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Sample records for proteasome inhibitor mg132-induced

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

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    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.

  2. MG132, a proteasome inhibitor, induces human pulmonary fibroblast cell death via increasing ROS levels and GSH depletion.

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    Park, Woo Hyun; Kim, Suhn Hee

    2012-04-01

    MG132 as a proteasome inhibitor can induce apoptotic cell death in lung cancer cells. However, little is known about the toxicological cellular effects of MG132 on normal primary lung cells. Here, we investigated the effects of N-acetyl cysteine (NAC) and vitamin C (well known antioxidants) or L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell death, reactive oxygen species (ROS) and glutathione (GSH). MG132 induced growth inhibition and death in HPF cells, accompanied by the loss of mitochondrial membrane potential (MMP; ∆ψm). MG132 increased ROS levels and GSH-depleted cell numbers in HPF cells. Both antioxidants, NAC and vitamin C, prevented growth inhibition, death and MMP (∆ψm) loss in MG132-treated HPF cells and also attenuated ROS levels in these cells. BSO showed a strong increase in ROS levels in MG132-treated HPF cells and slightly enhanced the growth inhibition, cell death, MMP (∆ψm) loss and GSH depletion. In addition, NAC decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. Furthermore, superoxide dismutase (SOD) 2, catalase (CTX) and GSH peroxidase (GPX) siRNAs enhanced HPF cell death by MG132, which was not correlated with ROS and GSH level changes. In conclusion, MG132 induced the growth inhibition and death of HPF cells, which were accompanied by increasing ROS levels and GSH depletion. Both NAC and vitamin C attenuated HPF cell death by MG132, whereas BSO slightly enhanced the death.

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

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    Skalniak, Lukasz; Dziendziel, Monika; Jura, Jolanta

    2014-10-01

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

  4. Combination therapy for hepatitis C virus with heat-shock protein 90 inhibitor 17-AAG and proteasome inhibitor MG132.

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    Ujino, Saneyuki; Yamaguchi, Saori; Shimotohno, Kunitada; Takaku, Hiroshi

    2010-03-09

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Here, we report a new and effective strategy for inhibiting HCV replication using an inhibitor of heat-shock protein 90, 17-AAG (17-allylamino-17demethoxygeldanamycin), and a proteasome inhibitor, MG132. To explore the virological basis of combination therapy, we analysed the effects of 17-AAG and MG132, singly and in combination on HCV replication in an HCV replicon cell system. In HCV replicon cells, HCV RNA replication was suppressed by 17-AAG in a dose-dependent manner. As shown in the present study, the 50% inhibitory concentration values were 0.82 nM for 17-AAG and 0.21 nM for MG132. Low concentrations of MG132 had strong synergistic inhibitory effects with low toxicity on HCV replicon cells. The results of this study suggest that the different effects and synergistic actions of 17-AAG and MG132 could provide a new therapeutic approach to HCV infection.

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

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    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

  6. Upregulated ROS production induced by the proteasome inhibitor MG-132 on XBP1 gene expression and cell apoptosis in Tca-8113 cells.

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    Chen, Hai-ying; Ren, Xiao-yan; Wang, Wei-hua; Zhang, Ying-xin; Chen, Shuang-feng; Zhang, Bin; Wang, Le-xin

    2014-07-01

    Exposure of Tca-8113 cells to proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG-132) causing apoptosis is associated with endoplasmic reticulum (ER) stress. X-box-binding protein-1 (XBP1) is an important regulator of a subset of genes active during ER stress, which is related to cell survival and is required for tumor growth. The present study is to evaluate the effect of MG-132 on ROS production, XBP1 gene expression, tumor necrosis factor receptor-associated factor 2 (TRAF2), ASK1 and c-jun protein expression in tongue squamous cell carcinoma cell line Tca-8113 cells. ROS production was measured by reactive oxygen species assay. X-box binding protein-1 (XBP1) mRNA was analyzed by real-time-PCR, TRAF2, ASK1 and c-jun protein were investigated by western blot and immunocytochemistry respectively. The result indicated that ROS production, TRAF2, ASK1 and c-jun were elevated in MG-132 treated cells. Giving ROS scavenger N-acetyl-L-cysteine (NAC) largely prevented the effects of MG-132. Furthermore, treating with MG-132 lead to decreased XBP1 mRNA expression but could not completely block the expression of XBP1. Taken together, these findings provide the evidence that MG-132 induced ER stress lead to Tca-8113 cells apoptosis through ROS generation and TRAF2-ASK1-JNK signal pathway activation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. MG132 as a proteasome inhibitor induces cell growth inhibition and cell death in A549 lung cancer cells via influencing reactive oxygen species and GSH level.

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    Han, Yong Hwan; Park, Woo Hyun

    2010-07-01

    Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In the present study, we evaluated the effects of MG132 on the growth of A549 lung cancer cells in relation to cell growth, ROS and glutathione (GSH) levels. Treatment with MG132 inhibited the growth of A549 cells with an IC(50) of approximately 20 microM at 24 hours. DNA flow cytometric analysis indicated that 0.5 approximately 30 microM MG132 induced a G1 phase arrest of the cell cycle in A549 cells. Treatment with 10 or 30 microM MG132 also induced apoptosis, as evidenced by sub-G1 cells and annexin V staining cells. This was accompanied by the loss of mitochondrial membrane potential (MMP; Delta psi m). The intracellular ROS levels including O(2) (*-) were strongly increased in 10 or 30 microM MG132-treated A549 cells but were down-regulated in 0.1, 0.5 or 1 microM MG132-treated cells. Furthermore, 10 or 30 microM MG132 increased mitochondrial O(2) (*- ) level but 0.1, 0.5 or 1 microM MG132 decreased that. In addition, 10 or 30 microM MG132 induced GSH depletion in A549 cells. In conclusion, MG132 inhibited the growth of human A549 cells via inducing the cell cycle arrest as well as triggering apoptosis, which was in part correlated with the changes of ROS and GSH levels. Our present data provide important information on the anti-growth mechanisms of MG132 in A549 lung cancer cells in relation to ROS and GSH.

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

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    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.

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

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    Saji, Chiaki [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Higashi, Chizuka; Niinaka, Yasufumi [Faculty of Medicine, University of Yamanashi, Chuoh-shi 409-3898 (Japan); Yamada, Koji [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Noguchi, Kohji [Faculty of Pharmacy, Keio University, 1-5-30 Shiba-koen, Minato-ku, Tokyo 105-8512 (Japan); Fujimuro, Masahiro, E-mail: fuji2@mb.kyoto-phu.ac.jp [Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-Shichonocho 1, Yamashinaku, Kyoto 607-8412 (Japan)

    2011-12-02

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

  10. The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

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    Pajonk, Frank; Ophoven, Arndt van; Weissenberger, Christian; McBride, William H

    2005-01-01

    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. 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. 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. We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132

  11. Inhibition effect of proteasome inhibitor MG132 combined with X-ray irradiation on cell growth, metastasis and cycle distribution of human lung adenocarcinoma cells

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    Liu Jing; Tang Yiting; Zhou Jundong; Zhang Shuyu; Cao Han; Wu Jinchang; Luo Judong; Chen Guanglie; Cao Jianping

    2014-01-01

    Objective: To study the effects of proteasome inhibitor MG132 on the growth, metastasis, and cell cycle distribution of human lung adenocarcinoma cells A549 irradiated by X-rays. Methods: After treatment of MG132 and irradiation,cell proliferation was detected by MTT assay. Survival was measured by clonogenic assay. Cell migration ability was detected by the Scratch migration assay. Cell invasion ability was detected by transwell migration assay. Cell cycle distribution were analyzed by flow cytometry assay. Protein expression was detected by Western blot assay. Results: MG132 alone inhibited cell growth in a dose-and time-dependent manner. MG132 in combination with radiation significantly suppressed the growth, migration and invasion of A549 cells compared to the control (F =554.78, 954.64, P<0.01). MG132 enhanced radiation-induced G 1 -arrest (t =4.44, 12.41, 3.52, 6.72, P<0.05). The G 1 cell cycle distribution rate of MG132 plus RT group was increased to (71.05 ± 4.17)%. The expressions of MMP-2, MMP-9 and Cyclin D1 were significantly suppressed by MG132 in combination with radiation, while the expression of P53 was up-regulated. Conclusions: MG132 inhibits cell growth, migration and invasion ability, and induces G 1 cell cycle arrest of A549 cells treated with MG132 in combination with radiation, in which the down-regulation of MMPs and Cyclin D1 and up-regulation of P53 may be involved. (authors)

  12. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

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    Tawa, N E; Odessey, R; Goldberg, A L

    1997-07-01

    Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlysosomal protein breakdown by up to 50% (P protein synthesis or amino acid pools, but improved overall protein balance in the muscle. Upon treatment with MG132, ubiquitin-conjugated proteins accumulated in the muscle. The inhibition of muscle proteolysis correlated with efficacy against the proteasome, although these agents could also inhibit calpain-dependent proteolysis induced with Ca2+. These inhibitors had much larger effects on proteolysis in atrophying muscles than in controls. In the denervated soleus undergoing atrophy, the increase in ATP-dependent proteolysis was reduced 70% by MG132 (P muscle proteolysis induced by administering thyroid hormones was reduced 40-70% by the inhibitors. Finally, in rats made septic by cecal puncture, the increase in muscle proteolysis was completely blocked by MG132. Thus, the enhanced proteolysis in many catabolic states (including denervation, hyperthyroidism, and sepsis) is due to a proteasome-dependent pathway, and inhibition of proteasome function may be a useful approach to reduce muscle wasting.

  13. Resveratrol protects leukemic cells against cytotoxicity induced by proteasome inhibitors via induction of FOXO1 and p27Kip1

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    Niu, Xiao-Fang; Liu, Bao-Qin; Du, Zhen-Xian; Gao, Yan-Yan; Li, Chao; Li, Ning; Guan, Yifu; Wang, Hua-Qin

    2011-01-01

    It was reported recently that resveratrol could sensitize a number of cancer cells to the antitumoral effects of some conventional chemotherapy drugs. The current study was designed to investigate whether resveratrol could sensitize leukemic cells to proteasome inhibitors. Leukemic cells were treated with MG132 alone or in combination with resveratrol. Cell viability was investigated using MTT assay, and induction of apoptosis and cell cycle distribution was measured using flow cytometry. Western blot and real-time RT-PCR were used to investigate the expression of FOXO1 and p27 Kip1 . CHIP was performed to investigate the binding of FOXO1 to the p27 Kip1 promoter. Resveratrol strongly reduced cytotoxic activities of proteasome inhibitors against leukemic cells. MG132 in combination with resveratrol caused cell cycle blockade at G1/S transition via p27 Kip1 accumulation. Knockdown of p27 Kip1 using siRNA dramatically attenuated the protective effects of resveratrol on cytotoxic actions of proteasome inhibitors against leukemic cells. Resveratrol induced FOXO1 expression at the transcriptional level, while MG132 increased nuclear distribution of FOXO1. MG132 in combination with resveratrol caused synergistic induction of p27 Kip1 through increased recruitment of FOXO1 on the p27 Kip1 promoter. Resveratrol may have the potential to negate the cytotoxic effects of proteasome inhibitors via regulation of FOXO1 transcriptional activity and accumulation of p27 Kip1

  14. FoxM1 is a general target for proteasome inhibitors.

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    Uppoor G Bhat

    2009-08-01

    Full Text Available Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties.

  15. A robust ex vivo model for evaluation of induction of apoptosis by rhTRAIL in combination with proteasome inhibitor MG132 in human premalignant cervical explants

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    Hougardy, Brigitte M. T.; Reesink-Peters, Nathalie; van den Heuvel, Fiona A. J.; ten Hoor, Klaske A.; Hollema, Harry; de Vries, Elisabeth G. E.; de Jong, Steven; van der Zee, Ate G. J.

    2008-01-01

    Development of medical therapies for high-grade cervical intraepithelial neoplasia (CIN II/III) is hampered by the lack of CIN II/III cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding to its receptors DR4 or DR5. Proteasome inhibition by MG132

  16. Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

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    Wu, William Ka Kei; Volta, Viviana; Cho, Chi Hin; Wu, Ya Chun; Li, Hai Tao; Yu, Le; Li, Zhi Jie; Sung, Joseph Jao Yiu

    2009-01-01

    Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of 35 S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.

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

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    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.

  18. Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

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    Matsumoto, Hotaru; Saitoh, Hisato

    2016-01-01

    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.

  19. Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

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    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.

  20. The Proteasome Inhibitor MG-132 Protects Hypoxic SiHa Cervical Carcinoma Cells after Cyclic Hypoxia/Reoxygenation from Ionizing Radiation

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    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.

  1. Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.

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    Tawa, N E; Odessey, R; Goldberg, A L

    1997-01-01

    Several observations have suggested that the enhanced proteolysis and atrophy of skeletal muscle in various pathological states is due primarily to activation of the ubiquitin-proteasome pathway. To test this idea, we investigated whether peptide aldehyde inhibitors of the proteasome, N-acetyl-leucyl-leucyl-norleucinal (LLN), or the more potent CBZ-leucyl-leucyl-leucinal (MG132) suppressed proteolysis in incubated rat skeletal muscles. These agents (e.g., MG132 at 10 microM) inhibited nonlyso...

  2. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

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    Lombardo, Tomás; Cavaliere, Victoria; Costantino, Susana N.; Kornblihtt, Laura; Alvarez, Elida M.; Blanco, Guillermo A.

    2012-01-01

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O 2 − ) levels. Our results showed that combined arsenite + MG132 produced low levels of O 2 − at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O 2 − levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O 2 − levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O 2 − at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O 2 − production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O 2 − levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect associated with superoxide levels as assessed by flow cytometry. ► Synergism

  3. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

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    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  4. The influence of proteasome inhibitor MG132, external radiation and unlabeled antibody on the tumor uptake and biodistribution of 188Re-labeled anti-E6 C1P5 antibody in cervical cancer in mice

    Science.gov (United States)

    Phaeton, Rébécca; Wang, Xing Guo; Einstein, Mark H.; Goldberg, Gary L.; Casadevall, Arturo; Dadachova, Ekaterina

    2009-01-01

    Background Human Papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer and >95% of all cervical cancers have detectable HPV sequences. We have recently demonstrated the efficacy of radioimmunotherapy (RIT) which targeted viral oncoprotein E6 in treatment of experimental cervical cancer We hypothesized that pre-treatment of tumor cells with various agents which cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors. Methods HPV-16 positive CasKi cells were treated in vitro with up to 6 Gy of external radiation, or proteasome inhibitor MG-132 or unlabeled anti-E6 antibody C1P5 and cell death was assessed. Biodistribution of 188Rhenium (188Re)-labeled C1P5 antibody was performed in both control and radiation MG-132 treated CasKi tumor-bearing nude mice. Results . 188Re-C1P5 antibody demonstrated tumor specificity and very low uptake and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pre-treatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by our previous in vivo studies in CasKi tumor model. Conclusion We demonstrated that pre-treatment of cervical tumors with proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate non-viable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor further development of RIT of cervical cancers targeting viral antigens. PMID:20127955

  5. Synergism between arsenite and proteasome inhibitor MG132 over cell death in myeloid leukaemic cells U937 and the induction of low levels of intracellular superoxide anion

    Energy Technology Data Exchange (ETDEWEB)

    Lombardo, Tomás [Laboratorio de Immunotoxicologia (LaITO), IDEHU-CONICET, Hospital de Clínicas, José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires (Argentina); Cavaliere, Victoria; Costantino, Susana N. [Laboratorio de Inmunología Tumoral (LIT), IDEHU-CONICET, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires (Argentina); Kornblihtt, Laura [Servicio de Hematología, Hospital de Clínicas, José de San Martín (UBA), Buenos Aires (Argentina); Alvarez, Elida M. [Laboratorio de Inmunología Tumoral (LIT), IDEHU-CONICET, Facultad de Farmacia y Bioquímica, UBA, Buenos Aires (Argentina); Blanco, Guillermo A., E-mail: gblanco@ffyb.uba.ar [Laboratorio de Immunotoxicologia (LaITO), IDEHU-CONICET, Hospital de Clínicas, José de San Martín, Universidad de Buenos Aires (UBA), Buenos Aires (Argentina)

    2012-02-01

    Increased oxygen species production has often been cited as a mechanism determining synergism on cell death and growth inhibition effects of arsenic-combined drugs. However the net effect of drug combination may not be easily anticipated solely from available knowledge of drug-induced death mechanisms. We evaluated the combined effect of sodium arsenite with the proteasome inhibitor MG132, and the anti-leukaemic agent CAPE, on growth-inhibition and cell death effect in acute myeloid leukaemic cells U937 and Burkitt's lymphoma-derived Raji cells, by the Chou–Talalay method. In addition we explored the association of cytotoxic effect of drugs with changes in intracellular superoxide anion (O{sub 2}{sup −}) levels. Our results showed that combined arsenite + MG132 produced low levels of O{sub 2}{sup −} at 6 h and 24 h after exposure and were synergic on cell death induction in U937 cells over the whole dose range, although the combination was antagonistic on growth inhibition effect. Exposure to a constant non-cytotoxic dose of 80 μM hydrogen peroxide together with arsenite + MG132 changed synergism on cell death to antagonism at all effect levels while increasing O{sub 2}{sup −} levels. Arsenite + hydrogen peroxide also resulted in antagonism with increased O{sub 2}{sup −} levels in U937 cells. In Raji cells, arsenite + MG132 also produced low levels of O{sub 2}{sup −} at 6 h and 24 h but resulted in antagonism on cell death and growth inhibition. By contrast, the combination arsenite + CAPE showed high levels of O{sub 2}{sup −} production at 6 h and 24 h post exposure but resulted in antagonism over cell death and growth inhibition effects in U937 and Raji cells. We conclude that synergism between arsenite and MG132 in U937 cells is negatively associated to O{sub 2}{sup −} levels at early time points after exposure. -- Highlights: ► Arsenic combined cytotoxic and anti-proliferative effects by Chou–Talalay method. ► Cytotoxic effect

  6. Geldanamycin-induced degradation of Chk1 is mediated by proteasome

    International Nuclear Information System (INIS)

    Nomura, M.; Nomura, N.; Yamashita, J.

    2005-01-01

    Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3 h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24 h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells

  7. MG132 plus apoptosis antigen-1 (APO-1) antibody cooperate to restore p53 activity inducing autophagy and p53-dependent apoptosis in HPV16 E6-expressing keratinocytes.

    Science.gov (United States)

    Lagunas-Martínez, Alfredo; García-Villa, Enrique; Arellano-Gaytán, Magaly; Contreras-Ochoa, Carla O; Dimas-González, Jisela; López-Arellano, María E; Madrid-Marina, Vicente; Gariglio, Patricio

    2017-01-01

    The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

  8. Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3β

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► MG132 induces the phosphorylation of GSK3β Ser9 and, to a lesser extent, of GSK3β Thr390 . ► MG132 induces dephosphorylation of p70S6K Thr389 and phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 dephosphorylates GSK3β Ser9 and phosphorylates GSK3β Thr390 . ► Inactivation of p38 phosphorylates p70S6K Thr389 and increases the phosphorylation of p70S6K Thr421/Ser424 . ► Inactivation of p38 decreases autophagy and increases apoptosis induced by MG132. -- Abstract: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70 kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser 9 and, to a lesser extent, Thr 390 , the dephosphorylation of p70S6K at Thr 389 , and the phosphorylation of p70S6K at Thr 421 and Ser 424 . The specific p38 inhibitor SB203080 reduced the p-GSK3β Ser9 and autophagy through the phosphorylation of p70S6K Thr389 ; however, it augmented the levels of p-ERK, p-GSK3β Thr390 , and p-70S6K Thr421/Ser424 induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK Ser9 /p70S6K Thr380 and ERK/GSK3β Thr390 /p70S6K Thr421/Ser424 kinase signaling, which is involved in cell survival and cell death.

  9. Transcriptional upregulation of BAG3 upon proteasome inhibition

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  10. Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

    Science.gov (United States)

    Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

    2017-01-01

    In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

  11. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Cheol-Hee [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Department of Pharmacology, College of Medicine, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of); Lee, Byung-Hoon [College of Pharmacy and Multiscreening Center for Drug Development, Seoul National University, Seoul 151-742 (Korea, Republic of); Ahn, Sang-Gun [Department of Pathology, College of Dentistry, Chosun University, Gwangju 501-759 (Korea, Republic of); Oh, Seon-Hee, E-mail: oshccw@hanmail.net [Research Center for Resistant Cells, Chosun University, Seosuk-dong, Dong-gu, Gwangju 501-759 (Korea, Republic of)

    2012-02-24

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

  13. Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

    Science.gov (United States)

    Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

    2005-03-01

    In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

  14. Apoptosis and radiosensitization of Hodgkin cells by proteasome inhibition

    International Nuclear Information System (INIS)

    Pajonk, Frank; Pajonk, Katja; McBride, William H.

    2000-01-01

    Purpose: Malignant cells from Hodgkin's disease have been reported to be defective in regulation of NF-κB activity. Ionizing radiation is known to activate NF-κB, and it has been suggested that this pathway may protect cells from apoptosis following exposure to radiation and other therapeutic agents. Defective NF-κB regulation in Hodgkin cells could therefore dictate the response of this disease to therapy, as well as be responsible for maintaining the malignant phenotype. The purpose of this study was to explore whether NF-κB activity could be modulated in Hodgkin cells and whether it determines the response of these cells to treatment with ionizing radiation and/or dexamethasone. Methods and Materials: Activation of NF-κB in cells is accomplished in large part by degradation of its inhibitor IκB through the 26s proteasome. HD-My-Z Hodgkin cells were treated with the proteasome inhibitor MG-132 or transduced with a dominant negative super-repressor IκBα. Clonogenic survival, apoptosis, proteasome activity, and NF-κB binding activity were monitored in response to ionizing radiation and/or dexamethasone treatment. Results: HD-My-Z Hodgkin cells had modest NF-κB levels but, unlike other cell types, did not decrease their level of constitutively active NF-κB in response to proteasome inhibition with MG-132. In contrast, transduction with a non-phosphorable IκBα construct abolished expression. MG-132 did, however, induce apoptosis in HD-My-Z cells and sensitized them to ionizing radiation. Dexamethasone treatment had no effect on NF-κB activity or clonogenic survival of Hodgkin cells, but protected them from irradiation. Conclusion: We conclude that inhibition of 26s proteasome activity can induce apoptosis in HD-My-Z Hodgkin cells and radiosensitize them, in spite of the fact that their constitutively active NF-κB levels are unaltered. The proteasome may be a promising new therapeutic target for intervention in this disease. In contrast, the use of

  15. 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

    2013-09-01

    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.

  16. TRAIL and proteasome inhibitors combination induces a robust apoptosis in human malignant pleural mesothelioma cells through Mcl-1 and Akt protein cleavages

    International Nuclear Information System (INIS)

    Yuan, Bao-Zhu; Chapman, Joshua; Ding, Min; Wang, Junzhi; Jiang, Binghua; Rojanasakul, Yon; Reynolds, Steven H

    2013-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive malignancy closely associated with asbestos exposure and extremely resistant to current treatments. It exhibits a steady increase in incidence, thus necessitating an urgent development of effective new treatments. Proteasome inhibitors (PIs) and TNFα-Related Apoptosis Inducing Ligand (TRAIL), have emerged as promising new anti-MPM agents. To develop effective new treatments, the proapoptotic effects of PIs, MG132 or Bortezomib, and TRAIL were investigated in MPM cell lines NCI-H2052, NCI-H2452 and NCI-H28, which represent three major histological types of human MPM. Treatment with 0.5-1 μM MG132 alone or 30 ng/mL Bortezomib alone induced a limited apoptosis in MPM cells associated with the elevated Mcl-1 protein level and hyperactive PI3K/Akt signaling. However, whereas 10–20 ng/ml TRAIL alone induced a limited apoptosis as well, TRAIL and PI combination triggered a robust apoptosis in all three MPM cell lines. The robust proapoptotic activity was found to be the consequence of a positive feedback mechanism-governed amplification of caspase activation and cleavage of both Mcl-1 and Akt proteins, and exhibited a relative selectivity in MPM cells than in non-tumorigenic Met-5A mesothelial cells. The combinatorial treatment using TRAIL and PI may represent an effective new treatment for MPMs

  17. Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination.

    Science.gov (United States)

    Radwan, Marta; Wilkinson, David J; Hui, Wang; Destrument, Auriane P M; Charlton, Sarah H; Barter, Matt J; Gibson, Beth; Coulombe, Josée; Gray, Douglas A; Rowan, Andrew D; Young, David A

    2015-08-01

    To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA). Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6). Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression. Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are

  18. Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Sansom, Owen J; Porecha, Nehal; Raich, Natacha; Du, Liqin; Maher, Joseph F

    2010-02-01

    In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

  19. Antitumorigenic effect of proteasome inhibitors on insulinoma cells

    DEFF Research Database (Denmark)

    Størling, Joachim; Allaman-Pillet, Nathalie; Karlsen, Allan E

    2004-01-01

    inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse betaTC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also...

  20. 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.

  1. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    Science.gov (United States)

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos. (c) 2009 Wiley-Liss, Inc.

  2. Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction

    International Nuclear Information System (INIS)

    He, Li-Cai; Xu, Han-Zhang; Gu, Zhi-Min; Liu, Chuan-Xu; Chen, Guo-Qiang; Wang, Yue-Fei; Wen, Dong-Hua; Wu, Ying-Li

    2011-01-01

    Research highlights: → Chemotherapeutic drugs or UV treatment reduces Ikaros prior to caspase-3 activation. → Etoposide treatment does not alter the mRNA but shortens the half-life of Ikaros. → MG132 or epoxomicin but not calpeptin inhibits etoposide-induced Ikaros degradation. → Overexpression of Ikaros accelerates etoposide-induced apoptosis in NB4 cells. -- Abstract: Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3 h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

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

    Science.gov (United States)

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

    2016-06-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast-osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO-A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post-translational degradation. We found that exogenous treatment of MLO-A5 and osteocytic IDG-SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome-selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin-A) produced similar dose-dependent increases in E11 protein levels in MLO-A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO-A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome-mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. © 2015 The Authors. Journal of

  4. Implication of altered ubiquitin-proteasome system and ER stress in the muscle atrophy of diabetic rats.

    Science.gov (United States)

    Reddy, S Sreenivasa; Shruthi, Karnam; Prabhakar, Y Konda; Sailaja, Gummadi; Reddy, G Bhanuprakash

    2018-02-01

    Skeletal muscle is adversely affected in type-1 diabetes, and excessively stimulated ubiquitin-proteasome system (UPS) was found to be a leading cause of muscle wasting or atrophy. The role of endoplasmic reticulum (ER) stress in muscle atrophy of type-1 diabetes is not known. Hence, we investigated the role of UPS and ER stress in the muscle atrophy of chronic diabetes rat model. Diabetes was induced with streptozotocin (STZ) in male Sprague-Dawley rats and were sacrificed 2- and 4-months thereafter to collect gastrocnemius muscle. In another experiment, 2-months post-STZ-injection diabetic rats were treated with MG132, a proteasome inhibitor, for the next 2-months and gastrocnemius muscle was collected. The muscle fiber cross-sectional area was diminished in diabetic rats. The expression of UPS components: E1, MURF1, TRIM72, UCHL1, UCHL5, ubiquitinated proteins, and proteasome activity were elevated in the diabetic rats indicating activated UPS. Altered expression of ER-associated degradation (ERAD) components and increased ER stress markers were detected in 4-months diabetic rats. Proteasome inhibition by MG132 alleviated alterations in the UPS and ER stress in diabetic rat muscle. Increased UPS activity and ER stress were implicated in the muscle atrophy of diabetic rats and proteasome inhibition exhibited beneficiary outcome. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. A new structural class of proteasome inhibitors that prevent NF-kappa B activation.

    Science.gov (United States)

    Lum, R T; Kerwar, S S; Meyer, S M; Nelson, M G; Schow, S R; Shiffman, D; Wick, M M; Joly, A

    1998-05-01

    The multicatalytic proteinase or proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of many proteins involved in important regulatory cellular processes. We have identified a novel class of inhibitors of the chymotrypsin-like proteolytic activity of the 20S proteasome that exhibit IC50 values ranging from 0.1 to 0.5 microgram/mL (0.1 to 1 microM). In cell proliferation assays, these compounds inhibit growth with an IC50 ranging from 5 to 10 micrograms/mL (10-20 microM). A representative member of this class of inhibitors was tested in other biological assays. CVT-634 (5-methoxy-1-indanone-3-acetyl-leu-D-leu-1-indanylamide) prevented lipopolysaccharide (LPS), tumor necrosis factor (TNF)-, and phorbol ester-induced activation of nuclear factor kappa B (NF-kappa B) in vitro by preventing signal-induced degradation of I kappa B-alpha. In these studies, the I kappa B-alpha that accumulated was hyperphosphorylated, indicating that CVT-634 did not inhibit I kappa B-alpha kinase, the enzyme responsible for signal-induced phosphorylation of I kappa B-alpha. In vivo studies indicated that CVT-634 prevented LPS-induced TNF synthesis in a murine macrophage cell line. In addition, in mice pretreated with CVT-634 at 25 and 50 mg/kg and subsequently treated with LPS, serum TNF levels were significantly lower (225 +/- 59 and 83 +/- 41 pg/mL, respectively) than in those mice that were treated only with LPS (865 +/- 282 pg/mL). These studies suggest that specific inhibition of the chymotrypsin-like activity of the proteasome is sufficient to prevent signal-induced NF-kappa B activation and that the proteasome is a novel target for the identification of agents that may be useful in the treatment of diseases whose etiology is dependent upon the activation of NF-kappa B.

  6. Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells.

    Science.gov (United States)

    Or, Chi-Hung R; Chang, Yachu; Lin, Wei-Cheng; Lee, Wee-Chyan; Su, Hong-Lin; Cheung, Muk-Wing; Huang, Chang-Po; Ho, Cheesang; Chang, Chia-Che

    2016-12-27

    Colorectal cancer is the third most common cancer worldwide. Aberrant overexpression of antiapoptotic BCL-2 (B-cell lymphoma 2) family proteins is closely linked to tumorigenesis and poor prognosis in colorectal cancer. Obatoclax is an inhibitor targeting all antiapoptotic BCL-2 proteins. A previous study has described the antiproliferative action of obatoclax in one human colorectal cancer cell line without elucidating the underlying mechanisms. We herein reported that, in a panel of human colorectal cancer cell lines, obatoclax inhibits cell proliferation, suppresses clonogenicity, and induces G₁-phase cell cycle arrest, along with cyclin D1 downregulation. Notably, ectopic cyclin D1 overexpression abrogated clonogenicity suppression but also G₁-phase arrest elicited by obatoclax. Mechanistically, pre-treatment with the proteasome inhibitor MG-132 restored cyclin D1 levels in all obatoclax-treated cell lines. Cycloheximide chase analyses further revealed an evident reduction in the half-life of cyclin D1 protein by obatoclax, confirming that obatoclax downregulates cyclin D1 through induction of cyclin D1 proteasomal degradation. Lastly, threonine 286 phosphorylation of cyclin D1, which is essential for initiating cyclin D1 proteasomal degradation, was induced by obatoclax in one cell line but not others. Collectively, we reveal a novel anticancer mechanism of obatoclax by validating that obatoclax targets cyclin D1 for proteasomal degradation to downregulate cyclin D1 for inducing antiproliferation.

  7. 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…

  8. 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

  9. 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.

  10. A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

    Science.gov (United States)

    Hakozaki, T; Laughlin, T; Zhao, S; Wang, J; Deng, D; Jewell-Motz, E; Elstun, L

    2013-07-01

    2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation. © 2013 The Authors BJD © 2013 British Association of Dermatologists.

  11. Trial Watch: Proteasomal inhibitors for anticancer therapy.

    Science.gov (United States)

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called "ubiquitin-proteasome system" (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients.

  12. The novel Akt inhibitor API-1 induces c-FLIP degradation and synergizes with TRAIL to augment apoptosis independent of Akt inhibition.

    Science.gov (United States)

    Li, Bo; Ren, Hui; Yue, Ping; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

    2012-04-01

    API-1 (pyrido[2,3-d]pyrimidines) is a novel small-molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation and has promising preclinical antitumor activity. In this study, we reveal a novel function of API-1 in regulation of cellular FLICE-inhibitory protein (c-FLIP) levels and TRAIL-induced apoptosis, independent of Akt inhibition. API-1 effectively induced apoptosis in tested cancer cell lines including activation of caspase-8 and caspase-9. It reduced the levels of c-FLIP without increasing the expression of death receptor 4 (DR4) or DR5. Accordingly, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c-FLIP did not attenuate API-1-induced apoptosis but inhibited its ability to enhance TRAIL-induced apoptosis. These data indicate that downregulation of c-FLIP mediates enhancement of TRAIL-induced apoptosis by API-1 but is not sufficient for API-1-induced apoptosis. API-1-induced reduction of c-FLIP could be blocked by the proteasome inhibitor MG132. Moreover, API-1 increased c-FLIP ubiquitination and decreased c-FLIP stability. These data together suggest that API-1 downregulates c-FLIP by facilitating its ubiquitination and proteasome-mediated degradation. Because other Akt inhibitors including API-2 and MK2206 had minimal effects on reducing c-FLIP and enhancement of TRAIL-induced apoptosis, it is likely that API-1 reduces c-FLIP and enhances TRAIL-induced apoptosis independent of its Akt-inhibitory activity. 2012 AACR

  13. Discovery of an Inhibitor of the Proteasome Subunit Rpn11.

    Science.gov (United States)

    Perez, Christian; Li, Jing; Parlati, Francesco; Rouffet, Matthieu; Ma, Yuyong; Mackinnon, Andrew L; Chou, Tsui-Fen; Deshaies, Raymond J; Cohen, Seth M

    2017-02-23

    The proteasome plays a crucial role in degradation of normal proteins that happen to be constitutively or inducibly unstable, and in this capacity it plays a regulatory role. Additionally, it degrades abnormal/damaged/mutant/misfolded proteins, which serves a quality-control function. Inhibitors of the proteasome have been validated in the treatment of multiple myeloma, with several FDA-approved therapeutics. Rpn11 is a Zn 2+ -dependent metalloisopeptidase that hydrolyzes ubiquitin from tagged proteins that are trafficked to the proteasome for degradation. A fragment-based drug discovery (FBDD) approach was utilized to identify fragments with activity against Rpn11. Screening of a library of metal-binding pharmacophores (MBPs) revealed that 8-thioquinoline (8TQ, IC 50 value ∼2.5 μM) displayed strong inhibition of Rpn11. Further synthetic elaboration of 8TQ yielded a small molecule compound (35, IC 50 value ∼400 nM) that is a potent and selective inhibitor of Rpn11 that blocks proliferation of tumor cells in culture.

