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Sample records for e3 ubiquitin ligases

  1. E3 Ubiquitin Ligases Neurobiological Mechanisms: Development to Degeneration

    Directory of Open Access Journals (Sweden)

    Arun Upadhyay

    2017-05-01

    Full Text Available Cells regularly synthesize new proteins to replace old or damaged proteins. Deposition of various aberrant proteins in specific brain regions leads to neurodegeneration and aging. The cellular protein quality control system develop various defense mechanisms against the accumulation of misfolded and aggregated proteins. The mechanisms underlying the selective recognition of specific crucial protein or misfolded proteins are majorly governed by quality control E3 ubiquitin ligases mediated through ubiquitin-proteasome system. Few known E3 ubiquitin ligases have shown prominent neurodevelopmental functions, but their interactions with different developmental proteins play critical roles in neurodevelopmental disorders. Several questions are yet to be understood properly. How E3 ubiquitin ligases determine the specificity and regulate degradation of a particular substrate involved in neuronal proliferation and differentiation is certainly the one, which needs detailed investigations. Another important question is how neurodevelopmental E3 ubiquitin ligases specifically differentiate between their versatile range of substrates and timing of their functional modulations during different phases of development. The premise of this article is to understand how few E3 ubiquitin ligases sense major molecular events, which are crucial for human brain development from its early embryonic stages to throughout adolescence period. A better understanding of these few E3 ubiquitin ligases and their interactions with other potential proteins will provide invaluable insight into disease mechanisms to approach toward therapeutic interventions.

  2. Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases

    National Research Council Canada - National Science Library

    Riley, B E; Lougheed, J C; Callaway, K; Velasquez, M; Brecht, E; Nguyen, L; Shaler, T; Walker, D; Yang, Y; Regnstrom, K; Diep, L; Zhang, Z; Chiou, S; Bova, M; Artis, D R; Yao, N; Baker, J; Yednock, T; Johnston, J A

    2013-01-01

    Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection...

  3. Molecular insights into RBR E3 ligase ubiquitin transfer mechanisms.

    Science.gov (United States)

    Dove, Katja K; Stieglitz, Benjamin; Duncan, Emily D; Rittinger, Katrin; Klevit, Rachel E

    2016-08-01

    RING-in-between-RING (RBR) ubiquitin (Ub) ligases are a distinct class of E3s, defined by a RING1 domain that binds E2 Ub-conjugating enzyme and a RING2 domain that contains an active site cysteine similar to HECT-type E3s. Proposed to function as RING/HECT hybrids, details regarding the Ub transfer mechanism used by RBRs have yet to be defined. When paired with RING-type E3s, E2s perform the final step of Ub ligation to a substrate. In contrast, when paired with RBR E3s, E2s must transfer Ub onto the E3 to generate a E3~Ub intermediate. We show that RBRs utilize two strategies to ensure transfer of Ub from the E2 onto the E3 active site. First, RING1 domains of HHARI and RNF144 promote open E2~Ubs. Second, we identify a Ub-binding site on HHARI RING2 important for its recruitment to RING1-bound E2~Ub. Mutations that ablate Ub binding to HHARI RING2 also decrease RBR ligase activity, consistent with RING2 recruitment being a critical step for the RBR Ub transfer mechanism. Finally, we demonstrate that the mechanism defined here is utilized by a variety of RBRs.

  4. Nuclear targeting of an endosomal E3 ubiquitin ligase.

    Science.gov (United States)

    Bocock, Jeffrey P; Carmicle, Stephanie; Madamba, Egbert; Erickson, Ann H

    2010-06-01

    Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post-translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12-myristate 13-acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full-length protein and a membrane-embedded C-terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant-negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress-induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase-active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.

  5. A design principle underlying the paradoxical roles of E3 ubiquitin ligases

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    Lee, Daewon; Kim, Minjin; Cho, Kwang-Hyun

    2014-07-01

    E3 ubiquitin ligases are important cellular components that determine the specificity of proteolysis in the ubiquitin-proteasome system. However, an increasing number of studies have indicated that E3 ubiquitin ligases also participate in transcription. Intrigued by the apparently paradoxical functions of E3 ubiquitin ligases in both proteolysis and transcriptional activation, we investigated the underlying design principles using mathematical modeling. We found that the antagonistic functions integrated in E3 ubiquitin ligases can prevent any undesirable sustained activation of downstream genes when E3 ubiquitin ligases are destabilized by unexpected perturbations. Interestingly, this design principle of the system is similar to the operational principle of a safety interlock device in engineering systems, which prevents a system from abnormal operation unless stability is guaranteed.

