drug for pancreatic cancer. © 2013. Published by Elsevier Inc. All rights reserved.

  2. Complex Mediation

    DEFF Research Database (Denmark)

    Bødker, Susanne; Andersen, Peter Bøgh

    2005-01-01

    This article has its starting point in a large number of empirical findings regarding computer-mediated work. These empirical findings have challenged our understanding of the role of mediation in such work; on the one hand as an aspect of communication and cooperation at work and on the other hand...... as an aspect of human engagement with instruments of work. On the basis of previous work in activity-theoretical and semiotic human—computer interaction, we propose a model to encompass both of these aspects. In a dialogue with our empirical findings we move on to propose a number of types of mediation...... that have helped to enrich our understanding of mediated work and the design of computer mediation for such work....

  3. A three-part signal governs differential processing of Gli1 and Gli3 proteins by the proteasome.

    Science.gov (United States)

    Schrader, Erin K; Harstad, Kristine G; Holmgren, Robert A; Matouschek, Andreas

    2011-11-11

    The Gli proteins are the transcriptional effectors of the mammalian Hedgehog signaling pathway. In an unusual mechanism, the proteasome partially degrades or processes Gli3 in the absence of Hedgehog pathway stimulation to create a Gli3 fragment that opposes the activity of the full-length protein. In contrast, Gli1 is not processed but degraded completely, despite considerable homology with Gli3. We found that these differences in processing can be described by defining a processing signal that is composed of three parts: the zinc finger domain, an adjacent linker sequence, and a degron. Gli3 processing is inhibited when any one component of the processing signal is disrupted. We show that the zinc fingers are required for processing only as a folded structure and that the location but not the identity of the processing degron is critical. Within the linker sequence, regions of low sequence complexity play a crucial role, but other sequence features are also important. Gli1 is not processed because two components of the processing signal, the linker sequence and the degron, are ineffective. These findings provide new insights into the molecular elements that regulate Gli protein processing by the proteasome.

  4. A Three-part Signal Governs Differential Processing of Gli1 and Gli3 Proteins by the Proteasome*

    Science.gov (United States)

    Schrader, Erin K.; Harstad, Kristine G.; Holmgren, Robert A.; Matouschek, Andreas

    2011-01-01

    The Gli proteins are the transcriptional effectors of the mammalian Hedgehog signaling pathway. In an unusual mechanism, the proteasome partially degrades or processes Gli3 in the absence of Hedgehog pathway stimulation to create a Gli3 fragment that opposes the activity of the full-length protein. In contrast, Gli1 is not processed but degraded completely, despite considerable homology with Gli3. We found that these differences in processing can be described by defining a processing signal that is composed of three parts: the zinc finger domain, an adjacent linker sequence, and a degron. Gli3 processing is inhibited when any one component of the processing signal is disrupted. We show that the zinc fingers are required for processing only as a folded structure and that the location but not the identity of the processing degron is critical. Within the linker sequence, regions of low sequence complexity play a crucial role, but other sequence features are also important. Gli1 is not processed because two components of the processing signal, the linker sequence and the degron, are ineffective. These findings provide new insights into the molecular elements that regulate Gli protein processing by the proteasome. PMID:21921029

  5. The transcription factor STAT2 enhances proteasomal degradation of RCAN1 through the ubiquitin E3 ligase FBW7.

    Science.gov (United States)

    Lee, Jee Won; Kang, Hye Seon; Lee, Jae Youn; Lee, Eun Jung; Rhim, Hyewhon; Yoon, Joo Heon; Seo, Su Ryeon; Chung, Kwang Chul

    2012-04-06

    Down syndrome is the most common genetic disorder and is characterized by three copies of chromosome 21. Regulator of calcineurin 1 (RCAN1) is located close to the Down syndrome critical region (distal part of chromosome 21), and its product functions as an endogenous inhibitor of calcineurin signaling. RCAN1 protein stability is regulated by several inflammatory signaling factors, though the underlying mechanisms remain incompletely understood. Here, we report that RCAN1 interacts with the inflammation-linked transcription factor, signal transducer and activator of transcription 2 (STAT2) in mammalian cells. STAT2 overexpression decreased levels of RCAN1 protein. Decreases in RCAN1 were blocked by a proteasome inhibitor, indicating that STAT2 regulates RCAN1 degradation via the ubiquitin-proteasome system. Co-immunoprecipitation/immunoblot analyses showed that STAT2 enhanced RCAN1 ubiquitination through the ubiquitin E3 ligase FBW7. This pathway appeared to be physiologically relevant, as treatment of cells with interferon-α reduced RCAN1 levels through the activation of STAT2 and FBW7. Together, these results suggest that STAT2 influences diverse cellular processes linked to RCAN1 by negatively affecting RCAN1 protein stability. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Ceramide PC102 inhibits melanin synthesis via proteasomal degradation of microphthalmia-associated transcription factor and tyrosinase.

    Science.gov (United States)

    Jeong, Hyo-Soon; Choi, Hye-Ryung; Yun, Hye-Young; Baek, Kwang Jin; Kwon, Nyoun Soo; Park, Kyoung-Chan; Kim, Dong-Seok

    2013-03-01

    A few types of ceramide are reported to decrease melanin synthesis. In the present study, we examined the effects of an artificial ceramide analog, PC102, on melanogenesis using a spontaneously immortalized melanocyte cell line (Mel-Ab). PC102 is currently used as a moisturizing additive in a variety of cosmetics. Our data showed that PC102 inhibited melanin production and tyrosinase activity in a dose-dependent manner, but did not directly affect tyrosinase activity. Microphthalmia-associated transcription factor (MITF), tyrosinase, and β-catenin protein levels decreased after 48 h of PC102 treatment. In contrast, PC102 did not decrease MITF, tyrosinase, and β-catenin mRNA levels. Therefore, we investigated whether the decrease in MITF and tyrosinase by PC102 is due to proteasomal degradation. MG132, a proteasomal inhibitor, completely abolished tyrosinase downregulation due to PC102 and partially reduced the downregulation of MITF and β-catenin due to PC102. Moreover, MG132 abrogated the inhibition of melanin synthesis by PC102. Taken together, our data suggest that PC102 may inhibit melanin synthesis through MITF and tyrosinase degradation.

  7. Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Anouk C.M. Platteel

    2017-08-01

    Full Text Available Proteasome-catalyzed peptide splicing (PCPS generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

  8. The Architecture of the Anbu Complex Reflects an Evolutionary Intermediate at the Origin of the Proteasome System.

    Science.gov (United States)

    Fuchs, Adrian C D; Alva, Vikram; Maldoner, Lorena; Albrecht, Reinhard; Hartmann, Marcus D; Martin, Jörg

    2017-06-06

    Proteasomes are self-compartmentalizing proteases that function at the core of the cellular protein degradation machinery in eukaryotes, archaea, and some bacteria. Although their evolutionary history is under debate, it is thought to be linked to that of the bacterial protease HslV and the hypothetical bacterial protease Anbu (ancestral beta subunit). Here, together with an ex