  14. 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

  15. 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

    2016-01-01

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

  16. 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.

  17. Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes.

    Science.gov (United States)

    Zhang, Fang Fang; Morioka, Norimitsu; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

    2015-09-04

    Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 μM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

    Science.gov (United States)

    Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

    2014-08-15

    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

  19. Studies on terrein as a new class of proteasome inhibitors

    International Nuclear Information System (INIS)

    Demasi, M.; Felicio, A.L.; Lima, C.; Pacheco, A.O.; Leite, H.G.; Andrade, L.H.

    2010-01-01

    The proteasome is an intracellular multicatalytic protease involved in the cell cycle regulation, signaling response, antigen presentation and apoptosis. Since proteasome inhibitors promote cell death by apoptosis, they have been proposed as new anti-tumoral drugs. Terrein, a secondary metabolite secreted by the fungus Aspergillus terreus, was firstly described in 1935. In the present work we report that terrein isolated through the screening for inhibitors of the 20S proteasome showed inhibitory effect upon both chymotrypsin- and trypsin-like activities of the multicatalytic core particle, the 20S proteasome. Despite of the high inhibitory concentration determined in vitro, that verified by incubating cells (fibroblasts and a pulmonary tumor cell line) in the presence of terrein was 4-fold lower indicating the proteasome as a selective intracellular target. Moreover, terrein promoted apoptotic cell death on both fibroblasts and pulmonary tumor cell line tested. Although terrein concentrations (mM range) necessary to elicit apoptosis in the cellular models herein tried were high when compared to those (muM and nM range) of other inhibitors recently described, its chemical structure is not correlated to any other inhibitor reported thus far. Therefore, the present results point out for the possibility of exploring terrein as a new molecular fragment for the development of synthetic proteasome inhibitors. (author)

  20. E1AF degradation by a ubiquitin-proteasome pathway

    International Nuclear Information System (INIS)

    Takahashi, Akiko; Higashino, Fumihiro; Aoyagi, Mariko; Yoshida, Koichi; Itoh, Miyuki; Kobayashi, Masanobu; Totsuka, Yasunori; Kohgo, Takao; Shindoh, Masanobu

    2005-01-01

    E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function

  1. Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4.

    Science.gov (United States)

    Tsubouchi, Kazuya; Araya, Jun; Minagawa, Shunsuke; Hara, Hiromichi; Ichikawa, Akihiro; Saito, Nayuta; Kadota, Tsukasa; Sato, Nahoko; Yoshida, Masahiro; Kurita, Yusuke; Kobayashi, Kenji; Ito, Saburo; Fujita, Yu; Utsumi, Hirofumi; Yanagisawa, Haruhiko; Hashimoto, Mitsuo; Wakui, Hiroshi; Yoshii, Yutaka; Ishikawa, Takeo; Numata, Takanori; Kaneko, Yumi; Asano, Hisatoshi; Yamashita, Makoto; Odaka, Makoto; Morikawa, Toshiaki; Nakayama, Katsutoshi; Nakanishi, Yoichi; Kuwano, Kazuyoshi

    2017-08-03

    Accumulation of profibrotic myofibroblasts is involved in the process of fibrosis development during idiopathic pulmonary fibrosis (IPF) pathogenesis. TGFB (transforming growth factor β) is one of the major profibrotic cytokines for myofibroblast differentiation and NOX4 (NADPH oxidase 4) has an essential role in TGFB-mediated cell signaling. Azithromycin (AZM), a second-generation antibacterial macrolide, has a pleiotropic effect on cellular processes including proteostasis. Hence, we hypothesized that AZM may regulate NOX4 levels by modulating proteostasis machineries, resulting in inhibition of TGFB-associated lung fibrosis development. Human lung fibroblasts (LF) were used to evaluate TGFB-induced myofibroblast differentiation. With respect to NOX4 regulation via proteostasis, assays for macroautophagy/autophagy, the unfolded protein response (UPR), and proteasome activity were performed. The potential anti-fibrotic property of AZM was examined by using bleomycin (BLM)-induced lung fibrosis mouse models. TGFB-induced NOX4 and myofibroblast differentiation were clearly inhibited by AZM treatment in LF. AZM-mediated NOX4 reduction was restored by treatment with MG132, a proteasome inhibitor. AZM inhibited autophagy and enhanced the UPR. Autophagy inhibition by AZM was linked to ubiquitination of NOX4 via increased protein levels of STUB1 (STIP1 homology and U-box containing protein 1), an E3 ubiquitin ligase. An increased UPR by AZM was associated with enhanced proteasome activity. AZM suppressed lung fibrosis development induced by BLM with concomitantly reduced NOX4 protein levels and enhanced proteasome activation. These results suggest that AZM suppresses NOX4 by promoting proteasomal degradation, resulting in inhibition of TGFB-induced myofibroblast differentiation and lung fibrosis development. AZM may be a candidate for the treatment of the fibrotic lung disease IPF.

  2. A free radical-generating system regulates AβPP metabolism/processing: involvement of the ubiquitin/proteasome and autophagy/lysosome pathways.

    Science.gov (United States)

    Recuero, María; Munive, Victor A; Sastre, Isabel; Aldudo, Jesús; Valdivieso, Fernando; Bullido, María J

    2013-01-01

    Oxidative stress is an early event in the pathogenesis of Alzheimer's disease (AD). We previously reported that, in SK-N-MC cells, the xanthine/xanthine oxidase (X-XOD) free radical generating system regulates the metabolism/processing of the amyloid-β protein precursor (AβPP). Oxidative stress alters the two main cellular proteolytic machineries, the ubiquitin/proteasome (UPS) and the autophagy/lysosome systems, and recent studies have established connections between the malfunctioning of these and the pathogenesis of AD. The aim of the present work was to examine the involvement of these proteolytic systems in the regulation of AβPP metabolism by X-XOD. The proteasome inhibitor MG132 was found to accelerate the metabolism/processing of AβPP promoted by X-XOD because it significantly enhances the secretion of α-secretase-cleaved soluble AβPP and also the levels of both carboxy-terminal fragments (CTFs) produced by α- and β-secretase. Further, MG132 modulated the intracellular accumulation of holo-AβPP and/or AβPP CTFs. This indicates that the X-XOD modulation of AβPP metabolism/processing involves the UPS pathway. With respect to the autophagy/lysosome pathway, the AβPP processing and intracellular location patterns induced by X-XOD treatment closely resembled those produced by the lysosome inhibitor ammonium chloride. The present results suggest that the regulation of AβPP metabolism/processing by mild oxidative stress requires UPS activity with a simultaneous reduction in that of the autophagy/lysosome system.

  3. 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.

  4. Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

    Energy Technology Data Exchange (ETDEWEB)

    Strom, Bjorn O. [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway); Paulsen, Ragnhild E., E-mail: r.e.paulsen@farmasi.uio.no [Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo (Norway)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

  5. Ubiquitin-dependent system controls radiation induced apoptosis

    International Nuclear Information System (INIS)

    Delic, J.; Magdelenat, H.; Glaisner, S.; Magdelenat, H.; Maciorowski, Z.

    1997-01-01

    The selective proteolytic pathway, dependent upon 'N-end rule' protein recognition/ubiquitination and on the subsequent proteasome dependent processing of ubiquitin conjugates, operates in apoptosis induced by γ-irradiation. The proteasome inhibitor peptide aldehyde, MG132, efficiently induced apoptosis and was also able (at doses lower than those required for apoptosis induction) to potentiate apoptosis induced by DNA damage. Its specificity is suggested by the induction of the ubiquitin (UbB and UbC) and E1 (ubiquitin activating enzyme) genes and by an altered ubiquitination pattern. More selectively, a di-peptide competitor of the 'N-end rule' of ubiquitin dependent protein processing inhibited radiation induced apoptosis. This inhibition is also followed by an altered ubiquitination pattern and by activation of Poly (ADP-ribose) polymerase (PARP). These data strongly suggest that early apoptosis radiation induced events are controlled by ubiquitin-dependent proteolytic processing. (author)

  6. Proteomic characterization of an isolated fraction of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells might offer clues to aggresomes as a cellular defensive response against proteasome inhibition by PSI

    Directory of Open Access Journals (Sweden)

    Li Xing'an

    2010-08-01

    Full Text Available Abstract Background Cooperation of constituents of the ubiquitin proteasome system (UPS with chaperone proteins in degrading proteins mediate a wide range of cellular processes, such as synaptic function and neurotransmission, gene transcription, protein trafficking, mitochondrial function and metabolism, antioxidant defence mechanisms, and apoptotic signal transduction. It is supposed that constituents of the UPS and chaperone proteins are recruited into aggresomes where aberrant and potentially cytotoxic proteins may be sequestered in an inactive form. Results To determinate the proteomic pattern of synthetic proteasome inhibitor (PSI-induced inclusions in PC12 cells after proteasome inhibition by PSI, we analyzed a fraction of PSI-induced inclusions. A proteomic feature of the isolated fraction was characterized by identification of fifty six proteins including twenty previously reported protein components of Lewy bodies, twenty eight newly identified proteins and eight unknown proteins. These proteins, most of which were recognized as a profile of proteins within cellular processes mediated by the UPS, a profile of constituents of the UPS and a profile of chaperone proteins, are classed into at least nine accepted categories. In addition, prolyl-4-hydroxylase beta polypeptide, an endoplasmic reticulum member of the protein disulfide isomerase family, was validated in the developmental process of PSI-induced inclusions in the cells. Conclusions It is speculated that proteomic characterization of an isolated fraction of PSI-induced inclusions in PC12 cells might offer clues to appearance of aggresomes serving as a cellular defensive response against proteasome inhibition.

  7. 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.

  8. 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.

  9. Sensitive detection of proteasomal activation using the Deg-On mammalian synthetic gene circuit.

    Science.gov (United States)

    Zhao, Wenting; Bonem, Matthew; McWhite, Claire; Silberg, Jonathan J; Segatori, Laura

    2014-04-08

    The ubiquitin proteasome system (UPS) has emerged as a drug target for diverse diseases characterized by altered proteostasis, but pharmacological agents that enhance UPS activity have been challenging to establish. Here we report the Deg-On system, a genetic inverter that translates proteasomal degradation of the transcriptional regulator TetR into a fluorescent signal, thereby linking UPS activity to an easily detectable output, which can be tuned using tetracycline. We demonstrate that this circuit responds to modulation of UPS activity in cell culture arising from the inhibitor MG-132 and activator PA28γ. Guided by predictive modelling, we enhanced the circuit's signal sensitivity and dynamic range by introducing a feedback loop that enables self-amplification of TetR. By linking UPS activity to a simple and tunable fluorescence output, these genetic inverters will enable a variety of applications, including screening for UPS activating molecules and selecting for mammalian cells with different levels of proteasome activity.

  10. BAX/BAK–Independent Mitoptosis during Cell Death Induced by Proteasome Inhibition?

    OpenAIRE

    Lomonosova, Elena; Ryerse, Jan; Chinnadurai, G.

    2009-01-01

    Proteasome inhibitors induce rapid death of cancer cells. We show that in epithelial cancer cells, such death is associated with dramatic and simultaneous up-regulation of several BH3-only proteins, including BIK, BIM, MCL-1S, NOXA, and PUMA, as well as p53. Elevated levels of these proteins seem to be the result of direct inhibition of their proteasomal degradation, induction of transcription, and active translation. Subsequent cell death is independent of BAX, and probably BAK, and proceeds...

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

    Science.gov (United States)

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

    2014-09-01

    The identification of the key role of the eukaryotic 26S proteasome in regulated intracellular proteolysis and its importance as a target in many pathological conditions wherein the proteasomal activity is defective (e.g., malignancies, autoimmune diseases, neurodegenerative diseases, etc.) prompted several research groups to the development of specific inhibitors of this multicatalytic complex with the aim of obtaining valid drug candidates. In regard to the anticancer therapy, the peptide boronate bortezomib (Velcade®) represents the first molecule approved by FDA for the treatment of multiple myeloma in 2003 and mantle cell lymphoma in 2006. Since then, a plethora of molecules targeting the proteasome have been identified as potential anticancer agents and a few of them reached clinical trials or are already in the market (i.e., carfilzomib; Kyprolis®). In most cases, the design of new proteasome inhibitors (PIs) takes into account a proven peptide or pseudopeptide motif as a base structure and places other chemical entities throughout the peptide skeleton in such a way to create an efficacious network of interactions within the catalytic sites. The purpose of this review is to provide an in-depth look at the current state of the research in the field of peptide-based PIs, specifically those ones that might find an application as anticancer agents. © 2014 Wiley Periodicals, Inc.

  12. Discovery and development of inhibitors selective for human constitutive proteasome and immunoproteasome active sites

    NARCIS (Netherlands)

    Xin, B.

    2017-01-01

    This thesis describes the design and development of subunit‐selective inhibitors of particular catalytically active subunits of human constitutive proteasomes and immunoproteasomes. Most existing proteasome inhibitors are oligopeptides composed of 2‐4 amino acid residues, N‐terminally

  13. [The mechanisms of p21WAF1/Cip-1 expression in MOLT-4 cell line induced by TSA].

    Science.gov (United States)

    Song, Yi; Liu, Mei-Ju; Zhao, Guo-Wei; Qian, Jun-Jie; Dong, Yan; Liu, Hua; Sun, Guo-Jing; Mei, Zhu-Zhong; Liu, Bin; Tian, Bao-Lei; Sun, Zhi-Xian

    2005-04-01

    To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.

  14. Proteasome phosphorylation regulates cocaine-induced sensitization.

    Science.gov (United States)

    Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

    2018-04-01

    Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Model-Informed Drug Development for Ixazomib, an Oral Proteasome Inhibitor.

    Science.gov (United States)

    Gupta, Neeraj; Hanley, Michael J; Diderichsen, Paul M; Yang, Huyuan; Ke, Alice; Teng, Zhaoyang; Labotka, Richard; Berg, Deborah; Patel, Chirag; Liu, Guohui; van de Velde, Helgi; Venkatakrishnan, Karthik

    2018-02-15

    Model-informed drug development (MIDD) was central to the development of the oral proteasome inhibitor ixazomib, facilitating internal decisions (switch from body surface area (BSA)-based to fixed dosing, inclusive phase III trials, portfolio prioritization of ixazomib-based combinations, phase III dose for maintenance treatment), regulatory review (model-informed QT analysis, benefit-risk of 4 mg dose), and product labeling (absolute bioavailability and intrinsic/extrinsic factors). This review discusses the impact of MIDD in enabling patient-centric therapeutic optimization during the development of ixazomib. © 2017 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

  16. BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein.

    Science.gov (United States)

    Chiappetta, Gennaro; Basile, Anna; Arra, Claudio; Califano, Daniela; Pasquinelli, Rosa; Barbieri, Antonio; De Simone, Veronica; Rea, Domenica; Giudice, Aldo; Pezzullo, Luciano; De Laurenzi, Vincenzo; Botti, Gerardo; Losito, Simona; Conforti, Daniela; Turco, Maria Caterina

    2012-01-01

    Anaplastic thyroid tumors (ATC) express high levels of BAG3, a member of the BAG family of cochaperone proteins that is involved in regulating cell apoptosis through multiple mechanisms. The objective of the study was the investigation of the influence of B-cell lymphoma-2-associated athanogene 3 (BAG3) on ATC growth. We investigated the effects of BAG3 down-modulation, obtained by using a specific small interfering RNA, on in vitro and in vivo growth of the human ATC cell line 8505C. Because BRAF protein plays an important role in ATC cell growth, we analyzed the effects of BAG3 down-modulation on BRAF protein levels. Furthermore, by using a proteasome inhibitor, we verified whether BAG3-mediated regulation of BRAF levels involved a proteasome-dependent mechanism. BAG3 down-modulation significantly inhibits ATC growth in vitro and in vivo. BAG3 coimmunoprecipitates with BRAF protein, and its down-modulation results in a significant reduction of BRAF protein levels, which can be reverted by incubation with the proteasome inhibitor MG132. BAG3 protein sustains ATC growth in vitro and in vivo. The underlying molecular mechanism appears to rely on BAG3 binding to BRAF, thus protecting it from proteasome-dependent degradation. These results are in line with the reported ability of BAG3 to interfere with the proteasomal delivery of a number of other client proteins.

  17. 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.

  18. Molecular mechanisms for synergistic effect of proteasome inhibitors with platinum-based therapy in solid tumors.

    Science.gov (United States)

    Chao, Angel; Wang, Tzu-Hao

    2016-02-01

    The successful development of the proteasome inhibitor bortezomib as an anticancer drug has improved survival in patients with multiple myeloma. With the emergence of the newly US Food and Drug Administration-approved proteasome inhibitor carfilzomib, ongoing trials are investigating this compound and other proteasome inhibitors either alone or in combination with other chemotherapy drugs. However, in solid tumors, the efficacy of proteasome inhibitors has not lived up to expectations. Results regarding the potential clinical efficacy of bortezomib combined with other agents in the treatment of solid tumors are eagerly awaited. Recent identification of the molecular mechanisms (involving apoptosis and autophagy) by which bortezomib and cisplatin can overcome chemotherapy resistance and sensitize tumor cells to anticancer therapy can provide insights into the development of novel therapeutic strategies for patients with solid malignancies. Copyright © 2016. Published by Elsevier B.V.

  19. Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

    Science.gov (United States)

    Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

    2012-01-01

    Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

  20. Electroacupuncture Confers Antinociceptive Effects via Inhibition of Glutamate Transporter Downregulation in Complete Freund's Adjuvant-Injected Rats

    Directory of Open Access Journals (Sweden)

    Ha-Neui Kim

    2012-01-01

    Full Text Available When we evaluated changes of glial fibrillary acidic protein (GFAP and two glutamate transporter (GTs by immunohistochemistry, expression of GFAP showed a significant increase in complete Freund's adjuvant (CFA-injected rats; however, this expression was strongly inhibited by electroacupuncture (EA stimulation. Robust downregulation of glutamate-aspartate transporter (GLAST and glutamate transporter-1 (GLT-1 was observed in CFA-injected rats; however, EA stimulation resulted in recovery of this expression. Double-labeling staining showed co-localization of a large proportion of GLAST or GLT-1 with GFAP. Using Western blot, we confirmed protein expression of two GTs, but no differences in the mRNA content of these GTs were observed. Because EA treatment resulted in strong inhibition of CFA-induced proteasome activities, we examined the question of whether thermal sensitivities and GTs expression could be regulated by proteasome inhibitor MG132. CFA-injected rats co-treated with EA and MG132 showed a significantly longer thermal sensitivity, compared with CFA-injected rats with or without MG132. Both EA and MG132 blocked CFA-induced GLAST and GLT-1 downregulation within the spinal cord. These results provide evidence for involvement of GLAST and GLT-1 in response to activation of spinal astrocytes in an EA antinociceptive effect. Antinociceptive effect of EA may be induced via proteasome-mediated regulation of spinal GTs.

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

    Science.gov (United States)

    Gräwert, Melissa Ann; Groll, Michael

    2012-02-01

    Cancer is the No. 2 cause of death in the Western world and one of the most expensive diseases to treat. Thus, it is not surprising, that every major pharmaceutical and biotechnology company has a blockbuster oncology product. In 2003, Millennium Pharmaceuticals entered the race with Velcade®, a first-in-class proteasome inhibitor that has been approved by the FDA for treatment of multiple myeloma and its sales have passed the billion dollar mark. Velcade®'s extremely toxic boronic acid pharmacophore, however, contributes to a number of severe side effects. Nevertheless, the launching of this product has validated the proteasome as a target in fighting cancer and further proteasome inhibitors have entered the market as anti-cancer drugs. Additionally, proteasome inhibitors have found application as crop protection agents, anti-parasitics, immunosuppressives, as well as in new therapies for muscular dystrophies and inflammation. Many of these compounds are based on microbial metabolites. In this review, we emphasize the important role of the structural elucidation of the various unique binding mechanisms of these compounds that have been optimized throughout evolution to target the proteasome. Based on this knowledge, medicinal chemists have further optimized these natural products, resulting in potential drugs with reduced off-target activities. This journal is © The Royal Society of Chemistry 2012

  2. 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.

  3. Inhibition of nuclear factor-κB and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma

    International Nuclear Information System (INIS)

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

    2005-01-01

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

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

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    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.

  5. Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits

    DEFF Research Database (Denmark)

    Acosta-Alvear, Diego; Cho, Min Y; Wild, Thomas

    2015-01-01

    Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM...

  6. Neocortex and allocortex respond differentially to cellular stress in vitro and aging in vivo.

    Directory of Open Access Journals (Sweden)

    Jessica M Posimo

    Full Text Available In Parkinson's and Alzheimer's diseases, the allocortex accumulates aggregated proteins such as synuclein and tau well before neocortex. We present a new high-throughput model of this topographic difference by microdissecting neocortex and allocortex from the postnatal rat and treating them in parallel fashion with toxins. Allocortical cultures were more vulnerable to low concentrations of the proteasome inhibitors MG132 and PSI but not the oxidative poison H2O2. The proteasome appeared to be more impaired in allocortex because MG132 raised ubiquitin-conjugated proteins and lowered proteasome activity in allocortex more than neocortex. Allocortex cultures were more vulnerable to MG132 despite greater MG132-induced rises in heat shock protein 70, heme oxygenase 1, and catalase. Proteasome subunits PA700 and PA28 were also higher in allocortex cultures, suggesting compensatory adaptations to greater proteasome impairment. Glutathione and ceruloplasmin were not robustly MG132-responsive and were basally higher in neocortical cultures. Notably, neocortex cultures became as vulnerable to MG132 as allocortex when glutathione synthesis or autophagic defenses were inhibited. Conversely, the glutathione precursor N-acetyl cysteine rendered allocortex resilient to MG132. Glutathione and ceruloplasmin levels were then examined in vivo as a function of age because aging is a natural model of proteasome inhibition and oxidative stress. Allocortical glutathione levels rose linearly with age but were similar to neocortex in whole tissue lysates. In contrast, ceruloplasmin levels were strikingly higher in neocortex at all ages and rose linearly until middle age. PA28 levels rose with age and were higher in allocortex in vivo, also paralleling in vitro data. These neo- and allocortical differences have implications for the many studies that treat the telencephalic mantle as a single unit. Our observations suggest that the topographic progression of protein

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

    Science.gov (United States)

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

    2016-03-29

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

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

    Science.gov (United States)

    Hui, Kwai Fung; Tam, Kam Pui; Chiang, Alan Kwok Shing

    2017-11-21

    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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2016-06-07

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

  11. 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.

  12. Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

    Science.gov (United States)

    Ghosh, Rajeshwary; Goswami, Sumanta K; Feitoza, Luis Felipe B B; Hammock, Bruce; Gomes, Aldrin V

    2016-11-15

    One of the most common nonsteroidal anti-inflammatory drugs (NSAIDs) used worldwide, diclofenac (DIC), has been linked to increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which DIC causes CVD is unknown. Proteasome activities were studied in hearts, livers, and kidneys from male Swiss Webster mice treated with either 100mg/kg DIC for 18h (acute treatment) or 10mg/kg DIC for 28days (chronic treatment). Cultured H9c2 cells and neonatal cardiomyocytes were also treated with different concentrations of DIC and proteasome function, cell death and ROS generation studied. Isolated mouse heart mitochondria were utilized to determine the effect of DIC on various electron transport chain complex activities. DIC significantly inhibited the chymotrypsin-like proteasome activity in rat cardiac H9c2 cells, murine neonatal cardiomyocytes, and mouse hearts, but did not affect proteasome subunit expression levels. Proteasome activity was also affected in liver and kidney tissues from DIC treated animals. The levels of polyubiquitinated proteins increased in hearts from DIC treated mice. Importantly, the levels of oxidized proteins increased while the β5i immunoproteasome activity decreased in hearts from DIC treated mice. DIC increased ROS production and cell death in H9c2 cells and neonatal cardiomyocytes while the cardioprotective NSAID, aspirin, had no effect on ROS levels or cell viability. DIC inhibited mitochondrial Complex III, a major source of ROS, and impaired mitochondrial membrane potential suggesting that mitochondria are the major sites of ROS generation. These results suggest that DIC induces cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

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    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

  14. Mechanism of Action of Bortezomib and the New Proteasome Inhibitors on Myeloma Cells and the Bone Microenvironment: Impact on Myeloma-Induced Alterations of Bone Remodeling

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    Fabrizio Accardi

    2015-01-01

    Full Text Available Multiple myeloma (MM is characterized by a high capacity to induce alterations in the bone remodeling process. The increase in osteoclastogenesis and the suppression of osteoblast formation are both involved in the pathophysiology of the bone lesions in MM. The proteasome inhibitor (PI bortezomib is the first drug designed and approved for the treatment of MM patients by targeting the proteasome. However, recently novel PIs have been developed to overcome bortezomib resistance. Interestingly, several preclinical data indicate that the proteasome complex is involved in both osteoclast and osteoblast formation. It is also evident that bortezomib either inhibits osteoclast differentiation induced by the receptor activator of nuclear factor kappa B (NF-κB ligand (RANKL or stimulates the osteoblast differentiation. Similarly, the new PIs including carfilzomib and ixazomib can inhibit bone resorption and stimulate the osteoblast differentiation. In a clinical setting, PIs restore the abnormal bone remodeling by normalizing the levels of bone turnover markers. In addition, a bone anabolic effect was described in responding MM patients treated with PIs, as demonstrated by the increase in the osteoblast number. This review summarizes the preclinical and clinical evidence on the effects of bortezomib and other new PIs on myeloma bone disease.

  15. Assessing Subunit Dependency of the Plasmodium Proteasome Using Small Molecule Inhibitors and Active Site Probes

    NARCIS (Netherlands)

    Li, H.; Linden, W.A. van der; Verdoes, M.; Florea, B.I.; McAllister, F.E.; Govindaswamy, K.; Elias, J.E.; Bhanot, P.; Overkleeft, H.S.; Bogyo, M.

    2014-01-01

    The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to

  16. Proteasome inhibitors block DNA repair and radiosensitize non-small cell lung cancer.

    Directory of Open Access Journals (Sweden)

    Kyle R Cron

    Full Text Available Despite optimal radiation therapy (RT, chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80-90% decrease in homologous recombination (HR, a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.

  17. MicroRNA-132 protects hippocampal neurons against oxygen-glucose deprivation-induced apoptosis.

    Science.gov (United States)

    Sun, Zu-Zhen; Lv, Zhan-Yun; Tian, Wen-Jing; Yang, Yan

    2017-09-01

    Hypoxic-ischemic brain injury (HIBI) results in death or long-term neurologic impairment in both adults and children. In this study, we investigated the effects of microRNA-132 (miR-132) dysregulation on oxygen-glucose deprivation (OGD)-induced apoptosis in fetal rat hippocampal neurons, in order to reveal the therapeutic potential of miR-132 on HIBI. MiR-132 dysregulation was induced prior to OGD exposure by transfection of primary fetal rat hippocampal neurons with miR-132 mimic or miR-132 inhibitor. The effects of miR-132 overexpression and suppression on OGD-stimulated hippocampal neurons were evaluated by detection of cell viability, apoptotic cells rate, and the expression of apoptosis-related proteins. Besides, TargetScan database and dual luciferase activity assay were used to seek a target gene of miR-132. As a result, miR-132 was highly expressed in hippocampal neurons following 2 h of OGD exposure. MiR-132 overexpression significantly increased OGD-diminished cell viability and reduced OGD-induced apoptosis at 12, 24, and 48 h post-OGD. MiR-132 overexpression significantly down-regulated the expressions of Bax, cytochrome c, and caspase-9, but up-regulated BCl-2. Caspase-3 activity was also significantly decreased by miR-132 overexpression. Furthermore, FOXO3 was a direct target of miR-132, and it was negatively regulated by miR-132. To conclude, our results provide evidence that miR-132 protects hippocampal neurons against OGD injury by inhibiting apoptosis.

  18. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

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    Yunwon Moon

    Full Text Available This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2 and severe hypoxia (0.1% O2. We found that chenodeoxy cholic acid (CDCA reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR, a CDCA receptor and its target gene, Small heterodimer partner (SHP are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

  19. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

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    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.

  20. BAG3-dependent noncanonical autophagy induced by proteasome inhibition in HepG2 cells.

    Science.gov (United States)

    Liu, Bao-Qin; Du, Zhen-Xian; Zong, Zhi-Hong; Li, Chao; Li, Ning; Zhang, Qiang; Kong, De-Hui; Wang, Hua-Qin

    2013-06-01

    Emerging lines of evidence have shown that blockade of ubiquitin-proteasome system (UPS) activates autophagy. The molecular players that regulate the relationship between them remain to be elucidated. Bcl-2 associated athanogene 3 (BAG3) is a member of the BAG co-chaperone family that regulates the ATPase activity of heat shock protein 70 (HSP70) chaperone family. Studies on BAG3 have demonstrated that it plays multiple roles in physiological and pathological processes, including antiapoptotic activity, signal transduction, regulatory role in virus infection, cell adhesion and migration. Recent studies have attracted much attention on its role in initiation of autophagy. The current study, for the first time, demonstrates that proteasome inhibitors elicit noncanonical autophagy, which was not suppressed by inhibitors of class III phosphatidylinositol 3-kinase (PtdIns3K) or shRNA against Beclin 1 (BECN1). In addition, we demonstrate that BAG3 is ascribed to activation of autophagy elicited by proteasome inhibitors and MAPK8/9/10 (also known as JNK1/2/3 respectively) activation is also implicated via upregulation of BAG3. Moreover, we found that noncanonical autophagy mediated by BAG3 suppresses responsiveness of HepG2 cells to proteasome inhibitors.

  1. Calcitonin Gene-Related Peptide Induces HIV-1 Proteasomal Degradation in Mucosal Langerhans Cells.

    Science.gov (United States)

    Bomsel, Morgane; Ganor, Yonatan

    2017-12-01

    The neuroimmune dialogue between peripheral neurons and Langerhans cells (LCs) within mucosal epithelia protects against incoming pathogens. LCs rapidly internalize human immunodeficiency virus type 1 (HIV-1) upon its sexual transmission and then trans -infect CD4 + T cells. We recently found that the neuropeptide calcitonin gene-related peptide (CGRP), secreted mucosally from peripheral neurons, inhibits LC-mediated HIV-1 trans -infection. In this study, we investigated the mechanism of CGRP-induced inhibition, focusing on HIV-1 degradation in LCs and its interplay with trans -infection. We first show that HIV-1 degradation occurs in endolysosomes in untreated LCs, and functionally blocking such degradation with lysosomotropic agents results in increased trans -infection. We demonstrate that CGRP acts via its cognate receptor and at a viral postentry step to induce faster HIV-1 degradation, but without affecting the kinetics of endolysosomal degradation. We reveal that unexpectedly, CGRP shifts HIV-1 degradation from endolysosomes toward the proteasome, providing the first evidence for functional HIV-1 proteasomal degradation in LCs. Such efficient proteasomal degradation significantly inhibits the first phase of trans -infection, and proteasomal, but not endolysosomal, inhibitors abrogate CGRP-induced inhibition. Together, our results establish that CGRP controls the HIV-1 degradation mode in LCs. The presence of endogenous CGRP within innervated mucosal tissues, especially during the sexual response, to which CGRP contributes, suggests that HIV-1 proteasomal degradation predominates in vivo Hence, proteasomal, rather than endolysosomal, HIV-1 degradation in LCs should be enhanced clinically to effectively restrict HIV-1 trans -infection. IMPORTANCE During sexual transmission, HIV-1 is internalized and degraded in LCs, the resident antigen-presenting cells in mucosal epithelia. Yet during trans -infection, infectious virions escaping degradation are transferred

  2. Paclitaxel-induced apoptosis is BAK-dependent, but BAX and BIM-independent in breast tumor.

    Directory of Open Access Journals (Sweden)

    Anna V Miller

    Full Text Available Paclitaxel (Taxol-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim(-/- MEFs (mouse embryonic fibroblasts, the bim(-/- mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak (-/- MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax(-/- MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.

  3. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    International Nuclear Information System (INIS)

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-01-01

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway

  4. The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

    Science.gov (United States)

    Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

    2013-11-22

    The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

  5. High glucose induces activation of NF-κB inflammatory signaling through IκBα sumoylation in rat mesangial cells

    International Nuclear Information System (INIS)

    Huang, Wei; Xu, Ling; Zhou, Xueqin; Gao, Chenlin; Yang, Maojun; Chen, Guo; Zhu, Jianhua; Jiang, Lan; Gan, Huakui; Gou, Fang; Feng, Hong; Peng, Juan; Xu, Yong

    2013-01-01

    Highlights: •The expression of SUMO1, SUMO2/3 under high glucose was obviously enhanced. •High glucose induced degradation of IκBα and activation of NF-κB pathway. •Sumoylation of IκBα in high glucose were significantly decreased. •The proteasome inhibitor MG132 could partially revert the degradation of IκBα. -- Abstract: The posttranslational modification of proteins by small ubiquitin-like modifiers (SUMOs) has emerged as an important regulatory mechanism for the alteration of protein activity, stability, and cellular localization. The latest research demonstrates that sumoylation is extensively involved in the regulation of the nuclear factor κB (NF-κB) pathway, which plays a critical role in the regulation of inflammation and contributes to fibrosis in diabetic nephropathy (DN). However, the role of sumoylation in the regulation of NF-κB signaling in DN is still unclear. In the present study, we cultured rat glomerular mesangial cells (GMCs) stimulated by high glucose and divided GMCs into six groups: normal glucose group (5.6 mmol/L), high glucose groups (10, 20, and 30 mmol/L), mannitol group (i.e., osmotic control group), and MG132 intervention group (30 mmol/L glucose with MG132, a proteasome inhibitor). The expression of SUMO1, SUMO2/3, IκBα, NF-κBp65, and monocyte chemotactic protein 1 (MCP-1) was measured by Western blot, reverse-transcription polymerase chain reaction, and indirect immunofluorescence laser scanning confocal microscopy. The interaction between SUMO1, SUMO2/3, and IκBα was observed by co-immunoprecipitation. The results showed that the expression of SUMO1 and SUMO2/3 was dose- and time-dependently enhanced by high glucose (p < 0.05). However, the expression of IκBα sumoylation in high glucose was significantly decreased compared with the normal glucose group (p < 0.05). The expression of IκBα was dose- and time-dependently decreased, and NF-κBp65 and MCP-1 were increased under high glucose conditions, which

  6. 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.

  7. On the role of proteasomes in cell biology and proteasome inhibition as a novel frontier in the development of immunosuppressants.

    Science.gov (United States)

    Wu, Jiangping

    2002-11-01

    The proteasome, a large protease complex in cells, is the major machinery for protein degradation. It was previously considered a humble garbage collector, performing housekeeping duties to remove misfolded or spent proteins. Until recently, the interests of immunologists in proteasomes were focused largely on its role in antigen processing. Its real importance in cell biology has only been revealed contemporarily due to the availability of relatively specific inhibitors. It has now become increasingly clear that many aspects of immune responses highly depend on proper proteasome activity. Recently, a proteasome inhibitor has been successfully used to prevent acute as well as ongoing heart allograft rejection in mice. Such inhibitors are also efficacious in treating several autoimmune diseases, such as arthritis, psoriasis, and probably type I diabetes, in animal models. Phase II and III clinical trials of proteasome inhibitors in treating various tumors have shown promising results, and the side-effects of these drugs are tolerable. Therefore, proteasome inhibition represents a new and promising frontier in immunosuppressant development.

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

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    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.

  9. Design, synthesis and docking studies of novel dipeptidyl boronic acid proteasome inhibitors constructed from αα- and αβ-amino acids.

    Science.gov (United States)

    Shi, Jingmiao; Lei, Meng; Wu, Wenkui; Feng, Huayun; Wang, Jia; Chen, Shanshan; Zhu, Yongqiang; Hu, Shihe; Liu, Zhaogang; Jiang, Cheng

    2016-04-15

    A series of novel dipeptidyl boronic acid proteasome inhibitors constructed from αα- and αβ-amino acids were designed and synthesized. Their structures were elucidated by (1)H NMR, (13)C NMR, LC-MS and HRMS. These compounds were evaluated for their β5 subunit inhibitory activities of human proteasome. The results showed that dipeptidyl boronic acid inhibitors composed of αα-amino acids were as active as bortezomib. Interestingly, the activities of those derived from αβ-amino acids lost completely. Of all the inhibitors, compound 22 (IC50=4.82 nM) was the most potent for the inhibition of proteasome activity. Compound 22 was also the most active against three MM cell lines with IC50 values less than 5 nM in inhibiting cell growth assays. Molecular docking studies displayed that 22 fitted very well in the β5 subunit active pocket of proteasome. Copyright © 2016. Published by Elsevier Ltd.

  10. BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: implications for a proteasome-to-autophagy switch.

    Science.gov (United States)

    Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F; Brunsting, Jeanette F; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H; Carra, Serena

    2014-09-01

    Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the "BAG-instructed proteasomal to autophagosomal switch and sorting" (BIPASS).

  11. Targeting proteasomes in infectious organisms to combat disease.

    Science.gov (United States)

    Bibo-Verdugo, Betsaida; Jiang, Zhenze; Caffrey, Conor R; O'Donoghue, Anthony J

    2017-05-01

    Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents. © 2017 Federation of European Biochemical Societies.

  12. Proteasome inhibitors activate autophagy involving inhibition of PI3K-Akt-mTOR pathway as an anti-oxidation defense in human RPE cells.

    Directory of Open Access Journals (Sweden)

    Bingrong Tang

    Full Text Available The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE, ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA or epoxomicin (Epo, at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3 from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf. Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt, a downstream target of phosphatidylinositol-3-kinases (PI3K, and mammalian target of rapamycin (mTOR in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3 or 4-hydroxynonenal (4-HNE. Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.

  13. Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38MAPK, and JNK Kinase Pathways

    Directory of Open Access Journals (Sweden)

    Nicoletta Marchesi

    2018-01-01

    Full Text Available RNA-binding protein dysregulation and altered expression of proteins involved in the autophagy/proteasome pathway play a role in many neurodegenerative disease onset/progression, including age-related macular degeneration (AMD. HuR/ELAVL1 is a master regulator of gene expression in human physiopathology. In ARPE-19 cells exposed to the proteasomal inhibitor MG132, HuR positively affects at posttranscriptional level p62 expression, a stress response gene involved in protein aggregate clearance with a role in AMD. Here, we studied the early effects of the proautophagy AICAR + MG132 cotreatment on the HuR-p62 pathway. We treated ARPE-19 cells with Erk1/2, AMPK, p38MAPK, PKC, and JNK kinase inhibitors in the presence of AICAR + MG132 and evaluated HuR localization/phosphorylation and p62 expression. Two-hour AICAR + MG132 induces both HuR cytoplasmic translocation and threonine phosphorylation via the Erk1/2 pathway. In these conditions, p62 mRNA is loaded on polysomes and its translation in de novo protein is favored. Additionally, for the first time, we report that JNK can phosphorylate HuR, however, without modulating its localization. Our study supports HuR’s role as an upstream regulator of p62 expression in ARPE-19 cells, helps to understand better the early events in response to a proautophagy stimulus, and suggests that modulation of the autophagy-regulating kinases as potential therapeutic targets for AMD may be relevant.

  14. 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.

  15. Scopadulciol, Isolated from Scoparia dulcis, Induces β-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells.

    Science.gov (United States)

    Fuentes, Rolly G; Toume, Kazufumi; Arai, Midori A; Sadhu, Samir K; Ahmed, Firoj; Ishibashi, Masami

    2015-04-24

    Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased β-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of β-catenin induced by 1. The 1-induced degradation of β-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of β-catenin, which resulted in the inhibition of TCF/β-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

  16. 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.

  17. Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors

    NARCIS (Netherlands)

    Hiroi, Toyoko; Deming, Clayton B.; Zhao, Haige; Hansen, Baranda S.; Arkenbout, Elisabeth K.; Myers, Thomas J.; McDevitt, Michael A.; Rade, Jeffrey J.

    2009-01-01

    OBJECTIVE: Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathological thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We

  18. Endoplasmic reticulum stress as a novel mechanism in amiodarone-induced destructive thyroiditis.

    Science.gov (United States)

    Lombardi, Angela; Inabnet, William Barlow; Owen, Randall; Farenholtz, Kaitlyn Ellen; Tomer, Yaron

    2015-01-01

    Amiodarone (AMIO) is one of the most effective antiarrhythmic drugs available; however, its use is limited by a serious side effect profile, including thyroiditis. The mechanisms underlying AMIO thyroid toxicity have been elusive; thus, identification of novel approaches in order to prevent thyroiditis is essential in patients treated with AMIO. Our aim was to evaluate whether AMIO treatment could induce endoplasmic reticulum (ER) stress in human thyroid cells and the possible implications of this effect in AMIO-induced destructive thyroiditis. Here we report that AMIO, but not iodine, significantly induced the expression of ER stress markers including Ig heavy chain-binding protein (BiP), phosphoeukaryotic translation initiation factor 2α (eIF2α), CCAAT/enhancer-binding protein homologous protein (CHOP) and spliced X-box binding protein-1 (XBP-1) in human thyroid ML-1 cells and human primary thyrocytes. In both experimental systems AMIO down-regulated thyroglobulin (Tg) protein but had little effect on Tg mRNA levels, suggesting a mechanism involving Tg protein degradation. Indeed, pretreatment with the specific proteasome inhibitor MG132 reversed AMIO-induced down-regulation of Tg protein levels, confirming a proteasome-dependent degradation of Tg protein. Corroborating our findings, pretreatment of ML-1 cells and human primary thyrocytes with the chemical chaperone 4-phenylbutyric acid completely prevented the effect of AMIO on both ER stress induction and Tg down-regulation. We identified ER stress as a novel mechanism contributing to AMIO-induced destructive thyroiditis. Our data establish that AMIO-induced ER stress impairs Tg expression via proteasome activation, providing a valuable therapeutic avenue for the treatment of AMIO-induced destructive thyroiditis.

  19. Oxidative Stress Posttranslationally Regulates the Expression of Ha-Ras and Ki-Ras in Cultured Astrocytes

    Directory of Open Access Journals (Sweden)

    Samantha Messina

    2012-01-01

    Full Text Available Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.

  20. Synergistic efficacy in human ovarian cancer cells by histone deacetylase inhibitor TSA and proteasome inhibitor PS-341.

    Science.gov (United States)

    Fang, Yong; Hu, Yi; Wu, Peng; Wang, Beibei; Tian, Yuan; Xia, Xi; Zhang, Qinghua; Chen, Tong; Jiang, Xuefeng; Ma, Quanfu; Xu, Gang; Wang, Shixuan; Zhou, Jianfeng; Ma, Ding; Meng, Li

    2011-05-01

    Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.

  1. BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta

    Science.gov (United States)

    Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F; Brunsting, Jeanette F; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H; Carra, Serena

    2014-01-01

    Eukaryotic cells use autophagy and the ubiquitin–proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested to be involved in this switch. However, at present it is still unknown whether and to what extent BAG3 can indeed reroute proteasomal clients to the autophagosomal pathway. Here, we show that BAG3 induces the sequestration of ubiquitinated clients into cytoplasmic puncta colabeled with canonical autophagy linkers and markers. Following proteasome inhibition, BAG3 upregulation significantly contributes to the compensatory activation of autophagy and to the degradation of the (poly)ubiquitinated proteins. BAG3 binding to the ubiquitinated clients occurs through the BAG domain, in competition with BAG1, another BAG family member, that normally directs ubiquitinated clients to the proteasome. Therefore, we propose that following proteasome impairment, increasing the BAG3/BAG1 ratio ensures the “BAG-instructed proteasomal to autophagosomal switch and sorting” (BIPASS). PMID:25046115

  2. Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

    Science.gov (United States)

    Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

    2013-09-01

    Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

  3. Degradation of Mcl-1 through GSK-3β Activation Regulates Apoptosis Induced by Bufalin in Non-Small Cell Lung Cancer H1975 Cells

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    Xiao-hong Kang

    2017-04-01

    Full Text Available Background/Aims: Mcl-1, an anti-apoptotic Bcl-2 family member, is often overexpressed in non-small cell lung cancer (NSCLC. Bufalin has been reported to induce apoptosis in various tumor cells. However, there is no report showing that bufalin could downregulate Mcl-1 expression in NSCLC. Methods: Cell proliferation was analyzed by cell counting kit-8 (CCK-8 assay in H1975 cells. Cell apoptosis was detected by flow cytometry. Mcl-1 mRNA was detected by RT-PCR. The expression of apoptosis-associated proteins in H1975 cells was detected by western blotting. The levels of Mcl-1 ubiquitination and NOXA were analyzed by Immunoprecipitation assay. Results: Cell growth was inhibited by bufalin in a time and dose-dependent manner. Bufalin induced apoptosis in NSCLC cells by activating caspase cascades and downregulating Mcl-1 expression. However, overexpression of Mcl-1 diminished bufalin-induced apoptosis. Furthermore, bufalin did not reduce Mcl-1 mRNA expression in H1975 cells, but strongly promoted Mcl-1 protein degradation. Proteasome inhibitor MG132 markedly prevented the degradation of Mcl-1 and blocked bufalin-induced Mcl-1 reduction. Bufalin did not significantly affect NOXA protein levels, but downregulated the expression of p-GSK-3β. GSK-3 inhibitor and GSK-3β siRNA resulted in increased levels of Mcl-1 and reversed the bufalin-induced Mcl-1 degradation. Conclusion: Bufalin induced cell apoptosis in H1975 cells may be through downregulation of Mcl-1. Proteasomal degradation of Mcl-1 via GSK-3β activation was involved in bufalin-induced apoptosis.

  4. Prospective iterative trial of proteasome inhibitor-based desensitization.

    Science.gov (United States)

    Woodle, E S; Shields, A R; Ejaz, N S; Sadaka, B; Girnita, A; Walsh, R C; Alloway, R R; Brailey, P; Cardi, M A; Abu Jawdeh, B G; Roy-Chaudhury, P; Govil, A; Mogilishetty, G

    2015-01-01

    A prospective iterative trial of proteasome inhibitor (PI)-based therapy for reducing HLA antibody (Ab) levels was conducted in five phases differing in bortezomib dosing density and plasmapheresis timing. Phases included 1 or 2 bortezomib cycles (1.3 mg/m(2) × 6-8 doses), one rituximab dose and plasmapheresis. HLA Abs were measured by solid phase and flow cytometry (FCM) assays. Immunodominant Ab (iAb) was defined as highest HLA Ab level. Forty-four patients received 52 desensitization courses (7 patients enrolled in multiple phases): Phase 1 (n = 20), Phase 2 (n = 12), Phase 3 (n = 10), Phase 4 (n = 5), Phase 5 (n = 5). iAb reductions were observed in 38 of 44 (86%) patients and persisted up to 10 months. In Phase 1, a 51.5% iAb reduction was observed at 28 days with bortezomib alone. iAb reductions increased with higher bortezomib dosing densities and included class I, II, and public antigens (HLA DRβ3, HLA DRβ4 and HLA DRβ5). FCM median channel shifts decreased in 11/11 (100%) patients by a mean of 103 ± 54 mean channel shifts (log scale). Nineteen out of 44 patients (43.2%) were transplanted with low acute rejection rates (18.8%) and de novo DSA formation (12.5%). In conclusion, PI-based desensitization consistently and durably reduces HLA Ab levels providing an alternative to intravenous immune globulin-based desensitization. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

  5. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  6. Angiotensin II Regulates Th1 T Cell Differentiation Through Angiotensin II Type 1 Receptor-PKA-Mediated Activation of Proteasome.

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    Qin, Xian-Yun; Zhang, Yun-Long; Chi, Ya-Fei; Yan, Bo; Zeng, Xiang-Jun; Li, Hui-Hua; Liu, Ying

    2018-01-01

    Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (β1, β1i, β2i and β5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.

  7. Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes.

    Science.gov (United States)

    Sutovsky, Peter; Manandhar, Gaurishankar; Laurincik, Jozef; Letko, Juraj; Caamaño, Jose Nestor; Day, Billy N; Lai, Liangxue; Prather, Randall S; Sharpe-Timms, Kathy L; Zimmer, Randall; Sutovsky, Miriam

    2005-03-01

    Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.

  8. 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

  9. Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo.

    Science.gov (United States)

    Schnerch, Dominik; Schüler, Julia; Follo, Marie; Felthaus, Julia; Wider, Dagmar; Klingner, Kathrin; Greil, Christine; Duyster, Justus; Engelhardt, Monika; Wäsch, Ralph

    2017-03-28

    Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

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

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    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.

  11. Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB.

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    Yao Dai

    Full Text Available Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1, with IC₅₀ in the range of 1-2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be

  12. Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

    Science.gov (United States)

    Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

    2013-01-01

    In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

  13. 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

  14. SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

    International Nuclear Information System (INIS)

    Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

    2012-01-01

    Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

  15. VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage.

    Science.gov (United States)

    Monsalve, Diana M; Campillo-Marcos, Ignacio; Salzano, Marcella; Sanz-García, Marta; Cantarero, Lara; Lazo, Pedro A

    2016-04-01

    NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1alpha/2alpha is mediated through chelation of iron.

    Science.gov (United States)

    Park, Sung-Soo; Bae, Insoo; Lee, Yong J

    2008-04-15

    Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 that is the key regulator of cellular response to low oxygen tension. Under normoxic conditions, HIF-1alpha is continuously degraded by the ubiquitin-proteasome pathway through pVHL (von Hippel-Lindau tumor suppressor protein). Under hypoxic conditions, HIF-1alpha is stabilized and induces the transcription of HIF-1 target genes. Quercetin, a flavonoid with anti-oxidant, anti-inflammatory, and kinase modulating properties, has been found to induce HIF-1alpha accumulation and VEGF secretion in normoxia. In this study, the molecular mechanisms of quercetin-mediated HIF-1alpha accumulation were investigated. Previous studies have shown that, in addition to being induced by hypoxia, HIF-1alpha can be induced through the phosphatidylinositol 3-kinase (PI3K)/Akt and p53 signaling pathways. But our study revealed, through p53 mutant-type as well as p53 null cell lines, that neither the PI3K/Akt nor the p53 signaling pathway is required for quercetin-induced HIF-1alpha accumulation. And we observed that HIF-1alpha accumulated by quercetin is not ubiquitinated and the interaction of HIF-1alpha with pVHL is reduced, compared with HIF-1alpha accumulated by the proteasome inhibitor MG132. The use of quercetin's analogues showed that only quercetin and galangin induce HIF-1/2alpha accumulation and this effect is completely reversed by additional iron ions. This is because quercetin and galangin are able to chelate cellular iron ions that are cofactors of HIF-1/2alpha proline hydroxylase (PHD). These data suggest that quercetin inhibits the ubiquitination of HIF-1/2alpha in normoxia by hindering PHD through chelating iron ions.

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

    International Nuclear Information System (INIS)

    Honma, Yuichi; Harada, Masaru

    2013-01-01

    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

  18. Cytokine-induced oxidative stress in cardiac inflammation and heart failure – how the ubiquitin proteasome system targets this vicious cycle

    Directory of Open Access Journals (Sweden)

    Antje eVoigt

    2013-03-01

    Full Text Available The ubiquitin proteasome system (UPS is critical for the regulation of many intracellular processes necessary for cell function and survival. The absolute requirement of the UPS for the maintenance of protein homeostasis and thereby for the regulation of protein quality control is reflected by the fact that deviation of proteasome function from the norm was reported in cardiovascular pathologies. Inflammation is a major factor contributing to cardiac pathology. Herein, cytokines induce protein translation and the production of free radicals, thereby challenging the cellular protein equilibrium. Here, we discuss current knowledge on the mechanisms of UPS-functional adaptation in response to oxidative stress in cardiac inflammation. The increasing pool of oxidant-damaged degradation-prone proteins in cardiac pathology accounts for the need for enhanced protein turnover by the UPS. This process is accomplished by an up-regulation of the ubiquitylation machinery and the induction of immunoproteasomes. Thereby, the inflamed heart muscle is cleared from accumulating misfolded proteins. Current advances on immunoproteasome-specific inhibitors in this field question the impact of the proteasome as a therapeutic target in heart failure.

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

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    Jitesh D Kawedia

    Full Text Available 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.

  20. 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. PMID:23469202

  1. Suppression of 19S proteasome subunits marks emergence of an altered cell state in diverse cancers.

    Science.gov (United States)

    Tsvetkov, Peter; Sokol, Ethan; Jin, Dexter; Brune, Zarina; Thiru, Prathapan; Ghandi, Mahmoud; Garraway, Levi A; Gupta, Piyush B; Santagata, Sandro; Whitesell, Luke; Lindquist, Susan

    2017-01-10

    The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.

  2. p53 mutations promote proteasomal activity.

    Science.gov (United States)

    Oren, Moshe; Kotler, Eran

    2016-07-27

    p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.

  3. D-Glucosamine down-regulates HIF-1{alpha} through inhibition of protein translation in DU145 prostate cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jee-Young; Park, Jong-Wook; Suh, Seong-Il [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of); Baek, Won-Ki, E-mail: wonki@dsmc.or.kr [Chronic Disease Research Center, School of Medicine, Keimyung University, 194 Dongsan-Dong, Jung-Gu, Daegu 700-712 (Korea, Republic of)

    2009-04-24

    D-Glucosamine has been reported to inhibit proliferation of cancer cells in culture and in vivo. In this study we report a novel response to D-glucosamine involving the translation regulation of hypoxia inducible factor (HIF)-1{alpha} expression. D-Glucosamine caused a decreased expression of HIF-1{alpha} under normoxic and hypoxic conditions without affecting HIF-1{alpha} mRNA expression in DU145 prostate cancer cells. D-Glucosamine inhibited HIF-1{alpha} accumulation induced by proteasome inhibitor MG132 and prolyl hydroxylase inhibitor DMOG suggesting D-glucosamine reduces HIF-1{alpha} protein expression through proteasome-independent pathway. Metabolic labeling assays indicated that D-glucosamine inhibits translation of HIF-1{alpha} protein. In addition, D-glucosamine inhibited HIF-1{alpha} expression induced by serum stimulation in parallel with inhibition of p70S6K suggesting D-glucosamine inhibits growth factor-induced HIF-1{alpha} expression, at least in part, through p70S6K inhibition. Taken together, these results suggest that D-glucosamine inhibits HIF-1{alpha} expression through inhibiting protein translation and provide new insight into a potential mechanism of the anticancer properties of D-glucosamine.

  4. Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells.

    Science.gov (United States)

    Dron, Michel; Dandoy-Dron, Françoise; Farooq Salamat, Muhammad Khalid; Laude, Hubert

    2009-08-01

    Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrP(C)) and abnormal (PrP(Sc)) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrP(C), leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP(26K), accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP(26K) species converts into the highly proteinase K-resistant PrP(Sc). In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrP(Sc) that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrP(C) from the natural promoter, proteasomal impairment may affect both the process of PrP(C) biosynthesis and the subcellular sites of PrP(Sc) accumulation, despite the fact that these two effects could essentially be disconnected.

  5. Up-stream events in the nuclear factor κB activation cascade in response to sparsely ionizing radiation

    Science.gov (United States)

    Hellweg, Christine E.; Langen, Britta; Klimow, Galina; Ruscher, Roland; Schmitz, Claudia; Baumstark-Khan, Christa; Reitz, Günther

    2009-10-01

    Radiation is a potentially limiting factor for manned long-term space missions. Prolonged exposure to galactic cosmic rays may shorten the healthy life-span after return to Earth due to cancer induction. During the mission, a solar flare can be life threatening. For better risk estimation and development of appropriate countermeasures, the study of the cellular radiation response is necessary. Since apoptosis may be a mechanism the body uses to eliminate damaged cells, the induction by cosmic radiation of the nuclear anti-apoptotic transcription factor nuclear factor κB (NF-κB) could influence the cancer risk of astronauts exposed to cosmic radiation by improving the survival of radiation-damaged cells. In previous studies using a screening assay for the detection of NF-κB-dependent gene induction (HEK-pNF-κB-d2EGFP/Neo cells), the activation of this transcription factor by heavy ions was shown [Baumstark-Khan, C., Hellweg, C.E., Arenz, A., Meier, M.M. Cellular monitoring of the nuclear factor kappa B pathway for assessment of space environmental radiation. Radiat. Res. 164, 527-530, 2005]. Studies with NF-κB inhibitors can map functional details of the NF-κB pathway and the influence of radiation-induced NF-κB activation on various cellular outcomes such as survival or cell cycle arrest. In this work, the efficacy and cytotoxicity of four different NF-κB inhibitors, caffeic acid phenethyl ester (CAPE), capsaicin, the proteasome inhibitor MG-132, and the cell permeable peptide NF-κB SN50 were analyzed using HEK-pNF-κB-d2EGFP/Neo cells. In the recommended concentration range, only CAPE displayed considerable cytotoxicity. CAPE and capsaicin partially inhibited NF-κB activation by the cytokine tumor necrosis factor α. MG-132 completely abolished the activation and was therefore used for experiments with X-rays. NF-κB SN-50 could not reduce NF-κB dependent expression of the reporter destabilized Enhanced Green Fluorescent Protein (d2EGFP). MG-132

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

    Science.gov (United States)

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

    2017-05-01

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

  7. Phase I Trial Using the Proteasome Inhibitor Bortezomib and Concurrent Chemoradiotherapy for Head-and-Neck Malignancies

    Energy Technology Data Exchange (ETDEWEB)

    Kubicek, Gregory J. [Department of Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Department of Radiation Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA (United States); Axelrod, Rita S. [Department of Medical Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Machtay, Mitchell [Department of Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Department of Radiation Oncology, Case Western Reserve University, Cleveland, OH (United States); Ahn, Peter H.; Anne, Pramila R. [Department of Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Fogh, Shannon [Department of Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Department of Radiation Oncology, University of California, San Francisco, San Francisco, CA (United States); Cognetti, David [Department of Otolaryngology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Myers, Thomas J. [EMD Serono, Rockland, MA (United States); Curran, Walter J. [Department of Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States); Dicker, Adam P., E-mail: Adam.dicker@jeffersonhospital.org [Department of Radiation Oncology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA (United States)

    2012-07-15

    Purpose: Advanced head-and-neck cancer (HNC) remains a difficult disease to cure. Proteasome inhibitors such as bortezomib have the potential to improve survival over chemoradiotherapy alone. This Phase I dose-escalation study examined the potential of bortezomib in combination with cisplatin chemotherapy and concurrent radiation in the treatment of locally advanced and recurrent HNC. Methods and Materials: Eligible patients received cisplatin once weekly at 30 mg/m{sup 2} per week and bortezomib along with concurrent radiation. Bortezomib was given on Days 1, 4, 8, and 11 every 3 weeks, with an initial starting dose of 0.7 mg/m{sup 2} and escalation levels of 1.0 and 1.3 mg/m{sup 2}. Dose escalation was performed only after assessment to rule out any dose-limiting toxicity. Results: We enrolled 27 patients with HNC, including 17 patients with recurrent disease who had received prior irradiation. Patients received bortezomib dose levels of 0.7 mg/m{sup 2} (7 patients), 1.0 mg/m{sup 2} (10 patients), and 1.3 mg/m{sup 2} (10 patients). No Grade 5 toxicities, 3 Grade 4 toxicities (all hematologic and considered dose-limiting toxicities), and 39 Grade 3 toxicities (in 20 patients) were observed. With a median follow-up of 7.4 months, the overall median survival was 24.7 months (48.4 months for advanced HNC patients and 15.4 months for recurrent HNC patients). Conclusion: Bortezomib in combination with radiation therapy and cisplatin chemotherapy is safe in the treatment of HNC with a bortezomib maximum tolerated dose of 1.0 mg/m{sup 2} in patients previously treated for HNC and 1.3 mg/m{sup 2} in radiation-naive patients.

  8. Concurrent Inhibition of Pim and FLT3 Kinases Enhances Apoptosis of FLT3-ITD Acute Myeloid Leukemia Cells through Increased Mcl-1 Proteasomal Degradation.

    Science.gov (United States)

    Kapoor, Shivani; Natarajan, Karthika; Baldwin, Patrick R; Doshi, Kshama A; Lapidus, Rena G; Mathias, Trevor J; Scarpa, Mario; Trotta, Rossana; Davila, Eduardo; Kraus, Manfred; Huszar, Dennis; Tron, Adriana E; Perrotti, Danilo; Baer, Maria R

    2018-01-01

    Purpose: fms -like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor-induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G 1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234-47. ©2017 AACR . ©2017 American Association for Cancer Research.

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

    Science.gov (United States)

    Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

    2015-01-01

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

  10. SPR imaging biosensor for the 20S proteasome: Sensor development and application to measurement of proteasomes in human blood plasma

    International Nuclear Information System (INIS)

    Gorodkiewicz, E.; Sankiewicz, A.; Ostrowska, H.

    2011-01-01

    The 20S proteasome is a multicatalytic enzyme complex responsible for intracellular protein degradation in mammalian cells. Its antigen level or enzymatic activity in blood plasma are potentially useful markers for various malignant and nonmalignant diseases. We have developed a method for highly selective determination of the 20S proteasome using a Surface Plasmon Resonance Imaging (SPRI) technique. It is based on the highly selective interaction between the proteasome's catalytic β5 subunit and immobilized inhibitors (the synthetic peptide PSI and epoxomicin). Inhibitor concentration and pH were optimized. Analytical responses, linear ranges, accuracy, precision and interferences were investigated. Biosensors based on either PSI and epoxomicin were found to be suitable for quantitative determination of the proteasome, with a precision of ±10% for each, and recoveries of 102% and 113%, respectively, and with little interference by albumin, trypsin, chymotrypsin, cathepsin B and papain. The proteasome also was determined in plasma of healthy subjects and of patients suffering from acute leukemia. Both biosensors gave comparable results (2860 ng.mL -1 on average for control, and 42300 ng.mL -1 on average for leukemia patients). (author)

  11. 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.

  12. Insulin-like Growth Factor-I Mediates Neuroprotection in Proteasome Inhibition-Induced Cytotoxicity in SH-SY5Y Cells

    Science.gov (United States)

    Cheng, Benxu; Maffi, Shivani Kaushal; Martinez, Alex Anthony; Acosta, Yolanda P Villarreal; Morales, Liza D; Roberts, James L

    2011-01-01

    The proteasome is an enzyme complex responsible for targeted intracellular proteolysis. Alterations in proteasome-mediated protein clearance have been implicated in the pathogenesis of aging, Alzheimer's disease (AD) and Parkinson's disease (PD). In such diseases, proteasome inhibition may contribute to formation of abnormal protein aggregates, which in turn activate intracellular unfolded protein responses that cause oxidative stress and apoptosis. In this study, we investigated the protective effect of Insulin-like Growth Factor-I (IGF-1) for neural SH-SY5Y cells treated with the proteasomal inhibitor, Epoxomicin, In SH-SY5Y cells, Epoxomicin treatment results in accumulation of intracellular ubiquitinated proteins and cytochrome c release from damaged mitochondria, leading to cell death, in Epoxomicin time- and dose-dependent manner. In cells treated with small amounts of IGF-1, the same dosages of Epoxomicin reduced both mitochondrial damage (cytochrome c release) and reduced caspase-3 activation and PARP cleavage, both of which are markers of apoptosis. Notably, however, IGF-1-treated SH-SY5Y cells still contained ubiquitinated protein aggregates. This result indicates that IGF-1 blocks the downstream apoptotic consequences of Epoxomicin treatment leading to decreased proteasome function. Clues as to the mechanism for this protective effect come from (a) increased AKT phosphorylation observed in IGF-1-protected cells, vs. cells exposed to Epoxomicin without IGF-1, and (b) reduction of IGF-1 protection by pretreatment of the cells with LY294002 (an inhibitor of PI3-kinase). Together these findings suggest that activation of PI3/AKT pathways by IGF-1 is involved in IGF-1 neuroprotection against apoptosis following proteasome inhibition. PMID:21545837

  13. Proteasome Inhibitor YSY01A Abrogates Constitutive STAT3 Signaling via Down-regulation of Gp130 and JAK2 in Human A549 Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Wei Huang

    2017-08-01

    Full Text Available Proteasome inhibition interfering with many cell signaling pathways has been extensively explored as a therapeutic strategy for cancers. Proteasome inhibitor YSY01A is a novel agent that has shown remarkable anti-tumor effects; however, its mechanisms of action are not fully understood. Here we report that YSY01A is capable of suppressing cancer cell survival by induction of apoptosis. Paradoxically, we find that YSY01A abrogates constitutive activation of STAT3 via proteasome-independent degradation of gp130 and JAK2, but not transcriptional regulation, in human A549 non-small cell lung cancer cells. The reduction in gp130 and JAK2 can be restored by co-treatment with 3-methyladenine, an early-stage autophagy lysosome and type I/III PI3K inhibitor. YSY01A also effectively inhibits cancer cell migration and lung xenograft tumor growth with little adverse effect on animals. Thus, our findings suggest that YSY01A represents a promising candidate for further development of novel anticancer therapeutics targeting the proteasome.

  14. nNOS inhibitors attenuate methamphetamine-induced dopaminergic neurotoxicity but not hyperthermia in mice.

    Science.gov (United States)

    Itzhak, Y; Martin, J L; Ail, S F

    2000-09-11

    Methamphetamine (METH)-induced dopaminergic neurotoxicity is associated with hyperthermia. We investigated the effect of several neuronal nitric oxide synthase (nNOS) inhibitors on METH-induced hyperthermia and striatal dopaminergic neurotoxicity. Administration of METH (5 mg/kg; q. 3 h x 3) to Swiss Webster mice produced marked hyperthermia and 50-60% depletion of striatal dopaminergic markers 72 h after METH administration. Pretreatment with the nNOS inhibitors S-methylthiocitrulline (SMTC; 10 mg/kg) or 3-bromo-7-nitroindazole (3-Br-7-NI; 20 mg/kg) before each METH injection did not affect the persistent hyperthermia produced by METH, but afforded protection against the depletion of dopaminergic markers. A low dose (25 mg/kg) of the nNOS inhibitor 7-nitroindazole (7-NI) did not affect METH-induced hyperthermia, but a high dose (50 mg/kg) produced significant hypothermia. These findings indicate that low dose of selective nNOS inhibitors protect against METH-induced neurotoxicity with no effect on body temperature and support the hypothesis that nitric oxide (NO) and peroxynitrite have a major role in METH-induced dopaminergic neurotoxicity.

  15. 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.

  16. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance

    International Nuclear Information System (INIS)

    Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

    2013-01-01

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells

  17. Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance.

    Science.gov (United States)

    Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

    2013-05-10

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.

  18. The use of activity based protein profiling to study proteasome biology

    NARCIS (Netherlands)

    Paniagua Soriano, Guillem

    2016-01-01

    The work described in this thesis focuses on the characterization of proteasome directed activity-based probes (ABPs) as well as on the adaptation mechanisms that make multiple myeloma derived cell lines resistant against proteasome inhibitors (PIs).

  19. Gpn3 is polyubiquitinated on lysine 216 and degraded by the proteasome in the cell nucleus in a Gpn1-inhibitable manner.

    Science.gov (United States)

    Méndez-Hernández, Lucía E; Robledo-Rivera, Angelica Y; Macías-Silva, Marina; Calera, Mónica R; Sánchez-Olea, Roberto

    2017-11-01

    Gpn1 associates with Gpn3, and both are required for RNA polymerase II nuclear targeting. Global studies have identified by mass spectrometry that human Gpn3 is ubiquitinated on lysines 189 and 216. Our goals here were to determine the type, physiological importance, and regulation of Gpn3 ubiquitination. After inhibiting the proteasome with MG132, Gpn3-Flag was polyubiquitinated on K216, but not K189, in HEK293T cells. Gpn3-Flag exhibited nucleo-cytoplasmic shuttling, but polyubiquitination and proteasomal degradation of Gpn3-Flag occurred only in the cell nucleus. Polyubiquitination-deficient Gpn3-Flag K216R displayed a longer half-life than Gpn3-Flag in two cell lines. Interestingly, Gpn1-EYFP inhibited Gpn3-Flag polyubiquitination in a dose-dependent manner. In conclusion, Gpn1-inhibitable, nuclear polyubiquitination on lysine 216 regulates the half-life of Gpn3 by tagging it for proteasomal degradation. © 2017 Federation of European Biochemical Societies.

  20. Celecoxib promotes c-FLIP degradation through Akt-independent inhibition of GSK3.

    Science.gov (United States)

    Chen, Shuzhen; Cao, Wei; Yue, Ping; Hao, Chunhai; Khuri, Fadlo R; Sun, Shi-Yong

    2011-10-01

    Celecoxib is a COX-2 inhibitor that reduces the risk of colon cancer. However, the basis for its cancer chemopreventive activity is not fully understood. In this study, we defined a mechanism of celecoxib action based on degradation of cellular FLICE-inhibitory protein (c-FLIP), a major regulator of the death receptor pathway of apoptosis. c-FLIP protein levels are regulated by ubiquitination and proteasome-mediated degradation. We found that celecoxib controlled c-FLIP ubiquitination through Akt-independent inhibition of glycogen synthase kinase-3 (GSK3), itself a candidate therapeutic target of interest in colon cancer. Celecoxib increased the levels of phosphorylated GSK3, including the α and β forms, even in cell lines, where phosphorylated Akt levels were not increased. Phosphoinositide 3-kinase inhibitors abrogated Akt phosphorylation as expected but had no effect on celecoxib-induced GSK3 phosphorylation. In contrast, protein kinase C (PKC) inhibitors abolished celecoxib-induced GSK3 phosphorylation, implying that celecoxib influenced GSK3 phosphorylation through a mechanism that relied upon PKC and not Akt. GSK3 blockade either by siRNA or kinase inhibitors was sufficient to attenuate c-FLIP levels. Combining celecoxib with GSK3 inhibition enhanced attenuation of c-FLIP and increased apoptosis. Proteasome inhibitor MG132 reversed the effects of GSK3 inhibition and increased c-FLIP ubiquitination, confirming that c-FLIP attenuation was mediated by proteasomal turnover as expected. Our findings reveal a novel mechanism through which the regulatory effects of c-FLIP on death receptor signaling are controlled by GSK3, which celecoxib acts at an upstream level to control independently of Akt.