  6. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Boomsma, Wouter Krogh; Nielsen, Sofie Vincents; Lindorff-Larsen, Kresten

    2016-01-01

    The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2...... ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions...... of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we...

  7. Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

    Science.gov (United States)

    Plechanovová, Anna; Jaffray, Ellis; Tatham, Michael H.; Naismith, James H.; Hay, Ronald T.

    2012-01-01

    SUMMARY Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate. PMID:22842904

  8. MDM2 E3 ubiquitin ligase mediates UT-A1 urea transporter ubiquitination and degradation.

    Science.gov (United States)

    Chen, Guangping; Huang, Haidong; Fröhlich, Otto; Yang, Yuan; Klein, Janet D; Price, S Russ; Sands, Jeff M

    2008-11-01

    UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT-A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation.

  9. Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4.

    Science.gov (United States)

    Lear, Travis; McKelvey, Alison C; Rajbhandari, Shristi; Dunn, Sarah R; Coon, Tiffany A; Connelly, William; Zhao, Joe Y; Kass, Daniel J; Zhang, Yingze; Liu, Yuan; Chen, Bill B

    2016-05-30

    The E3 small ubiquitin-like modifier (SUMO) protein ligase protein inhibitor of activated STAT 4 (PIAS4) is a pivotal protein in regulating the TGFβ pathway. In this study, we discovered a new protein isoform encoded by KIAA0317, termed fibrosis-inducing E3 ligase 1 (FIEL1), which potently stimulates the TGFβ signaling pathway through the site-specific ubiquitination of PIAS4. FIEL1 targets PIAS4 using a double locking mechanism that is facilitated by the kinases PKCζ and GSK3β. Specifically, PKCζ phosphorylation of PIAS4 and GSK3β phosphorylation of FIEL1 are both essential for the degradation of PIAS4. FIEL1 protein is highly expressed in lung tissues from patients with idiopathic pulmonary fibrosis (IPF), whereas PIAS4 protein levels are significantly reduced. FIEL1 overexpression significantly increases fibrosis in a bleomycin murine model, whereas FIEL1 knockdown attenuates fibrotic conditions. Further, we developed a first-in-class small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice. This study provides a basis for IPF therapeutic intervention by modulating PIAS4 protein abundance.

  10. Types of Ubiquitin Ligases.

    Science.gov (United States)

    Morreale, Francesca Ester; Walden, Helen

    2016-03-24

    Ubiquitination is a post-translational modification of proteins involved in a variety of cellular processes. Ubiquitination requires the sequential action of three enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin ligases). This SnapShot highlights the main types of E3 ubiquitin ligases, which can be classified in three families depending on the presence of characteristic domains and on the mechanism of ubiquitin transfer to the substrate protein.

  11. RBR E3 ubiquitin ligases: new structures, new insights, new questions.

    Science.gov (United States)

    Spratt, Donald E; Walden, Helen; Shaw, Gary S

    2014-03-15

    The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.

  12. A Bacterial Inhibitor of Host Programmed Cell Death Defenses is an E3 Ubiquitin Ligase

    Energy Technology Data Exchange (ETDEWEB)

    Janjusevic,R.; Abramovitch, R.; Martin, G.; Stebbins, C.

    2005-01-01

    The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.

  13. ATLs and BTLs, plant-specific and general eukaryotic structurally-related E3 ubiquitin ligases.

    Science.gov (United States)

    Guzmán, Plinio

    2014-02-01

    Major components of the ubiquitin proteasome system are the enzymes that operate on the transfer of ubiquitin to selected target substrate, known as ubiquitin ligases. The RING finger is a domain that is present in key classes of ubiquitin ligases. This domain coordinates the interaction with a suitable E2 conjugase and the transfer of ubiquitin from the E2 to protein targets. Additional domains coupled to the same polypeptide are important for modulating the function of these ubiquitin ligases. Plants contain several types of E3 ubiquitin ligases that in many cases have expanded as multigene families. Some families are specific to the plant lineage, whereas others may have a common ancestor among plants and other eukaryotic lineages. Arabidopsis Tóxicos en Levadura (ATLs) and BCA2 zinc finger ATLs (BTLs) are two families of ubiquitin ligases that share some common structural features. These are intronless genes that encode a highly related RING finger domain, and yet during evolutionary history, their mode of gene expansion and function is rather different. In each of these two families, the co-occurrence of transmembrane helices or C2/C2 (BZF finger) domains with a selected variation on the RING finger has been subjected to strong selection pressure in order to preserve their unique domain architectures during evolution.

