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Ubiquitin Ligase (ubiquitin + ligase)
Kinds of Ubiquitin Ligase Selected AbstractsA tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer's disease-relevant insultsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2003Takako Niikura Abstract Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S -transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways. [source] Ubiquitination of E3 ubiquitin ligase TRIM5, and its potential roleFEBS JOURNAL, Issue 7 2008Keiko Yamauchi HIV-1 efficiently infects susceptible cells and causes AIDS in humans. Although HIV can also enter the cells of Old World monkeys, it encounters a block before reverse transcription. Data have shown that this species-specific restriction is mediated by tripartite motif (TRIM)5,, whose molecular function is still undefined. Here, we show that TRIM5, functions as a RING-finger-type E3 ubiquitin ligase both in vitro and in vivo and ubiquitinates itself in cooperation with the E2 ubiquitin-conjugating enzyme UbcH5B. In addition to the self-ubiquitination, we show that TRIM5, is ubiquitinated by another E3 ubiquitin ligase, Ro52, and deubiquitinated by YopJ, one of the pathogenic proteins derived from Yersinia species. Thus, the ubiquitination of TRIM5, is catalyzed by itself and Ro52 and downregulated by YopJ. Unexpectedly, although TRIM5, is ubiquitinated, our results have revealed that the proteasome inhibitors MG115 and MG132 do not stabilize it in HeLa cells, suggesting that the ubiquitination of TRIM5, does not lead to proteasomal degradation. Importantly, TRIM5, is clearly conjugated by a single ubiquitin molecule (monoubiquitination). Our monoubiquitin-fusion assay suggests that monoubiquitination is a signal for TRIM5, to translocate from cytoplasmic bodies to the cytoplasm. [source] Identification and functional characterization of an Src homology domain 3 domain-binding site on CblFEBS JOURNAL, Issue 23 2006Archana Sanjay§ Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP(540,551), which corresponds to residues 540,551 of Cbl, inhibited the binding of a GST,Src SH3 fusion protein to Cbl, whereas RDLAPPAPPPDR(540,551) did not, suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543,548 reduced Src binding to the Cbl 479,636 fragment significantly more than mutating the prolines in the PPVPPR(494,499) motif, which was previously reported to bind Src SH3. Mutating Cbl prolines 543,548 to alanines substantially reduced Src binding to Cbl, Src-induced phosphorylation of Cbl, and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast, disabling the tyrosine kinase-binding domain of full-length Cbl by mutating glycine 306 to glutamic acid, and thereby preventing the previously described binding of the tyrosine kinase-binding domain to the Src phosphotyrosine 416, had no effect on Cbl phosphorylation, the inhibition of Src activity by full-length Cbl, or bone resorption. These data indicate that the Cbl RDLPPPP(540,546) sequence is a functionally important binding site for Src. [source] Rsp5 is required for the nuclear export of mRNA of HSF1 and MSN2/4 under stress conditions in Saccharomyces cerevisiaeGENES TO CELLS, Issue 2 2008Yutaka Haitani Rsp5 is an essential and multi-functional E3 ubiquitin ligase in Saccharomyces cerevisiae. We previously isolated the Ala401Glu rsp5 mutant that is hypersensitive to various stresses. In rsp5A401E cells, the transcription of the stress protein genes was defective. To understand the mechanism by which Rsp5 regulates the expression of stress proteins, we analyzed the expression and localization of two major transcription factors, Hsf1 and Msn2/4, required for stress protein gene expression in S. cerevisiae. The mRNA levels of HSF1 and MSN2/4 in rsp5A401E cells were slightly lower than those of wild-type cells. An interesting finding is that the protein levels of HSF1 and Msn2/4 were remarkably defective in rsp5A401E cells after exposure to temperature up-shift and ethanol, although these proteins are mainly localized in the nucleus under these stress conditions. We also showed that the mRNAs of HSF1 and MSN2/4 were accumulated in the nucleus of rsp5A401E cells after exposure to temperature up-shift and ethanol, and even under non-stress conditions, suggesting that Rsp5 is required for the nuclear export of these mRNAs. These results indicate that, in response to environmental stresses, Rsp5 primarily regulates the expression of Hsf1 and Msn2/4 at the post-transcriptional level and is involved in the repair system of stress-induced abnormal proteins. [source] Lafora disease in the Indian population: EPM2A and NHLRC1 gene mutations and their impact on subcellular localization of laforin and malin,HUMAN MUTATION, Issue 6 2008Shweta Singh Abstract Lafora disease (LD) is a fatal form of teenage-onset autosomal recessive progressive myoclonus epilepsy. LD is more common among geographic isolates and in populations with a higher rate of consanguinity. Mutations in two genes, EPM2A encoding laforin phosphatase, and NHLRC1 encoding malin ubiquitin ligase, have been shown to cause the LD. We describe here a systematic analysis of the EPM2A and the NHLRC1 gene sequences in 20,LD families from the Indian population. We identified 12 distinct mutations in 15,LD families. The identified novel mutations include 4 missense mutations (K140N, L310W, N148Y, and E210,K) and a deletion of exon 3 for EPM2A, and 4 missense mutations (S22R, L279P, L279P, and L126P) and a single base-pair insertional mutation (612insT) for NHLRC1. The EPM2A gene is known to encode two laforin isoforms having distinct carboxyl termini; a major isoform localized in the cytoplasm, and a minor isoform that targeted the nucleus. We show here that the effect of the EPM2A gene mutation L310W was limited to the cytoplasmic isoform of laforin, and altered its subcellular localization. We have also analyzed the impact of NHLRC1 mutations on the subcellular localization of malin. Of the 6 distinct mutants tested, three targeted the nucleus, one formed perinuclear aggregates, and two did not show any significant difference in the subcellular localization as compared to the wild-type malin. Our results suggest that the altered subcellular localization of mutant proteins of the EPM2A and NHLRC1 genes could be one of the molecular bases of the LD phenotype. © 2008 Wiley-Liss, Inc. [source] The degradation of cell cycle regulators by SKP2/CKS1 ubiquitin ligase is genetically controlled in rodent liver cancer and contributes to determine the susceptibility to the diseaseINTERNATIONAL JOURNAL OF CANCER, Issue 5 2010Diego F. Calvisi Abstract Previous work showed a genetic control of cell cycle deregulation during hepatocarcinogenesis. We now evaluated in preneoplastic lesions, dysplastic nodules and hepatocellular carcinoma (HCC), chemically induced in genetically susceptible F344 and resistant Brown Norway (BN) rats, the role of cell cycle regulating proteins in the determination of a phenotype susceptible to HCC development. p21WAF1, p27KIP1, p57KIP2 and p130 mRNA levels increased in fast growing lesions of F344 rats. Lower/no increases occurred in slowly growing lesions of BN rats. A similar behavior of RassF1A mRNA was previously found in the 2 rat strains. However, p21WAF1, p27KIP1, p57KIP, p130 and RassF1A proteins exhibited no change/low increase in the lesions of F344 rats and consistent rise in dysplastic nodules and HCC of BN rats. Increase in Cks1-Skp2 ligase and ubiquitination of cell cycle regulators occurred in F344 but not in BN rat lesions, indicating that posttranslational modifications of cell cycle regulators are under genetic control and contribute to determine a phenotype susceptible to HCC. Moreover, proliferation index of 60 human HCCs was inversely correlated with protein levels but not with mRNA levels of P21WAF1, P27KIP1, P57KIP2 and P130, indicating a control of human HCC proliferation by posttranslational modifications of cell cycle regulators. [source] Phosphorylation by COP9 Signalosome-Associated CK2 Promotes Degradation of p27 during the G1 Cell Cycle PhaseISRAEL JOURNAL OF CHEMISTRY, Issue 2 2006Xiaohua Huang The cell cycle regulator p27Kip1 (p27) is controlled by 26S proteasome-mediated proteolysis by two different pathways. From the S till the G2 phase of the cell cycle, degradation of p27 takes place in the nucleus and is initiated by CDK2-dependent phosphorylation of threonine 187 with subsequent ubiquitination by the SCFSkp2 ubiquitin ligase. During the G1 cell cycle phase (G1), p27 breakdown is cytosolic and is initiated by nuclear export with subsequent ubiquitination by a RING finger ligase called kip1 ubiquitination complex. Here we show that the COP9 signalosome (CSN) is a regulator of p27 proteolysis during G1. The CSN interacts with p27 and the CSN-associated kinase CK2 phosphorylates p27 at two regions. One is central to the protein (amino acids 101,113), and the other was mapped near to the C-terminus (amino acids 170,189). Elimination of the putative C-terminal phosphorylation sites stabilizes ectopic p27 towards proteasomal degradation and abolishes CSN,p27 binding. Inhibition of CSN-associated kinase activity by curcumin attenuates loss of p27 upon cell cycle re-entry. Similar but not additive effects of the phosphoinositol-3-kinase blocker LY 290042 may point to a common pathway of CSN-associated CK2 and protein kinase B/Akt (Akt) in regulating p27 abundance. Akt is found in Flag pulldowns of lysates obtained from cells permanently expressing Flag-tagged CSN2, indicating that Akt is a novel kinase associated with the CSN. Thus, the CSN seems to regulate p27 proteolysis at G1 downstream of Ras-mediated signal pathways. [source] ,4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)-binding proteinJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2010William J. McDonald Abstract Mammalian ,4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target-of-rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell-cycle progression. ,4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using ,4 as bait in yeast two-hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of ,4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)-binding protein (PABP), suggesting that PABP may also interact with ,4. PABP recruits translation factors to the poly(A)-tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between ,4 and EDD in rat Nb2 T-lymphoma and human MCF-7 breast cancer cell lines. ,4 also interacted with PABP in Nb2, MCF-7 and the human Jurkat T-leukemic and K562 myeloma cell lines. COS-1 cells, transfected with Flag-tagged-pSG5-EDD, gave a (Flag)-EDD,,4 immunocomplex. Furthermore, deletion mutants of ,4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C-terminal region of ,4, independent of the ,4-PP2Ac binding site. Therefore, in addition to PP2Ac, ,4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell-cycle progression. J. Cell. Biochem. 