  1. Proteasome inhibitor carfilzomib interacts synergistically with histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells.

    Science.gov (United States)

    Gao, Minjie; Gao, Lu; Tao, Yi; Hou, Jun; Yang, Guang; Wu, Xiaosong; Xu, Hongwei; Tompkins, Van S; Han, Ying; Wu, Huiqun; Zhan, Fenghuang; Shi, Jumei

    2014-06-01

    In the present study, we investigated the interactions between proteasome inhibitor carfilzomib (CFZ) and histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells. Coexposure of cells to minimally lethal concentrations of CFZ with very low concentration of vorinostat resulted in synergistic antiproliferative effects and enhanced apoptosis in Jurkat T-leukemia cells, accompanied with the sharply increased reactive oxygen species (ROS), the striking decrease in the mitochondrial membrane potential (MMP), the increased release of cytochrome c, the enhanced activation of caspase-9 and -3, and the cleavage of PARP. The combined treatment of Jurkat cells pre-treated with ROS scavengers N-acetylcysteine (NAC) significantly blocked the loss of mitochondrial membrane potential, suggesting that ROS generation was a former event of the loss of mitochondrial membrane potential. Furthermore, NAC also resulted in a marked reduction in apoptotic cells, indicating a critical role for increased ROS generation by combined treatment. In addition, combined treatment arrested the cell cycle in G2-M phase. These results imply that CFZ interacted synergistically with vorinostat in Jurkat T-leukemia cells, which raised the possibility that the combination of carfilzomib with vorinostat may represent a novel strategy in treating T-cell Leukemia. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

  2. Phase 1 clinical trial of the novel proteasome inhibitor marizomib with the histone deacetylase inhibitor vorinostat in patients with melanoma, pancreatic and lung cancer based on in vitro assessments of the combination.

    Science.gov (United States)

    Millward, Michael; Price, Timothy; Townsend, Amanda; Sweeney, Christopher; Spencer, Andrew; Sukumaran, Shawgi; Longenecker, Angie; Lee, Lonnie; Lay, Ana; Sharma, Girish; Gemmill, Robert M; Drabkin, Harry A; Lloyd, G Kenneth; Neuteboom, Saskia T C; McConkey, David J; Palladino, Michael A; Spear, Matthew A

    2012-12-01

    Combining proteasome and histone deacetylase (HDAC) inhibition has been seen to provide synergistic anti-tumor activity, with complementary effects on a number of signaling pathways. The novel bi-cyclic structure of marizomib with its unique proteasome inhibition, toxicology and efficacy profiles, suggested utility in combining it with an HDAC inhibitor such as vorinostat. Thus, in this study in vitro studies assessed the potential utility of combining marizomib and vorinostat, followed by a clinical trial with the objectives of assessing the recommended phase 2 dose (RP2D), pharmacokinetics (PK), pharmacodynamics (PD), safety and preliminary anti-tumor activity of the combination in patients. Combinations of marizomib and vorinostat were assessed in vitro. Subsequently, in a Phase 1 clinical trial patients with melanoma, pancreatic carcinoma or Non-small Cell Lung Cancer (NSCLC) were given escalating doses of weekly marizomib in combination with vorinostat 300 mg daily for 16 days in 28 day cycles. In addition to standard safety studies, proteasome inhibition and pharmacokinetics were assayed. Marked synergy of marizomib and vorinostat was seen in tumor cell lines derived from patients with NSCLC, melanoma and pancreatic carcinoma. In the clinical trial, 22 patients were enrolled. Increased toxicity was not seen with the combination. Co-administration did not appear to affect the PK or PD of either drug in comparison to historical data. Although no responses were demonstrated using RECIST criteria, 61% of evaluable patients demonstrated stable disease with 39% having decreases in tumor measurements. Treatment of multiple tumor cell lines with marizomib and vorinostat resulted in a highly synergistic antitumor activity. The combination of full dose marizomib with vorinostat is tolerable in patients with safety findings consistent with either drug alone.

  3. BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta : Implications for a proteasome-to-autophagy switch

    NARCIS (Netherlands)

    Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F.; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena

    Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested

  4. BAG3 induces the sequestration of proteasomal clients into cytoplasmic puncta: implications for a proteasome-to-autophagy switch

    NARCIS (Netherlands)

    Minoia, Melania; Boncoraglio, Alessandra; Vinet, Jonathan; Morelli, Federica F.; Brunsting, Jeanette F.; Poletti, Angelo; Krom, Sabine; Reits, Eric; Kampinga, Harm H.; Carra, Serena

    2014-01-01

    Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Upon proteasomal impairment, cells switch to autophagy to ensure proper clearance of clients (the proteasome-to-autophagy switch). The HSPA8 and HSPA1A cochaperone BAG3 has been suggested

  5. Molecular basis for the regulation of hypoxia-inducible factor-1α levels by 2-deoxy-D-ribose.

    Science.gov (United States)

    Ikeda, Ryuji; Tabata, Sho; Tajitsu, Yusuke; Nishizawa, Yukihiko; Minami, Kentaro; Furukawa, Tatsuhiko; Yamamoto, Masatatsu; Shinsato, Yoshinari; Akiyama, Shin-Ichi; Yamada, Katsushi; Takeda, Yasuo

    2013-09-01

    The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.

  6. 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.

  7. 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.

  8. 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.

  9. Histone deacetylase inhibitor, Trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Charles Coombes R

    2006-02-01

    Full Text Available Abstract Background Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3β phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1 transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus. Results Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3β-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3β/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells. Conclusion We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3β-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

  10. Silibinin induces apoptosis via calpain-dependent AIF nuclear translocation in U87MG human glioma cell death

    Directory of Open Access Journals (Sweden)

    Kim Yong K

    2011-04-01

    Full Text Available Abstract Background Silibinin, a natural polyphenolic flavonoid, has been reported to induce cell death in various cancer cell types. However, the molecular mechanism is not clearly defined. Our previous study showed that silibinin induces glioma cell death and its effect was effectively prevented by calpain inhibitor. The present study was therefore undertaken to examine the role of calpain in the silibinin-induced glioma cell death. Methods U87MG cells were grown on well tissue culture plates and cell viability was measured by MTT assay. ROS generation and △ψm were estimated using the fluorescence dyes. PKC activation and Bax expression were measured by Western blot analysis. AIF nuclear translocation was determined by Western blot and immunocytochemistry. Results Silibinin induced activation of calpain, which was blocked by EGTA and the calpain inhibitor Z-Leu-Leu-CHO. Silibinin caused ROS generation and its effect was inhibited by calpain inhibitor, the general PKC inhibitor GF 109203X, the specific PKCδ inhibitor rottlerin, and catalase. Silibinin-induce cell death was blocked by calpain inhibitor and PKC inhibitors. Silibinin-induced PKCδ activation and disruption of △ψm were prevented by the calpain inhibitor. Silibinin induced AIF nuclear translocation and its effect was prevented by calpain inhibitor. Transfection of vector expressing microRNA of AIF prevented the silibinin-induced cell death. Conclusions Silibinin induces apoptotic cell death through a calpain-dependent mechanism involving PKC, ROS, and AIF nuclear translocation in U87MG human glioma cells.

  11. Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells.

    Science.gov (United States)

    Song, X; Kim, S-Y; Zhou, Z; Lagasse, E; Kwon, Y T; Lee, Y J

    2013-04-04

    Colorectal cancer is the third leading cause of cancer-related mortality in the world; the main cause of death of colorectal cancer is hepatic metastases, which can be treated with hyperthermia using isolated hepatic perfusion (IHP). In this study, we report that mild hyperthermia potently reduced cellular FLIP(long), (c-FLIP(L)), a major regulator of the death receptor (DR) pathway of apoptosis, thereby enhancing humanized anti-DR4 antibody mapatumumab (Mapa)-mediated mitochondria-independent apoptosis. We observed that overexpression of c-FLIP(L) in CX-1 cells abrogated the synergistic effect of Mapa and hyperthermia, whereas silencing of c-FLIP in CX-1 cells enhanced Mapa-induced apoptosis. Hyperthermia altered c-FLIP(L) protein stability without concomitant reductions in FLIP mRNA. Ubiquitination of c-FLIP(L) was increased by hyperthermia, and proteasome inhibitor MG132 prevented heat-induced downregulation of c-FLIP(L). These results suggest the involvement of the ubiquitin-proteasome system in this process. We also found lysine residue 195 (K195) to be essential for c-FLIP(L) ubiquitination and proteolysis, as mutant c-FLIP(L) lysine 195 arginine (arginine replacing lysine) was left virtually un-ubiquitinated and was refractory to hyperthermia-triggered degradation, and thus partially blocked the synergistic effect of Mapa and hyperthermia. Our observations reveal that hyperthermia transiently reduced c-FLIP(L) by proteolysis linked to K195 ubiquitination, which contributed to the synergistic effect between Mapa and hyperthermia. This study supports the application of hyperthermia combined with other regimens to treat colorectal hepatic metastases.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Gotoh, Eiko; Kawamata, Natsuko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Uchihara, Yoshie [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Iwanari, Hiroko [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sugiyama, Akira; Kawamura, Takeshi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032 (Japan); Mochizuki, Yasuhiro [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Tanaka, Toshiya [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sakai, Juro [Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Hamakubo, Takao [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); and others

    2015-10-02

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  14. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    International Nuclear Information System (INIS)

    Tachibana, Keisuke; Gotoh, Eiko; Kawamata, Natsuko; Ishimoto, Kenji; Uchihara, Yoshie; Iwanari, Hiroko; Sugiyama, Akira; Kawamura, Takeshi; Mochizuki, Yasuhiro; Tanaka, Toshiya; Sakai, Juro; Hamakubo, Takao; Kodama, Tatsuhiko

    2015-01-01

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

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

  16. Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation*

    Science.gov (United States)

    Choi, Seung-Won; Song, Jun-Kyu; Yim, Ye-Seal; Yun, Ho-Geun; Chun, Kyung-Hee

    2015-01-01

    Autophagy is a conserved process that contributes to cell homeostasis. It is well known that induction mainly occurs in response to nutrient starvation, such as starvation of amino acids and insulin, and its mechanisms have been extensively characterized. However, the mechanisms behind cellular glucose deprivation-induced autophagy are as of now poorly understood. In the present study, we determined a mechanism by which glucose deprivation induced the PKC-dependent proteasomal degradation of β-catenin, leading to autophagy. Glucose deprivation was shown to cause a sub-G1 transition and enhancement of the LC3-II protein levels, whereas β-catenin protein underwent degradation in a proteasome-dependent manner. Moreover, the inhibition of GSK3β was unable to abolish the glucose deprivation-mediated β-catenin degradation or up-regulation of LC3-II protein levels, which suggested GSK3β-independent protein degradation. Intriguingly, the inhibition of PKCα using a pharmacological inhibitor and transfection of siRNA for PKCα was observed to effectively block glucose deprivation-induced β-catenin degradation as well as the increase in LC3-II levels and the accumulation of a sub-G1 population. Together, our results demonstrated a molecular mechanism by which glucose deprivation can induce the GSK3β-independent protein degradation of β-catenin, leading to autophagy. PMID:25691573

  17. Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?

    Science.gov (United States)

    Caldeira, Margarida V; Salazar, Ivan L; Curcio, Michele; Canzoniero, Lorella M T; Duarte, Carlos B

    2014-01-01

    The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Defining Translational Reprogramming in Tuberous Sclerosis Complex

    Science.gov (United States)

    2016-07-01

    global protein synthesis.15 Pre- exposure of cells to a proline analog L-azetidine-2- car - boxylic acid (AZC) and a proteasome inhibitor MG132 markedly...mediated method for the efficient creation of large, synthetic RNAs. RNA 12, 2014– 2019 (2006). 28. Pelletier, J. & Sonenberg, N. Internal initiation of...luciferin (1mM, Regis Tech ) was added into the culture medium immediately after transfection. Luciferase activity was monitored and recorded using

  19. FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

    Science.gov (United States)

    Liu, Yanying; Qiao, Fangfang; Leiferman, Patricia C; Ross, Alan; Schlenker, Evelyn H; Wang, Hongmin

    2017-11-15

    Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Bortezomib partially improves laminin α2 chain-deficient muscular dystrophy.

    Science.gov (United States)

    Körner, Zandra; Fontes-Oliveira, Cibely C; Holmberg, Johan; Carmignac, Virginie; Durbeej, Madeleine

    2014-05-01

    Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. Overexpression of microRNA-132 enhances the radiosensitivity of cervical cancer cells by down-regulating Bmi-1.

    Science.gov (United States)

    Liu, Gui-Feng; Zhang, Shu-Hua; Li, Xue-Feng; Cao, Li-Yan; Fu, Zhan-Zhao; Yu, Shao-Nan

    2017-10-06

    We examined the effects of microRNA-132 (miR-132) on Bmi-1 expression and radiosensitivity in HeLa, SiHa, and C33A cervical cancer (CC) cells and 104 CC patients. MiR-132 expression was decreased and Bmi-1 expression was increased in tumor tissues compared to adjacent normal tissues and in radiotherapy-resistant patients compared to radiotherapy-sensitive patients. MiR-132 expression and Bmi-1 mRNA expression were also negatively correlated in tumor tissues. HeLa, SiHa, and C33A cells were divided into blank, miR-132 negative control (NC), miR-132 inhibitor, miR-132 mimics, siBmi-1, and miR-132 inhibitor + siBmi-1 groups, after which expression of miR-132 and Bmi-1, and the interaction between them and cell survival, proliferation, and apoptosis were examined. Bmi-1 was confirmed as a target of miRNA-132. Survival was higher and apoptosis lower in the miR-132 inhibitor group than the blank group after various doses of radiation. By contrast, survival was lower and apoptosis higher in the miRNA-132 mimics and siBmi-1 groups than in the blank group. Moreover, miR-132 expression increased and Bmi-1 mRNA expression decreased in each group at radiation doses of 6 and 8 Gy. Finally, co-administration of radiotherapy and exogenous miR-132 inhibited the growth of HeLa cell transplant-induced tumors in nude mice more effectively than radiotherapy alone. These results suggest overexpression of miR-132 enhances the radiosensitivity of CC cells by down-regulating Bmi-1 and that miR-132 may be a useful new target for the treatment of CC.

  2. Down-regulation of cellular FLICE-inhibitory protein (Long Form contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

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    Wang Qilin

    2012-12-01

    Full Text Available Abstract Background Cellular FLICE-Inhibitory Protein (long form, c-FLIPL is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90 either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP-mediated mechanisms. Methods Calu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90. Results c-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB, c-FLIPL level declined further and there was a higher degree of caspase activation. Conclusion We have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets

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

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    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.

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

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    Uozumi, Naoki; 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)

    2016-05-27

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

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

    International Nuclear Information System (INIS)

    Uozumi, Naoki; Matsumoto, Hotaru; Saitoh, Hisato

    2016-01-01

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

  6. Impaired methylation as a novel mechanism for proteasome suppression in liver cells

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    Osna, Natalia A., E-mail: nosna@UNMC.edu [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); White, Ronda L.; Donohue, Terrence M. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); Beard, Michael R. [Department of Molecular Biosciences, University of Adelaide (Australia); Tuma, Dean J.; Kharbanda, Kusum K. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States)

    2010-01-08

    The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.

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

    OpenAIRE

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

    2010-01-01

    Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degra...

  8. Potential role of proteasome on c-jun related signaling in hypercholesterolemia induced atherosclerosis

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    Erdi Sozen

    2014-01-01

    Full Text Available Atherosclerosis and its complications are major causes of death all over the world. One of the major risks of atherosclerosis is hypercholesterolemia. During atherosclerosis, oxidized low density lipoprotein (oxLDL regulates CD36-mediated activation of c-jun amino terminal kinase-1 (JNK1 and modulates matrix metalloproteinase (MMP induction which stimulates inflammation with an invasion of monocytes. Additionally, inhibition of proteasome leads to an accumulation of c-jun and phosphorylated c-jun and activation of activator protein-1 (AP-1 related increase of MMP expression. We have previously reported a significant increase in cluster of differentiation 36 (CD36 mRNA levels in hypercholesterolemic rabbits and shown that vitamin E treatment prevented the cholesterol induced increase in CD36 mRNA expression. In the present study, our aim is to identify the signaling molecules/transcription factors involved in the progression of atherosclerosis following CD36 activation in an in vivo model of hypercholesterolemic (induced by 2% cholesterol containing diet rabbits. In this direction, proteasomal activities by fluorometry and c-jun, phospo c-jun, JNK1, MMP-9 expressions by quantitative RT-PCR and immunoblotting were tested in aortic tissues. The effects of vitamin E on these changes were also investigated in this model. As a result, c-jun was phosphorylated following decreased proteasomal degradation in hypercholesterolemic group. MMP-9 expression was also increased in cholesterol group rabbits contributing to the development of atherosclerosis. In addition, vitamin E showed its effect by decreasing MMP-9 levels and phosphorylation of c-jun.

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

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    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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  12. Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

    Science.gov (United States)

    Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

    2002-05-15

    Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.

  13. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

    Science.gov (United States)

    Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

    2016-06-01

    Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.

  14. SYVN1, an ERAD E3 Ubiquitin Ligase, Is Involved in GABAAα1 Degradation Associated with Methamphetamine-Induced Conditioned Place Preference

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    Dong-Liang Jiao

    2017-10-01

    Full Text Available Abuse of methamphetamine (METH, a powerful addictive amphetamine-type stimulants (ATS, is becoming a global public health problem. The gamma-aminobutyric acid (GABAergic system plays a critical role in METH use disorders. By using rat METH conditioned place preference (CPP model, we previously demonstrated that METH-associated rewarding memory formation was associated with the reduction of GABAAα1 expression in the dorsal straitum (Dstr, however, the underlying mechanism was unclear. In the present study, we found that METH-induced CPP formation was accompanied by a significant increase in the expression of Synovial apoptosis inhibitor 1 (SYVN1, an endoplasmic reticulum (ER-associated degradation (ERAD E3 ubiquitin ligase, in the Dstr. The siRNA knockdown of SYVN1 significantly increased GABAAα1 protein levels in both primary cultured neurons and rodent Dstr. Inhibition of proteasomal activity by MG132 and Lactacystin significantly increased GABAAα1 protein levels. We further found that SYVN1 knockdown increased GABAAα1 in the intra-ER, but not in the extra-ER. Accordingly, endoplasmic reticulum stress (ERS-associated Glucose-regulated protein 78 (GRP78 and C/EBP homologous protein (CHOP increased. Thus, this study revealed that SYVN1, as the ERAD E3 ubiquitin ligase, was associated with Dstr GABAAα1 degradation induced by METH conditioned pairing.

  15. MDM2 regulates hypoxic hypoxia-inducible factor 1α stability in an E3 ligase, proteasome, and PTEN-phosphatidylinositol 3-kinase-AKT-dependent manner.

    Science.gov (United States)

    Joshi, Shweta; Singh, Alok R; Durden, Donald L

    2014-08-15

    Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. MDM2 Regulates Hypoxic Hypoxia-inducible Factor 1α Stability in an E3 Ligase, Proteasome, and PTEN-Phosphatidylinositol 3-Kinase-AKT-dependent Manner*

    Science.gov (United States)

    Joshi, Shweta; Singh, Alok R.; Durden, Donald L.

    2014-01-01

    Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α–HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. PMID:24982421

  17. Influence of cholinesterase inhibitors, donepezil and rivastigmine on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference in rats.

    Science.gov (United States)

    Gawel, Kinga; Labuz, Krzysztof; Jenda, Malgorzata; Silberring, Jerzy; Kotlinska, Jolanta H

    2014-07-15

    The influence of systemic administration of cholinesterase inhibitors, donepezil and rivastigmine on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference (CPP) was examined in rats. Additionally, this study aimed to compare the effects of donepezil, which selectively inhibits acetylcholinesterase, and rivastigmine, which inhibits both acetylcholinesterase and butyrylcholinesterase on morphine reward. Morphine-induced CPP (unbiased method) was induced by four injections of morphine (5 mg/kg, i.p.). Donepezil (0.5, 1, and 3 mg/kg, i.p.) or rivastigmine (0.03, 0.5, and 1 mg/kg, i.p.) were given 20 min before morphine during conditioning phase and 20 min before the expression or reinstatement of morphine-induced CPP. Our results indicated that both inhibitors of cholinesterase attenuated the acquisition and expression of morphine CPP. The results were more significant after rivastigmine due to a broader inhibitory spectrum of this drug. Moreover, donepezil (1 mg/kg) and rivastigmine (0.5 mg/kg) attenuated the morphine CPP reinstated by priming injection of 5mg/kg morphine. These properties of both cholinesterase inhibitors were reversed by mecamylamine (3 mg/kg, i.p.), a nicotinic acetylcholine receptor antagonist but not scopolamine (0.5 mg/kg, i.p.), a muscarinic acetylcholine receptor antagonist. All effects of cholinesterase inhibitors were observed at the doses that had no effects on locomotor activity of animals. Our results suggest beneficial role of cholinesterase inhibitors in reduction of morphine reward and morphine-induced seeking behavior. Finally, we found that the efficacy of cholinesterase inhibitors in attenuating reinstatement of morphine CPP provoked by priming injection may be due to stimulation of nicotinic acetylcholine receptors. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Acquisition and reinstatement of ethanol-induced conditioned place preference in rats: Effects of the cholinesterase inhibitors donepezil and rivastigmine.

    Science.gov (United States)

    Gawel, Kinga; Labuz, Krzysztof; Gibula-Bruzda, Ewa; Jenda, Malgorzata; Marszalek-Grabska, Marta; Silberring, Jerzy; Kotlinska, Jolanta H

    2016-07-01

    The present study examined the influence of the cholinesterase inhibitors donepezil (a selective inhibitor of acetylcholinesterase) and rivastigmine (also an inhibitor of butyrylcholinesterase) on the acquisition and reinstatement of ethanol-induced conditioned place preference (CPP) in rats. Before the CPP procedure, animals received a single injection of ethanol (0.5 g/kg, 10% w/v, intraperitoneally [i.p.]) for 15 days. The ethanol-induced CPP (biased method) was developed by four injections of ethanol (0.5 g/kg, 10% w/v, i.p.) every second day. Control rats received saline instead of ethanol. Donepezil (0.5, 1 or 3 mg/kg, i.p.) or rivastigmine (0.03, 0.5 or 1 mg/kg, i.p.) were administered before ethanol during conditioning or before the reinstatement of ethanol-induced CPP. The cholinesterase inhibitors were equally effective in increasing (dose dependently) the acquisition of ethanol-induced CPP. Furthermore, priming injections of both inhibitors reinstated (cross-reinstatement) the ethanol-induced CPP with similar efficacy. These effects of both cholinesterase inhibitors were reversed by mecamylamine (3 mg/kg, i.p.), a nicotinic acetylcholine receptor antagonist, but not by scopolamine (0.5 mg/kg, i.p.), a muscarinic acetylcholine receptor antagonist. Thus, our results show that the cholinergic system is involved in the reinforcing properties of ethanol, and nicotinic acetylcholine receptors play an important role in the relapse to ethanol-seeking behaviour. © The Author(s) 2016.

  19. Evolution of extra-nigral damage predicts behavioural deficits in a rat proteasome inhibitor model of Parkinson's disease.

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    Anthony C Vernon

    2011-02-01

    Full Text Available Establishing the neurological basis of behavioural dysfunction is key to provide a better understanding of Parkinson's disease (PD and facilitate development of effective novel therapies. For this, the relationships between longitudinal structural brain changes associated with motor behaviour were determined in a rat model of PD and validated by post-mortem immunohistochemistry. Rats bearing a nigrostriatal lesion induced by infusion of the proteasome inhibitor lactacystin into the left-medial forebrain bundle and saline-injected controls underwent magnetic resonance imaging (MRI at baseline (prior to surgery and 1, 3 and 5 weeks post-surgery with concomitant motor assessments consisting of forelimb grip strength, accelerating rotarod, and apormorphine-induced rotation. Lactacystin-injected rats developed early motor deficits alongside decreased ipsilateral cortical volumes, specifically thinning of the primary motor (M1 and somatosensory cortices and lateral ventricle hypertrophy (as determined by manual segmentation and deformation-based morphometry. Although sustained, motor dysfunction and nigrostriatal damage were maximal by 1 week post-surgery. Additional volume decreases in the ipsilateral ventral midbrain; corpus striatum and thalamus were only evident by week 3 and 5. Whilst cortical MRI volume changes best predicted the degree of motor impairment, post-mortem tyrosine hydroxylase immunoreactivity in the striatum was a better predictor of motor behaviour overall, with the notable exception of performance in the accelerating rotarod, in which, M1 cortical thickness remained the best predictor. These results highlight the importance of identifying extra-nigral regions of damage that impact on behavioural dysfunction from damage to the nigrostriatal system.

  20. The anti-apoptotic activity of BAG3 is restricted by caspases and the proteasome.

    Directory of Open Access Journals (Sweden)

    Victoria M Virador

    Full Text Available Caspase-mediated cleavage and proteasomal degradation of ubiquitinated proteins are two independent mechanisms for the regulation of protein stability and cellular function. We previously reported BAG3 overexpression protected ubiquitinated clients, such as AKT, from proteasomal degradation and conferred cytoprotection against heat shock. We hypothesized that the BAG3 protein is regulated by proteolysis.Staurosporine (STS was used as a tool to test for caspase involvement in BAG3 degradation. MDA435 and HeLa human cancer cell lines exposed to STS underwent apoptosis with a concomitant time and dose-dependent loss of BAG3, suggesting the survival role of BAG3 was subject to STS regulation. zVAD-fmk or caspase 3 and 9 inhibitors provided a strong but incomplete protection of both cells and BAG3 protein. Two putative caspase cleavage sites were tested: KEVD (BAG3(E345A/D347A within the proline-rich center of BAG3 (PXXP and the C-terminal LEAD site (BAG3(E516A/D518A. PXXP deletion mutant and BAG3(E345A/D347A, or BAG3(E516A/D518A respectively slowed or stalled STS-mediated BAG3 loss. BAG3, ubiquitinated under basal growth conditions, underwent augmented ubiquitination upon STS treatment, while there was no increase in ubiquitination of the BAG3(E516A/D518A caspase-resistant mutant. Caspase and proteasome inhibition resulted in partial and independent protection of BAG3 whereas inhibitors of both blocked BAG3 degradation. STS-induced apoptosis was increased when BAG3 was silenced, and retention of BAG3 was associated with cytoprotection.BAG3 is tightly controlled by selective degradation during STS exposure. Loss of BAG3 under STS injury required sequential caspase cleavage followed by polyubiquitination and proteasomal degradation. The need for dual regulation of BAG3 in apoptosis suggests a key role for BAG3 in cancer cell resistance to apoptosis.

  1. Effects of diuretics on sodium-dependent glucose cotransporter 2 inhibitor-induced changes in blood pressure in obese rats suffering from the metabolic syndrome.

    Science.gov (United States)

    Rahman, Asadur; Kittikulsuth, Wararat; Fujisawa, Yoshihide; Sufiun, Abu; Rafiq, Kazi; Hitomi, Hirofumi; Nakano, Daisuke; Sohara, Eisei; Uchida, Shinichi; Nishiyama, Akira

    2016-05-01

    Experiments were carried out to investigate whether diuretics (hydrochlorothiazide + furosemide) impact on the effects of a sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor on glucose metabolism and blood pressure (BP) in metabolic syndrome SHR/NDmcr-cp(+/+) rats (SHRcp). Male 13-week-old SHRcp were treated with: vehicle; the SGLT2-inhibitor luseogliflozin (10 mg/kg per day); diuretics (hydrochlorothiazide; 10 mg/kg/day + furosemide; 5 mg/kg per day); or luseogliflozin + diuretics (n = 5-8 for each group) daily by oral gavage for 5 weeks. BP and glucose metabolism were evaluated by a telemetry system and oral glucose tolerance test, respectively. Vehicle-treated SHRcp developed nondipper type hypertension (dark vs. light-period mean arterial pressure: 148.6 ± 0.7 and 148.0 ± 0.7 mmHg, respectively, P = 0.2) and insulin resistance. Compared with vehicle-treated animals, luseogliflozin-treated rats showed an approximately 4000-fold increase in urinary excretion of glucose and improved glucose metabolism. Luseogliflozin also significantly decreased BP and turned the circadian rhythm of BP from a nondipper to dipper pattern (dark vs. light-period mean arterial pressure: 138.0 ± 1.6 and 132.0 ± 1.3 mmHg, respectively, P diuretics did not influence luseogliflozin-induced improvement of glucose metabolism and circadian rhythm of BP in SHRcp. These data suggest that a SGLT2 inhibitor elicits its beneficial effects on glucose metabolism and hypertension in study participants with metabolic syndrome undergoing treatment with diuretics.

  2. 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.

  3. A novel and selective poly (ADP-ribose polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

    Directory of Open Access Journals (Sweden)

    Lauren E Ta

    Full Text Available Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose polymerase (PARP inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888 would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice.An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p. injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment.Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

  4. A novel and selective poly (ADP-ribose) polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

    Science.gov (United States)

    Ta, Lauren E; Schmelzer, James D; Bieber, Allan J; Loprinzi, Charles L; Sieck, Gary C; Brederson, Jill D; Low, Philip A; Windebank, Anthony J

    2013-01-01

    Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice. An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment. Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

  5. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo

    DEFF Research Database (Denmark)

    Nudelman, Aaron Samuel; DiRocco, Derek P; Lambert, Talley J

    2010-01-01

    Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its......, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h...

  6. Impact of High Glucose and Proteasome Inhibitor MG132 on Histone H2A and H2B Ubiquitination in Rat Glomerular Mesangial Cells

    Directory of Open Access Journals (Sweden)

    Chenlin Gao

    2013-01-01

    Full Text Available Background. Hyperglycemia plays a pivotal role in the development of diabetic nephropathy (DN and may be related to epigenetic metabolic memory. One of the most crucial epigenetic mechanisms is histone modification, which is associated with the expression of a fibrosis factor in vascular injury. Aim .In this study, we investigated the ubiquitination of histones H2A and H2B to explore the epigenetic mechanisms of DN. Materials and Methods. The GMCs were cultured as follows: normal group, high glucose group, mannitol group, and intervention group. After 12 hr, 24 hr, and 48 hr, histones ubiquitination, transforming growth factor-β (TGF-β, and fibronectin (FN were measured using WB, RT-PCR, and IF. Result. High glucose can induce the upregulation of FN. H2A ubiquitination in GMCs increased in high glucose group (P<0.01, whereas it decreased significantly in intervention group (P<0.05. In contrast, H2B ubiquitination decreased with an increasing concentration of glucose, but it was recovered in the intervention group (P<0.05. Expression of TGF-β changed in response to abnormal histone ubiquitination. Conclusions. The high glucose may induce H2A ubiquitination and reduce H2B ubiquitination in GMCs. The changes of histone ubiquitination may be due in part to DN by activating TGF-β signaling pathway.

  7. Inulin significantly improves serum magnesium levels in proton pump inhibitor-induced hypomagnesaemia.

    Science.gov (United States)

    Hess, M W; de Baaij, J H F; Broekman, M; Bisseling, T M; Haarhuis, B; Tan, A; Te Morsche, R; Hoenderop, J G J; Bindels, R J M; Drenth, J P H

    2016-06-01

    Proton pump inhibitors (PPI) are among the most widely prescribed drugs to treat gastric acid-related disorders. PPI-induced hypomagnesaemia, a defect in intestinal absorption of Mg(2+) , can be a severe side effect of chronic PPI use. To restore serum Mg(2+) concentrations in PPI-induced hypomagnesaemia patients by dietary supplementation with inulin fibres. Eleven patients with PPI-induced hypomagnesaemia and 10 controls were treated with inulin (20 g/day). Each trial consisted of two cycles of 14-day inulin treatment followed by a washout period of 14 days. Patients continued to use their PPI. Serum Mg(2+) levels served as the primary endpoint. Inulin significantly enhanced serum Mg(2+) levels from 0.60 to 0.68 mmol/L in PPI-induced hypomagnesaemia patients, and from 0.84 to 0.93 mmol/L in controls. As a consequence 24 h urinary Mg(2+) excretion was significantly increased in patients with PPI-induced hypomagnesaemia (0.3-2.2 mmol/day). Symptoms related to hypomagnesaemia, including muscle cramps and paraesthesia, were reduced during intervention with inulin. Inulin increases serum Mg(2+) concentrations under PPI maintenance in patients with PPI-induced hypomagnesaemia. © 2016 John Wiley & Sons Ltd.

  8. Gamma rays induced mutation for low phytic acid and trypsin inhibitor content in soybean

    International Nuclear Information System (INIS)

    Gupta, S.K.; Manjaya, J.G.

    2017-01-01

    Soybean (Glycine max (L.) Merrill) is an important source of vegetable protein and is used as a food, feed and health supplement. However, consumption of soybean as food is limited because of the presence of many anti-nutritional factors. Trypsin inhibitors and phytic acid are two major anti-nutritional factors present in soybean that need to be removed for increasing the soybean consumption as food. Trypsin inhibitor is known to inhibit the trypsin/chymotrpsin activity and phytic acid reduces the bioavailability of essential micronutrients in digestive tract, resulting in adverse effect on health. Therefore, developing soybean cultivars having low trypsin inhibitors and phytic acid content is highly desirable. Soybean cultivar JS 93-05 was irradiated with 250 Gy gamma rays to induce mutation for various morphological and biochemical characters. A large number of mutants with altered morphological characters were identified. Ninety true breeding mutant lines in M6 generation were screened for trypsin inhibitor and phytic acid content. The phytic acid content was estimated using modified colorimetric method and trypsin inhibitor concentration was estimated using BAPNA as substrate in colorimetric method. The phytic acid content in the mutants varied from 7.59 to 24.14 mg g -1 . Two mutants lines TSG - 62 (7.59 mg g -1 ) and TSG - 66 (9.62 mg g -1 ) showed significant low phytic acid content as compared to the parent JS 93-05 (20.19 mg g -1 ). The trypsin inhibitor concentration in the mutants varied from 19.92 to 53.64 TIU mg -1 and one mutant line (TSG -14) was found with the lowest trypsin inhibitor concentration of 19.92 TIU mg -1 compared to parent JS 93-05 (50.90 TIU mg -1 ). The mutant lines identified in this study will serve as important genetic resources for developing low phytic acid and low trypsin inhibitor cultivars in soybean. (author)

  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

    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 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. The Author(s). Published by S. Karger AG, Basel.

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

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    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

  11. Isolation, characterization, and genetic complementation of a cellular mutant resistant to retroviral infection

    Science.gov (United States)

    Agarwal, Sumit; Harada, Josephine; Schreifels, Jeffrey; Lech, Patrycja; Nikolai, Bryan; Yamaguchi, Tomoyuki; Chanda, Sumit K.; Somia, Nikunj V.

    2006-01-01

    By using a genetic screen, we have isolated a mammalian cell line that is resistant to infection by retroviruses that are derived from the murine leukemia virus, human immunodeficiency virus type 1, and feline immunodeficiency virus. We demonstrate that the cell line is genetically recessive for the resistance, and hence it is lacking a factor enabling infection by retroviruses. The block to infection is early in the life cycle, at the poorly understood uncoating stage. We implicate the proteasome at uncoating by completely rescuing the resistant phenotype with the proteasomal inhibitor MG-132. We further report on the complementation cloning of a gene (MRI, modulator of retrovirus infection) that can also act to reverse the inhibition of infection in the mutant cell line. These data implicate a role for the proteasome during uncoating, and they suggest that MRI is a regulator of this activity. Finally, we reconcile our findings and other published data to suggest a model for the involvement of the proteasome in the early phase of the retroviral life cycle. PMID:17043244

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

    International Nuclear Information System (INIS)

    Kwak, Mi-Kyoung; Kensler, Thomas W.

    2006-01-01

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

  13. Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma.

    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

    2014-08-01

    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.

  14. Human sperm degradation of zona pellucida proteins contributes to fertilization.

    Science.gov (United States)

    Saldívar-Hernández, Analilia; González-González, María E; Sánchez-Tusié, Ana; Maldonado-Rosas, Israel; López, Pablo; Treviño, Claudia L; Larrea, Fernando; Chirinos, Mayel

    2015-09-02

    The mammalian oocyte extracellular matrix known as the zona pellucida (ZP) acts as a barrier to accomplish sperm fusion with the female gamete. Although penetration of the ZP is a limiting event to achieve fertilization, this is one of the least comprehended stages of gamete interaction. Even though previous studies suggest that proteases of sperm origin contribute to facilitate the passage of sperm through the ZP, in human this process is not yet fully understood. The aim of this study was to determine the ability of human sperm to degrade recombinant human ZP (rhZPs) proteins and to characterize the proteases involved in this process. Purified rhZP2, rhZP3 and rhZP4 proteins were incubated with capacitated sperm and the proteolytic activity was determined by Western blot analysis. To further characterize the proteases involved, parallel incubations were performed in the presence of the protease inhibitors o-phenanthroline, benzamidine and MG-132 meant to block the activity of metalloproteases, serine proteases and the proteasome, respectively. Additionally, protease inhibitors effect on sperm-ZP binding was evaluated by hemizona assay. The results showed that rhZPs were hydrolyzed in the presence of capacitated sperm. O-phenanthroline inhibited the degradation of rhZP3, MG-132 inhibited the degradation of rhZP4 and benzamidine inhibited the degradation of the three proteins under investigation. Moreover, hemizona assays demonstrated that sperm proteasome inhibition impairs sperm interaction with human native ZP. This study suggests that sperm proteasomes could participate in the degradation of ZP, particularly of the ZP4 protein. Besides, metalloproteases may be involved in specific degradation of ZP3 while serine proteases may contribute to unspecific degradation of the ZP. These findings suggest that localized degradation of ZP proteins by sperm is probably involved in ZP penetration and may be of help in understanding the mechanisms of fertilization in humans.

  15. 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.

  16. Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells.

    Science.gov (United States)

    Xiao, Dong; Johnson, Candace S; Trump, Donald L; Singh, Shivendra V

    2004-05-01

    Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, offers significant protection against cancer in animals induced by a variety of carcinogens. The present study demonstrates that PEITC suppresses proliferation of PC-3 cells in a dose-dependent manner by causing G(2)-M-phase cell cycle arrest and apoptosis. Interestingly, phenyl isothiocyanate (PITC), which is a structural analogue of PEITC but lacks the -CH(2) spacers that link the aromatic ring to the -N=C=S group, neither inhibited PC-3 cell viability nor caused cell cycle arrest or apoptosis. These results indicated that even a subtle change in isothiocyanate (ITC) structure could have a significant impact on its biological activity. The PEITC-induced cell cycle arrest was associated with a >80% reduction in the protein levels of cyclin-dependent kinase 1 (Cdk1) and cell division cycle 25C (Cdc25C; 24 h after treatment with 10 micro M PEITC), which led to an accumulation of Tyr(15) phosphorylated (inactive) Cdk1. On the other hand, PITC treatment neither reduced protein levels of Cdk1 or Cdc25C nor affected Cdk1 phosphorylation. The PEITC-induced decline in Cdk1 and Cdc25C protein levels and cell cycle arrest were significantly blocked on pretreatment of PC-3 cells with proteasome inhibitor lactacystin. A 24 h exposure of PC-3 cells to 10 micro M PEITC, but not PITC, resulted in about 56% and 44% decrease in the levels of antiapoptotic proteins Bcl-2 and Bcl-X(L), respectively. However, ectopic expression of Bcl-2 failed to alter sensitivity of PC-3 cells to growth inhibition or apoptosis induction by PEITC. Treatment of cells with PEITC, but not PITC, also resulted in cleavage of procaspase-3, procaspase-9, and procaspase-8. Moreover, the PEITC-induced apoptosis was significantly attenuated in the presence of general caspase inhibitor and specific inhibitors of caspase-8 and caspase-9. In conclusion, our data indicate that PEITC-induced cell cycle arrest in PC-3 cells is likely due

  17. 1,2,3,4,6-Penta-O-Galloyl-Beta-D-Glucopyranoside Inhibits Proliferation of Multiple Myeloma Cells Accompanied with Suppression of MYC Expression

    Directory of Open Access Journals (Sweden)

    Duurenjargal Tseeleesuren

    2018-02-01

    Full Text Available Multiple myeloma (MM still remains an incurable disease, therefore discovery of novel drugs boosts the therapeutics for MM. The natural compound 1,2,3,4,6-Penta-O-galloyl-beta-D-glucopyranoside (PGG has been shown to exhibit antitumor activities against various cancer cells. Here, we aim to evaluate antitumor effects of PGG on MM cell lines. PGG inhibited the growth of three different MM cell lines in a dose- and time-dependent manner. Cell cycle analysis revealed that PGG treatment caused cell cycle arrest in G1 phase. It also induced apoptosis which was indicated by significant increases of Annexin V positive cells, caspase 3/7 activity, and cleaved caspase 3 expression in PGG treated MM cell. Since MYC is frequently hyperactivated in MM and inhibition of MYC leads to MM cell death. We further demonstrated that PGG decreased MYC expression in protein and mRNA levels and reversed the mRNA expression of MYC target genes such as p21, p27, and cyclin D2. In addition, PGG also reduced protein expression of DEPTOR which is commonly overexpressed in MM. Unexpectedly, PGG antagonized the cytotoxic effect of bortezomib in the combination treatment. However, PGG treatment sensitized MM cells to another proteasome inhibitor MG132 induced cytotoxicity. Moreover, MYC inhibitor JQ1 enhanced the cytotoxic effect of bortezomib on MM cells. Our findings raised concerns about the combinatory use of bortezomib with particular types of chemicals. The evidence also provide useful insights into the combination of MYC and proteasome-inhibitors for MM therapy. Finally, PGG has a therapeutic potential for treatment of MM and further development is mandatory.

  18. Nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance

    International Nuclear Information System (INIS)

    Kraus, M; Bader, J; Overkleeft, H; Driessen, C

    2013-01-01

    HIV protease inhibitors (HIV-PI) are oral drugs for HIV treatment. HIV-PI have antitumor activity via induction of ER-stress, inhibition of phospho-AKT (p-AKT) and the proteasome, suggesting antimyeloma activity. We characterize the effects of all approved HIV-PI on myeloma cells. HIV-PI were compared regarding cytotoxicity, proteasome activity, ER-stress induction and AKT phosphorylation using myeloma cells in vitro. Nelfinavir is the HIV-PI with highest cytotoxic activity against primary myeloma cells and with an IC 50 near therapeutic drug blood levels (8–14 μM), irrespective of bortezomib sensitivity. Only nelfinavir inhibited intracellular proteasome activity in situ at drug concentrations <40 μℳ. Ritonavir, saquinavir and lopinavir inhibited p-AKT comparable to nelfinavir, and showed similar synergistic cytotoxicity with bortezomib against bortezomib-sensitive cells. Nelfinavir had superior synergistic activity with bortezomib/carfilzomib in particular against bortezomib/carfilzomib-resistant myeloma cells. It inhibited not only the proteasomal β1/β5 active sites, similar to bortezomib/carfilzomib, but in addition the β2 proteasome activity not targeted by bortezomib/carfilzomib. Additional inhibition of β2 proteasome activity is known to sensitize cells for bortezomib and carfilzomib. Nelfinavir has unique proteasome inhibiting activity in particular on the bortezomib/carfilzomib-insensitive tryptic (β2) proteasome activity in intact myeloma cells, and is active against bortezomib/carfilzomib-resistant myeloma cells in vitro

  19. Method of oocyte activation affects cloning efficiency in pigs.

    Science.gov (United States)

    Whitworth, Kristin M; Li, Rongfeng; Spate, Lee D; Wax, David M; Rieke, August; Whyte, Jeffrey J; Manandhar, Gaurishankar; Sutovsky, Miriam; Green, Jonathan A; Sutovsky, Peter; Prather, Randall S

    2009-05-01

    The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca(2+) followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P NT1 (71.4%, n = 28; P 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. (c) 2008 Wiley-Liss, Inc.

  20. Oxidative stress reduces levels of dysbindin-1A via its PEST domain.

    Science.gov (United States)

    Yap, Mei-Yi Alicia; Lo, Yew-Long; Talbot, Konrad; Ong, Wei-Yi

    2014-12-01

    Oxidative stress resulting from the generation of reactive oxygen species has been proposed as an etiological factor in schizophrenia. The present study tests the hypothesis that oxidative stress can affect levels of dysbindin-1A, encoded by Dtnbp1, a genetic risk factor for schizophrenia, via its PEST domain. In vitro studies on SH-SY5Y cells indicate that oxidative stress triggers proteasomal degradation of dysbindin-1A, and that this requires interactions with its PEST domain, which may be a TRIM32 target. We specifically found (a) that oxidative stress induced in SH-SY5Y cells by 500 µM hydrogen peroxide reduced levels of full-length dysbindin-1, but did not reduce levels of that protein lacking its PEST domain and (b) that levels of full-length dysbindin-1, but not dysbindin-1 lacking its PEST domain, were higher in cells treated with the proteasome inhibitor MG132. Oxidative stress thus emerges as the first known cellular factor regulating dysbindin-1 isoforms with PEST domains. These findings are consistent with the previously noted fact that phosphorylation of PEST domains often marks proteins for proteasomal degradation, and raises the possibility that treatments reducing oxidative stress in the brain, especially during development, may lower schizophrenia risk. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Nitric oxide maintains cell survival of Trichomonas vaginalis upon iron depletion.

    Science.gov (United States)

    Cheng, Wei-Hung; Huang, Kuo-Yang; Huang, Po-Jung; Hsu, Jo-Hsuan; Fang, Yi-Kai; Chiu, Cheng-Hsun; Tang, Petrus

    2015-07-25

    Iron plays a pivotal role in the pathogenesis of Trichomonas vaginalis, the causative agent of highly prevalent human trichomoniasis. T. vaginalis resides in the vaginal region, where the iron concentration is constantly changing. Hence, T. vaginalis must adapt to variations in iron availability to establish and maintain an infection. The free radical signaling molecules reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been proven to participate in iron deficiency in eukaryotes. However, little is known about the roles of these molecules in iron-deficient T. vaginalis. T. vaginalis cultured in iron-rich and -deficient conditions were collected for all experiments in this study. Next generation RNA sequencing was conducted to investigate the impact of iron on transcriptome of T. vaginalis. The cell viabilities were monitored after the trophozoites treated with the inhibitors of nitric oxide (NO) synthase (L-NG-monomethyl arginine, L-NMMA) and proteasome (MG132). Hydrogenosomal membrane potential was measured using JC-1 staining. We demonstrated that NO rather than ROS accumulates in iron-deficient T. vaginalis. The level of NO was blocked by MG132 and L-NMMA, indicating that NO production is through a proteasome and arginine dependent pathway. We found that the inhibition of proteasome activity shortened the survival of iron-deficient cells compared with untreated iron-deficient cells. Surprisingly, the addition of arginine restored both NO level and the survival of proteasome-inhibited cells, suggesting that proteasome-derived NO is crucial for cell survival under iron-limited conditions. Additionally, NO maintains the hydrogenosomal membrane potential, a determinant for cell survival, emphasizing the cytoprotective effect of NO on iron-deficient T. vaginalis. Collectively, we determined that NO produced by the proteasome prolonged the survival of iron-deficient T. vaginalis via maintenance of the hydrogenosomal functions. The findings in this

  2. SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

    Science.gov (United States)

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

    2010-06-01

    Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Saturated fatty acid palmitate negatively regulates autophagy by promoting ATG5 protein degradation in meniscus cells.

    Science.gov (United States)

    Mallik, Aritra; Yammani, Raghunatha R

    2018-07-20

    Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

    Science.gov (United States)

    Patrick, B A; Jaiswal, A K

    2012-10-04

    Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

  5. Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

    Energy Technology Data Exchange (ETDEWEB)

    Denamur, Sophie; Boland, Lidvine [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Beyaert, Maxime [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Verstraeten, Sandrine L. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Fillet, Marianne [University of Liege, CIRM, Department of Pharmacy, Laboratory for the Analysis of Medicines, Quartier Hopital, Avenue Hippocrate, 15, B36, Tower 4, 4000 Liège 1 (Belgium); Tulkens, Paul M. [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium); Bontemps, Françoise [Université catholique de Louvain, de Duve Institute, Laboratory of Physiological Chemistry, UCL B1.75.08, avenue Hippocrate, 75 B -1200 Brussels (Belgium); Mingeot-Leclercq, Marie-Paule [Université catholique de Louvain, Louvain Drug Research Institute, Cellular and Molecular Pharmacology, UCL B1.73.05, avenue E. Mounier, 73 – B1200 Brussels (Belgium)

    2016-10-15

    Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

  6. Subcellular mechanisms involved in apoptosis induced by aminoglycoside antibiotics: Insights on p53, proteasome and endoplasmic reticulum

    International Nuclear Information System (INIS)

    Denamur, Sophie; Boland, Lidvine; Beyaert, Maxime; Verstraeten, Sandrine L.; Fillet, Marianne; Tulkens, Paul M.; Bontemps, Françoise; Mingeot-Leclercq, Marie-Paule

    2016-01-01

    Gentamicin, an aminoglycoside used to treat severe bacterial infections, may cause acute renal failure. In the renal cell line LLC-PK1, gentamicin accumulates in lysosomes, induces alterations of their permeability, and triggers the mitochondrial pathway of apoptosis via activation of caspase-9 and -3 and changes in Bcl-2 family proteins. Early ROS production in lysosomes has been associated with gentamicin induced lysosomal membrane permeabilization. In order to better understand the multiple interconnected pathways of gentamicin-induced apoptosis and ensuing renal cell toxicity, we investigated the effect of gentamicin on p53 and p21 levels. We also studied the potential effect of gentamicin on proteasome by measuring the chymotrypsin-, trypsin- and caspase-like activities, and on endoplasmic reticulum by determining phopho-eIF2α, caspase-12 activation and GRP78 and 94. We observed an increase in p53 levels, which was dependent on ROS production. Accumulation of p53 resulted in accumulation of p21 and of phospho-eIF2α. These effects could be related to an impairment of proteasome as we demonstrated an inhibition of trypsin-and caspase-like activities. Moderate endoplasmic reticulum stress could also participate to cellular toxicity induced by gentamicin, with activation of caspase-12 without change in GRP74 and GRP98. All together, these data provide new mechanistic insights into the apoptosis induced by aminoglycoside antibiotics on renal cell lines. - Highlights: • Gentamicin induces apoptosis through p53 pathway. • Gentamicin inhibits proteosomal activity. • Gentamicin activates caspase-12.

  7. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

    Science.gov (United States)

    Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

    2014-01-01

    Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

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

    Science.gov (United States)

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

    2015-01-01

    The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31y) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although HSP72 and HSP40 (Hdj1) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging. PMID:25018089

  9. SOCS-1 localizes to the microtubule organizing complex-associated 20S proteasome.

    Science.gov (United States)

    Vuong, Bao Q; Arenzana, Teresita L; Showalter, Brian M; Losman, Julie; Chen, X Peter; Mostecki, Justin; Banks, Alexander S; Limnander, Andre; Fernandez, Neil; Rothman, Paul B

    2004-10-01

    The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unknown. In this report, SOCS-1 is found to colocalize and biochemically copurify with the microtubule organizing complex (MTOC) and its associated 20S proteasome. The SOCS-1 SH2 domain is required for the localization of SOCS-1 to the MTOC. Overexpression of SOCS-1 targets Jak1 in an SH2-dependent manner to a perinuclear distribution resembling the MTOC-associated 20S proteasome. Analysis of MTOCs fractionated from SOCS-1-deficient cells demonstrates that SOCS-1 may function redundantly to regulate the localization of Jak1 to the MTOC. Nocodazole inhibits the protein turnover of SOCS-1, demonstrating that the minus-end transport of SOCS-1 to the MTOC-associated 20S proteasome is required to regulate SOCS-1 protein levels. These data link SOCS-1 directly with the proteasome pathway and suggest another function for the SH2 domain of SOCS-1 in the regulation of Jak/STAT signaling.

  10. Carbonic anhydrase inhibitor attenuates ischemia-reperfusion induced acute lung injury.

    Directory of Open Access Journals (Sweden)

    Chou-Chin Lan

    Full Text Available Ischemia-reperfusion (IR-induced acute lung injury (ALI is implicated in several clinical conditions including lung transplantation, cardiopulmonary bypass surgery, re-expansion of collapsed lung from pneumothorax or pleural effusion and etc. IR-induced ALI remains a challenge in the current treatment. Carbonic anhydrase has important physiological function and influences on transport of CO2. Some investigators suggest that CO2 influences lung injury. Therefore, carbonic anhydrase should have the role in ALI. This study was undertaken to define the effect of a carbonic anhydrase inhibitor, acetazolamide (AZA, in IR-induced ALI, that was conducted in a rat model of isolated-perfused lung with 30 minutes of ischemia and 90 minutes of reperfusion. The animals were divided into six groups (n = 6 per group: sham, sham + AZA 200 mg/kg body weight (BW, IR, IR + AZA 100 mg/kg BW, IR + AZA 200 mg/kg BW and IR+ AZA 400 mg/kg BW. IR caused significant pulmonary micro-vascular hyper-permeability, pulmonary edema, pulmonary hypertension, neutrophilic sequestration, and an increase in the expression of pro-inflammatory cytokines. Increases in carbonic anhydrase expression and perfusate pCO2 levels were noted, while decreased Na-K-ATPase expression was noted after IR. Administration of 200mg/kg BW and 400mg/kg BW AZA significantly suppressed the expression of pro-inflammatory cytokines (TNF-α, IL-1, IL-6 and IL-17 and attenuated IR-induced lung injury, represented by decreases in pulmonary hyper-permeability, pulmonary edema, pulmonary hypertension and neutrophilic sequestration. AZA attenuated IR-induced lung injury, associated with decreases in carbonic anhydrase expression and pCO2 levels, as well as restoration of Na-K-ATPase expression.

  11. Bacterial proteasome activator bpa (rv3780) is a novel ring-shaped interactor of the mycobacterial proteasome.

    Science.gov (United States)

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

    2014-01-01

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

  12. The heat shock protein 90 inhibitor, 17-AAG, attenuates thioacetamide induced liver fibrosis in mice.

    Science.gov (United States)

    Abu-Elsaad, Nashwa M; Serrya, Marwa S; El-Karef, Amr M; Ibrahim, Tarek M

    2016-04-01

    Heat shock protein 90 (Hsp90) is proposed to be involved in liver disorders. This study was conducted to test effect of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90, on attenuating thioacetamide induced liver fibrosis in vivo. Four groups of Swiss albino male mice (CD-1 strain) were used as follows: control group; thioacetamide group (received 100mg/kg thioacetamide, ip injection, 3 times/week for 8 weeks); thioacetamide plus 17-AAG groups (received 100mg/kg thioacetamide, ip injection, 3 times/week for 8 weeks plus 25 or 50mg/kg 17-AAG, ip injection, 5 days/week along the last 4 weeks). Fibrosis was quantified by measuring hydroxyproline level and by morphometry and oxidative stress biomarkers were assigned. Relative hepatic mRNA expressions of α-smooth muscle actin (α-SMA), collagen-1-alpha-1 (Col1A1) and tissue inhibitor metalloproteinase-1 (TIMP-1) mRNAs were measured by RT-PCR. Levels of the apoptotic markers caspase-3, factor related apoptosis (Fas) and Hsp-90 were assigned in tissue homogenate. 17-AAG (50mg/kg) significantly decreased fibrosis percentage significantly (pAAG (50mg/kg) compared to other groups. The Hsp90 inhibitor, 17-AAG, can attenuate thioacetamide hepatotoxicity through oxidative stress counterbalance, reducing stellate cells activity and inducing apoptosis. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  13. 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.

  14. Melatonin-Induced Temporal Up-Regulation of Gene Expression Related to Ubiquitin/Proteasome System (UPS in the Human Malaria Parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Fernanda C. Koyama

    2014-12-01

    Full Text Available There is an increasing understanding that melatonin and the ubiquitin/ proteasome system (UPS interact to regulate multiple cellular functions. Post-translational modifications such as ubiquitination are important modulators of signaling processes, cell cycle and many other cellular functions. Previously, we reported a melatonin-induced upregulation of gene expression related to ubiquitin/proteasome system (UPS in Plasmodium falciparum, the human malaria parasite, and that P. falciparum protein kinase 7 influences this process. This implies a role of melatonin, an indolamine, in modulating intraerythrocytic development of the parasite. In this report we demonstrate by qPCR analysis, that melatonin induces gene upregulation in nine out of fourteen genes of the UPS, consisting of the same set of genes previously reported, between 4 to 5 h after melatonin treatment. We demonstrate that melatonin causes a temporally controlled gene expression of UPS members.

  15. Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner.

    Directory of Open Access Journals (Sweden)

    John Meshki

    Full Text Available U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R. Substance P (SP, the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.

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

    Science.gov (United States)

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

    2014-09-01

    The impairment of protein degradation via the ubiquitin-proteasome system (UPS) is present in sporadic Parkinson's disease (PD), and might play a key role in selective degeneration of vulnerable dopamine (DA) neurons in the substantia nigra pars compacta (SN). Further evidence for a causal role of dysfunctional UPS in familial PD comes from mutations in parkin, which results in a loss of function of an E3-ubiquitin-ligase. In a mouse model, genetic inactivation of an essential component of the 26S proteasome lead to widespread neuronal degeneration including DA midbrain neurons and the formation of alpha-synuclein-positive inclusion bodies, another hallmark of PD. Studies using pharmacological UPS inhibition in vivo had more mixed results, varying from extensive degeneration to no loss of DA SN neurons. However, it is currently unknown whether UPS impairment will affect the neurophysiological functions of DA midbrain neurons. To answer this question, we infused a selective proteasome inhibitor into the ventral midbrain in vivo and recorded single DA midbrain neurons 2 weeks after the proteasome challenge. We found a selective increase in the mean in vivo firing frequencies of identified DA SN neurons in anesthetized mice, while those in the ventral tegmental area (VTA) were unaffected. Our results demonstrate that a single-hit UPS inhibition is sufficient to induce a stable and selective hyperexcitability phenotype in surviving DA SN neurons in vivo. This might imply that UPS dysfunction sensitizes DA SN neurons by enhancing 'stressful pacemaking'. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  17. A genome-wide siRNA screen identifies proteasome addiction as a vulnerability of basal-like triple-negative breast cancer cells

    Science.gov (United States)

    Petrocca, Fabio; Altschuler, Gabriel; Tan, Shen Mynn; Mendillo, Marc L.; Yan, Haoheng; Jerry, D. Joseph; Kung, Andrew L.; Hide, Winston; Ince, Tan A.; Lieberman, Judy

    2013-01-01

    Summary Basal-like triple negative breast cancers (TNBC) have poor prognosis. To identify basal-like TNBC dependencies, a genome-wide siRNA lethality screen compared two human breast epithelial cell lines transformed with the same genes - basal-like BPLER and myoepithelial HMLER. Expression of the screen’s 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Proteasome genes were overrepresented hits. Basal-like TNBC lines were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiating cell function and macrometastasis. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence. PMID:23948298

  18. Modeling proteasome dynamics in Parkinson's disease

    International Nuclear Information System (INIS)

    Sneppen, Kim; Lizana, Ludvig; Jensen, Mogens H; Pigolotti, Simone; Otzen, Daniel

    2009-01-01

    In Parkinson's disease (PD), there is evidence that α-synuclein (αSN) aggregation is coupled to dysfunctional or overburdened protein quality control systems, in particular the ubiquitin–proteasome system. Here, we develop a simple dynamical model for the on-going conflict between αSN aggregation and the maintenance of a functional proteasome in the healthy cell, based on the premise that proteasomal activity can be titrated out by mature αSN fibrils and their protofilament precursors. In the presence of excess proteasomes the cell easily maintains homeostasis. However, when the ratio between the available proteasome and the αSN protofilaments is reduced below a threshold level, we predict a collapse of homeostasis and onset of oscillations in the proteasome concentration. Depleted proteasome opens for accumulation of oligomers. Our analysis suggests that the onset of PD is associated with a proteasome population that becomes occupied in periodic degradation of aggregates. This behavior is found to be the general state of a proteasome/chaperone system under pressure, and suggests new interpretations of other diseases where protein aggregation could stress elements of the protein quality control system

  19. 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.

  20. Discovery of a novel orally active PDE-4 inhibitor effective in an ovalbumin-induced asthma murine model.

    Science.gov (United States)

    Kwak, Hyun Jeong; Nam, Ji Yeon; Song, Jin Sook; No, Zaesung; Yang, Sung Don; Cheon, Hyae Gyeong

    2012-06-15

    Phosphodiesterase-4 (PDE-4) is responsible for metabolizing adenosine 3',5'-cyclic monophosphate that reduces the activation of a wide range of inflammatory cells including eosinophils. PDE-4 inhibitors are under development for the treatment of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Herein, we report a novel PDE-4 inhibitor, PDE-423 (3-[1-(3-cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid), which shows good in vitro and in vivo oral activities. PDE-423 exhibited in vitro IC(50)s of 140 nM and 550 nM in enzyme assay and cell-based assay, respectively. In vivo study using ovalbumin-induced asthmatic mice revealed that PDE-423 reduced methacholine-stimulated airway hyperreactivity in a dose-dependent manner by once daily oral administration (ED(50)=18.3 mg/kg), in parallel with decreased eosinophil peroxidase activity and improved lung histology. In addition, PDE-423 was effective in diminishing lipopolysaccharide-induced neutrophilia in vivo as well as in vitro. Oral administration of PDE-423 (100 mg/kg) had no effect on the duration of xylazine/ketamine-induced anesthesia and did not induce vomiting incidence in ferrets up to the dose of 1000 mg/kg. The present study indicates that a novel PDE-4 inhibitor, PDE-423, has good pharmacological profiles implicating this as a potential candidate for the development of a new anti-asthmatic drug. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

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    Przemysław Karpowicz

    Full Text Available The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.

  2. Histone deacetylase inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats

    OpenAIRE

    Lee, Eunjo; Song, Min-ji; Lee, Hae-Ahm; Kang, Seol-Hee; Kim, Mina; Yang, Eun Kyoung; Lee, Do Young; Ro, Seonggu; Cho, Joong Myung; Kim, Inkyeom

    2016-01-01

    CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats...

  3. CacyBP/SIP promotes the proliferation of colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Huihong Zhai

    Full Text Available CacyBP/SIP is a component of the ubiquitin pathway and is overexpressed in several transformed tumor tissues, including colon cancer, which is one of the most common cancers worldwide. It is unknown whether CacyBP/SIP promotes the proliferation of colon cancer cells. This study examined the expression level, subcellular localization, and binding activity of CacyBP/SIP in human colon cancer cells in the presence and absence of the hormone gastrin. We found that CacyBP/SIP was expressed in a high percentage of colon cancer cells, but not in normal colonic surface epithelium. CacyBP/SIP promoted the cell proliferation of colon cancer cells under both basal and gastrin stimulated conditions as shown by knockdown studies. Gastrin stimulation triggered the translocation of CacyBP/SIP to the nucleus, and enhanced interaction between CacyBP/SIP and SKP1, a key component of ubiquitination pathway which further mediated the proteasome-dependent degradation of p27kip1 protein. The gastrin induced reduction in p27kip1 was prevented when cells were treated with the proteasome inhibitor MG132. These results suggest that CacyBP/SIP may be promoting growth of colon cancer cells by enhancing ubiquitin-mediated degradation of p27kip1.

  4. Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

    International Nuclear Information System (INIS)

    Hristov, K.; Mitev, V.; Knox, K.

    2006-01-01

    Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

  5. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE2 induced pain model

    International Nuclear Information System (INIS)

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D.; Trindade da Silva, Carlos Antonio; Morisseau, Christophe; Hammock, Bruce D.

    2015-01-01

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE 2 was monitored. While OME treatment by itself exhibited variable effects on PGE 2 induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.

  6. 15,16-Dihydrotanshinone I, a Compound of Salvia miltiorrhiza Bunge, Induces Apoptosis through Inducing Endoplasmic Reticular Stress in Human Prostate Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Mao-Te Chuang

    2011-01-01

    Full Text Available 5,16-dihydrotanshinone I (DHTS is extracted from Salvia miltiorrhiza Bunge (tanshen root and was found to be the most effective compound of tanshen extracts against breast cancer cells in our previous studies. However, whether DHTS can induce apoptosis through an endoplasmic reticular (ER stress pathway was examined herein. In this study, we found that DHTS significantly inhibited the proliferation of human prostate DU145 carcinoma cells and induced apoptosis. DHTS was able to induce ER stress as evidenced by the upregulation of glucose regulation protein 78 (GRP78/Bip and CAAT/enhancer binding protein homologous protein/growth arrest- and DNA damage-inducible gene 153 (CHOP/GADD153, as well as increases in phosphorylated eukaryotic initiation factor 2α (eIF2α, c-jun N-terminal kinase (JNK, and X-box-binding protein 1 (XBP1 mRNA splicing forms. DHTS treatment also caused significant accumulation of polyubiquitinated proteins and hypoxia-inducible factor (HIF-1α, indicating that DHTS might be a proteasome inhibitor that is known to induce ER stress or enhance apoptosis caused by the classic ER stress-dependent mechanism. Moreover, DHTS-induced apoptosis was reversed by salubrinal, an ER stress inhibitor. Results suggest that DHTS can induce apoptosis of prostate carcinoma cells via induction of ER stress and/or inhibition of proteasome activity, and may have therapeutic potential for prostate cancer patients.

  7. 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.

  8. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  9. A selective phosphodiesterase 10A inhibitor reduces l-dopa-induced dyskinesias in parkinsonian monkeys.

    Science.gov (United States)

    Beck, Goichi; Maehara, Shunsuke; Chang, Phat Ly; Papa, Stella M

    2018-03-06

    Phosphodiesterase 10A is a member of the phosphodiesterase family whose brain expression is restricted to the striatum. Phosphodiesterase 10A regulates cyclic adenosine monophosphate and cyclic guanosine monophosphate, which mediate responses to dopamine receptor activation, and the levels of these cyclic nucleotides are decreased in experimental models of l-dopa-induced dyskinesia. The elevation of cyclic adenosine monophosphate/cyclic guanosine monophosphate levels by phosphodiesterase 10A inhibition may thus be targeted to reduce l-dopa-induced dyskinesia. The present study was aimed at determining the potential antidyskinetic effects of phosphodiesterase 10A inhibitors in a primate model of Parkinson's disease (PD). The experiments performed in this model were also intended to provide translational data for the design of future clinical trials. Five MPTP-treated macaques with advanced parkinsonism and reproducible l-dopa-induced dyskinesia were used. MR1916, a selective phosphodiesterase 10A inhibitor, at doses 0.0015 to 0.05 mg/kg, subcutaneously, or its vehicle (control test) was coadministered with l-dopa methyl ester acutely (predetermined optimal and suboptimal subcutaneous doses) and oral l-dopa chronically as daily treatment for 5 weeks. Standardized scales were used to assess motor disability and l-dopa-induced dyskinesia by blinded examiners. Pharmacokinetics was also examined. MR1916 consistently reduced l-dopa-induced dyskinesia in acute tests of l-dopa optimal and suboptimal doses. Significant effects were present with every MR1916 dose tested, but the most effective was 0.015 mg/kg. None of the MR1916 doses tested affected the antiparkinsonian action of l-dopa at the optimal dose. The anti-l-dopa-induced dyskinesia effect of MR1916 (0.015 mg/kg, subcutaneously) was sustained with chronic administration, indicating that tolerance did not develop over the 5-week treatment. No adverse effects were observed after MR1916 administration acutely or

  10. The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System

    Directory of Open Access Journals (Sweden)

    Stephen R. Armstrong

    2016-12-01

    Full Text Available The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.

  11. House dust mite induces expression of intercellular adhesion molecule-1 in EoL-1 human eosinophilic leukemic cells.

    Science.gov (United States)

    Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn; Shin, Myeong Heon

    2007-10-01

    The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-kappaB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-kappaB and JNK.

  12. The human immunodeficiency virus protease inhibitor ritonavir is potentially active against urological malignancies

    Directory of Open Access Journals (Sweden)

    Sato A

    2015-04-01

    Full Text Available Akinori Sato Department of Urology, National Defense Medical College, Tokorozawa, Japan Abstract: The human immunodeficiency virus protease inhibitor ritonavir has recently been shown to have antineoplastic activity, and its use in urological malignancies is under investigation with an eye toward drug repositioning. Ritonavir is thought to exert its antineoplastic activity by inhibiting multiple signaling pathways, including the Akt and nuclear factor-kappaB pathways. It can increase the amount of unfolded proteins in the cell by inhibiting both the proteasome and heat shock protein 90. Combinations of ritonavir with agents that increase the amount of unfolded proteins, such as proteasome inhibitors, histone deacetylase inhibitors, or heat shock protein 90 inhibitors, therefore, induce endoplasmic reticulum stress cooperatively and thereby kill cancer cells effectively. Ritonavir is also a potent cytochrome P450 3A4 and P-glycoprotein inhibitor, increasing the intracellular concentration of combined drugs by inhibiting their degradation and efflux from cancer cells and thereby enhancing their antineoplastic activity. Furthermore, riotnavir’s antineoplastic activity includes modulation of immune system activity. Therapies using ritonavir are thus an attractive new approach to cancer treatment and, due to their novel mechanisms of action, are expected to be effective against malignancies that are refractory to current treatment strategies. Further investigations using ritonavir are expected to find new uses for clinically available drugs in the treatment of urological malignancies as well as many other types of cancer. Keywords: drug repositioning, novel treatment

  13. Omeprazole increases the efficacy of a soluble epoxide hydrolase inhibitor in a PGE{sub 2} induced pain model

    Energy Technology Data Exchange (ETDEWEB)

    Goswami, Sumanta Kumar; Inceoglu, Bora; Yang, Jun; Wan, Debin; Kodani, Sean D. [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Trindade da Silva, Carlos Antonio [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Department of Genetics and Biochemistry, Federal University of Uberlandia, MG (Brazil); Morisseau, Christophe [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States); Hammock, Bruce D., E-mail: bdhammock@ucdavis.edu [Department of Entomology and Nematology, UC Davis Comprehensive Cancer Center, University of California, Davis, CA (United States)

    2015-12-15

    Epoxyeicosatrienoic acids (EETs) are potent endogenous analgesic metabolites produced from arachidonic acid by cytochrome P450s (P450s). Metabolism of EETs by soluble epoxide hydrolase (sEH) reduces their activity, while their stabilization by sEH inhibition decreases both inflammatory and neuropathic pain. Here, we tested the complementary hypothesis that increasing the level of EETs through induction of P450s by omeprazole (OME), can influence pain related signaling by itself, and potentiate the anti-hyperalgesic effect of sEH inhibitor. Rats were treated with OME (100 mg/kg/day, p.o., 7 days), sEH inhibitor TPPU (3 mg/kg/day, p.o.) and OME (100 mg/kg/day, p.o., 7 days) + TPPU (3 mg/kg/day, p.o., last 3 days of OME dose) dissolved in vehicle PEG400, and their effect on hyperalgesia (increased sensitivity to pain) induced by PGE{sub 2} was monitored. While OME treatment by itself exhibited variable effects on PGE{sub 2} induced hyperalgesia, it strongly potentiated the effect of TPPU in the same assay. The significant decrease in pain with OME + TPPU treatment correlated with the increased levels of EETs in plasma and increased activities of P450 1A1 and P450 1A2 in liver microsomes. The results show that reducing catabolism of EETs with a sEH inhibitor yielded a stronger analgesic effect than increasing generation of EETs by OME, and combination of both yielded the strongest pain reducing effect under the condition of this study. - Highlights: • The soluble epoxide hydrolase (sEH) inhibitor TPPU is anti-hyperalgesic. • Omeprazole potentiates the anti-hyperalgesic actions of TPPU. • This potentiation is associated with increased P450 activity. • The potentiation is associated with an increase in fatty acid epoxide/diol ratio. • Joint use of sEH inhibitors and P450 inducers could result in drug–drug interactions.

  14. 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.

  15. Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer.

    Science.gov (United States)

    Khurana, Namrata; Kim, Hogyoung; Chandra, Partha K; Talwar, Sudha; Sharma, Pankaj; Abdel-Mageed, Asim B; Sikka, Suresh C; Mondal, Debasis

    2017-11-01

    Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (PAR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those expressing AR-V7.

  16. Angiotensin converting enzyme inhibitors mitigate collagen synthesis induced by a single dose of radiation to the whole thorax

    International Nuclear Information System (INIS)

    Kma, L.; Gao, F.; Fish, B.L.; Moulder, J.E.; Jacobs, E.R.; Medhora, M.

    2012-01-01

    Our long-term goal is to use angiotensin converting enzyme (ACE) inhibitors to mitigate the increase in lung collagen synthesis that is induced by irradiation to the lung, which could result from accidental exposure or radiological terrorism. Rats (WAG/RijCmcr) were given a single dose of 13 Gy (dose rate of 1.43 Gy/min) of X-irradiation to the thorax. Three structurally-different ACE inhibitors, captopril, enalapril and fosinopril were provided in drinking water beginning 1 week after irradiation. Rats that survived acute pneumonitis (at 6-12 weeks) were evaluated monthly for synthesis of lung collagen. Other endpoints included breathing rate, wet to dry lung weight ratio, and analysis of lung structure. Treatment with captopril (145-207 mg/m 2 /day) or enalapril (19-28 mg/m 2 /day), but not fosinopril (19-28 mg/m 2 /day), decreased morbidity from acute pneumonitis. Lung collagen in the surviving irradiated rats was increased over that of controls by 7 months after irradiation. This increase in collagen synthesis was not observed in rats treated with any of the three ACE inhibitors. Analysis of the lung morphology at 7 months supports the efficacy of ACE inhibitors against radiation-induced fibrosis. The effectiveness of fosinopril against fibrosis, but not against acute pneumonitis, suggests that pulmonary fibrosis may not be a simple consequence of injury during acute pneumonitis. In summary, three structurally-different ACE inhibitors mitigate the increase in collagen synthesis 7 months following irradiation of the whole thorax and do so, even when therapy is started one week after irradiation. (author)

  17. Regulation of repp86 stability by human Siah2

    International Nuclear Information System (INIS)

    Szczepanowski, Monika; Adam-Klages, Sabine; Kruse, Marie-Luise; Pollmann, Marc; Klapper, Wolfram; Parwaresch, Reza; Heidebrecht, Hans-Juergen

    2007-01-01

    Human repp86 is a nuclear protein that is expressed in a tightly limited period of time during the cell cycle and plays an essential role in its progression. Manipulation of repp86 expression by reduction of endogenous repp86 or overexpression of exogenous repp86 results in cell cycle arrest. We found that repp86 interacts with human Siah2, which is a known mediator for proteasomal degradation. Siah2 failed to interact with repp86 lacking the first 67 N-terminal amino acids. Overexpression of Siah2 reduced endogenous and exogenous repp86 at the protein level without affecting its mRNA, as shown by cotransfection and RT-PCR experiments. Furthermore, MG-132-a specific inhibitor of the proteasome-blocked the degradation of repp86 in Siah2 overexpressing cells. Moreover, transiently transfected Siah2 abrogated the mitotic arrest in repp86 overexpressing cells. Our data show that Siah2 is an important mediator of repp86 protein degradation

  18. 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...... inhibitor. We identified subgroups of putative SUMO2 targets that were either degraded or stabilized by EPOX upon SUMO2 conjugation in response to replication stress. Interestingly, the subgroup of proteins degraded upon SUMO2 conjugation was enriched in proteins playing roles in DNA damage repair...

  19. Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties.

    Science.gov (United States)

    Fuchs, Adrian C D; Maldoner, Lorena; Hipp, Katharina; Hartmann, Marcus D; Martin, Jörg

    2018-01-19

    Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Structural Basis for the Species-Selective Binding of N,C-Capped Dipeptides to the Mycobacterium tuberculosis Proteasome

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Hao-Chi [Van Andel Research Institute, Grand Rapids, Michigan 49503, United States; Singh, Pradeep K.; Fan, Hao; Wang, Rong [NMR; Sukenick, George [NMR; Nathan, Carl; Lin, Gang; Li, Huilin [Van Andel Research Institute, Grand Rapids, Michigan 49503, United States

    2016-12-27

    The Mycobacterium tuberculosis (Mtb) 20S proteasome is vital for the pathogen to survive under nitrosative stress in vitro and to persist in mice. To qualify for drug development, inhibitors targeting Mtb 20S must spare both the human constitutive proteasome (c-20S) and immunoproteasome (i-20S). We recently reported members of a family of noncovalently binding dipeptide proteasome inhibitors that are highly potent and selective for Mtb 20S over human c-20S and i-20S. To understand the structural basis of their potency and selectivity, we have studied the structure–activity relationship of six derivatives and solved their cocrystal structures with Mtb 20S. The dipeptide inhibitors form an antiparallel β-strand with the active site β-strands. Selectivity is conferred by several features of Mtb 20S relative to its mouse counterparts, including a larger S1 pocket, additional hydrogen bonds in the S3 pocket, and hydrophobic interactions in the S4 pocket. Serine-20 and glutamine-22 of Mtb 20S interact with the dipeptides and confer Mtb-specific inhibition over c-20S and i-20S. The Mtb 20S and mammalian i-20S have a serine-27 that interacts strongly with the dipeptides, potentially explaining the higher inhibitory activity of the dipeptides toward i-20S over c-20S. This detailed structural knowledge will aid in optimizing the dipeptides as anti-tuberculosis drugs.

  1. An anti-HIV-1 compound that increases steady-state expression of apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G.

    Science.gov (United States)

    Ejima, Tomohiko; Hirota, Mayuko; Mizukami, Tamio; Otsuka, Masami; Fujita, Mikako

    2011-10-01

    Human apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) 3G (A3G) is an antiviral protein that blocks HIV-1 replication. However, the antiviral activity of A3G is overcome by the HIV-1 protein Vif. This inhibitory function of Vif is related to its ability to degrade A3G in the proteasome. This finding prompted us to examine the activities of 4-(dimethylamino)-2,6-bis[(N-(2-[(2-nitrophenyl)dithio]ethyl)amino)methyl]pyridine (SN-2) and SN-3. We found that 5 µM SN-2 increases the expression of A3G to a level much higher than that observed in the absence of Vif, without affecting the level of Vif expression. The proteasome inhibitor MG-132 increased the level of both A3G and Vif expression. These results demonstrate that A3G is ubiquitinated and degraded in the proteasome by a factor other than Vif, and that SN-2 selectively inhibits these processes. Furthermore, 5 µM SN-2 significantly inhibited the MAGI cell infectivity of wild-type HIV-1. These findings may contribute to the development of a novel anti-HIV-1 drug.

  2. 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.

  3. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

    Energy Technology Data Exchange (ETDEWEB)

    Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Ghazawi, Feras [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); MacPherson, Paul, E-mail: pmacpherson@toh.on.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Division of Infectious Diseases, The Ottawa Hospital General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada)

    2016-11-15

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

  4. HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

    International Nuclear Information System (INIS)

    Sugden, Scott; Ghazawi, Feras; MacPherson, Paul

    2016-01-01

    HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

  5. Misfolded Gβ is recruited to cytoplasmic dynein by Nudel for efficient clearance

    Institute of Scientific and Technical Information of China (English)

    Yihan Wan; Zhenye Yang; Jing Guo; Qiangge Zhang; Liyong Zeng; Wei Song; Yue Xiao; Xueliang Zhu

    2012-01-01

    The Gβγ heterodimer is an important signal transducer.Gβ,however,is prone to misfolding due to its requirement for Gγ and chaperones for proper folding.How cells dispose of misfolded Gβ (mfGβ) is not clear.Here,we showed that mfGβ was able to be polyubiquitinated and subsequently degraded by the proteasome.It was sequestered in aggresomes after the inhibition of the proteasome activity with MG132.Sustained activation of Gβγ signaling further elevated cellular levels of the ubiquitinated Gβ.Moreover,Nudel,a regulator of cytoplasmic dynein,the microtubule minus end-directed motor,directly interacted with both the unubiquitinated and ubiquitinated mfGβ.Increasing the levels of both mfGβ and Nudel promoted the association of Gβ with both Nudel and dynein,resulting in robust aggresome formation in a dynein-dependent manner.Depletion of Nudel by RNAi reduced the dynein-associated mfGβ,impaired the MG132-induced aggresome formation,and markedly prolonged the half-life of nascent Gβ.Therefore,cytosolic mfGβ is recruited to dynein by Nudel and transported to the centrosome for rapid sequestration and degradation.Such a process not only eliminates mfGβ efficiently for the control of protein quality,but may also help to terminate the Gβγ signaling.

  6. Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

    International Nuclear Information System (INIS)

    Geelen, Caroline MM van; Pennarun, Bodvael; Le, Phuong TK; Vries, Elisabeth GE de; Jong, Steven de

    2011-01-01

    rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5). Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies. Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane. SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN-gamma could also increase DR5-mediated apoptosis in SW948

  7. Modulation of TRAIL resistance in colon carcinoma cells: Different contributions of DR4 and DR5

    Directory of Open Access Journals (Sweden)

    de Vries Elisabeth GE

    2011-01-01

    Full Text Available Abstract Background rhTRAIL is a therapeutic agent, derived from the TRAIL cytokine, which induces apoptosis in cancer cells by activating the membrane death receptors 4 and 5 (DR4 and DR5. Here, we investigated each receptor's contribution to rhTRAIL sensitivity and rhTRAIL resistance. We assessed whether agonistic DR4 or DR5 antibodies could be used to circumvent rhTRAIL resistance, alone or in combination with various chemotherapies. Methods Our study was performed in an isogenic model comprised of the SW948 human colon carcinoma cell line and its rhTRAIL resistant sub-line SW948-TR. Effects of rhTRAIL and agonistic DR4/DR5 antibodies on cell viability were measured using MTT assays and identification of morphological changes characteristic of apoptosis, after acridine orange staining. Sensitivity to the different death receptor ligands was stimulated using pretreatment with the cytokine IFN-gamma and the proteasome inhibitor MG-132. To investigate the mechanisms underlying the changes in rhTRAIL sensitivity, alterations in expression levels of targets of interest were measured by Western blot analysis. Co-immunoprecipitation was used to determine the composition of the death-inducing signalling complex at the cell membrane. Results SW948 cells were sensitive to all three of the DR-targeting agents tested, although the agonistic DR5 antibody induced only weak caspase 8 cleavage and limited apoptosis. Surprisingly, agonistic DR4 and DR5 antibodies induced equivalent DISC formation and caspase 8 cleavage at the level of their individual receptors, suggesting impairment of further caspase 8 processing upon DR5 stimulation. SW948-TR cells were cross-resistant to all DR-targeting agents as a result of decreased caspase 8 expression levels. Caspase 8 protein expression was restored by MG-132 and IFN-gamma pretreatment, which also re-established sensitivity to rhTRAIL and agonistic DR4 antibody in SW948-TR. Surprisingly, MG-132 but not IFN

  8. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    Science.gov (United States)

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  10. Dietary Inulin Fibers Prevent Proton-Pump Inhibitor (PPI)-Induced Hypocalcemia in Mice.

    Science.gov (United States)

    Hess, Mark W; de Baaij, Jeroen H F; Gommers, Lisanne M M; Hoenderop, Joost G J; Bindels, René J M

    2015-01-01

    Proton-pump inhibitor-induced hypomagnesemia (PPIH) is the most recognized side effect of proton-pump inhibitors (PPIs). Additionally, PPIH is associated with hypocalcemia and hypokalemia. It is hypothesized that PPIs reduce epithelial proton secretion and thereby increase the pH in the colon, which may explain the reduced absorption of and Mg2+ and Ca2+. Fermentation of dietary oligofructose-enriched inulin fibers by the microflora leads to acidification of the intestinal lumen and by this enhances mineral uptake. This study aimed, therefore, to improve mineral absorption by application of dietary inulin to counteract PPIH. Here, C57BL/J6 mice were supplemented with omeprazole and/or inulin. Subsequently, Mg2+ and Ca2+ homeostasis was assessed by means of serum, urine and fecal electrolyte measurements. Moreover, the mRNA levels of magnesiotropic and calciotropic genes were examined in the large intestine and kidney by real-time PCR. Treatment with omeprazole significantly reduced serum Mg2+ and Ca2+ levels. However, concomitant addition of dietary inulin fibers normalized serum Ca2+ but not serum Mg2+ concentrations. Inulin abolished enhanced expression of Trpv6 and S100g in the colon by omeprazole. Additionally, intestinal and renal mRNA levels of the Trpm6 gene were reduced after inulin intake. This study suggests that dietary inulin counteracts reduced intestinal Ca2+ absorption upon PPI treatment. In contrast, inulin did not increase intestinal absorption of Mg2+ sufficiently to recover serum Mg2+. The clinical potential of dietary inulin treatment should be the subject of future studies.

  11. Preventative and therapeutic effects of a GABA transporter 1 inhibitor administered systemically in a mouse model of paclitaxel-induced neuropathic pain

    Directory of Open Access Journals (Sweden)

    Willias Masocha

    2016-12-01

    Full Text Available Background There is a dearth of drugs to manage a dose-limiting painful peripheral neuropathy induced by paclitaxel in some patients during the treatment of cancer. Gamma-aminobutyric acid transporter-1 (GAT-1 whose expression is increased in the brain and spinal cord during paclitaxel-induced neuropathic pain (PINP might be a potential therapeutic target for managing PINP. Thus, our aim was to evaluate if systemic administration of a GAT-1 inhibitor ameliorates PINP. Methods The reaction latency to thermal stimuli (hot plate test; at 55 °C and cold stimuli (cold plate test; at 4 °C of female BALB/c mice was recorded before and after intraperitoneal treatment with paclitaxel, its vehicle, and/or a selective GAT-1 inhibitor NO-711. The effects of NO-711 on motor coordination were evaluated using the rotarod test at a constant speed of 4 rpm or accelerating mode from 4 rpm to 40 rpm over 5 min. Results The coadministration of paclitaxel with NO-711 3 mg/kg prevented the development of paclitaxel-induced thermal hyperalgesia and cold allodynia at day 7 after drug treatment. NO-711 at 3 mg/kg produced antihyperalgesic activity up to 1 h and antiallodynic activity up to 2 h in mice with established paclitaxel-induced thermal hyperalgesia and cold allodynia. No motor deficits were observed with NO-711 at a dose of 3 mg/kg, whereas a higher dose 5 mg/kg caused motor impairment and reduced mean time spent on the rotarod at a constant speed of 4 rpm. However, at a rotarod accelerating mode from 4 rpm to 40 rpm over 5 min, NO-711 3 mg/kg caused motor impairment up to 1 h, but had recovered by 2 h. Conclusions These results show that systemic administration of the GAT-1 inhibitor NO-711 has preventative and therapeutic activity against paclitaxel-induced thermal hyperalgesia and cold allodynia. NO-711’s antiallodynic effects, but not antihyperalgesic effects, were independent of its motor impairment/sedation properties. Thus, low doses of GAT-1

  12. Mitigation of radiation-induced lung fibrosis by angiotensin converting enzyme inhibitors

    International Nuclear Information System (INIS)

    Kma, Lakhan; Gao, Feng; Jacobs, Elizabeth R.; Medhora, Meetha; Fish, Brian L.; Moulder, John E.

    2014-01-01

    The aim of this study was to test the mitigating potential of angiotensin converting enzyme inhibitors (ACEi) against radiation-induced pulmonary fibrosis, which could result from accidental exposure or radiological terrorism. Rats (WAG/RijCmcr) were exposed to a single dose of 13 Gy of X-irradiation to the whole thorax, at the dose rate of 1.43 Gy/min. Three structurally-different ACEi's, captopril (145-207 mg/m 2 /day), enalapril (19-28 mg/m 2 /day) and fosinopril (19-28 mg/m 2 /day) were administered in drinking water beginning 1 week after whole thoracic irradiation. Rats that survived acute pneumonitis (6-12 weeks) were accessed monthly after irradiation for the effects on lung structure and function. Endpoints included breathing rate, wet:dry weight ratio, collagen content and histolopathological studies. Treatment with captopril or enalapril, but not fosinopril, beginning 1 week after 13 Gy X-irradiation improved survival of rats. Mortality of 30-35% was observed with administration of captopril or enalapril compared to 70% for 13 Gy alone. All three ACEi's attenuated radiation-induced lung fibrosis at 7 months after irradiation based on histological indices and measurement of lung collagen. After whole-thoracic irradiation, ACEi's mitigate radiation induced pulmonary fibrosis based on histological and biochemical endpoints. These treatments were effective even when administration was not started until one week after irradiation. Our findings support the therapeutic potential of ACEi's against chronic radiation induced lung injury. (author)

  13. 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.

  14. Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors

    International Nuclear Information System (INIS)

    Chintharlapalli, Sudhakar; Papineni, Sabitha; Lei, Ping; Pathi, Satya; Safe, Stephen

    2011-01-01

    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. 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. 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. 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

  15. Impairment of the Ubiquitin-Proteasome Pathway by Methyl N-(6-Phenylsulfanyl-1H-benzimidazol-2-yl)carbamate Leads to a Potent Cytotoxic Effect in Tumor Cells

    Science.gov (United States)

    Dogra, Nilambra; Mukhopadhyay, Tapas

    2012-01-01

    In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and IκBα were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1α, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells. PMID:22745125

  16. Reduced Airway Hyperresponsiveness by Phosphodiesterase 3 and 4 Inhibitors in Guinea-Pigs

    Directory of Open Access Journals (Sweden)

    Nöella Germain

    1999-01-01

    Full Text Available The aim of the present study was to compare the effects of selective phosphodiesterase (PDE 3, 4 and 5 inhibitors on antigen-induced airway hyperresponsiveness in sensitized guinea-pigs. When the sensitized guinea-pigs were orally pre-treated with the selective PDE4 inhibitor, Ro 20-1724 (30 mg/kg, and studied 48 h after OA, a significant reduction (p<0.01 of the leftward shift of the dose-response curve to ACh was noted, whereas it was ineffective at the lower dose (10 mg/kg. Administration of the selective PDE3 inhibitor, milrinone (30 mg/kg also elicited a significant reduction (p<0.01 of the airway hyperresponsiveness, whereas the PDE5 inhibitor zaprinast (30 mg/kg was ineffective. These results show that both PDE3 and PDE4 inhibitors are able to inhibit the antigen-induced airway hyperresponsiveness in sensitized guinea-pigs and support the potential utility of selective PDE inhibitors in the treatment of asthma.

  17. Impact of clinically tested NEP/ACE inhibitors on tumor uptake of [(111)In-DOTA]MG11-first estimates for clinical translation.

    Science.gov (United States)

    Kaloudi, Aikaterini; Nock, Berthold A; Lymperis, Emmanouil; Valkema, Roelf; Krenning, Eric P; de Jong, Marion; Maina, Theodosia

    2016-12-01

    We have recently shown that treatment of mice with the neutral endopeptidase (NEP) inhibitor phosphoramidon (PA) improves the bioavailability and tumor uptake of biodegradable radiopeptides. For the truncated gastrin radiotracer [(111)In-DOTA]MG11 ([(DOTA)DGlu(10)]gastrin(10-17)), this method led to impressively high tumor-to-kidney ratios. Translation of this concept in the clinic requires the use of certified NEP inhibitors, such as thiorphan (TO) and its orally administered prodrug racecadotril (Race). Besides NEP, angiotensin-converting enzyme (ACE) has also been implicated in the catabolism of gastrin analogs. In the present study, we first compared the effects induced by NEP inhibition (using PA, TO, or Race) and/or by ACE inhibition (using lisinopril, Lis) on the biodistribution profile of [(111)In-DOTA]MG11 in mice. In addition, we compared the efficacy of PA and TO at different administered doses to enhance tumor uptake. [(111)In-DOTA]MG11 was coinjected with (a) vehicle, (b) PA (300 μg), (c) TO (150 μg), (d) Lis (100 μg), (e) PA (300 μg) plus Lis (100 μg), or (f) 30-40 min after intraperitoneal (ip) injection of Race (3 mg) in SCID mice bearing AR42J xenografts. In addition, [(111)In-DOTA]MG11 was coinjected with vehicle, or with progressively increasing amounts of PA (3, 30, or 300 μg) or TO (1.5, 15, and 150 μg) in SCID mice bearing twin A431-CCK2R(+/-) tumors. In all above cases, biodistribution was conducted at 4 h postinjection (pi). During NEP inhibition, the uptake of [(111)In-DOTA]MG11 in the AR42J tumors impressively increased from 1.8 ± 1.0 % ID/g (controls) to 15.3 ± 4.7 % ID/g (PA) and 12.3 ± 3.6 % ID/g (TO), while with Race tumor values reached 6.8 ± 2.8 % ID/g. Conversely, Lis had no effect on tumor uptake and no additive effect when coinjected with PA. During the dose dependence study in mice, PA turned out to be more efficacious in enhancing tumor uptake of [(111)In-DOTA]MG11 in the CCK2R

  18. Exposure to histone deacetylase inhibitors during Pavlovian conditioning enhances subsequent cue-induced reinstatement of operant behavior.

    Science.gov (United States)

    Ploense, Kyle L; Kerstetter, Kerry A; Wade, Matthew A; Woodward, Nicholas C; Maliniak, Dan; Reyes, Michael; Uchizono, Russell S; Bredy, Timothy W; Kippin, Tod E

    2013-06-01

    Histone deacetylase inhibitors (HDACIs) strengthen memory following fear conditioning and cocaine-induced conditioned place preference. Here, we examined the effects of two nonspecific HDACIs, valproic acid (VPA) and sodium butyrate (NaB), on appetitive learning measured by conditioned stimulus (CS)-induced reinstatement of operant responding. Rats were trained to lever press for food reinforcement and then injected with VPA (50-200 mg/kg, i.p.), NaB (250-1000 mg/kg, i.p.), or saline vehicle (1.0 ml/kg), 2 h before receiving pairings of noncontingent presentation of food pellets preceded by a tone+light cue CS. Rats next underwent extinction of operant responding followed by response-contingent re-exposure to the CS. Rats receiving VPA (100 mg/kg) or NaB (1000 mg/kg) before conditioning displayed significantly higher cue-induced reinstatement than did saline controls. Rats that received either vehicle or VPA (100 mg/kg) before a conditioning session with a randomized relation between presentation of food pellets and the CS failed to show subsequent cue-induced reinstatement with no difference between the two groups. These findings indicate that, under certain contexts, HDACIs strengthen memory formation by specifically increasing the associative strength of the CS, not through an increasing motivation to seek reinforcement. © 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.

  19. Long Non-coding RNA, PANDA, Contributes to the Stabilization of p53 Tumor Suppressor Protein.

    Science.gov (United States)

    Kotake, Yojiro; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Niida, Hiroyuki; Naemura, Madoka; Kitagawa, Masatoshi

    2016-04-01

    P21-associated noncoding RNA DNA damage-activated (PANDA) is induced in response to DNA damage and represses apoptosis by inhibiting the function of nuclear transcription factor Y subunit alpha (NF-YA) transcription factor. Herein, we report that PANDA affects regulation of p53 tumor-suppressor protein. U2OS cells were transfected with PANDA siRNAs. At 72 h post-transfection, cells were subjected to immunoblotting and quantitative reverse transcription-polymerase chain reaction. Depletion of PANDA was associated with decreased levels of p53 protein, but not p53 mRNA. The stability of p53 protein was markedly reduced by PANDA silencing. Degradation of p53 protein by silencing PANDA was prevented by treatment of MG132, a proteasome inhibitor. Moreover, depletion of PANDA prevented accumulation of p53 protein, as a result of DNA damage, induced by the genotoxic agent etoposide. These results suggest that PANDA stabilizes p53 protein in response to DNA damage, and provide new insight into the regulatory mechanisms of p53. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  20. SOCS-1 Localizes to the Microtubule Organizing Complex-Associated 20S Proteasome

    OpenAIRE

    Vuong, Bao Q.; Arenzana, Teresita L.; Showalter, Brian M.; Losman, Julie; Chen, X. Peter; Mostecki, Justin; Banks, Alexander S.; Limnander, Andre; Fernandes, Neil; Rothman, Paul B.

    2005-01-01

    The regulation of cytokine signaling is critical for controlling cellular proliferation and activation during an immune response. SOCS-1 is a potent inhibitor of Jak kinase activity and of signaling initiated by several cytokines. SOCS-1 protein levels are tightly regulated, and recent data suggest that SOCS-1 may regulate the protein levels of some signaling proteins by the ubiquitin proteasome pathway; however, the cellular mechanism by which SOCS-1 directs proteins for degradation is unkno...

  1. Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

    Science.gov (United States)

    Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

    2011-10-21

    Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

  2. Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

    Science.gov (United States)

    Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

    2011-01-01

    Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

  3. Lifetime measurements and decay spectroscopy of 132I

    Directory of Open Access Journals (Sweden)

    Bhattacharyya S.

    2014-03-01

    Full Text Available The low-lying states of odd-odd 132I, the 3p-3h nucleus with respect to the doubly magic 132Sn, have been characterized from decay spectroscopy. The neutron rich Iodine and Tellurium isotopes have been produced as fission product of alpha-induced fission of 235U and radiochemically separated. The life-time of the first excited state of 132I have been precisely measured using LaBr3(Ce scintillators from the decay of 132Te. The IT decay of the high spin isomer (8- in 132I has been measured with a Low Energy Photon Spectrometer (LEPS of segmented planar Ge detector.

  4. Selective Inducible Nitric Oxide Synthase Inhibitor Reversed Zinc Chloride-Induced Spatial Memory Impairment via Increasing Cholinergic Marker Expression.

    Science.gov (United States)

    Tabrizian, Kaveh; Azami, Kian; Belaran, Maryam; Soodi, Maliheh; Abdi, Khosrou; Fanoudi, Sahar; Sanati, Mehdi; Mottaghi Dastjerdi, Negar; Soltany Rezaee-Rad, Mohammad; Sharifzadeh, Mohammad

    2016-10-01

    Zinc, an essential micronutrient and biochemical element of the human body, plays structural, catalytic, and regulatory roles in numerous physiological functions. In the current study, the effects of a pretraining oral administration of zinc chloride (10, 25, and 50 mg/kg) for 14 consecutive days and post-training bilateral intra-hippocampal infusion of 1400W as a selective inducible nitric oxide synthase (iNOS) inhibitor (10, 50, and 100 μM/side), alone and in combination, on the spatial memory retention in Morris water maze (MWM) were investigated. Animals were trained for 4 days and tested 48 h after completion of training. Also, the molecular effects of these compounds on the expression of choline acetyltransferase (ChAT), as a cholinergic marker in the CA1 region of the hippocampus and medial septal area (MSA), were evaluated. Behavioral and molecular findings of this study showed that a 2-week oral administration of zinc chloride (50 mg/kg) impaired spatial memory retention in MWM and decreased ChAT expression. Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400W revealed a significant increase in ChAT immunoreactivity. Furthermore, post-training bilateral intra-hippocampal infusion of 1400W into the CA1 region of the hippocampus reversed zinc chloride-induced spatial memory impairment in MWM and significantly increased ChAT expression in comparison with zinc chloride-treated animals. Taken together, these results emphasize the role of selective iNOS inhibitors in reversing zinc chloride-induced spatial memory deficits via modulation of cholinergic marker expression.

  5. Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

    Science.gov (United States)

    Crow, Marni S; Cristea, Ileana M

    2017-04-01

    The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral

  6. β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS.

    Directory of Open Access Journals (Sweden)

    Zhiying Yu

    Full Text Available β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl-β-elemene (DX1, 13-(cis-3,5-dimethyl-1-piperazinyl-β-elemene (DX2, 13-(4-ethyl-1-piperazinyl-β-elemene (DX3, 13-(4-isopropyl-1-piperazinyl-β-elemene (DX4 and 13-piperazinyl-β-elemene (DX5, were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H(2O(2, a decrease of mitochondrial membrane potential (MMP, and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H(2O(2 production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H(2O(2-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP. The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H(2O(2 which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways.

  7. Impaired proteasome function in sporadic amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kabashi, Edor; Agar, Jeffrey N; Strong, Michael J; Durham, Heather D

    2012-06-01

    Abstract The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS.

  8. Proteasomal and lysosomal protein degradation and heart disease.

    Science.gov (United States)

    Wang, Xuejun; Robbins, Jeffrey

    2014-06-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". Copyright © 2013 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wei [Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, Xi' an 710032 (China); Fu, Jianfang [Department of Endocrinology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Shun [Reproductive Medicine Center, Department of Gynecology and Obstetrics, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Zhao, Jie [Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, Xi' an 710032 (China); Xie, Nianlin, E-mail: xienianlin@126.com [Department of Thoracic Surgery, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Cai, Guoqing, E-mail: firstchair@fmmu.edu.cn [Department of Gynaecology and Obstetrics, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China)

    2015-06-01

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

  11. Protective effects of a squalene synthase inhibitor, lapaquistat acetate (TAK-475), on statin-induced myotoxicity in guinea pigs

    International Nuclear Information System (INIS)

    Nishimoto, Tomoyuki; Ishikawa, Eiichiro; Anayama, Hisashi; Hamajyo, Hitomi; Nagai, Hirofumi; Hirakata, Masao; Tozawa, Ryuichi

    2007-01-01

    High-dose statin treatment has been recommended as a primary strategy for aggressive reduction of LDL cholesterol levels and protection against coronary artery disease. The effectiveness of high-dose statins may be limited by their potential for myotoxic side effects. There is currently little known about the molecular mechanisms of statin-induced myotoxicity. Previously we showed that T-91485, an active metabolite of the squalene synthase inhibitor lapaquistat acetate (lapaquistat: a previous name is TAK-475), attenuated statin-induced cytotoxicity in human skeletal muscle cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A. Biochem. Pharmacol. 66, 2133-2139]. In the current study, we investigated the effects of lapaquistat administration on statin-induced myotoxicity in vivo. Guinea pigs were treated with either high-dose cerivastatin (1 mg/kg) or cerivastatin together with lapaquistat (30 mg/kg) for 14 days. Treatment with cerivastatin alone decreased plasma cholesterol levels by 45% and increased creatine kinase (CK) levels by more than 10-fold (a marker of myotoxicity). The plasma CK levels positively correlated with the severity of skeletal muscle lesions as assessed by histopathology. Co-administration of lapaquistat almost completely prevented the cerivastatin-induced myotoxicity. Administration of mevalonolactone (100 mg/kg b.i.d.) prevented the cerivastatin-induced myotoxicity, confirming that this effect is directly related to HMG-CoA reductase inhibition. These results strongly suggest that cerivastatin-induced myotoxicity is due to depletion of mevalonate derived isoprenoids. In addition, squalene synthase inhibition could potentially be used clinically to prevent statin-induced myopathy

  12. The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

    DEFF Research Database (Denmark)

    Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

    2017-01-01

    -induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p...

  13. Inhibitors of Deubiquitinating Enzymes Block HIV-1 Replication and Augment the Presentation of Gag-Derived MHC-I Epitopes.

    Science.gov (United States)

    Setz, Christian; Friedrich, Melanie; Rauch, Pia; Fraedrich, Kirsten; Matthaei, Alina; Traxdorf, Maximilian; Schubert, Ulrich

    2017-08-12

    In recent years it has been well established that two major constituent parts of the ubiquitin proteasome system (UPS)-the proteasome holoenzymes and a number of ubiquitin ligases-play a crucial role, not only in virus replication but also in the regulation of the immunogenicity of human immunodeficiency virus type 1 (HIV-1). However, the role in HIV-1 replication of the third major component, the deubiquitinating enzymes (DUBs), has remained largely unknown. In this study, we show that the DUB-inhibitors (DIs) P22077 and PR-619, specific for the DUBs USP7 and USP47, impair Gag processing and thereby reduce the infectivity of released virions without affecting viral protease activity. Furthermore, the replication capacity of X4- and R5-tropic HIV-1 NL4-3 in human lymphatic tissue is decreased upon treatment with these inhibitors without affecting cell viability. Most strikingly, combinatory treatment with DIs and proteasome inhibitors synergistically blocks virus replication at concentrations where mono-treatment was ineffective, indicating that DIs can boost the therapeutic effect of proteasome inhibitors. In addition, P22077 and PR-619 increase the polyubiquitination of Gag and thus its entry into the UPS and the major histocompatibility complex (MHC)-I pathway. In summary, our data point towards a model in which specific inhibitors of DUBs not only interfere with virus spread but also increase the immune recognition of HIV-1 expressing cells.

  14. 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.

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

    Science.gov (United States)

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

    2016-12-06

    Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. 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.

  17. Exposure in utero to 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) impairs sperm function and alters testicular apoptosis-related gene expression in rat offspring

    International Nuclear Information System (INIS)

    Hsu, P.-C.; Pan, M.-H.; Li, L.-A.; Chen, C.-J.; Tsai, S.-S.; Guo, Y.L.

    2007-01-01

    Toxicity of the polychlorinated biphenyls (PCBs) depends on their molecular structure. Mechanisms by prenatal exposure to a non-dioxin-like PCB, 2,2',3,4',5',6-hexachlorobiphenyl (PCB 132) that may act on reproductive pathways in male offspring are relatively unknown. The purpose was to determine whether epididymal sperm function and expression of apoptosis-related genes were induced or inhibited by prenatal exposure to PCB 132. Pregnant rats were treated with a single dose of PCB 132 at 1 or 10 mg/kg on gestational day 15. Male offspring were killed and the epididymal sperm counts, motility, velocity, reactive oxygen species (ROS) generation, sperm-oocyte penetration rate (SOPR), testicular histopathology, apoptosis-related gene expression and caspase activation were assessed on postnatal day 84. Prenatal exposure to PCB 132 with a single dose of 1 or 10 mg/kg decreased cauda epididymal weight, epididymal sperm count and motile epididymal sperm count in adult offspring. The spermatozoa of PCB 132-exposed offspring produced significantly higher levels of ROS than the controls; ROS induction and SOPR reduction were dose-related. In the low-dose PCB 132 group, p53 was significantly induced and caspase-3 was inhibited. In the high-dose group, activation of caspase-3 and -9 was significantly increased, while the expressions of Fas, Bax, bcl-2, and p53 genes were significantly decreased. Gene expression and caspase activation data may provide insight into the mechanisms by which exposure to low-dose or high-dose PCB 132 affects reproduction in male offspring in rats. Because the doses of PCB 132 administered to the dams were approximately 625-fold in low-dose group and 6250-fold higher in high-dose group than the concentration in human tissue levels, the concentrations are not biologically or environmentally relevant. Further studies using environmentally relevant doses are needed for hazard identification

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

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    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.

  19. Antiatherogenic effect of quercetin is mediated by proteasome inhibition in the aorta and circulating leukocytes.

    Science.gov (United States)

    Pashevin, Denis A; Tumanovska, Lesya V; Dosenko, Victor E; Nagibin, Vasyl S; Gurianova, Veronika L; Moibenko, Alexey A

    2011-01-01

    Quercetin, a plant-derived flavonoid, has attracted considerable attention as promising compound for heart disease prevention and therapy. It has been linked to decreased mortality from heart disease and decreased incidence of stroke. Here, we report new data showing the angioprotective properties of quercetin mediated by its effect on proteasomal proteolysis. This study was designed to investigate the ability of quercetin to modulate proteasomal activity in a rabbit model of cholesterol-induced atherosclerosis. First, we show proteasomal trypsin-like (TL) activity increased up to 2.4-fold, chymotrypsin-like (CTL) activity increased by up to 43% and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activity increased by up to 10% after 8 weeks of a cholesterol-rich diet. A single intravenous injection of the water-soluble form of quercetin (Corvitin) significantly decreased proteasomal TL activity 1.85-fold in monocytes, and decreased the CTL and PGPH activities more than 2-fold in polymorphonuclear leukocytes (PMNL) after 2 h. Prolonged administration (1 month) of Corvitin to animals following a cholesterol-rich diet significantly decreased all types of proteolytic proteasome activities both in tissues and in circulating leukocytes and was associated with the reduction of atherosclerotic lesion areas in the aorta. Additionally, the pharmacological form of quercetin (Quertin) was shown to have an antiatherogenic effect and an ability to inhibit proteasome activities.

  20. Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

    Science.gov (United States)

    Steely, Andrea M; Willoughby, Jamin A; Sundar, Shyam N; Aivaliotis, Vasiliki I; Firestone, Gary L

    2017-10-01

    Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

  1. Selinexor (KPT-330) Induces Tumor Suppression through Nuclear Sequestration of IκB and Downregulation of Survivin.

    Science.gov (United States)

    Nair, Jayasree S; Musi, Elgilda; Schwartz, Gary K

    2017-08-01

    Purpose: Selinexor, a small molecule that inhibits nuclear export protein XPO1, has demonstrated efficacy in solid tumors and hematologic malignancies with the evidence of clinical activity in sarcoma as a single agent. Treatment options available are very few, and hence the need to identify novel targets and strategic therapies is of utmost importance. Experimental Design: The mechanistic effects of selinexor in sarcomas as a monotherapy and in combination with proteasome inhibitor, carfilzomib, across a panel of cell lines in vitro and few in xenograft mouse models were investigated. Results: Selinexor induced IκB nuclear localization as a single agent, and the effect was enhanced by stabilization of IκB when pretreated with the proteasome inhibitor carfilzomib. This stabilization and retention of IκB in the nucleus resulted in inhibition of NFκB and transcriptional suppression of the critical antiapoptotic protein, survivin. Treatment of carfilzomib followed by selinexor caused selinexor-sensitive and selinexor-resistant cell lines to be more sensitive to selinexor as determined by an increase in apoptosis. This was successfully demonstrated in the MPNST xenograft model with enhanced tumor suppression. Conclusions: The subcellular distributions of IκB and NFκB are indicative of carcinogenesis. Inhibition of XPO1 results in intranuclear retention of IκB, which inhibits NFκB and thereby provides a novel mechanism for drug therapy in sarcoma. This effect can be further enhanced in relatively selinexor-resistant sarcoma cell lines by pretreatment with the proteasome inhibitor carfilzomib. Because of these results, a human clinical trial with selinexor in combination with a proteasome inhibitor is planned for the treatment of sarcoma. Clin Cancer Res; 23(15); 4301-11. ©2017 AACR . ©2017 American Association for Cancer Research.

  2. 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.

  3. Modulation of oxazolone-induced hypersensitivity in mice by selective PDE inhibitors

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    I. Moodley

    1995-01-01

    Full Text Available The effects of PDE inhibitors on oxazolone-induced contact hypersensitivity (CS were studied in mice. Rolipram, Ro 20-1724 and theophylline dose dependently inhibited CS but none caused >53% inhibition. ED30 values at 24 h before challenge for rolipram, Ro 20-1724 and theophylline were 2.1, 5.4 and 30.4 mg/kg, p.o., respectively. Milrinone and SKF 94836 at 30 mg/kg caused a small, but significant inhibition of 13% and 18%, respectively, although the inhibition (8% caused by zaprinast was not significant. Betamethasone (10 mg/kg, p.o. caused a marked inhibition (80% as did indomethacin (65% at 5 mg/kg, p.o.. Rolipram and Ro 20-1724 inhibited proliferation of mouse lymphoblasts with IC50 values of 0.08 μM and 0.83 μM, respectively. In contrast, zaprinast caused only a weak inhibition (IC50 = 119 μM of lymphocyte proliferation, whereas SKF 94836 and theophylline failed to cause any significant inhibition at 100 μM (26% and 2%, respectively. These findings suggest that PDE IV isozymes play a principal role in mediating CS by inhibiting lymphocyte activation.

  4. Inhibition of Rac1 activity induces G1/S phase arrest through the GSK3/cyclin D1 pathway in human cancer cells.

    Science.gov (United States)

    Liu, Linna; Zhang, Hongmei; Shi, Lei; Zhang, Wenjuan; Yuan, Juanli; Chen, Xiang; Liu, Juanjuan; Zhang, Yan; Wang, Zhipeng

    2014-10-01

    Rac1 has been shown to regulate the cell cycle in cancer cells. Yet, the related mechanism remains unclear. Thus, the present study aimed to investigate the mechanism involved in the regulation of G1/S phase transition by Rac1 in cancer cells. Inhibition of Rac1 by inhibitor NSC23766 induced G1/S phase arrest and inhibited the proliferation of A431, SW480 and U2-OS cells. Suppression of GSK3 by shRNA partially rescued G1/S phase arrest and inhibition of proliferation. Incubation of cells with NSC23766 reduced p-AKT and inactivated p-GSK3α and p-GSK3β, increased p-cyclin D1 expression and decreased the level of cyclin D1 protein. Consequently, cyclin D1 targeting transcriptional factor E2F1 expression, which promotes G1 to S phase transition, was also reduced. In contrast, constitutive active Rac1 resulted in increased p-AKT and inactivated p-GSK3α and p-GSK3β, decreased p-cyclin D1 expression and enhanced levels of cyclin D1 and E2F1 expression. Moreover, suppression of GSK3 did not alter p-AKT or Rac1 activity, but decreased p-cyclin D1 and increased total cyclin D1 protein. However, neither Rac1 nor GSK3 inhibition altered cyclin D1 at the RNA level. Moreover, after inhibition of Rac1 or GSK3 following proteasome inhibitor MG132 treatment, cyclin D1 expression at the protein level remained constant, indicating that Rac1 and GSK3 may regulate cyclin D1 turnover through phosphorylation and degradation. Therefore, our findings suggest that inhibition of Rac1 induces cell cycle G1/S arrest in cancer cells by regulation of the GSK3/cyclin D1 pathway.

  5. Chaperoning Proteins for Destruction: Diverse Roles of Hsp70 Chaperones and their Co-Chaperones in Targeting Misfolded Proteins to the Proteasome

    Directory of Open Access Journals (Sweden)

    Ayala Shiber

    2014-07-01

    Full Text Available Molecular chaperones were originally discovered as heat shock-induced proteins that facilitate proper folding of proteins with non-native conformations. While the function of chaperones in protein folding has been well documented over the last four decades, more recent studies have shown that chaperones are also necessary for the clearance of terminally misfolded proteins by the Ub-proteasome system. In this capacity, chaperones protect misfolded degradation substrates from spontaneous aggregation, facilitate their recognition by the Ub ligation machinery and finally shuttle the ubiquitylated substrates to the proteasome. The physiological importance of these functions is manifested by inefficient proteasomal degradation and the accumulation of protein aggregates during ageing or in certain neurodegenerative diseases, when chaperone levels decline. In this review, we focus on the diverse roles of stress-induced chaperones in targeting misfolded proteins to the proteasome and the consequences of their compromised activity. We further discuss the implications of these findings to the identification of new therapeutic targets for the treatment of amyloid diseases.

  6. Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease

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    Tingting Chen

    2017-11-01

    Full Text Available Evidence continues to accumulate that pesticides are the leading candidates of environmental toxins that may contribute to the pathogenesis of Parkinson’s disease. The mechanisms, however, remain largely unclear. According to epidemiological studies, we selected nine representative pesticides (paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate, tebufenpyrad, trichlorphon and carbaryl which are commonly used in China and detected the effects of the pesticides on mitochondria and ubiquitin-proteasome system (UPS function. Our results reveal that all the nine studied pesticides induce morphological changes of mitochondria at low concentrations. Paraquat, rotenone, chlorpyrifos, pendimethalin, endosulfan, fenpyroximate and tebufenpyrad induced mitochondria fragmentation. Furthermore, some of them (paraquat, rotenone, chlorpyrifos, fenpyroximate and tebufenpyrad caused a significant dose-dependent decrease of intracellular ATP. Interestingly, these pesticides which induce mitochondria dysfunction also inhibit 26S and 20S proteasome activity. However, two out of the nine pesticides, namely trichlorphon and carbaryl, were found not to cause mitochondrial fragmentation or functional damage, nor inhibit the activity of the proteasome, which provides significant guidance for selection of pesticides in China. Moreover, our results demonstrate a potential link between inhibition of mitochondria and the UPS, and pesticide-induced Parkinsonism.

  7. Tofacitinib for Psoriatic Arthritis in Patients with an Inadequate Response to TNF Inhibitors.

    Science.gov (United States)

    Gladman, Dafna; Rigby, William; Azevedo, Valderilio F; Behrens, Frank; Blanco, Ricardo; Kaszuba, Andrzej; Kudlacz, Elizabeth; Wang, Cunshan; Menon, Sujatha; Hendrikx, Thijs; Kanik, Keith S

    2017-10-19

    Tofacitinib is an oral Janus kinase inhibitor that is under investigation for the treatment of psoriatic arthritis. We evaluated tofacitinib in patients with active psoriatic arthritis who had previously had an inadequate response to tumor necrosis factor (TNF) inhibitors. In this 6-month randomized, placebo-controlled, double-blind, phase 3 trial, we randomly assigned 395 patients, in a 2:2:1:1 ratio, to four regimens: 5 mg of tofacitinib administered orally twice daily (132 patients); 10 mg of tofacitinib twice daily (132 patients); placebo, with a switch to 5 mg of tofacitinib twice daily at 3 months (66 patients); or placebo, with a switch to 10 mg of tofacitinib twice daily at 3 months (65 patients). Data from the patients who received placebo during the first 3 months of the trial were pooled. The primary end points were the percentage of patients who had at least 20% improvement according to the criteria of the American College of Rheumatology (ACR20 response) and the change from baseline score on the Health Assessment Questionnaire-Disability Index (HAQ-DI; scores range from 0 to 3, with higher scores indicating greater disability) at the month 3 analysis. At 3 months, the rates of ACR20 response were 50% with the 5-mg dose of tofacitinib and 47% with the 10-mg dose, as compared with 24% with placebo (Ptofacitinib continuously and in 6% who received the 10-mg dose continuously. Over the course of 6 months, there were four serious infections, three herpes zoster infections, one myocardial infarction, and one ischemic stroke among the patients who received tofacitinib continuously. Elevations of aspartate and alanine aminotransferase concentrations of three or more times the upper limit of the normal range occurred in more patients who received tofacitinib continuously than in patients who received placebo followed by tofacitinib. In this trial involving patients with active psoriatic arthritis who had had an inadequate response to TNF inhibitors, tofacitinib

  8. Proteasomes in the archaea: from structure to function.

    Science.gov (United States)

    Maupin-Furlow, J A; Wilson, H L; Kaczowka, S J; Ou, M S

    2000-09-01

    Survival of cells is critically dependent on their ability to rapidly adapt to changes in the natural environment no matter how 'extreme'the habitat. An interplay between protein folding and hydrolysis is emerging as a central mechanism for stress survival and proper cell function. In eucaryotic cells, most proteins destined for destruction are covalently modified by the ubiquitin-system and then degraded in an energy-dependent mechanism by the 26S proteasome, a multicatalytic protease. The 26S proteasome is composed of a 20S proteolytic core and 19S cap (PA700) regulator which includes six AAA+ ATPase subunits. Related AAA+ proteins and 20S proteasomes are found in the archaea and Gram positive actinomycetes. In general, 20S proteasomes form a barrel-shaped nanocompartment with narrow openings which isolate rather non-specific proteolytic active-sites to the interior of the cylinder and away from interaction with cytosolic proteins. The proteasome-associated AAA+ proteins are predicted to form ring-like structures which unfold substrate proteins for entry into the central proteolytic 20S chamber resulting in an energy-dependent and processive destruction of the protein. Detailed biochemical and biophysical analysis as well as identification of proteasomes in archaea with developed genetic tools are providing a foundation for understanding the biological role of the proteasome in these unusual organisms.

  9. 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.

  10. Response of the Ubiquitin-Proteasome System to Memory Retrieval After Extended-Access Cocaine or Saline Self-Administration.

    Science.gov (United States)

    Werner, Craig T; Milovanovic, Mike; Christian, Daniel T; Loweth, Jessica A; Wolf, Marina E

    2015-12-01

    The ubiquitin-proteasome system (UPS) has been implicated in the retrieval-induced destabilization of cocaine- and fear-related memories in Pavlovian paradigms. However, nothing is known about its role in memory retrieval after self-administration of cocaine, an operant paradigm, or how the length of withdrawal from cocaine may influence retrieval mechanisms. Here, we examined UPS activity after an extended-access cocaine self-administration regimen that leads to withdrawal-dependent incubation of cue-induced cocaine craving. Controls self-administered saline. In initial experiments, memory retrieval was elicited via a cue-induced seeking/retrieval test on withdrawal day (WD) 50-60, when craving has incubated. We found that retrieval of cocaine- and saline-associated memories produced similar increases in polyubiquitinated proteins in the nucleus accumbens (NAc), compared with rats that did not undergo a seeking/retrieval test. Measures of proteasome catalytic activity confirmed similar activation of the UPS after retrieval of saline and cocaine memories. However, in a subsequent experiment in which testing was conducted on WD1, proteasome activity in the NAc was greater after retrieval of cocaine memory than saline memory. Analysis of other brain regions confirmed that effects of cocaine memory retrieval on proteasome activity, relative to saline memory retrieval, depend on withdrawal time. These results, combined with prior studies, suggest that the relationship between UPS activity and memory retrieval depends on training paradigm, brain region, and time elapsed between training and retrieval. The observation that mechanisms underlying cocaine memory retrieval change depending on the age of the memory has implications for development of memory destabilization therapies for cue-induced relapse in cocaine addicts.

  11. Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

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    Gili Ben-Nissan

    2014-09-01

    Full Text Available For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by the core 20S proteasome itself. Degradation by the 20S proteasome does not require ubiquitin tagging or the presence of the 19S regulatory particle; rather, it relies on the inherent structural disorder of the protein being degraded. Thus, proteins that contain unstructured regions due to oxidation, mutation, or aging, as well as naturally, intrinsically unfolded proteins, are susceptible to 20S degradation. Unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome, relatively little is known about the means by which 20S-mediated proteolysis is controlled. Here, we describe our current understanding of the regulatory mechanisms that coordinate 20S proteasome-mediated degradation, and highlight the gaps in knowledge that remain to be bridged.

  12. Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes.

    Science.gov (United States)

    Ververis, Katherine; Rodd, Annabelle L; Tang, Michelle M; El-Osta, Assam; Karagiannis, Tom C

    2011-12-01

    Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

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

    Directory of Open Access Journals (Sweden)

    Johanna Abrigo

    2016-01-01

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

  14. The neuronal nitric oxide synthase inhibitor, 7-nitroindazole, protects against methamphetamine-induced neurotoxicity in vivo.

    Science.gov (United States)

    Itzhak, Y; Ali, S F

    1996-10-01

    The present study was undertaken to investigate whether the relatively selective neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), protects against methamphetamine (METH)-induced neurotoxicity. Male Swiss Webster mice received the following treatments (i.p.; q 3 h x 3): (a) vehicle/saline, (b) 7-NI (25 mg/kg)/saline, (c) vehicle/METH (5 mg/kg), and (d) 7-NI (25 mg/kg)/METH (5 mg/kg). On the second day, groups (a) and (b) received two vehicle injections, and groups (c) and (d) received two 7-NI injections (25 mg/kg, each). Administration of vehicle/METH resulted in 68, 44, and 55% decreases in the concentration of dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, respectively, and a 48% decrease in the number of [3H]mazindol binding sites in the striatum compared with control values. Treatment with 7-NI (group d) provided full protection against the depletion of dopamine and its metabolites and the loss of dopamine transporter binding sites. Administration of 7-NI/saline (group b) affected neither the tissue concentration of dopamine and its metabolites nor the binding parameters of [3H] mazindol compared with control values. 7-NI had no significant effect on animals' body temperature, and it did not affect METH-induced hyperthermia. These findings indicate a role for nitric oxide in methamphetamine-induced neurotoxicity and also suggest that blockade of NOS may be beneficial for the management of Parkinson's disease.

  15. CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.

    Science.gov (United States)

    Cheong, Jit Kong; Gunaratnam, Lakshman; Hsu, Stephen I-Hong

    2008-04-25

    Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.

  16. Proteasomal and Lysosomal Protein Degradation and Heart Disease

    OpenAIRE

    Wang, Xuejun; Robbins, Jeffrey

    2013-01-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out ...

  17. Targeting proteasomes in infectious organisms to combat disease

    OpenAIRE

    Bibo-Verdugo, B; Jiang, Z; Caffrey, CR; O'Donoghue, AJ

    2017-01-01

    Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein...

  18. VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle.

    Science.gov (United States)

    Cantarero, Lara; Sanz-García, Marta; Vinograd-Byk, Hadar; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A

    2015-06-12

    Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics.

  19. Mechanisms promoting and inhibiting the process of proteasomal degradation of cells

    Directory of Open Access Journals (Sweden)

    Pedrycz Agnieszka

    2016-03-01

    Full Text Available Defects in the process of degradation of unneeded cellular proteins underlie many diseases. This article discusses one of the most important systems of removal of abnormal proteins. It describes the process of ubiquitination of proteins for proteasome degradation. It also describes the structure of the 26S and 20S proteasomes and the mechanism of ubiquitin-proteasome system. Proteasome proteolytic system is highly specialized and organized. Protease-proteasome 26S is particularly important for proper cell functioning. It recognizes and degrades marked proteins. Inhibition of proteasome pathway leads to cell cycle arrest and apoptosis.

  20. Morphological and metabolic changes in the nigro-striatal pathway of synthetic proteasome inhibitor (PSI-treated rats: a MRI and MRS study.

    Directory of Open Access Journals (Sweden)

    Stefano Delli Pizzi

    Full Text Available Systemic administration of a Synthetic Proteasome Inhibitor (PSI in rats has been described as able to provide a model of Parkinson's disease (PD, characterized by behavioral and biochemical modifications, including loss of dopaminergic neurons in the substantia nigra (SN, as assessed by post-mortem studies. With the present study we aimed to assess in-vivo by Magnetic Resonance (MR possible morphological and metabolic changes in the nigro-striatal pathway of PSI-treated rats. 10 animals were subcutaneously injected with PSI 6.0 mg/kg dissolved in DMSO 100%. Injections were made thrice weekly over the course of two weeks. 5 more animals injected with DMSO 100% with the same protocol served as controls. The animals underwent MR sessions before and at four weeks after the end of treatment with either PSI or vehicle. MR Imaging was performed to measure SN volume and Proton MR Spectroscopy ((1H-MRS was performed to measure metabolites changes at the striatum. Animals were also assessed for motor function at baseline and at 4 and 6 weeks after treatment. Dopamine and dopamine metabolite levels were measured in the striata at 6 weeks after treatment. PSI-treated animals showed volumetric reduction of the SN (p<0.02 at 4 weeks after treatment as compared to baseline. Immunofluorescence analysis confirmed MRI changes in SN showing a reduction of tyrosine hydroxylase expression as compared to neuron-specific enolase expression. A reduction of N-acetyl-aspartate/total creatine ratio (p = 0.05 and an increase of glutamate-glutamine-γ amminobutirrate/total creatine were found at spectroscopy (p = 0.03. At 6 weeks after treatment, PSI-treated rats also showed motor dysfunction compared to baseline (p = 0.02, accompanied by dopamine level reduction in the striatum (p = 0.02. Treatment with PSI produced morphological and metabolic modifications of the nigro-striatal pathway, accompanied by motor dysfunction. MR demonstrated to be a powerful mean to assess in

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

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    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.

  2. 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.

  3. Long Non-Coding RNA TUG1 Promotes Proliferation and Inhibits Apoptosis of Osteosarcoma Cells by Sponging miR-132-3p and Upregulating SOX4 Expression.

    Science.gov (United States)

    Li, Gang; Liu, Keyu; Du, Xinhui

    2018-03-01

    Long non-coding RNA taurine upregulated gene 1 (TUG1) is reported to be a vital regulator of the progression of various cancers. This study aimed to explore the exact roles and molecular mechanisms of TUG1 in osteosarcoma (OS) development. Real-time quantitative PCR was applied to detect the expressions of TUG1 and microRNA-132-3p (miR-132-3p) in OS tissues and cells. Western blot was performed to measure protein levels of sex determining region Y-box 4 (SOX4). Cell viability was assessed using XTT assay. Cell apoptosis was evaluated using flow cytometry and caspase-3 activity detection assays. Bioinformatics analysis and luciferase reporter experiments were employed to confirm relationships among TUG1, miR-132-3p, and SOX4. TUG1 was highly expressed in human OS tissues, OS cell lines, and primary OS cells. TUG1 knockdown hindered proliferation and induced apoptosis in human OS cell lines and primary OS cells. Moreover, TUG1 inhibited miR-132-3p expression by direct interaction, and introduction of miR-132-3p inhibitor partly abrogated the effect of TUG1 knockdown on the proliferation and apoptosis of OS cells. Furthermore, SOX4 was validated as a target of miR-132-3p. Further functional analyses revealed that miR-132-3p inhibited proliferation and induced apoptosis of OS cells, while this effect was greatly abated following SOX4 overexpression. Moreover, TUG1 knockdown suppressed proliferation and promoted apoptosis by upregulating miR-132-3p and downregulating SOX4 in primary OS cells. TUG1 facilitated proliferation and suppressed apoptosis by regulating the miR-132-3p/SOX4 axis in human OS cell lines and primary OS cells. This finding provides a potential target for OS therapy. © Copyright: Yonsei University College of Medicine 2018

  4. Identification and Characterisation of a Proteasome -

    DEFF Research Database (Denmark)

    Andersen, Katrine Mølgaard

    this domain. A txl1 yeast knockout mutant displays a synthetic growth defect with a cut8 knockout, whereas the txc1 knockout does not. In fission yeast, Cut8 is a nuclear protein that tethers the 26S proteasome to the nuclear membrane. In both wild type cells and in a cut8 null mutant Txl1 co......-down of Txnl1 in HeLa cells, whereas no stabilisation was detected in txl1¿ cells in S. pombe. In human cells, an active site Txnl1 mutant formed a mixed disulfide intermediate with eEF1A1, a reported substrate-recruiting factor of the 26S proteasome. Fission yeast Txl1 can reduce an oxidised proteasomal model...

  5. 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.

  6. 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.

  7. The Effect of Serine Protease Inhibitors on Airway Inflammation in a Chronic Allergen-Induced Asthma Mouse Model

    Directory of Open Access Journals (Sweden)

    Chih-Che Lin

    2014-01-01

    Full Text Available Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT, gabexate mesilate (FOY, and ulinastatin (UTI on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50 μL, 1 mg/mL at one-week intervals. Therapeutic doses of FUT (0.0625 mg/kg, FOY (20 mg/kg, or UTI (10,000 U/kg were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR, remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF-κB activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-κB activation in lung tissue.

  8. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    Science.gov (United States)

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  9. Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

    Directory of Open Access Journals (Sweden)

    Tomishima Yoshiro

    2013-01-01

    Full Text Available Abstract Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2 synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg. The effects of ozagrel (200 mg/kg treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL on cytochrome P450 2E1 (CYP2E1 activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos and C/EBP homologous protein (chop, but did not suppress B-cell lymphoma 2-like protein11 (bim expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest

  10. Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde

    Energy Technology Data Exchange (ETDEWEB)

    Ortega-Atienza, Sara; Green, Samantha E.; Zhitkovich, Anatoly, E-mail: anatoly_zhitkovich@brown.edu

    2015-07-15

    Formaldehyde (FA) is a human carcinogen with numerous sources of environmental and occupational exposures. This reactive aldehyde is also produced endogenously during metabolism of drugs and other processes. DNA–protein crosslinks (DPCs) are considered to be the main genotoxic lesions for FA. Accumulating evidence suggests that DPC repair in high eukaryotes involves proteolysis of crosslinked proteins. Here, we examined a role of the main cellular proteolytic machinery proteasomes in toxic responses of human lung cells to low FA doses. We found that transient inhibition of proteasome activity increased cytotoxicity and diminished clonogenic viability of FA-treated cells. Proteasome inactivation exacerbated suppressive effects of FA on DNA replication and increased the levels of the genotoxic stress marker γ-H2AX in normal human cells. A transient loss of proteasome activity in FA-exposed cells also caused delayed perturbations of cell cycle, which included G2 arrest and a depletion of S-phase populations at FA doses that had no effects in control cells. Proteasome activity diminished p53-Ser15 phosphorylation but was important for FA-induced CHK1 phosphorylation, which is a biochemical marker of DPC proteolysis in replicating cells. Unlike FA, proteasome inhibition had no effect on cell survival and CHK1 phosphorylation by the non-DPC replication stressor hydroxyurea. Overall, we obtained evidence for the importance of proteasomes in protection of human cells against biologically relevant doses of FA. Biochemically, our findings indicate the involvement of proteasomes in proteolytic repair of DPC, which removes replication blockage by these highly bulky lesions. - Highlights: • Proteasome inhibition enhances cytotoxicity of low-dose FA in human lung cells. • Active proteasomes diminish replication-inhibiting effects of FA. • Proteasome activity prevents delayed G2 arrest in FA-treated cells. • Proteasome inhibition exacerbates replication stress by FA in

  11. Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

    Science.gov (United States)

    Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

    2017-05-31

    Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

  12. Deltamethrin-induced testicular apoptosis in rats: the protective effect of nitric oxide synthase inhibitor.

    Science.gov (United States)

    El-Gohary, M; Awara, W M; Nassar, S; Hawas, S

    1999-01-01

    This study is the first to examine and characterize the testicular apoptosis which might be induced due to exposure of male rats to deltamethrin. Furthermore, the role which might be played by nitric oxide (NO), as well as the other reactive oxygen species (ROS) in controlling this testicular apoptosis was assessed. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis and cellular morphology on testicular tissue sections. It was found that administration of deltamethrin (1 mg/kg daily for 21 days) to animals resulted in characteristic DNA migration patterns (laddering), thereby providing evidence that apoptosis is the major mechanism of cell death in the testicular tissues. In addition, histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells, primary and secondary spermatocytes. These changes, in addition to the appearance of Sertoli cell vacuoles in deltamethrin-intoxicated animals, indicates the suppression of spermatogenesis. At the same time, the plasma levels of both NO and lipid peroxides measured as malondialdehyde (MDA) were found to be significantly increased in deltamethrin-treated animals. Administration of NO synthase (NOS) inhibitors such as N(G)-nitro monomethyl L-arginine hydrochloride (L-NMMA, 1 mg/kg) to rats 2 h before exposure to deltamethrin was effective in the reduction of the typically testicular apoptotic DNA fragmentation pattern and the associated histopathological changes. These findings may suggest that deltamethrin-induced testicular apoptosis is mediated by NO. Therefore, the pharmacological manipulation of apoptosis by selective NOS inhibitors such as L-NMMA may offer new possibilities for the control of deltamethrin-induced testicular dysfunction and infertility in the future.

  13. Decay of 132Cs and nuclear structure of 132Xe

    International Nuclear Information System (INIS)

    Cruz, M.T.F. da; Goldman, I.D.

    1989-02-01

    Gamma spectroscopy, coincidence and angular correlation experiments were performed, in an extensive study of 132 Cs decay. The population intensities of states in 132 Ba were measured, some upper limits were determined and the value of the transition to the 4 1 + state of 132 Ba has drastically changed. A new gamma transition of 688 KeV was detected and located in the level scheme of 132 Xe. The angular correlation measurements were performed with a Ge(Li)-NaI(Tl) spectrometer and helped in choosing the spin value for the 1804 KeV state of 132 Xe. Kumar-Baranger-type calculations were performed for the 132 Xe fairly good results. The resulting potential energy surface for 132 Xe is very different from that of Gneuss and Greiner model, suggesting the need for any quadrupole moment measurement for this nucleus. (author) [pt

  14. The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies.

    Science.gov (United States)

    Ehm, Patrick; Nalaskowski, Marcus M; Wundenberg, Torsten; Jücker, Manfred

    2015-01-01

    The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in haematopoietic cells. By converting PI(3,4,5)P3 to PtdIns(3,4)P2 at the plasma membrane, SHIP1 modifies PI3-kinase mediated signaling. We have recently demonstrated that SHIP1 is a nucleo-cytoplasmic shuttling protein and SHIP1 nuclear puncta partially colocalize with FLASH, a component of nuclear bodies. In this study, we demonstrate that endogenous SHIP1 localizes to intranucleolar regions of both normal and leukemic haematopoietic cells. In addition, we report that ectopically expressed SHIP1 accumulates in nucleolar cavities and colocalizes with the tumor suppressor protein p53 and components of PML nuclear bodies (e.g. SP100, SUMO-1 and CK2). Moreover, SHIP1 also colocalizes in nucleolar cavities with components of the ubiquitin-proteasome pathway. By using confocal microscopy data, we generated 3D-models revealing the enormous extent of the SHIP1 aggresomes in the nucleolus. Furthermore, treatment of cells with the proteasome inhibitor MG132 causes an enlargement of nucleolar SHIP1 containing structures. Unexpectedly, this accumulation can be partially prevented by treatment with the inhibitor of nuclear protein export Leptomycin B. In recent years, several proteins aggregating in nucleolar cavities were shown to be key factors of neurodegenerative diseases and cancerogenesis. Our findings support current relevance of nuclear localized SHIP1.

  15. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    Science.gov (United States)

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

  16. 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.

  17. Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats.

    Science.gov (United States)

    Hosaka, E M; Santos, O F P; Seguro, A C; Vattimo, M F F

    2004-07-01

    The frequent use of nonsteroidal anti-inflammatory drugs (NSAID) in combination with gentamicin poses the additional risk of nephrotoxic renal failure. Cyclooxygenase-1 (COX-1) is the main enzyme responsible for the synthesis of renal vasodilator prostaglandins, while COX-2 participates predominantly in the inflammatory process. Both are inhibited by non-selective NSAID such as indomethacin. Selective COX-2 inhibitors such as rofecoxib seem to have fewer renal side effects than non-selective inhibitors. The objective of the present study was to determine whether the combined use of rofecoxib and gentamicin can prevent the increased renal injury caused by gentamicin and indomethacin. Male Wistar rats (250-300 g) were treated with gentamicin (100 mg/kg body weight, ip, N = 7), indomethacin (5 mg/kg, orally, N = 7), rofecoxib (1.4 mg/kg, orally, N = 7), gentamicin + rofecoxib (100 and 1.4 mg/kg, respectively) or gentamicin + indomethacin (100 and 5 mg/kg, respectively, N = 8) for 5 days. Creatinine clearance and alpha-glutathione-S-transferase concentrations were used as markers of renal injury. Animals were anesthetized with ether and sacrificed for blood collection. The use of gentamicin plus indomethacin led to worsened renal function (0.199 +/- 0.019 ml/min), as opposed to the absence of a nephrotoxic effect of rofecoxib when gentamicin plus rofexicob was used (0.242 +/- 0.011 ml/min). These results indicate that COX-2-selective inhibitors can be used as an alternative treatment to conventional NSAID, especially in situations in which risk factors for nephrotoxicity are present.

  18. Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats

    Directory of Open Access Journals (Sweden)

    Hosaka E.M.

    2004-01-01

    Full Text Available The frequent use of nonsteroidal anti-inflammatory drugs (NSAID in combination with gentamicin poses the additional risk of nephrotoxic renal failure. Cyclooxygenase-1 (COX-1 is the main enzyme responsible for the synthesis of renal vasodilator prostaglandins, while COX-2 participates predominantly in the inflammatory process. Both are inhibited by non-selective NSAID such as indomethacin. Selective COX-2 inhibitors such as rofecoxib seem to have fewer renal side effects than non-selective inhibitors. The objective of the present study was to determine whether the combined use of rofecoxib and gentamicin can prevent the increased renal injury caused by gentamicin and indomethacin. Male Wistar rats (250-300 g were treated with gentamicin (100 mg/kg body weight, ip, N = 7, indomethacin (5 mg/kg, orally, N = 7, rofecoxib (1.4 mg/kg, orally, N = 7, gentamicin + rofecoxib (100 and 1.4 mg/kg, respectively or gentamicin + indomethacin (100 and 5 mg/kg, respectively, N = 8 for 5 days. Creatinine clearance and alpha-glutathione-S-transferase concentrations were used as markers of renal injury. Animals were anesthetized with ether and sacrificed for blood collection. The use of gentamicin plus indomethacin led to worsened renal function (0.199 ± 0.019 ml/min, as opposed to the absence of a nephrotoxic effect of rofecoxib when gentamicin plus rofexicob was used (0.242 ± 0.011 ml/min. These results indicate that COX-2-selective inhibitors can be used as an alternative treatment to conventional NSAID, especially in situations in which risk factors for nephrotoxicity are present.

  19. 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.

  20. CCR 20th anniversary commentary: Preclinical study of proteasome inhibitor bortezomib in head and neck cancer.

    Science.gov (United States)

    Allen, Clint T; Conley, Barbara; Sunwoo, John B; Van Waes, Carter

    2015-03-01

    In a study published in the May 1, 2001, issue of Clinical Cancer Research, Sunwoo and colleagues provided evidence for proteasome inhibition of NF-κB and tumorigenesis, supporting early-phase clinical trials in solid malignancies of the upper aerodigestive tract. Subsequent clinical studies uncovered a dichotomy of responses in patients with hematopoietic and solid malignancies, and the mechanisms of resistance. ©2015 American Association for Cancer Research.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2001-01-01

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

  3. Changes in expression of proteasome in rats at different stages of atherosclerosis.

    Science.gov (United States)

    Ismawati; Oenzil, Fadil; Yanwirasti; Yerizel, Eti

    2016-06-01

    It has been suggested that proteasome system has a role in initiation, progression, and complication stages of atherosclerosis. Although there is still controversy, there has been no research that compares the expression of proteasome in tissue and serum at each of these stages. This study aimed to investigated the expression of proteasome at different stages of atherosclerosis using rat model. We measured the expression of aortic proteasome by immunohistochemical analyses and were then analyzed using ImageJ software for percentage of area and integrated density. We used Photoshop version 3.0 to analyze aortic proteasome expression as a comparison. We measured serum proteasome expression by enzyme linked immunosorbents assays. Kruskal-Wallis test was used to compare mean value of percentage of area and serum proteasome. Analysis of variance test was used to compare mean value of integrated density. Correlation test between vascular proteasome expression and serum proteasome expression was made using Spearman test. A P-value of 0.05 was considered statistically significant. Compared with normal, percentage of area was higher in initiation, progression, and complication. Compared with normal, integrated density was higher in initiation and further higher in progression and complication. Data from Image J is similar with data from Photoshop. Serum proteasome expression was higher in initiation compared with normal, and further higher in progression and complication. It was concluded that there were different vascular proteasome expression and serum proteasome expression at the stages of atherosclerosis. These results may be used in research into new marker and therapeutic target in atherosclerosis.

  4. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Bhupesh, E-mail: drbhupeshresearch@gmail.com; Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential

  5. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    International Nuclear Information System (INIS)

    Sharma, Bhupesh; Sharma, P.M.

    2013-01-01

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  6. Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae.

    Science.gov (United States)

    Yedidi, Ravikiran S; Fatehi, Amatullah K; Enenkel, Cordula

    The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

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

    Directory of Open Access Journals (Sweden)

    Karl A Rodriguez

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

  8. 26 S proteasomes function as stable entities

    DEFF Research Database (Denmark)

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

    2002-01-01

    , shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during...

  9. 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.

  10. 26 CFR 1.132-0 - Outline of regulations under section 132.

    Science.gov (United States)

    2010-04-01

    .... § 1.132-6 (b) Frequency. (1) Employee-measured frequency. (2) Employer-measured frequency. § 1.132-6 (c) Administrability. § 1.132-6 (d) Special rules. (1) Transit passes. (2) Occasional meal money or...) Benefits exceeding value and frequency limits. § 1.132-6 (e) Examples. (1) Benefits excludable from income...

  11. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    International Nuclear Information System (INIS)

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J.

    2006-01-01

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1α associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome

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

    Directory of Open Access Journals (Sweden)

    Seung-Wook Shin

    2012-11-01

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

  13. Screening of a Drug Library Identifies Inhibitors of Cell Intoxication by CNF1.

    Science.gov (United States)

    Mahtal, Nassim; Brewee, Clémence; Pichard, Sylvain; Visvikis, Orane; Cintrat, Jean-Christophe; Barbier, Julien; Lemichez, Emmanuel; Gillet, Daniel

    2018-04-06

    Cytotoxic necrotizing factor 1 (CNF1) is a toxin produced by pathogenic strains of Escherichia coli responsible for extra-intestinal infections. CNF1 deamidates Rac1, thereby triggering its permanent activation and worsening inflammatory reactions. Activated Rac1 is prone to proteasomal degradation. There is no targeted therapy against CNF1, despite its clinical relevance. In this work we developed a fluorescent cell-based immunoassay to screen for inhibitors of CNF1-induced Rac1 degradation among 1120 mostly approved drugs. Eleven compounds were found to prevent CNF1-induced Rac1 degradation, and five also showed a protective effect against CNF1-induced multinucleation. Finally, lasalocid, monensin, bepridil, and amodiaquine protected cells from both diphtheria toxin and CNF1 challenges. These data highlight the potential for drug repurposing to fight several bacterial infections and Rac1-based diseases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. 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

  15. Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

    Directory of Open Access Journals (Sweden)

    Kouichi Yamada

    2011-11-01

    Full Text Available When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s, which insert nucleotide(s opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF. In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells, and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−, UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

  16. 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

  17. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Zhen [Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Gan, Ye-Hua, E-mail: kqyehuagan@bjmu.edu.cn [Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China)

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  18. 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.

  19. Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1.

    Directory of Open Access Journals (Sweden)

    Albert Salehi

    Full Text Available BACKGROUND: A distinctive feature of type 2 diabetes is inability of insulin-secreting beta-cells to properly respond to elevated glucose eventually leading to beta-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of beta-cell dysfunction. PRINCIPAL FINDINGS: We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N(G-nitro-L-arginine methyl ester (L-NAME greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon. CONCLUSION: The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms.

  20. Podocytes degrade endocytosed albumin primarily in lysosomes.

    Science.gov (United States)

    Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, plysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.

  1. GSK-3 inhibitors induce chromosome instability

    Directory of Open Access Journals (Sweden)

    Staples Oliver D

    2007-08-01

    Full Text Available Abstract Background Several mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 – a protein kinase, which in concert with APC, targets β-catenin for proteolysis – and ask whether GSK-3 is required for accurate chromosome segregation. Results To probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3β. Cells deficient for GSK-3β exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3β repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation. Conclusion Thus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability.

  2. Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels

    Science.gov (United States)

    Hei, Hongya; Li, Fangping; Wang, Yunman; Peng, Wen; Zhang, Xuemei

    2015-01-01

    Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms. PMID:26672753

  3. Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels.

    Directory of Open Access Journals (Sweden)

    Qijing Chen

    Full Text Available Large conductance Ca2+-activated potassium channels (BK are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α and BK (α+β1 currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1. Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Sugiyama, Masaaki, E-mail: sugiyama@rri.kyoto-u.ac.jp [Research Reactor Institute, Kyoto University, Osaka 590-0494 (Japan); Sahashi, Hiroki [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Kurimoto, Eiji [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Faculty of Pharmacy, Meijo University, Nagoya 468-8503 (Japan); Takata, Shin-ichi [J-PARC Center, Japan Atomic Energy Agency, Ibaraki 319-1195 (Japan); Yagi, Hirokazu; Kanai, Keita; Sakata, Eri [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Minami, Yasufumi [Department of Biotechnology, Maebashi Institute of Technology, Gunma 371-0816 (Japan); Tanaka, Keiji [Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506 (Japan); Kato, Koichi, E-mail: kkatonmr@ims.ac.jp [Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787 (Japan); Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787 (Japan)

    2013-03-01

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

  6. The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster.

    Science.gov (United States)

    Pomatto, Laura C D; Wong, Sarah; Carney, Caroline; Shen, Brenda; Tower, John; Davies, Kelvin J A

    2017-04-01

    Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C ( Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H 2 O 2 ) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H 2 O 2 , and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H 2 O 2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.

  7. The 20S proteasome splicing activity discovered by SpliceMet.

    Directory of Open Access Journals (Sweden)

    Juliane Liepe

    2010-06-01

    Full Text Available The identification of proteasome-generated spliced peptides (PSP revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS. For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

  8. 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.

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

    International Nuclear Information System (INIS)

    Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

    2016-01-01

    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.

  10. 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.

  11. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment.

    Science.gov (United States)

    Hyer, Marc L; Milhollen, Michael A; Ciavarri, Jeff; Fleming, Paul; Traore, Tary; Sappal, Darshan; Huck, Jessica; Shi, Judy; Gavin, James; Brownell, Jim; Yang, Yu; Stringer, Bradley; Griffin, Robert; Bruzzese, Frank; Soucy, Teresa; Duffy, Jennifer; Rabino, Claudia; Riceberg, Jessica; Hoar, Kara; Lublinsky, Anya; Menon, Saurabh; Sintchak, Michael; Bump, Nancy; Pulukuri, Sai M; Langston, Steve; Tirrell, Stephen; Kuranda, Mike; Veiby, Petter; Newcomb, John; Li, Ping; Wu, Jing Tao; Powe, Josh; Dick, Lawrence R; Greenspan, Paul; Galvin, Katherine; Manfredi, Mark; Claiborne, Chris; Amidon, Benjamin S; Bence, Neil F

    2018-02-01

    The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

  12. Boosted protease inhibitor monotherapy versus boosted protease inhibitor plus lamivudine dual therapy as second-line maintenance treatment for HIV-1-infected patients in sub-Saharan Africa (ANRS12 286/MOBIDIP): a multicentre, randomised, parallel, open-label, superiority trial.

    Science.gov (United States)

    Ciaffi, Laura; Koulla-Shiro, Sinata; Sawadogo, Adrien Bruno; Ndour, Cheik Tidiane; Eymard-Duvernay, Sabrina; Mbouyap, Pretty Rosereine; Ayangma, Liliane; Zoungrana, Jacques; Gueye, Ndeye Fatou Ngom; Diallo, Mohamadou; Izard, Suzanne; Bado, Guillaume; Kane, Coumba Toure; Aghokeng, Avelin Fobang; Peeters, Martine; Girard, Pierre Marie; Le Moing, Vincent; Reynes, Jacques; Delaporte, Eric

    2017-09-01

    Despite satisfactory efficacy of WHO-recommended second-line antiretroviral treatment for patients with HIV in low-income countries, the need for simplified, low-cost, and less-toxic maintenance strategies remains high. We compared boosted protease inhibitor monotherapy with dual therapy with boosted protease inhibitor plus lamivudine in patients on second-line antiretrovial therapy (ART). We did a multicentre, randomised, parallel, open-label, superiority, trial in the HIV services of five hospitals in sub-Saharan Africa (Yaoundé, Cameroon; Dakar, Senegal; and Bobo Dioulasso, Burkina Faso). We recruited patients from the long-term, post-trial cohort of the ANRS 12169/2LADY study that compared the efficacy of three second-line combinations based on boosted protease inhibitors. Participants for our study were HIV-1 infected with multiple mutations including M184V, at first-line failure, aged 18 years and older, on boosted protease inhibitor plus two nucleoside reverse transcriptase inhibitors (NRTI) for at least 48 weeks with at least 48 weeks follow-up in the 2LADY trial, with two viral load measurements of less than 200 copies per mL in the previous 6 months, CD4 counts of more than 100 cells per μL, adherence of at least 90%, and no change to ART in the past 3 months. We randomly assigned participants (1:1) to receive either monotherapy with their boosted protease inhibitor (once-daily darunavir 800 mg [two 400 mg tablets] boosted with ritonavir 100 mg [one tablet] or coformulation of lopinavir 200 mg with ritonavir 50 mg [two tablets taken twice per day]) or to boosted protease inhibitor plus once-daily lamivudine 300 mg (one 300 mg tablet or two 150 mg tablets). Computer-generated randomisation was stratified by study site and viral load at screening (treatment allocation was not masked from clinicians or patients]. Patients had follow-up visits at weeks 4 and 12, and every 3 months until 96 weeks; if viral load exceeded 500 copies per mL at any visit, NRTI

  13. Assessment of 105 Patients with Angiotensin Converting Enzyme-Inhibitor Induced Angioedema

    DEFF Research Database (Denmark)

    Rasmussen, Eva Rye; von Buchwald, Christian; Wadelius, Mia

    2017-01-01

    Objective. To asses a cohort of 105 consecutive patients with angiotensin converting enzyme-inhibitor induced angioedema with regard to demographics, risk factors, family history of angioedema, hospitalization, airway management, outcome, and use of diagnostic codes used for the condition. Study...... gender was associated with a significantly higher risk of angiotensin converting enzyme-inhibitor induced angioedema. 6.7% had a positive family history of angioedema. Diabetes seemed to be a protective factor with regard to angioedema. 95% experienced angioedema of the head and neck. 4.7% needed...... Design. Cohort study. Methods. This was a retrospective cohort study of 105 patients with angiotensin converting enzyme-inhibitor induced angioedema in the period 1995-2014. Results. The cohort consisted of 67 females and 38 males (F : M ratio 1.8), with a mean age of 63 [range 26-86] years. Female...

  14. Regression of fibrosis and reversal of cirrhosis in rats by galectin inhibitors in thioacetamide-induced liver disease.

    Directory of Open Access Journals (Sweden)

    Peter G Traber

    Full Text Available Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan or GM-CT-01 (galactomannan. In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip or GM-CT-01 (180 mg/kg ip given once weekly during weeks 8-11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis.

  15. 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.

  16. Bladder cancer detection using a peptide substrate of the 20S proteasome.

    Science.gov (United States)

    Gruba, Natalia; Wysocka, Magdalena; Brzezińska, Magdalena; Dębowski, Dawid; Sieńczyk, Marcin; Gorodkiewicz, Ewa; Guszcz, Tomasz; Czaplewski, Cezary; Rolka, Krzysztof; Lesner, Adam

    2016-08-01

    The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods. © 2016 Federation of European Biochemical Societies.

  17. Effects of a Natural Prolyl Oligopeptidase Inhibitor, Rosmarinic Acid, on Lipopolysaccharide-Induced Acute Lung Injury in Mice

    Directory of Open Access Journals (Sweden)

    Miaomiao Wei

    2012-03-01

    Full Text Available Rosmarinic acid (RA, a polyphenolic phytochemical, is a natural prolyl oligopeptidase inhibitor. In the present study, we found that RA exerted potent anti-inflammatory effects in in vivo models of acute lung injury (ALI induced by lipopolysaccharide (LPS. Mice were pretreated with RA one hour before challenge with a dose of 0.5 mg/kg LPS. Twenty-four hours after LPS was given, bronchoalveolar lavage fluid (BALF was obtained to measure pro-inflammatory mediator and total cell counts. RA significantly decreased the production of LPS-induced TNF-a, IL-6, and IL-1β compare with the LPS group. When pretreated with RA (5, 10, or 20 mg/kg the lung wet-to-dry weight (W/D ratio of the lung tissue and the number of total cells, neutrophils and macrophages in the BALF were decreased significantly. Furthermore, RA may enhance oxidase dimutase (SOD activity during the inflammatory response to LPS-induced ALI. And we further demonstrated that RA exerts anti-inflammation effect in vivo models of ALI through suppresses ERK/MAPK signaling in a dose dependent manner. These studies have important implications for RA administration as a potential treatment for ALI.

  18. 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.

  19. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

    Science.gov (United States)

    Koues, Olivia I; Mehta, Ninad T; Truax, Agnieszka D; Dudley, R Kyle; Brooks, Jeanne K; Greer, Susanna F

    2010-02-04

    Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription. Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL)/complex of proteins associated with Set I (COMPASS) complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

  20. Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation

    Directory of Open Access Journals (Sweden)

    Koues Olivia I

    2010-02-01

    Full Text Available Abstract Background Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC-II and class II transactivator (CIITA promoters, implicating Sug1 in events required to initiate mammalian transcription. Results Our previous studies indicate that H3 lysine 4 trimethylation at cytokine-inducible MHC-II and CIITA promoters is dependent on proteolytic-independent functions of 19S ATPases. In this report, we show that multiple common subunits of the mixed lineage leukemia (MLL/complex of proteins associated with Set I (COMPASS complexes bind to the inducible MHC-II and CIITA promoters; that overexpressing a single common MLL/COMPASS subunit significantly enhances promoter activity and MHC-II HLA-DRA expression; and that these common subunits are important for H3 lysine 4 trimethylation at MHC-II and CIITA promoters. In addition, we show that H3 lysine 27 trimethylation, which is inversely correlated with H3 lysine 4 trimethylation, is significantly elevated in the presence of diminished 19S ATPase Sug1. Conclusion Taken together, these experiments suggest that the 19S proteasome plays a crucial role in the initial reorganization of events enabling the relaxation of the repressive chromatin structure surrounding inducible promoters.

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

    Science.gov (United States)

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

    2015-05-07

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

  2. Bosutinib induced pleural effusions: Case report and review of tyrosine kinase inhibitors induced pulmonary toxicity

    Directory of Open Access Journals (Sweden)

    Natalia I. Moguillansky, MD

    2017-01-01

    Full Text Available Tyrosine kinase inhibitors are known to cause pulmonary complications. We report a case of bosutinib related bilateral pleural effusions in a patient with chronic myeloid leukemia. Characteristics of the pleural fluid are presented. We also discuss other tyrosine kinase inhibitors induced pulmonary toxicities, including pulmonary hypertension and interstitial lung disease.

  3. 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...

  4. NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin

    Directory of Open Access Journals (Sweden)

    Seung Un Seo

    2017-10-01

    Full Text Available Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4, human breast carcinoma (MDA-MB231, and human glioma (U87MG cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926. We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5 expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

  5. The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

    DEFF Research Database (Denmark)

    Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

    2017-01-01

    Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer......-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p...

  6. Aggresome formation is regulated by RanBPM through an interaction with HDAC6

    Directory of Open Access Journals (Sweden)

    Louisa M. Salemi

    2014-05-01

    Full Text Available In conditions of proteasomal impairment, the build-up of damaged or misfolded proteins activates a cellular response leading to the recruitment of damaged proteins into perinuclear aggregates called aggresomes. Aggresome formation involves the retrograde transport of cargo proteins along the microtubule network and is dependent on the histone deacetylase HDAC6. Here we show that ionizing radiation (IR promotes Ran-Binding Protein M (RanBPM relocalization into discrete perinuclear foci where it co-localizes with aggresome components ubiquitin, dynein and HDAC6, suggesting that the RanBPM perinuclear clusters correspond to aggresomes. RanBPM was also recruited to aggresomes following treatment with the proteasome inhibitor MG132 and the DNA-damaging agent etoposide. Strikingly, aggresome formation by HDAC6 was markedly impaired in RanBPM shRNA cells, but was restored by re-expression of RanBPM. RanBPM was found to interact with HDAC6 and to inhibit its deacetylase activity. This interaction was abrogated by a RanBPM deletion of its LisH/CTLH domain, which also prevented aggresome formation, suggesting that RanBPM promotes aggresome formation through an association with HDAC6. Our results suggest that RanBPM regulates HDAC6 activity and is a central regulator of aggresome formation.

  7. Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

    International Nuclear Information System (INIS)

    Ozaki, Kei-ichi; Minoda, Ai; Kishikawa, Futaba; Kohno, Michiaki

    2006-01-01

    Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated

  8. PARP inhibitors protect against sex- and AAG-dependent alkylation-induced neural degeneration.

    Science.gov (United States)

    Allocca, Mariacarmela; Corrigan, Joshua J; Fake, Kimberly R; Calvo, Jennifer A; Samson, Leona D

    2017-09-15

    Alkylating agents are commonly used to treat cancer. Although base excision repair (BER) is a major pathway for repairing DNA alkylation damage, under certain conditions, the initiation of BER produces toxic repair intermediates that damage healthy tissues. The initiation of BER by the alkyladenine DNA glycosylase (AAG, a.k.a. MPG) can mediate alkylation-induced cytotoxicity in specific cells in the retina and cerebellum of male mice. Cytotoxicity in both wild-type and Aag -transgenic ( AagTg ) mice is abrogated in the absence of Poly(ADP-ribose) polymerase-1 (PARP1). Here, we tested whether PARP inhibitors can also prevent alkylation-induced retinal and cerebellar degeneration in male and female WT and AagTg mice. Importantly, we found that WT mice display sex-dependent alkylation-induced retinal damage (but not cerebellar damage), with WT males being more sensitive than females. Accordingly, estradiol treatment protects males against alkylation-induced retinal degeneration. In AagTg male and female mice, the alkylation-induced tissue damage in both the retina and cerebellum is exacerbated and the sex difference in the retina is abolished. PARP inhibitors, much like Parp1 gene deletion, protect against alkylation-induced AAG-dependent neuronal degeneration in WT and AagTg mice, regardless of the gender, but their efficacy in preventing alkylation-induced neuronal degeneration depends on PARP inhibitor characteristics and doses. The recent surge in the use of PARP inhibitors in combination with cancer chemotherapeutic alkylating agents might represent a powerful tool for obtaining increased therapeutic efficacy while avoiding the collateral effects of alkylating agents in healthy tissues.

  9. The ubiquitin-proteasome system

    Indian Academy of Sciences (India)

    ... the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first 'tagged' by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome.

  10. The novel triple monoamine reuptake inhibitor tesofensine induces sustained weight loss and improves glycemic control in the diet-induced obese rat: comparison to sibutramine and rimonabant

    DEFF Research Database (Denmark)

    Hansen, Henrik H; Hansen, Gitte; Tang-Christensen, Mads

    2010-01-01

    Tesofensine, a novel triple monoamine reuptake inhibitor, produces a significant weight loss in humans. The present study aimed at characterizing the weight-reducing effects of tesofensine in a rat model of diet-induced obesity. Sibutramine and rimonabant were used as reference comparators....... Compared to baseline, long-term treatment with tesofensine (28 days, 1.0 or 2.5mg/kg, p.o.) resulted in a significant, dose-dependent and sustained weight loss of 5.7 and 9.9%, respectively. Sibutramine (7.5mg/kg, p.o.) treatment caused a sustained weight loss of 7.6%, whereas the employed dose...... of rimonabant (10mg/kg, p.o.) only produced a transient weight reduction. While all compounds exhibited a significant inhibitory effect on food intake which gradually wore off, the hypophagic effect of tesofensine was longer lasting than sibutramine and rimonabant. In contrast to tesofensine, the body weight...

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

    Science.gov (United States)

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

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. The histone deacetylase inhibitor, Vorinostat, represses hypoxia inducible factor 1 alpha expression through translational inhibition.

    Directory of Open Access Journals (Sweden)

    Darren M Hutt

    Full Text Available Hypoxia inducible factor 1α (HIF-1α is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor--eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.

  13. Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

    Directory of Open Access Journals (Sweden)

    Nigou Jérôme

    2007-08-01

    Full Text Available Abstract Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase. Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.

  14. Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

    Science.gov (United States)

    Kulms, D; Pöppelmann, B; Schwarz, T

    2000-05-19

    Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

  15. 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.

  16. Proteins interacting with the 26S proteasome

    DEFF Research Database (Denmark)

    Hartmann-Petersen, R; Gordon, C

    2004-01-01

    The 26S proteasome is the multi-protein protease that recognizes and degrades ubiquitinylated substrates targeted for destruction by the ubiquitin pathway. In addition to the well-documented subunit organization of the 26S holoenzyme, it is clear that a number of other proteins transiently...... associate with the 26S complex. These transiently associated proteins confer a number of different roles such as substrate presentation, cleavage of the multi-ubiquitin chain from the protein substrate and turnover of misfolded proteins. Such activities are essential for the 26S proteasome to efficiently...... fulfill its intracellular function in protein degradation....

  17. Selective Serotonin Reuptake Inhibitor-Induced Sexual Dysfunction in Adolescents: A Review.

    Science.gov (United States)

    Scharko, Alexander M.

    2004-01-01

    Objective: To review the existing literature on selective serotonin reuptake inhibitor (SSRI)-induced sexual dysfunction in adolescents. Method: A literature review of SSRI-induced adverse effects in adolescents focusing on sexual dysfunction was done. Nonsexual SSRI-induced adverse effects were compared in adult and pediatric populations.…

  18. A comparative study of neuroprotective effect of angiotensin converting enzyme inhibitors against scopolamine-induced memory impairments in rats

    Directory of Open Access Journals (Sweden)

    Talha Jawaid

    2015-01-01

    Full Text Available The comparative study of neuroprotective effect of angiotensin converting enzyme inhibitors against scopolamine-induced neuroinflammation in albino Wistar rats was studied. Male albino rats were administered with scopolamine to induce memory impairment. The standard nootropic agent, piracetam (200 mg/kg b.w., [i.p.], perindopril (0.1 mg/kg b.w., [i.p.], enalapril (0.1 mg/kg b.w., [i.p.], and ramipril (0.1 mg/kg b.w., [i.p.] were administered in different group of animals for 5 days. On 5 th day, scopolamine (1 mg/kg b.w., i.p. was administered after 60 min of the last dose of test drug. Memory function was evaluated in Morris water maze (MWM test and pole climbing test (PCT. Biochemical estimations like glutathione (GSH, malondialdehyde (MDA, and acetylcholinesterase activity in the brain were estimated after completion of behavior study. All three test groups shows improvement in learning and memory in comparison to control group. Perindopril treated group showed a more effective significant decrease in escape latency time and transfer latency time compared to enalapril and ramipril treated group on day 4 in MWM test and PCT, respectively. Perindopril shows a significant reduction in MDA level and acetylcholinesterase activity and a significant rise in GSH level compared to enalapril and ramipril. The finding of this study indicates that Perindopril is more effective in memory retention compared to enalapril and ramipril.

  19. Ubiquitin Proteasome System in Parkinson Disease: a keeper or a witness?

    Science.gov (United States)

    Martins-Branco, Diogo; Esteves, Ana R.; Santos, Daniel; Arduino, Daniela M.; Swerdlow, Russell H.; Oliveira, Catarina R.; Januario, Cristina; Cardoso, Sandra M.

    2014-01-01

    Objective The aim of this work was to evaluate the role of Ubiquitin-Proteasome System (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patients population we evaluated aSN levels in plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a down regulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomers levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified an ubiquitin-dependent clearance insufficiency with accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. PMID:22921536

  20. Efficacy and gastrointestinal tolerability of ML3403, a selective inhibitor of p38 MAP kinase and CBS-3595, a dual inhibitor of p38 MAP kinase and phosphodiesterase 4 in CFA-induced arthritis in rats.

    Science.gov (United States)

    Koch, Diana A; Silva, Rodrigo B M; de Souza, Alessandra H; Leite, Carlos E; Nicoletti, Natália F; Campos, Maria M; Laufer, Stefan; Morrone, Fernanda B

    2014-03-01

    Mitogen-activated protein kinase (MAPK) p38 inhibitors have entered the clinical phase, although many of them have failed due to high toxicity and lack of efficacy. In the present study we compared the effects of the selective p38 inhibitor ML3403 and the dual p38-PDE4 inhibitor CBS-3595, on inflammatory and nociceptive parameters in a model of polyarthritis in rats. Male Wistar rats (180-200 g) were used for the complete Freund's adjuvant (CFA)-induced arthritis model and they were evaluated at 14-21 days. We also analysed the effects of these pharmacological tools on liver and gastrointestinal toxicity and on cytokine levels. Repeated CBS-3595 (3 mg/kg) or ML3403 (10 mg/kg) administration produced significant anti-inflammatory actions in the chronic arthritis model induced by CFA. CBS-3595 and ML3403 treatment also markedly reduced the production of the proinflammatory cytokine IL-6 in the paw tissue, whereas it widely increased the levels of the anti-inflammatory cytokine IL-10. Moreover, CBS-3595 produced partial anti-allodynic effects in the CFA model at 4 and 8 days after treatment. Notably, ML3403 and CBS-3595 did not show marked signs of hepatoxicity, as supported by unaltered histological observations in the liver sections. Finally, both compounds were safe in the gastrointestinal tract, according to evaluation of intestinal biopsies. CBS-3595 displayed a superior profile regarding its anti-inflammatory effects. Thus p38 MAPK/PDE4 blocking might well constitute a relevant strategy for the treatment of RA.

  1. 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.

  2. 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.

  3. Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

    International Nuclear Information System (INIS)

    Rodríguez-Rigueiro, Teresa; Valladares-Ayerbes, Manuel; Haz-Conde, Mar; Aparicio, Luis A; Figueroa, Angélica

    2011-01-01

    The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells. Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed. Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The

  4. The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices

    NARCIS (Netherlands)

    Kistemaker, Loes E M; Oenema, Tjitske A; Baarsma, Hoeke A; Bos, I. Sophie T.; Schmidt, Martina; Facchinetti, Fabrizio; Civelli, Maurizio; Villetti, Gino; Gosens, Reinoud

    2017-01-01

    Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced

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

    Directory of Open Access Journals (Sweden)

    Nibedita Chattopadhyay

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

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Na+/Ca2+ exchange inhibitor, KB-R7943, attenuates contrast-induced acute 
kidney injury.

    Science.gov (United States)

    Yang, Dingwei; Yang, Dingping; Jia, Ruhan; Tan, Jin

    2013-01-01

    Intracellular Ca2+ overload is considered to be a key factor in contrast-induced acute kidney injury (CI-AKI). The Na+/Ca2+ exchanger (NCX) system is one of the main pathways of intracellular Ca2+ overload. We investigated the effects of KB-R7943, an inhibitor of the reverse mode of NCX, on CI-AKI in a rat model. Rats were divided into control group, CI-AKI group and pretreatment groups (with KB-R7943 dose of 5 or 10 mg/kg). CI-AKI was induced by diatrizoate administration in rats with cholesterol-supplemented diet for 8 weeks. Renal function and renal hemodynamics were determined 1 day following contrast medium administration. Renal histopathology was observed by light microscope. Renal tubular apoptosis was examined by TUNEL. Renal endothelin-1 (ET-1) was measured by radioimmunoassay. Renal malondialdehyde (MDA) and catalase (CAT) were measured as oxidative markers. Levels of serum creatinine (Scr), renal ET-1, MDA and CAT, and resistance index (RI) of renal blood vessels increased significantly in CI-AKI rats. The 
increases in Scr and RI of renal blood vessels induced by diatrizoate were suppressed significantly and 
dose-dependently by pretreatment with KB-R7943. Histopathological and TUNEL results showed that 
the contrast medium-induced severe renal tubular 
necrosis and apoptosis were significantly and dose-dependently attenuated by KB-R7943. KB-R7943 significantly suppressed the increment of renal ET-1 content and MDA and CAT level induced by contrast medium administration. Activation of the reverse mode of NCX, followed by ET-1 overproduction and increased oxidative stress, seems to play an important role in the pathogenesis of CI-AKI. The inhibitor of the reverse mode of NCX, KB-R7943, has renoprotective effects on CI-AKI.

  8. OsCESA9 conserved-site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice.

    Science.gov (United States)

    Li, Fengcheng; Xie, Guosheng; Huang, Jiangfeng; Zhang, Ran; Li, Yu; Zhang, Miaomiao; Wang, Yanting; Li, Ao; Li, Xukai; Xia, Tao; Qu, Chengcheng; Hu, Fan; Ragauskas, Arthur J; Peng, Liangcai

    2017-09-01

    Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Since cellulose synthase (CESA) gene was first identified, several dozen CESA mutants have been reported, but almost all mutants exhibit the defective phenotypes in plant growth and development. In this study, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P-CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%-41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cell walls compared with wild type. CESA co-IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low-DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3-fold and ethanol productivity by 34%-42%. This study has for the first time reported a direct modification for the low-DP cellulose production that has broad applications in biomass industries. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  9. NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

    Science.gov (United States)

    Seo, Seung Un; Kim, Tae Hwan; Kim, Dong Eun; Min, Kyoung-Jin; Kwon, Taeg Kyu

    2017-10-01

    Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Epithelial tissue hyperplasia induced by the RAF inhibitor PF-04880594 is attenuated by a clinically well-tolerated dose of the MEK inhibitor PD-0325901.

    Science.gov (United States)

    Torti, Vince R; Wojciechowicz, Donald; Hu, Wenyue; John-Baptiste, Annette; Evering, Winston; Troche, Gabriel; Marroquin, Lisa D; Smeal, Tod; Yamazaki, Shinji; Palmer, Cynthia L; Burns-Naas, Leigh Ann; Bagrodia, Shubha

    2012-10-01

    Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal-regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor-induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers. ©2012 AACR.

  11. 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.

  12. Saururus cernuus lignans-Potent sm