  14. The centrosomal E3 ubiquitin ligase FBXO31-SCF regulates neuronal morphogenesis and migration.

    Directory of Open Access Journals (Sweden)

    Mayur Vadhvani

    Full Text Available Neuronal development requires proper migration, polarization and establishment of axons and dendrites. Growing evidence identifies the ubiquitin proteasome system (UPS with its numerous components as an important regulator of various aspects of neuronal development. F-box proteins are interchangeable subunits of the Cullin-1 based E3 ubiquitin ligase, but only a few family members have been studied. Here, we report that the centrosomal E3 ligase FBXO31-SCF (Skp1/Cullin-1/F-box protein regulates neuronal morphogenesis and axonal identity. In addition, we identified the polarity protein Par6c as a novel interaction partner and substrate targeted for proteasomal degradation in the control of axon but not dendrite growth. Finally, we ascribe a role for FBXO31 in dendrite growth and neuronal migration in the developing cerebellar cortex. Taken together, we uncovered the centrosomal E3 ligase FBXO31-SCF as a novel regulator of neuronal development.

  15. Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

    2008-01-01

    The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...

  16. Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

    Directory of Open Access Journals (Sweden)

    Diego A Peralta

    Full Text Available Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7 gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

  17. Aβ-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

    Science.gov (United States)

    Rodrigues, Elizabeth M; Scudder, Samantha L; Goo, Marisa S; Patrick, Gentry N

    2016-02-03

    Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. Copyright © 2016 the authors 0270-6474/16/361590-06$15.00/0.

  18. HSV-1 ICP0: An E3 Ubiquitin Ligase That Counteracts Host Intrinsic and Innate Immunity

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    Mirna Perusina Lanfranca

    2014-05-01

    Full Text Available The herpes simplex virus type 1 (HSV-1 encoded E3 ubiquitin ligase, infected cell protein 0 (ICP0, is required for efficient lytic viral replication and regulates the switch between the lytic and latent states of HSV-1. As an E3 ubiquitin ligase, ICP0 directs the proteasomal degradation of several cellular targets, allowing the virus to counteract different cellular intrinsic and innate immune responses. In this review, we will focus on how ICP0’s E3 ubiquitin ligase activity inactivates the host intrinsic defenses, such as nuclear domain 10 (ND10, SUMO, and the DNA damage response to HSV-1 infection. In addition, we will examine ICP0’s capacity to impair the activation of interferon (innate regulatory mediators that include IFI16 (IFN γ-inducible protein 16, MyD88 (myeloid differentiation factor 88, and Mal (MyD88 adaptor-like protein. We will also consider how ICP0 allows HSV-1 to evade activation of the NF-κB (nuclear factor kappa B inflammatory signaling pathway. Finally, ICP0’s paradoxical relationship with USP7 (ubiquitin specific protease 7 and its roles in intrinsic and innate immune responses to HSV-1 infection will be discussed.

  19. The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.

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    Lamaze, Angélique; Lamouroux, Annie; Vias, Carine; Hung, Hsiu-Cheng; Weber, Frank; Rouyer, François

    2011-06-01

    In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

  20. The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis.

    Directory of Open Access Journals (Sweden)

    Masashi Kajiro

    Full Text Available Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.

  1. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

    Science.gov (United States)

    Fischer, Eric S; Böhm, Kerstin; Lydeard, John R; Yang, Haidi; Stadler, Michael B; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M; Tichkule, Ritesh B; Schebesta, Michael; Forrester, William C; Schirle, Markus; Hassiepen, Ulrich; Ottl, Johannes; Hild, Marc; Beckwith, Rohan E J; Harper, J Wade; Jenkins, Jeremy L; Thomä, Nicolas H

    2014-08-07

    In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.

  2. The substrate binding domains of human SIAH E3 ubiquitin ligases are now crystal clear

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qi; Wang, Zhongduo; Hou, Feng; Harding, Rachel; Huang, Xinyi; Dong, Aiping; Walker, John R.; Tong, Yufeng

    2017-01-01

    Seven in absentia homologs (SIAHs) comprise a family of highly conserved E3 ubiquitin ligases that play an important role in regulating signalling pathways in tumorigenesis, including the DNA damage repair and hypoxia response pathways. SIAH1 and SIAH2 have been found to function as a tumour repressor and a proto-oncogene, respectively, despite the high sequence identity of their substrate binding domains (SBDs). Ubiquitin-specific protease USP19 is a deubiquitinase that forms a complex with SIAHs and counteracts the ligase function. Much effort has been made to find selective inhibitors of the SIAHs E3 ligases. Menadione was reported to inhibit SIAH2 specifically. We used X-ray crystallography, peptide array, bioinformatic analysis, and biophysical techniques to characterize the structure and interaction of SIAHs with deubiquitinases and literature reported compounds. We solved the crystal structures of SIAH1 in complex with a USP19 peptide and of the apo form SIAH2. Phylogenetic analysis revealed the SIAH/USP19 complex is conserved in evolution. We demonstrated that menadione destabilizes both SIAH1 and SIAH2 non-specifically through covalent modification. The SBDs of SIAH E3 ligases are structurally similar with a subtle stability difference. USP19 is the only deubiquitinase that directly binds to SIAHs through the substrate binding pocket. Menadione is not a specific inhibitor for SIAH2. The crystallographic models provide structural insights into the substrate binding of the SIAH family E3 ubiquitin ligases that are critically involved in regulating cancer-related pathways. Our results suggest caution should be taken when using menadione as a specific SIAH2 inhibitor.

  3. Wwp2, an E3 Ubiquitin Ligase That Targets Transcription Factor Oct-4 for Ubiquitination

    Institute of Scientific and Technical Information of China (English)

    HuiMingXu; BingLiao; QianJunZhang; BeiBeiWang; Hui,Li; XiaoMinZhong; HuiZhenSheng; YingXinZhao; YingMingZhao; YingJin

    2005-01-01

    The POU transcription factor Oct-4 is a master regulator affecting the fate of pluripotent embryonic stem cells. However, the precise mechanisms by which the activation and expression of Oct-4 are regulated still remain to be elucidated. We describe here a novel murine ubiquitin ligase, Wwp2, that specifically interacts with Oct-4 and promotes its ubiquitination both in vivo and in vitro. Remarkably, the expression of a catalytically inactive point mutant of Wwp2 abolishes Oct-4 ubiquitination. Moreover, Wwp2 promotes Oct-4 degradation in the presence of overexpressed ubiquitin. The degradation is blocked by treatment with proteasome inhibitor. Fusion of a single ubiquitin to Oct-4 inactivates its transcriptional activity in a heterologous Oct-4-driven reporter system. Furthermore, overexpression of Wwp2 in embryonic stem cells significantly reduces the Oct-4-transcriptional activities. Collectively, we demonstrate for the first time that Oct-4 can be posttranslationatly modified by ubiquitination and that this modification dramatically suppresses its transcriptional activity. These results reveal that the functional status of Oct-4, in addition to its expression level, dictates its transcriptional activity, and the results open up a new avenue to understand how Oct-4 defines the fate of embryonic stem cells.

  4. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide

    OpenAIRE

    Fischer, Eric S.; Böhm, Kerstin; Lydeard, John R.; Yang, Haidi; Stadler, Michael B.; Cavadini, Simone; Nagel, Jane; Serluca, Fabrizio; Acker, Vincent; Lingaraju, Gondichatnahalli M.; Tichkule, Ritesh B.; Schebesta, Michael; Forrester, William C.; Schirle, Markus; Hassiepen, Ulrich

    2015-01-01

    In the 1950s the drug thalidomide administered as a sedative to pregnant women led to the birth of thousands of children with multiple defects. Despite its teratogenicity, thalidomide and its derivatives lenalidomide and pomalidomide (together known as Immunomodulatory Drugs: IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-dysplasia. IMiDs target the CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase and promote the ubiquitination of Ikaros/Aiolos transcription fac...

  5. The E3 Ligase CHIP Mediates Ubiquitination and Degradation of Mixed-Lineage Kinase 3

    OpenAIRE

    Blessing, Natalya A.; Brockman, April L.; Chadee, Deborah N.

    2014-01-01

    Mixed-lineage kinase 3 (MLK3) activates mitogen-activated protein kinase (MAPK) signaling pathways and has important functions in migration, invasion, proliferation, tumorigenesis, and apoptosis. We investigated the role of the E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) in the regulation of MLK3 protein levels. We show that CHIP interacts with MLK3 and, together with the E2 ubiquitin-conjugating enzyme UbcH5 (UbcH5a, -b, -c, or -d), ubiquitinates MLK3 in vitro. CHIP or Hs...

  6. Novel E3 ubiquitin ligases that regulate histone protein levels in the budding yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Rakesh Kumar Singh

    Full Text Available Core histone proteins are essential for packaging the genomic DNA into chromatin in all eukaryotes. Since multiple genes encode these histone proteins, there is potential for generating more histones than what is required for chromatin assembly. The positively charged histones have a very high affinity for negatively charged molecules such as DNA, and any excess of histone proteins results in deleterious effects on genomic stability and cell viability. Hence, histone levels are known to be tightly regulated via transcriptional, posttranscriptional and posttranslational mechanisms. We have previously elucidated the posttranslational regulation of histone protein levels by the ubiquitin-proteasome pathway involving the E2 ubiquitin conjugating enzymes Ubc4/5 and the HECT (Homologous to E6-AP C-Terminus domain containing E3 ligase Tom1 in the budding yeast. Here we report the identification of four additional E3 ligases containing the RING (Really Interesting New Gene finger domains that are involved in the ubiquitylation and subsequent degradation of excess histones in yeast. These E3 ligases are Pep5, Snt2 as well as two previously uncharacterized Open Reading Frames (ORFs YKR017C and YDR266C that we have named Hel1 and Hel2 (for Histone E3 Ligases respectively. Mutants lacking these E3 ligases are sensitive to histone overexpression as they fail to degrade excess histones and accumulate high levels of endogenous histones on histone chaperones. Co-immunoprecipitation assays showed that these E3 ligases interact with the major E2 enzyme Ubc4 that is involved in the degradation related ubiquitylation of histones. Using mutagenesis we further demonstrate that the RING domains of Hel1, Hel2 and Snt2 are required for histone regulation. Lastly, mutants corresponding to Hel1, Hel2 and Pep5 are sensitive to replication inhibitors. Overall, our results highlight the importance of posttranslational histone regulatory mechanisms that employ multiple E3

  7. RING finger palmitoylation of the endoplasmic reticulum Gp78 E3 ubiquitin ligase.

    Science.gov (United States)

    Fairbank, Maria; Huang, Kun; El-Husseini, Alaa; Nabi, Ivan R

    2012-07-30

    Gp78 is an E3 ubiquitin ligase within the endoplasmic reticulum-associated degradation pathway. We show that Flag-tagged gp78 undergoes sulfhydryl cysteine palmitoylation (S-palmitoylation) within the RING finger motif, responsible for its ubiquitin ligase activity. Screening of 19 palmitoyl acyl transferases (PATs) identified five that increased gp78 RING finger palmitoylation. Endoplasmic reticulum (ER)-localized Myc-DHHC6 overexpression promoted the peripheral ER distribution of Flag-gp78 while RING finger mutation and the palmitoylation inhibitor 2-bromopalmitate restricted gp78 to the central ER. Palmitoylation of RING finger cysteines therefore regulates gp78 distribution to the peripheral ER. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Role of SKP1-CUL1-F-Box-Protein (SCF) E3 Ubiquitin Ligases in Skin Cancer

    Institute of Scientific and Technical Information of China (English)

    Chuan-Ming Xie; Wenyi Wei; Yi Sun

    2013-01-01

    Many biological processes such as cell proliferation,differentiation,and cell death depend precisely on the timely synthesis and degradation of key regulatory proteins.While protein synthesis can be regulated at multiple levels,protein degradation is mainly controlled by the ubiquitin-proteasome system (UPS),which consists of two distinct steps:(1) ubiquitylation of targeted protein by E1 ubiquitin-activating enzyme,E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase,and (2) subsequent degradation by the 26S proteasome.Among all E3 ubiquitin ligases,the SCF (SKP1-CUL1-F-box protein) E3 ligases are the largest family and are responsible for the turnover of many key regulatory proteins.Aberrant regulation of SCF E3 ligases is associated with various human diseases,such as cancers,including skin cancer.In this review,we provide a comprehensive overview of all currently published data to define a promoting role of SCF E3 ligases in the development of skin cancer.The future directions in this area of research are also discussed with an ultimate goal to develop small molecule inhibitors of SCF E3 ligases as a novel approach for the treatment of human skin cancer.Furthermore,altered components or substrates of SCF E3 ligases may also be developed as the biomarkers for early diagnosis or predicting prognosis.

  9. The Nedd4-like ubiquitin E3 ligases target angiomotin/p130 to ubiquitin-dependent degradation.

    Science.gov (United States)

    Wang, Chenji; An, Jian; Zhang, Pingzhao; Xu, Chen; Gao, Kun; Wu, Di; Wang, Dejie; Yu, Hongxiu; Liu, Jun O; Yu, Long

    2012-06-01

    AMOT (angiomotin) is a membrane-associated protein that is expressed in ECs (endothelial cells) and controls migration, TJ (tight junction) formation, cell polarity and angiogenesis. Recent studies have revealed that AMOT and two AMOT-like proteins, AMOTL1 and AMOTL2, play critical roles in the Hippo pathway by regulating the subcellular localization of the co-activators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif). However, it has been unclear how AMOT is regulated. In the present study, we report that AMOT undergoes proteasomal degradation. We identify three members of Nedd4 (neural-precursor-cell-expressed developmentally down-regulated)-like ubiquitin E3 ligases, Nedd4, Nedd4-2 and Itch, as the ubiquitin E3 ligases for the long isoform of AMOT, AMOT/p130. We demonstrate that Nedd4, Nedd4-2 and Itch mediate poly-ubiquitination of AMOT/p130 in vivo. Overexpression of Nedd4, Nedd4-2 or Itch leads to AMOT/p130 proteasomal degradation. Knockdown of Nedd4, Nedd4-2 and Itch causes an accumulation of steady-state level of AMOT/p130. We also show that three L/P-PXY motifs of AMOT/p130 and the WW domains of Nedd4 mediate their interaction. Furthermore, Nedd4-like ubiquitin E3 ligases might compete with YAP for the binding to AMOT/p130, and subsequently targeting AMOT/p130 for ubiquitin-dependent degradation. Together, these observations reveal a novel post-translational regulatory mechanism of AMOT/p130.

  10. Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage

    Science.gov (United States)

    Kang, Ho Chul; Lee, Yun-Il; Shin, Joo-Ho; Andrabi, Shaida A.; Chi, Zhikai; Gagné, Jean-Philippe; Lee, Yunjong; Ko, Han Seok; Lee, Byoung Dae; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2011-01-01

    Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair. PMID:21825151

  11. RING E3 ligases

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Ryu, Moon Young; Kim, Jong Hum

    2017-01-01

    response pathways of plants through various molecular and genetic studies. In particular, it was recently discovered that ubiquitin proteasome system (UPS), a regulatory mechanism for protein turn over, is greatly involved in the stress responsive pathways. In the UPS, many E3 ligases play key roles...... in recognizing and tethering poly-ubiquitins on target proteins for subsequent degradation by the 26S proteasome. Here we discuss the roles of RING ligases that have been defined in related to abiotic stress responses in plants....

  12. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    Science.gov (United States)

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

  13. Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.

    Science.gov (United States)

    Xia, Tian; Dimitropoulou, Christiana; Zeng, Jingmin; Antonova, Galina N; Snead, Connie; Venema, Richard C; Fulton, David; Qian, Shuibing; Patterson, Cam; Papapetropoulos, Andreas; Catravas, John D

    2007-11-01

    The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.

  14. Lenalidomide Stabilizes the Erythropoietin Receptor by Inhibiting the E3 Ubiquitin Ligase RNF41.

    Science.gov (United States)

    Basiorka, Ashley A; McGraw, Kathy L; De Ceuninck, Leentje; Griner, Lori N; Zhang, Ling; Clark, Justine A; Caceres, Gisela; Sokol, Lubomir; Komrokji, Rami S; Reuther, Gary W; Wei, Sheng; Tavernier, Jan; List, Alan F

    2016-06-15

    In a subset of patients with non-del(5q) myelodysplastic syndrome (MDS), lenalidomide promotes erythroid lineage competence and effective erythropoiesis. To determine the mechanism by which lenalidomide promotes erythropoiesis, we investigated its action on erythropoietin receptor (EpoR) cellular dynamics. Lenalidomide upregulated expression and stability of JAK2-associated EpoR in UT7 erythroid cells and primary CD71+ erythroid progenitors. The effects of lenalidomide on receptor turnover were Type I cytokine receptor specific, as evidenced by coregulation of the IL3-Rα receptor but not c-Kit. To elucidate this mechanism, we investigated the effects of lenalidomide on the E3 ubiquitin ligase RNF41. Lenalidomide promoted EpoR/RNF41 association and inhibited RNF41 auto-ubiquitination, accompanied by a reduction in EpoR ubiquitination. To confirm that RNF41 is the principal target responsible for EpoR stabilization, HEK293T cells were transfected with EpoR and/or RNF41 gene expression vectors. Steady-state EpoR expression was reduced in EpoR/RNF41 cells, whereas EpoR upregulation by lenalidomide was abrogated, indicating that cellular RNF41 is a critical determinant of drug-induced receptor modulation. Notably, shRNA suppression of CRBN gene expression failed to alter EpoR upregulation, indicating that drug-induced receptor modulation is independent of cereblon. Immunohistochemical staining showed that RNF41 expression decreased in primary erythroid cells of lenalidomide-responding patients, suggesting that cellular RNF41 expression merits investigation as a biomarker for lenalidomide response. Our findings indicate that lenalidomide has E3 ubiquitin ligase inhibitory effects that extend to RNF41 and that inhibition of RNF41 auto-ubiquitination promotes membrane accumulation of signaling competent JAK2/EpoR complexes that augment Epo responsiveness. Cancer Res; 76(12); 3531-40. ©2016 AACR.

  15. E3 ubiquitin ligases Pellinos as regulators of pattern recognition receptor signaling and immune responses.

    Science.gov (United States)

    Medvedev, Andrei E; Murphy, Michael; Zhou, Hao; Li, Xiaoxia

    2015-07-01

    Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an interleukin-1 (IL-1) receptor-associated kinase homolog in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2, and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g. Pellino-1 being a negative regulator in T lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we summarize current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and tumor necrosis factor receptors, and discuss Pellinos roles in sepsis and infectious diseases, as well as in autoimmune, inflammatory, and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies.

  16. K48-linked KLF4 ubiquitination by E3 ligase Mule controls T-cell proliferation and cell cycle progression

    DEFF Research Database (Denmark)

    Hao, Zhenyue; Sheng, Yi; Duncan, Gordon S.

    2017-01-01

    T-cell proliferation is regulated by ubiquitination but the underlying molecular mechanism remains obscure. Here we report that Lys-48-linked ubiquitination of the transcription factor KLF4 mediated by the E3 ligase Mule promotes T-cell entry into S phase. Mule is elevated in T cells upon TCR...

  17. Evidence of an Antimicrobial Peptide Signature Encrypted in HECT E3 Ubiquitin Ligases

    Science.gov (United States)

    Candido-Ferreira, Ivan Lavander; Kronenberger, Thales; Sayegh, Raphael Santa Rosa; Batista, Isabel de Fátima Correia; da Silva Junior, Pedro Ismael

    2017-01-01

    The ubiquitin-proteasome pathway (UPP) is a hallmark of the eukaryotic cell. In jawed vertebrates, it has been co-opted by the adaptive immune system, where proteasomal degradation produces endogenous peptides for major histocompatibility complex class I antigen presentation. However, proteolytic products are also necessary for the phylogenetically widespread innate immune system, as they often play a role as host defense peptides (HDPs), pivotal effectors against pathogens. Here, we report the identification of the arachnid HDP oligoventin, which shares homology to a core member of the UPP, E3 ubiquitin ligases. Oligoventin has broad antimicrobial activity and shows strong synergy with lysozymes. Using computational and phylogenetic approaches, we show high conservation of the oligoventin signature in HECT E3s. In silico simulation of HECT E3s self-proteolysis provides evidence that HDPs can be generated by fine-tuned 26S proteasomal degradation, and therefore are consistent with the hypothesis that oligoventin is a cryptic peptide released by the proteolytic processing of an Nedd4 E3 precursor protein. Finally, we compare the production of HDPs and endogenous antigens from orthologous HECT E3s by proteasomal degradation as a means of analyzing the UPP coupling to metazoan immunity. Our results highlight the functional plasticity of the UPP in innate and adaptive immune systems as a possibly recurrent mechanism to generate functionally diverse peptides. PMID:28119686

  18. BICP0 and its RING finger domain act as ubiquitin E3 ligases in vitro

    Institute of Scientific and Technical Information of China (English)

    DIAO Lirong; QIAO Wentao; CHEN Qimin; WANG Chen; GENG Yunqi

    2005-01-01

    Bovine infected-cell protein 0 (BICP0) encoded by bovine herpes virus 1 (BHV-1) immediate early gene is necessary for efficient productive infection, in a large part because it activates all 3 classes of BHV-1 genes. It also has the ability to efficiently transactivate promoters that are not derived from BHV-1. To investigate the mechanism by which BICP0 achieves these effects, we expressed and purified BICP0 and its different mutants in E. coli. In vitro assays showed that both full-length BICP0 and its isolated RING finger domain induce the accumulation of polyubiquitin chains. Mutations within the RING finger region that abolish the in vitro ubiquitination activity also cause severe reductions in BICP0 activity in other assays. Based on these, we conclude that BICP0 has the potential to act as an E3 ubiquitin ligase during viral infection and its RING finger domain is necessary for this function. These strongly support the hypothesis that BICP0 might influence virus infection through its ability to interact with the ubiquitin-proteasome pathway.

  19. The E3 Ubiquitin Ligase TMEM129 Is a Tri-Spanning Transmembrane Protein

    Directory of Open Access Journals (Sweden)

    Michael L. van de Weijer

    2016-11-01

    Full Text Available Misfolded proteins from the endoplasmic reticulum (ER are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD pathway to downregulate human leukocyte antigen (HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129 as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo–Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins.

  20. [Suppression of E3 ubiquitin ligase Cbl-b in interleukin-1 signaling].

    Science.gov (United States)

    Yu, Jiang-Tian; Bu, Xin; Zhao, Hu; Su, Jin

    2015-08-25

    The present study aims to investigate the effect of Cbl-b, a member of E3 ubiquitin ligase family, on interleukin-1 (IL-1) pathway in synoviocytes. The protein expression levels of Cbl-b and IL-1-induced matrix metalloproteinase 13 (MMP-13) in synoviocytes were analyzed by Western blot. Collagen substrates were incubated with the conditioned medium collected from synoviocytes cultures and then subjected to SDS-PAGE for analysis of collagen degradation. The results showed that compared with wild-type cells, Cbl-b-deficient cells expressed more MMP-13 protein and had enhanced ability to degrade collagens under IL-1 stimulation. These data suggest that Cbl-b may negatively regulate IL-1-triggered degradation of collagen matrix in synoviocytes.

  1. Cullin4B/E3-ubiquitin ligase negatively regulates -catenin

    Indian Academy of Sciences (India)

    Rachana Tripathi; Satya Keerthi Kota; Usha K Srinivas

    2007-09-01

    -catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, -catenin is targeted to ubiquitin–proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in -catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased -catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and -catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of -catenin levels.

  2. Ret Finger Protein: An E3 Ubiquitin Ligase Juxtaposed to the XY Body in Meiosis

    Directory of Open Access Journals (Sweden)

    Isabelle Gillot

    2009-01-01

    Full Text Available During prophase I of male meiosis, the sex chromosomes form a compact structure called XY body that associates with the nuclear membrane of pachytene spermatocytes. Ret Finger Protein is a transcriptional repressor, able to interact with both nuclear matrix-associated proteins and double-stranded DNA. We report the precise and unique localization of Ret Finger Protein in pachytene spermatocytes, in which Ret Finger Protein takes place of lamin B1, between the XY body and the inner nuclear membrane. This localization of Ret Finger Protein does not seem to be associated with O-glycosylation or sumoylation. In addition, we demonstrate that Ret Finger Protein contains an E3 ubiquitin ligase activity. These observations lead to an attractive hypothesis in which Ret Finger Protein would be involved in the positioning and the attachment of XY body to the nuclear lamina of pachytene spermatocytes.

  3. E3 ubiquitin ligases as drug targets and prognostic biomarkers in melanoma

    Directory of Open Access Journals (Sweden)

    Kristina Bielskienė

    2015-01-01

    E3 ligases are of interest as drug targets for their ability to regulate proteins stability and functions. Compared to the general proteasome inhibitor bortezomib, which blocks the entire protein degradation, drugs that target a particular E3 ligase are expected to have better selectivity with less associated toxicity. Components of different E3 ligases complexes (FBW7, MDM2, RBX1/ROC1, RBX2/ROC2, cullins and many others are known as oncogenes or tumor suppressors in melanomagenesis. These proteins participate in regulation of different cellular pathways and such important proteins in cancer development as p53 and Notch. In this review we summarized published data on the role of known E3 ligases in the development of melanoma and discuss the inhibitors of E3 ligases as a novel approach for the treatment of malignant melanomas.

  4. CRL4A(CRBN) E3 ubiquitin ligase restricts BK channel activity and prevents epileptogenesis.

    Science.gov (United States)

    Liu, Jiye; Ye, Jia; Zou, Xiaolong; Xu, Zhenghao; Feng, Yan; Zou, Xianxian; Chen, Zhong; Li, