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

2006 ◽  
Vol 81 (3) ◽  
pp. 1174-1185 ◽  
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.

scholarly journals S-Nitrosylation of IRP2 Regulates Its Stability via the Ubiquitin-Proteasome Pathway

2004 ◽  
Vol 24 (1) ◽  
pp. 330-337 ◽  
Author(s):  
Sangwon Kim ◽  
Simon S. Wing ◽  
Prem Ponka
Keyword(s):  
Regulatory Protein ◽  
Untranslated Regions ◽  
Raw 264.7 Cells ◽  
Ubiquitin Proteasome Pathway ◽  
Functional Implications ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Iron Responsive Elements

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.

scholarly journals Oestrogen causes ATBF1 protein degradation through the oestrogen-responsive E3 ubiquitin ligase EFP

2012 ◽  
Vol 444 (3) ◽  
pp. 581-590 ◽  
Author(s):  
Xue-Yuan Dong ◽  
Xiaoying Fu ◽  
Songqing Fan ◽  
Peng Guo ◽  
Dan Su ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Feedback Loop ◽  
E3 Ubiquitin Ligase ◽  
Breast Development ◽  
Protein Levels ◽  
Ubiquitin Proteasome ◽  
Dependent Cell ◽  
Proteasome Pathway

We reported previously that the tumour suppressor ATBF1 (AT motif-binding factor 1) formed an autoregulatory feedback loop with oestrogen–ERα (oestrogen receptor α) signalling to regulate oestrogen-dependent cell proliferation in breast cancer cells. In this loop ATBF1 inhibits the function of oestrogen–ERα signalling, whereas ATBF1 protein levels are fine-tuned by oestrogen-induced transcriptional up-regulation as well as UPP (ubiquitin–proteasome pathway)-mediated protein degradation. In the present study we show that EFP (oestrogen-responsive finger protein) is an E3 ubiquitin ligase mediating oestrogen-induced ATBF1 protein degradation. Knockdown of EFP increases ATBF1 protein levels, whereas overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in oestrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumours the levels of ATBF1 protein are positively correlated with the levels of EFP protein, as both are directly up-regulated ERα target gene products. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for oestrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and oestrogen–ERα signalling and thus implicate ATBF1 in oestrogen-dependent breast development and carcinogenesis.

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

2018 ◽  
Vol Volume 10 ◽  
pp. 887-897 ◽  
Author(s):  
Zhaotao Wang ◽  
Guoyong Yu ◽  
Zhi Liu ◽  
Jianwei Zhu ◽  
Chen Chen ◽  
...  
Keyword(s):  
Ubiquitin Proteasome Pathway ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

scholarly journals Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1636 ◽  
Author(s):  
Chris D. Balak ◽  
Jesse M. Hunter ◽  
Mary E. Ahearn ◽  
David Wiley ◽  
Gennaro D'urso ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Diagnostic Assays ◽  
Missense Variants ◽  
Thioester Bond ◽  
In The Wild ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

Background: X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the UBA1 gene, which contains the active adenylation domain (AAD). Methods: In this study, our group characterized the three known missense variants in vitro. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions in vitro to determine possible biochemical effects of the missense variants. Results: Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. Conclusions: Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1’s enzymatic activity with Ubiquitin, to other roles such as altering UBA1 mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important in vivo binding partners.  These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations’ biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.

scholarly journals Hepatitis C Virus Core Protein Interacts with 14-3-3 Protein and Activates the Kinase Raf-1

2000 ◽  
Vol 74 (4) ◽  
pp. 1736-1741 ◽  
Author(s):  
Hiroshi Aoki ◽  
Junpei Hayashi ◽  
Mitsuhiko Moriyama ◽  
Yasuyuki Arakawa ◽  
Okio Hino
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Growth Regulation ◽  
Core Protein ◽  
Dependent Manner ◽  
Kinase Activation ◽  
Virus Core ◽  
Hcv Core Protein

ABSTRACT Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core–14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.

Abstract 369: Engineering Rrm2 to Elevate 2-deoxy-ATP and Improve Cardiomyocyte Function

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Jason D Murray ◽  
Xuan Guan ◽  
Farid Moussavi-Harami ◽  
Sigurast S Olafsson ◽  
Charles E Murry ◽  
...  
Keyword(s):  
Therapeutic Option ◽  
Induced Pluripotent Stem Cell ◽  
Left Ventricular ◽  
Ubiquitin Binding ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Induced Pluripotent ◽  
Cultured Cardiomyocytes

Overexpression of ribonucleotide reductase (RNR) in cardiomyocytes increases the amount of cytosolic 2-deoxy-ATP (dATP), which can be used by myosin and significantly increases contraction of cardiac muscle at all levels of calcium activation. Our group is working to develop enhanced dATP as a therapeutic option for heart failure. We have demonstrated that virally-mediated overexpression of RNR elevates dATP and increases the rate and magnitude of contraction and increases left ventricular contraction in normal hearts as well as rodent models of myocardial infarction and dilated cardiomyopathy. RNR is a heterotetramer containing two subunits, Rrm1 and Rrm2. While cardiomyocyte-specific overexpression of Rrm1 is stable, we have observed high variability in expression levels of the Rrm2 subunit in multiple disease models. We hypothesized that this variability was largely due to protein degradation via the ubiquitin-proteasome complex (UPC). We found that pharmacological inhibition of proteasome activity leads to increased expression of Rrm2 in virally-transduced cardiomyocytes in vitro. To confirm the hypothesis that the overexpressed Rrm2 is degraded via UPC-mediated degradation, we engineered mutations in specific ubiquitin-binding degrons of the Rrm2 gene. Transfecting human induced pluripotent stem cell-derived cardiomyocytes resulted in higher levels of Rrm2 than those overexpressing wild-type protein and resulted in higher levels of cytosolic dATP as measured by Liquid chromatography-mass spectrometry. Ongoing and planned experiments will compare the effects of this engineered mutation on Rrm2 overexpression, dATP production, and contractility in cultured cardiomyocytes. Our goal is to develop an improved RNR vector that will be resistant to degradation through the ubiquitin-proteasome pathway and therefore enable more stable and consistent RNR enzyme activity and deoxynucleotide levels of cardiomyocytes transduced in vivo.

scholarly journals Nanosized Particles Assembled by a Recombinant Virus Protein Are Able to Encapsulate Negatively Charged Molecules and Structured RNA

Polymers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 858
Author(s):  
Hemalatha Mani ◽  
Yi-Cheng Chen ◽  
Yen-Kai Chen ◽  
Wei-Lin Liu ◽  
Shih-Yen Lo ◽  
...  
Keyword(s):  
Self Assembly ◽  
Rna Binding ◽  
Core Protein ◽  
Average Diameter ◽  
Virus Protein ◽  
Force Microscopy ◽  
Atomic Force ◽  
Hcv Core Protein

RNA-based molecules have recently become hot candidates to be developed into therapeutic agents. However, successful applications of RNA-based therapeutics might require suitable carriers to protect the RNA from enzymatic degradation by ubiquitous RNases in vivo. Because of their better biocompatibility and biodegradability, protein-based nanoparticles are considered to be alternatives to their synthetic polymer-based counterparts for drug delivery. Hepatitis C virus (HCV) core protein has been suggested to be able to self-assemble into nucleocapsid-like particles in vitro. In this study, the genomic RNA-binding domain of HCV core protein consisting of 116 amino acids (p116) was overexpressed with E. coli for investigation. The recombinant p116 was able to assemble into particles with an average diameter of approximately 27 nm, as visualized by electron microscopy and atomic force microscopy. Measurements with fluorescence spectroscopy, flow cytometry, and fluorescence quenching indicated that the p116-assembled nanoparticles were able to encapsulate small anionic molecules and structured RNA. This study demonstrates methods that exploit the self-assembly nature of a virus-derived protein for nanoparticle production. This study also suggests that the virus-derived protein-assembled particles could possibly be developed into potential carriers for anionic molecular drugs and structured RNA-based therapeutics.

scholarly journals Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

2008 ◽  
Vol 19 (4) ◽  
pp. 1328-1336 ◽  
Author(s):  
Daisuke Morito ◽  
Kazuyoshi Hirao ◽  
Yukako Oda ◽  
Nobuko Hosokawa ◽  
Fuminori Tokunaga ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Yeast Cells ◽  
Glutathione S Transferase ◽  
Assembly Factor ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Membrane Spanning ◽  
Interfering Rna

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRΔF508, by specifically promoting ubiquitylation of CFTRΔF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRΔF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRΔF508 and catalyzes further polyubiquitylation of CFTRΔF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRΔF508.

scholarly journals Identification and Mechanistic Studies of a Novel Ubiquitin E1 Inhibitor

2012 ◽  
Vol 17 (4) ◽  
pp. 421-434 ◽  
Author(s):  
Dana Ungermannova ◽  
Seth J. Parker ◽  
Christopher G. Nasveschuk ◽  
Douglas A. Chapnick ◽  
Andrew J. Phillips ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Small Molecule Compound ◽  
Ubiquitin Proteasome ◽  
A Cell ◽  
Proteasome Pathway

Protein degradation via the ubiquitin-proteasome pathway is important for a diverse number of cellular processes ranging from cell signaling to development. Disruption of the ubiquitin pathway occurs in a variety of human diseases, including several cancers and neurological disorders. Excessive proteolysis of tumor suppressor proteins, such as p27, occurs in numerous aggressive human tumors. To discover small-molecule inhibitors that potentially prevent p27 degradation, we developed a series of screening assays, including a cell-based screen of a small-molecule compound library and two novel nucleotide exchange assays. Several small-molecule inhibitors, including NSC624206, were identified and subsequently verified to prevent p27 ubiquitination in vitro. The mechanism of NSC624206 inhibition of p27 ubiquitination was further unraveled using the nucleotide exchange assays and shown to be due to antagonizing ubiquitin activating enzyme (E1). We determined that NSC624206 and PYR-41, a recently reported inhibitor of ubiquitin E1, specifically block ubiquitin-thioester formation but have no effect on ubiquitin adenylation. These studies reveal a novel E1 inhibitor that targets a specific step of the E1 activation reaction. NSC624206 could, therefore, be potentially useful for the control of excessive ubiquitin-mediated proteolysis in vivo.

scholarly journals Characterization of SPP inhibitors suppressing propagation of HCV and protozoa

2017 ◽  
Vol 114 (50) ◽  
pp. E10782-E10791 ◽  
Author(s):  
Junki Hirano ◽  
Toru Okamoto ◽  
Yukari Sugiyama ◽  
Tatsuya Suzuki ◽  
Shinji Kusakabe ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Core Protein ◽  
Drug Resistant ◽  
Hcv Core Protein ◽  
Core Proteins ◽  
Secretase Inhibitor ◽  
Direct Acting

Signal peptide peptidase (SPP) is an intramembrane aspartic protease involved in the maturation of the core protein of hepatitis C virus (HCV). The processing of HCV core protein by SPP has been reported to be critical for the propagation and pathogenesis of HCV. Here we examined the inhibitory activity of inhibitors for γ-secretase, another intramembrane cleaving protease, against SPP, and our findings revealed that the dibenzoazepine-type structure in the γ-secretase inhibitors is critical for the inhibition of SPP. The spatial distribution showed that the γ-secretase inhibitor compound YO-01027 with the dibenzoazepine structure exhibits potent inhibiting activity against SPP in vitro and in vivo through the interaction of Val223 in SPP. Treatment with this SPP inhibitor suppressed the maturation of core proteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting antivirals. YO-01027 also efficiently inhibited the propagation of protozoa such asPlasmodium falciparumandToxoplasma gondii. These data suggest that SPP is an ideal target for the development of therapeutics not only against chronic hepatitis C but also against protozoiasis.

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