Paeoniflorin inhibits glioblastoma growth in vivo and in vitro: a role for the Triad3A-dependent ubiquitin proteasome pathway in TLR4 degradation

ABSTRACT Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.

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Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin–proteasome pathway

Biochemical Society Transactions ◽

10.1042/bst0330953 ◽

2005 ◽

Vol 33 (5) ◽

pp. 953-956 ◽

Cited By ~ 30

Author(s):

Q. He ◽

Y. Liu

Keyword(s):

Circadian Clock ◽

Major Function ◽

Ubiquitin Proteasome Pathway ◽

Mutant Strains ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

The Stability ◽

Clock Protein

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

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Interaction of U-box E3 ligase SNEV with PSMB4, the β7 subunit of the 20 S proteasome

Biochemical Journal ◽

10.1042/bj20041517 ◽

2005 ◽

Vol 388 (2) ◽

pp. 593-603 ◽

Cited By ~ 38

Author(s):

Marlies LÖSCHER ◽

Klaus FORTSCHEGGER ◽

Gustav RITTER ◽

Martina WOSTRY ◽

Regina VOGLAUER ◽

...

Keyword(s):

Immunofluorescence Microscopy ◽

E3 Ligase ◽

Proteasome Inhibition ◽

Ubiquitin Proteasome System ◽

E3 Ligases ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Recognition of specific substrates for degradation by the ubiquitin–proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the β7 subunit of the 20 S proteasome. Upon inhibition of proteasome activity, SNEV does not accumulate within the cells although its co-localization with the proteasome increases significantly. Since immunofluorescence microscopy also shows increased co-localization of SNEV with ubiquitin after proteasome inhibition, without SNEV being ubiquitinated by itself, we suggest that SNEV shows E3 ligase activity not only in vitro but also in vivo and escorts its substrate to the proteasome. Since the yeast homologue of SNEV, Prp19, also interacts with the yeast β7 subunit of the proteasome, this mechanism seems to be conserved during evolution. Therefore these results support the hypothesis that E3 ligases might generally be involved in substrate transport to the proteasome. Additionally, our results provide the first evidence for a physical link between components of the ubiquitin–proteasome system and the spliceosome.

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Astragalus polysaccharide, a component of traditional Chinese medicine, inhibits muscle cell atrophy (cachexia) in an in vivo and in vitro rat model of chronic renal failure by activating the ubiquitin-proteasome pathway

Experimental and Therapeutic Medicine ◽

10.3892/etm.2017.4492 ◽

2017 ◽

Vol 14 (1) ◽

pp. 91-96 ◽

Cited By ~ 5

Author(s):

Zhenbo Geng ◽

Lianbo Wei ◽

Chunhua Zhang ◽

Xiaohua Yan

Keyword(s):

Renal Failure ◽

Chronic Renal Failure ◽

Chinese Medicine ◽

Traditional Chinese Medicine ◽

Rat Model ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

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

Oncotarget ◽

10.18632/oncotarget.2317 ◽

2014 ◽

Vol 5 (17) ◽

pp. 7870-7885 ◽

Cited By ~ 50

Author(s):

Hsien-Yeh Hsu ◽

Tung-Yi Lin ◽

Yu-Chung Wu ◽

Shu-Ming Tsao ◽

Pai-An Hwang ◽

...

Keyword(s):

Lung Cancer ◽

Ubiquitin Proteasome Pathway ◽

Tgfβ Receptor ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

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The RING Domain of cIAP1 Mediates the Degradation of RING-bearing Inhibitor of Apoptosis Proteins by Distinct Pathways

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0107 ◽

2008 ◽

Vol 19 (7) ◽

pp. 2729-2740 ◽

Cited By ~ 42

Author(s):

Herman H. Cheung ◽

Stéphanie Plenchette ◽

Chris J. Kern ◽

Douglas J. Mahoney ◽

Robert G. Korneluk

Keyword(s):

Fas Ligand ◽

Ring Domain ◽

Inhibitor Of Apoptosis ◽

Inhibitor Of Apoptosis Proteins ◽

Down Regulation ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Apoptosis Proteins

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR–mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.