110: 1123,1129, 2010. Published 2010 Wiley-Liss, Inc. [source] Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2007Robert J. Nadeau Abstract Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGF-dependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when co-expressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels. J. Cell. Biochem. 100: 151,160, 2007. © 2006 Wiley-Liss, Inc. [source] Centrosome function in normal and tumor cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2006Satish Sankaran Abstract Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process. J. Cell. Biochem. 99: 1240,1250, 2006. © 2006 Wiley-Liss, Inc. [source] Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopyJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2010Gloria Funari Abstract Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour-suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down-regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N-terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein,protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti-cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd. [source] A ubiquitin ligase HRD1 promotes the degradation of Pael receptor, a substrate of ParkinJOURNAL OF NEUROCHEMISTRY, Issue 6 2006Tomohiro Omura Abstract It has been proposed that in autosomal recessive juvenile parkinsonism (AR-JP), a ubiquitin ligase (E3) Parkin, which is involved in endoplasmic reticulum-associated degradation (ERAD), lacks E3 activity. The resulting accumulation of Parkin-associated endothelin receptor-like receptor (Pael-R), a substrate of Parkin, leads to endoplasmic reticulum stress, causing neuronal death. We previously reported that human E3 HRD1 in the endoplasmic reticulum protects against endoplasmic reticulum stress-induced apoptosis. This study shows that HRD1 was expressed in substantia nigra pars compacta (SNC) dopaminergic neurons and interacted with Pael-R through the HRD1 proline-rich region, promoting the ubiquitylation and degradation of Pael-R. Furthermore, the disruption of endogenous HRD1 by small interfering RNA (siRNA) induced Pael-R accumulation and caspase-3 activation. We also found that ATF6 overexpression, which induced HRD1, accelerated and caused Pael-R degradation; the suppression of HRD1 expression by siRNA partially prevents this degradation. These results suggest that in addition to Parkin, HRD1 is also involved in the degradation of Pael-R. [source] Sequestosome 1/p62 shuttles polyubiquitinated tau for proteasomal degradationJOURNAL OF NEUROCHEMISTRY, Issue 1 2005Jeganathan Ramesh Babu Abstract Inclusions isolated from several neurodegenerative diseases, including Alzheimer's disease (AD), are characterized by ubiquitin-positive proteinaceous aggregates. Employing confocal and immunoelectron microscopy, we find that the ubiquitin-associating protein sequestosome1/p62, co-localizes to aggregates isolated from AD but not control brain, along with the E3 ubiquitin ligase, TRAF6. This interaction could be recapitulated by co-transfection in HEK293 cells. Employing both in vitro and in vivo approaches, tau was found to be a substrate of the TRAF6, possessing lysine 63 polyubiquitin chains. Moreover, tau recovered from brain of TRAF6 knockout mice, compared with wild type, was not ubiquitinated. Tau degradation took place through the ubiquitin,proteasome pathway and was dependent upon either the K63-polyubiquitin chains or upon p62. In brain lysates of p62 knockout mice, tau fails to co-interact with Rpt1, a proteasomal subunit, thereby indicating a requirement for p62 shuttling of tau to the proteasome. Our results demonstrate that p62 interacts with K63-polyubiquitinated tau through its UBA domain and serves a novel role in regulating tau proteasomal degradation. We propose a model whereby either a decline in p62 expression or a decrease in proteasome activity may contribute to accumulation of insoluble/aggregated K63-polyubiquitinated tau. [source] Alterations of hHrd1 expression are related to hyperphosphorylated tau in the hippocampus in Alzheimer's diseaseJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Hai-Long Hou Abstract The degradation of aberrantly phosphorylated tau in neurons plays an important role in the pathogenesis of Alzheimer's disease (AD). hHrd1 is a newly identified ubiquitin ligase involved in the endoplasmic reticulum (ER)-associated protein degradation. The expression and function of hHrd1 in AD brain remains elusive. In the present study, the expression of hHrd1 in AD hippocampus and the morphological relations between hHrd1 expression and pretangle formation were studied by using immunohistochemical single- and double-labeling methods. The results showed that hHrd1 was expressed in neurons and reactive astrocytes, especially in the CA2,CA4 hippocampal subfields. The ratio of hHrd1-positive neurons/astrocytes to total neurons/astrocytes was increased in the CA1 subfield in AD hippocampus compared with the age-matched controls (P < 0.05). Most Alz-50 labeled pretangles were colocalized with hHrd1, and the expression levels showed an inversed change, implied that hHrd1 might be associated with the degradation of hyperphosphorylated tau. © 2006 Wiley-Liss, Inc. [source] Regulation of embryonic endochondral ossification by Smurf2JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2008Qiuqian Wu Abstract Smurf2 is an E3 ubiquitin ligase that targets TGF-, receptor activated Smad2 and Smad3 for the proteasome in primary articular chondrocytes, thus stimulating their hypertrophic differentiation. Comparatively, how Smurf2 functions in growth plate chondrocytes in a developing long bone is an open question. In this study, we measured the mRNA levels of endogenous Smurf2 and type X collagen in chick growth plate at different embryonic stages to monitor the correlation between the level of Smurf2 expression and chondrocyte maturational stage. We found that high levels of Smurf2 were associated with the differentiative and proliferative stages, while Smurf2 levels were thereafter decreased as the chondrocytes matured toward hypertrophy. In addition, we injected Smurf2 -RCAS into chick wing buds at HH stage 20,23 and examined how the ectopic overexpression of Smurf2 in condensing chondrogenic mesenchyme affects the subsequent process of chondrocyte maturation and ossification during embryonic development. Histological analysis showed that overexpression of Smurf2 in a developing wing bud accelerated chondrocyte maturation and endochondral ossification, which may result from a decrease in TGF-, signaling in the infected chondrocytes with Smurf2 -RCAS. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:704,712, 2008 [source] Exploration of oligosaccharide-protein interactions in glycoprotein quality control by synthetic approachesTHE CHEMICAL RECORD, Issue 6 2006Shinya Hagihara Abstract High-mannose-type oligosaccharides, which are cotranslationally introduced to nascent polypeptides, play important roles in glycoprotein quality control. This process is highly complex, involving a number of lectins, chaperones, and glycan-processing enzymes. For example, calnexin and calreticulin (CRT) are molecular chaperones that recognize monoglucosylated forms of high-mannose-type glycans. UDP-glucose,:,glycoprotein glucosyltransferase (UGGT) only glucosylates high-mannose-type glycans attached to partially folded proteins. Fbs1 is a component of ubiquitin ligase that recognizes sugar chains. Although recent studies have clarified the properties of these proteins, most of them used oligosaccharides derived from natural sources, which contain structural heterogeneity. In order to gain a more precise understanding, we started our program to comprehensively synthesize high-mannose-type glycans associated with a protein quality control system. Additionally, investigation of artificial glycoproteins led us to the discovery of the first nonpeptidic substrate of UGGT. These synthetic oligosaccharide probes have allowed us to conduct quantitative evaluations of the activity and specificity of CRT, Fbs1, and UGGT. © 2007 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 6: 290,302; 2006: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.20088 [source] AtCHIP functions as an E3 ubiquitin ligase of protein phosphatase 2A subunits and alters plant response to abscisic acid treatmentTHE PLANT JOURNAL, Issue 4 2006Jinhua Luo Summary CHIP proteins are E3 ubiquitin ligases that promote degradation of Hsp70 and Hsp90 substrate proteins through the 26S proteasome in animal systems. A CHIP-like protein in Arabidopsis, AtCHIP, also has E3 ubiquitin ligase activity and has important roles to play under conditions of abiotic stress. In an effort to study the mode of action of AtCHIP in plant cells, proteins that physically interact with it were identified. Like its animal orthologs, AtCHIP interacts with a unique class of ubiquitin-conjugating enzymes (UBC or E2) that belongs to the stress-inducible UBC4/5 class in yeast. AtCHIP also interacts with other proteins, including an A subunit of protein phosphatase 2A (PP2A). This PP2A subunit appears to be a substrate of AtCHIP, because it can be ubiquitylated by AtCHIP in vitro and because the activity of PP2A is increased in AtCHIP -overexpressing plants in the dark or under low-temperature conditions. Unlike the rcn1 mutant, that has reduced PP2A activity due to a mutation in one of the A subunit genes of PP2A, AtCHIP -overexpressing plants are more sensitive to ABA treatment. Since PP2A was previously shown to be involved in low-temperature responses in plants, the low-temperature-sensitive phenotype observed in AtCHIP -overexpressing plants might be partly due to the change in PP2A activity. These data suggest that the E3 ubiquitin ligase AtCHIP may function upstream of PP2A in stress-responsive signal transduction pathways under conditions of low temperature or in the dark. [source] Repression of light signaling by Arabidopsis SPA1 involves post-translational regulation of HFR1 protein accumulationTHE PLANT JOURNAL, Issue 1 2005Jianping Yang Summary Arabidopsis uses two major classes of photoreceptors to mediate seedling de-etiolation. The cryptochromes (cry1 and cry2) absorb blue/ultraviolet-A light, whereas the phytochromes (phyA,phyE) predominantly regulate responses to red/far-red light. Arabidopsis COP1 represses light signaling by acting as an E3 ubiquitin ligase in the nucleus, and is responsible for targeted degradation of a number of photomorphogenesis-promoting factors, including HY5, LAF1, phyA, and HFR1. Distinct light signaling pathways initiated by multiple photoreceptors (including both phytochromes and cryptochromes) eventually converge on COP1, causing its inactivation and nuclear depletion. Arabidopsis SPA1, which encodes a protein structurally related to COP1, also represses light signaling under various light conditions. In this study, we present genetic evidence supporting that HFR1, which encodes a photomorphogenesis-promoting bHLH transcription factor, acts downstream of SPA1 and is required for different subsets of branch pathways of light signaling controlled by SPA1 under different light conditions. We show that SPA1 physically interacts with HFR1 in a yeast two-hybrid assay and an in vitro co-immunoprecipitation assay. We demonstrate that higher levels of HFR1 protein accumulate in the spa1 mutant background under various light conditions, including far-red, red, blue, and white light, whereas a marginal increase in HFR1 transcript level is only seen in dark- and far-red light-grown spa1-100 mutants. Together, our data suggest that repression of light signaling by Arabidopsis SPA1 likely involves post-translational regulation of HFR1 protein accumulation. [source] Activation of synoviolin promoter in rheumatoid synovial cells by a novel transcription complex of interleukin enhancer binding factor 3 and GA binding protein ,ARTHRITIS & RHEUMATISM, Issue 1 2009Toshihiko Izumi Objective Synoviolin is an E3 ubiquitin ligase, and its overexpression is implicated in the pathogenesis of rheumatoid arthritis (RA). We reported previously that Ets binding site 1 (EBS-1) within the synoviolin promoter is crucial for the expression of synoviolin, and GA binding protein (GABP) binds to this site. This study was undertaken to elucidate the precise mechanisms of transcriptional regulation via EBS-1. Methods We performed purification and identification of complex components that bind to EBS-1 and inspected their contributions to the transcriptional regulation of synoviolin in rheumatoid synovial cells. We biochemically purified proteins that had EBS-1 binding activity and identified the proteins using liquid chromatography tandem mass spectrometry analysis. The identified proteins were verified to recruit and form the complex on EBS-1 using electrophoretic mobility shift assay and coimmunoprecipitation assay. Furthermore, their transcription activities were tested by reporter assays and RNA interference experiments. Results We identified interleukin enhancer binding factor 3 (ILF-3) as a novel factor in the complex. ILF-3 was demonstrated to activate the synoviolin promoter via association with GABP, in rheumatoid synovial cells. In addition, further activation was observed with ILF-2 and GABP,, previously reported interactants of ILF-3 and GABP,, respectively. Moreover, ILF-3,knockdown experiments showed reduced expression of the synoviolin gene. Conclusion Our findings indicate that ILF-3, which has been known to regulate IL-2 expression in T cells, up-regulates synoviolin expression with GABP, in rheumatoid synovial cells. ILF-3 might be a target for RA treatment through its effect on IL-2 in T cells and synoviolin in rheumatoid synovial cells. [source] Overexpression of synoviolin in peripheral blood and synoviocytes from rheumatoid arthritis patients and continued elevation in nonresponders to infliximab treatmentARTHRITIS & RHEUMATISM, Issue 7 2006Myew-Ling Toh Objective Synoviolin is a novel E3 ubiquitin ligase that has been implicated in the pathogenesis of rheumatoid arthritis (RA). The purpose of this study was to examine the expression and regulation of synoviolin by tumor necrosis factor , (TNF,), both in vivo and in vitro. Methods A total of 54 RA patients and 23 healthy control subjects were analyzed before, 4 hours after the first infusion, and at week 22 of infliximab treatment. Clinical response was assessed by the American College of Rheumatology criteria for 20% improvement and the Disease Activity Score in 28 joints (DAS28) at 6 months. Synoviolin messenger RNA expression was measured by real-time reverse transcription,polymerase chain reaction in peripheral blood (PB) and fibroblast-like synoviocytes (FLS) and with and without TNF, or interleukin-1, (IL-1,) stimulation. Results Synoviolin expression was increased in whole PB obtained from RA patients as compared with that from healthy controls and was significantly reduced early and late after infliximab treatment in responders, but in not nonresponders. Reduction in synoviolin expression was associated with reduced levels of markers of disease activity, including C-reactive protein levels. Nonresponders to infliximab therapy had significantly higher synoviolin expression at baseline as compared with responders, and this elevation persisted despite infliximab therapy. PB CD14+ monocytes expressed increased synoviolin levels compared with CD3+ lymphocytes, and TNF, or IL-1, induced a further increase in expression in CD3+ cells. TNF, or IL-1, induced sustained synoviolin expression in RA FLS. Conclusion Elevated PB levels of synoviolin were identified in circulating PB mononuclear cells and were associated with nonresponse to infliximab treatment. Sustained up-regulation of synoviolin by IL-1, and TNF, may contribute to prolonged survival of immune cells and dysregulated FLS proliferation, leading to RA chronicity. [source] Regulation of TGF-, family signaling by E3 ubiquitin ligasesCANCER SCIENCE, Issue 11 2008Yasumichi Inoue Members of the transforming growth factor-, (TGF-,) family, including TGF-,, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Alterations in their downstream signaling pathways are associated with a range of human diseases like cancer. TGF-, family members transduce signals through membrane serine/threonine kinase receptors and intracellular Smad proteins. The ubiquitin,proteasome pathway, an evolutionarily conserved cascade, tightly regulates TGF-, family signaling. In this pathway, E3 ubiquitin ligases play a crucial role in the recognition and degradation of target proteins by the 26S proteasomes. Smad degradation regulates TGF-, family signaling; HECT (homologous to the E6-accessory protein C-terminus)-type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1), Smurf2, and a RING-type E3 ubiquitin ligase, ROC1-SCFFbw1a have been implicated in Smad degradation. Smurf1 and Smurf2 bind to TGF-, family receptors via the inhibitory Smads, Smad6 and Smad7, to induce their ubiquitin-dependent degradation. Arkadia, a RING-type E3 ubiquitin ligase, induces the ubiquitination and degradation of Smad7 and corepressors, c-Ski and SnoN, to enhance TGF-, family signaling. Abnormalities in E3 ubiquitin ligases that control components of TGF-, family signaling may lead to the development and progression of various cancers. (Cancer Sci 2008; 99: 2107,2112) [source] Basic mechanisms of high-risk human papillomavirus-induced carcinogenesis: Roles of E6 and E7 proteinsCANCER SCIENCE, Issue 10 2007Mako Narisawa-Saito Human papillomaviruses (HPV) are believed to be the primary causal agents for development of pre-neoplastic and malignant lesions of the uterine cervix, and high-risk types such as type 16 and 18 are associated with more than 90% of all cervical carcinomas. The E6 and E7 genes of HPV are thought to play causative roles, since E6 promotes the degradation of p53 through its interaction with E6AP, an E3 ubiquitin ligase, whereas E7 binds to the retinoblastoma protein (pRb) and disrupts its complex formation with E2F transcription factors. Although prophylactic vaccines have become available, it is still necessary to clarify the mechanisms of HPV-induced carcinogenesis because of the widespread nature of HPV infection. Approximately 493 000 new cases of cervical cancer are diagnosed each year with approximately 274 000 mortalities due to invasive cervical cancer. In the present article, the mechanisms of HPV16 E6- and E7-induced multistep carcinogenesis and recently identified functions of these onco-proteins are reviewed. (Cancer Sci 2007; 98: 1505,1511) [source] Jostling for Position: Optimizing Linker Location in the Design of Estrogen Receptor-Targeting PROTACsCHEMMEDCHEM, Issue 7 2010Kedra Cyrus Dr. Abstract Estrogen receptor-, (ER) antagonists have been widely used for breast cancer therapy. Despite initial responsiveness, hormone-sensitive ER-positive cancer cells eventually develop resistance to ER antagonists. It has been shown that in most of these resistant tumor cells, the ER is expressed and continues to regulate tumor growth. Recent studies indicate that tamoxifen initially acts as an antagonist, but later functions as an ER agonist, promoting tumor growth. This suggests that targeted ER degradation may provide an effective therapeutic approach for breast cancers, even those that are resistant to conventional therapies. With this in mind, we previously demonstrated that proteolysis targeting chimeras (PROTACs) effectively induce degradation of the ER as a proof-of-concept experiment. Herein we further refined the PROTAC approach to target the ER for degradation. The ER-targeting PROTACs are composed of an estradiol on one end and a hypoxia-inducing factor,1, (HIF-1,)-derived synthetic pentapeptide on the other. The pentapeptide is recognized by an E3 ubiquitin ligase called the von,Hippel Lindau tumor suppressor protein (pVHL), thereby recruiting the ER to this E3 ligase for ubiquitination and degradation. Specifically, the pentapeptide is attached at three different locations on estradiol to generate three different PROTAC types. With the pentapeptide linked through the C7, position of estradiol, the resulting PROTAC shows the most effective ER degradation and highest affinity for the estrogen receptor. This result provides an opportunity to develop a novel type of ER antagonist that may overcome the resistance of breast tumors to conventional drugs such as tamoxifen and fulvestrant (Faslodex). [source] Genetic variations associated with psoriasis and psoriatic arthritis found by genome-wide associationDERMATOLOGIC THERAPY, Issue 2 2010Kristina Callis Duffin ABSTRACT Psoriasis and psoriatic arthritis are immune disorders with a complex polygenic basis. HLA-Cw6, which lies in the major histocompatibility region on chromosome 6, is considered the major genetic determinant of psoriasis. Recent genome-wide association studies have identified new variants outside of the MHC with relevance to the immunology of psoriasis. Variants in or near genes that encode subunits of cytokines (IL12B, IL23A) or cytokine receptors (IL23R) are interesting given that the gene product of IL12B, p40, is the target of a recently approved monoclonal antibody therapy for psoriasis (ustekinumab). Association with psoriasis and psoriatic arthritis has been found in TNFAIP3 and TNFIP1, ubiquitin ligases in the NF-,B pathway, and IL13, a Th2 cytokine. Copy number variation of human beta-defensin and late cornified envelope genes also associate with psoriasis. Many of these genetic variations also associate with immune disorders considered psoriatic co-morbidities, including Crohn's disease and diabetes. [source] Evolutionary divergence of valosin-containing protein/cell division cycle protein 48 binding interactions among endoplasmic reticulum-associated degradation proteinsFEBS JOURNAL, Issue 5 2009Giacomo Morreale Endoplasmic reticulum (ER)-associated degradation (ERAD) is a cell-autonomous process that eliminates large quantities of misfolded, newly synthesized protein, and is thus essential for the survival of any basic eukaryotic cell. Accordingly, the proteins involved and their interaction partners are well conserved from yeast to mammals, and Saccharomyces cerevisiae is widely used as a model system with which to investigate this fundamental cellular process. For example, valosin-containing protein (VCP) and its yeast homologue cell division cycle protein 48 (Cdc48p), which help to direct polyubiquitinated proteins for proteasome-mediated degradation, interact with an equivalent group of ubiquitin ligases in mouse and in S. cerevisiae. A conserved structural motif for cofactor binding would therefore be expected. We report a VCP-binding motif (VBM) shared by mammalian ubiquitin ligase E4b (Ube4b),ubiquitin fusion degradation protein 2a (Ufd2a), hydroxymethylglutaryl reductase degradation protein 1 (Hrd1),synoviolin and ataxin 3, and a related sequence in Mr 78 000 glycoprotein,Amfr with slightly different binding properties, and show that Ube4b and Hrd1 compete for binding to the N-terminal domain of VCP. Each of these proteins is involved in ERAD, but none has an S. cerevisiae homologue containing the VBM. Some other invertebrate model organisms also lack the VBM in one or more of these proteins, in contrast to vertebrates, where the VBM is widely conserved. Thus, consistent with their importance in ERAD, evolution has developed at least two ways to bring these proteins together with VCP,Cdc48p. However, the differing molecular architecture of VCP,Cdc48p complexes indicates a key point of divergence in the molecular details of ERAD mechanisms. [source] AMP-activated protein kinase enhances the expression of muscle-specific ubiquitin ligases despite its activation of IGF-1/Akt signaling in C2C12 myotubesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Jun F. Tong Abstract Two muscle-specific ubiquitin ligases (UL), muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1), are crucial for myofibrillar protein breakdown. The insulin like growth factor-1 (IGF-1) pathway inhibits muscle UL expression through Akt-mediated inhibition of FoxO transcription factors, while AMP-activated protein kinase (AMPK) promotes UL expression. The underlying cellular mechanism, however, remains obscure. In this study, the effect of AMPK and its interaction with IGF-1 on ubiquitin ligases expression was investigated. C2C12 myotubes were treated with 0, 0.1, 0.3, and 1.0,mM 5-aminoimidazole-4-carboxamide-1-,- D -ribofuranoside (AICAR) in the presence or absence of 50,ng/ml IGF-1. IGF-1 activated Akt, which enhanced phosphorlytion of FoxO3a at Thr 318/321 and reduced the expression of UL. Intriguingly, though activation of AMPK by 0.3 and 1.0,mM AICAR synergized IGF-1-induced Akt activation, the expression of UL was not attenuated, but strengthened by AMPK activation. AICAR treatment decreased FoxO3a phosphorylation at 318/321 in the cytoplasm and induced FoxO3 nuclear relocation. mTOR inhibition increased basal MAFbx expression and reversed the inhibitory effect of IGF-1 on UL expression. In conclusion, our data show that AMPK activation by AICAR stimulates UL expression despite the activation of Akt signaling, which may be due to the possible antagonistic effect of FoxO phosphorylation by AMPK on phosphorylation by Akt. In addition, AMPK inhibition of mTOR may provide an additional explanation for the enhancement of UL expression by AMPK. J. Cell. Biochem. 108: 458,468, 2009. © 2009 Wiley-Liss, Inc. [source] ,B-crystallin is mutant B-RAF regulated and contributes to cyclin D1 turnover in melanocytic cellsPIGMENT CELL & MELANOMA RESEARCH, Issue 2 2010Rong Hu Summary The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, ,B-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that ,B-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate ,B-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. ,B-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of ,B-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that ,B-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, ,B-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage. [source] Antioxidant administration attenuates mechanical ventilation-induced rat diaphragm muscle atrophy independent of protein kinase B (PKB,Akt) signallingTHE JOURNAL OF PHYSIOLOGY, Issue 1 2007J. M. McClung Oxidative stress promotes controlled mechanical ventilation (MV)-induced diaphragmatic atrophy. Nonetheless, the signalling pathways responsible for oxidative stress-induced muscle atrophy remain unknown. We tested the hypothesis that oxidative stress down-regulates insulin-like growth factor-1,phosphotidylinositol 3-kinase,protein kinase B serine threonine kinase (IGF-1,PI3K,Akt) signalling and activates the forkhead box O (FoxO) class of transcription factors in diaphragm fibres during MV-induced diaphragm inactivity. Sprague,Dawley rats were randomly assigned to one of five experimental groups: (1) control (Con), (2) 6 h of MV, (3) 6 h of MV with infusion of the antioxidant Trolox, (4) 18 h of MV, (5) 18 h of MV with Trolox. Following 6 h and 18 h of MV, diaphragmatic Akt activation decreased in parallel with increased nuclear localization and transcriptional activation of FoxO1 and decreased nuclear localization of FoxO3 and FoxO4, culminating in increased expression of the muscle-specific ubiquitin ligases, muscle atrophy factor (MAFbx) and muscle ring finger-1 (MuRF-1). Interestingly, following 18 h of MV, antioxidant administration was associated with attenuation of MV-induced atrophy in type I, type IIa and type IIb/IIx myofibres. Collectively, these data reveal that the antioxidant Trolox attenuates MV-induced diaphragmatic atrophy independent of alterations in Akt regulation of FoxO transcription factors and expression of MAFbx or MuRF-1. Further, these results also indicate that differential regulation of diaphragmatic IGF-1,PI3K,Akt signalling exists during the early and late stages of MV. [source] AtCHIP functions as an E3 ubiquitin ligase of protein phosphatase 2A subunits and alters plant response to abscisic acid treatmentTHE PLANT JOURNAL, Issue 4 2006Jinhua Luo Summary CHIP proteins are E3 ubiquitin ligases that promote degradation of Hsp70 and Hsp90 substrate proteins through the 26S proteasome in animal systems. A CHIP-like protein in Arabidopsis, AtCHIP, also has E3 ubiquitin ligase activity and has important roles to play under conditions of abiotic stress. In an effort to study the mode of action of AtCHIP in plant cells, proteins that physically interact with it were identified. Like its animal orthologs, AtCHIP interacts with a unique class of ubiquitin-conjugating enzymes (UBC or E2) that belongs to the stress-inducible UBC4/5 class in yeast. AtCHIP also interacts with other proteins, including an A subunit of protein phosphatase 2A (PP2A). This PP2A subunit appears to be a substrate of AtCHIP, because it can be ubiquitylated by AtCHIP in vitro and because the activity of PP2A is increased in AtCHIP -overexpressing plants in the dark or under low-temperature conditions. Unlike the rcn1 mutant, that has reduced PP2A activity due to a mutation in one of the A subunit genes of PP2A, AtCHIP -overexpressing plants are more sensitive to ABA treatment. Since PP2A was previously shown to be involved in low-temperature responses in plants, the low-temperature-sensitive phenotype observed in AtCHIP -overexpressing plants might be partly due to the change in PP2A activity. These data suggest that the E3 ubiquitin ligase AtCHIP may function upstream of PP2A in stress-responsive signal transduction pathways under conditions of low temperature or in the dark. [source] Regulation of TGF-, family signaling by E3 ubiquitin ligasesCANCER SCIENCE, Issue 11 2008Yasumichi Inoue Members of the transforming growth factor-, (TGF-,) family, including TGF-,, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. Alterations in their downstream signaling pathways are associated with a range of human diseases like cancer. TGF-, family members transduce signals through membrane serine/threonine kinase receptors and intracellular Smad proteins. The ubiquitin,proteasome pathway, an evolutionarily conserved cascade, tightly regulates TGF-, family signaling. In this pathway, E3 ubiquitin ligases play a crucial role in the recognition and degradation of target proteins by the 26S proteasomes. Smad degradation regulates TGF-, family signaling; HECT (homologous to the E6-accessory protein C-terminus)-type E3 ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1), Smurf2, and a RING-type E3 ubiquitin ligase, ROC1-SCFFbw1a have been implicated in Smad degradation. Smurf1 and Smurf2 bind to TGF-, family receptors via the inhibitory Smads, Smad6 and Smad7, to induce their ubiquitin-dependent degradation. Arkadia, a RING-type E3 ubiquitin ligase, induces the ubiquitination and degradation of Smad7 and corepressors, c-Ski and SnoN, to enhance TGF-, family signaling. Abnormalities in E3 ubiquitin ligases that control components of TGF-, family signaling may lead to the development and progression of various cancers. (Cancer Sci 2008; 99: 2107,2112) [source] | ||||||||||||||||