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Abstract 37: Centrosome Destabilization Through the Ubiquitin-Proteasome Pathway Regulates Proplatelet Production

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.37 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Kellie R Machlus ◽

Prakrith Vijey ◽

Thomas Soussou ◽

Joseph E Italiano

Keyword(s):

Protein Degradation ◽

Proteasome Inhibitors ◽

Aurora Kinase ◽

Proteasome Activity ◽

Major Pathway ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Proplatelet Formation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

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c-Myc Proteolysis by the Ubiquitin-Proteasome Pathway: Stabilization of c-Myc in Burkitt's Lymphoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.20.7.2423-2435.2000 ◽

2000 ◽

Vol 20 (7) ◽

pp. 2423-2435 ◽

Cited By ~ 272

Author(s):

Mark A. Gregory ◽

Stephen R. Hann

Keyword(s):

Amino Acids ◽

Phosphorylation Site ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

Lymphoma Cells ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Burkitt’S Lymphoma Cells

ABSTRACT The c-Myc oncoprotein is a transcription factor which is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt's lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Here we demonstrate that proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein which are required for rapid proteolysis. We found that a centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. We found that c-Myc is significantly stabilized (two- to sixfold) in many Burkitt's lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitt's lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitt's lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitt's lymphoma cells.

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Reproductive Cytotoxicity Is Predicted by Magnetic Resonance Microscopy and Confirmed by Ubiquitin–Proteasome Immunohistochemistry in a Theophylline-Induced Model of Rat Testicular and Epididymal Toxicity

Microscopy and Microanalysis ◽

10.1017/s143192760505021x ◽

2005 ◽

Vol 11 (4) ◽

pp. 300-312 ◽

Cited By ~ 7

Author(s):

M.W. Tengowski ◽

P. Sutovsky ◽

L.W. Hedlund ◽

D.J. Guyot ◽

J.E. Burkhardt ◽

...

Keyword(s):

Magnetic Resonance ◽

Phosphodiesterase Inhibitor ◽

Magnetic Resonance Microscopy ◽

Vascular Perfusion ◽

Ubiquitin Proteasome Pathway ◽

Organ Systems ◽

Water Signal ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

This study investigated the testicular changes in the rat induced by the nonspecific phosphodiesterase inhibitor, theophylline using magnetic resonance microscopy (MRM) and ubiquitin immunostaining techniques.In vivoT1- and T2-weighted images were acquired at 2 T under anesthesia. Increased signal observed in the theophylline-treated rats suggests that leakage of MRM contrast was occurring.In vivoMRM results indicate that day 16 testis displayed an increased T1-weighted water signal in the area of the seminiferous tubule that decreased by day 32. These findings were validated by histopathology, suggesting thatin vivoMRM has the sensitivity to predict changes in testis and epididymal tissues. The participation of the ubiquitin system was investigated, using probes for various markers of the ubiquitin-proteasome pathway. MRM can be used to detect subtle changes in the vascular perfusion of organ systems, and the up-regulation/mobilization of ubiquitin-proteasome pathway may be one of the mechanisms used in theophylline-treated epididymis to remove damaged cells before storage in the cauda epididymis. The combined use ofin vivoMRM and subsequent tissue or seminal analysis for the presence of ubiquitin in longitudinal studies may become an important biomarker for assessing testis toxicities drug studies.

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E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

Journal of Virology ◽

10.1128/jvi.01684-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1174-1185 ◽

Cited By ~ 86

Author(s):

Masayuki Shirakura ◽

Kyoko Murakami ◽

Tohru Ichimura ◽

Ryosuke Suzuki ◽

Tetsu Shimoji ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Core Protein ◽

Protein Levels ◽

The Core ◽

Hcv Core Protein ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Viral Nucleocapsid

ABSTRACT Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

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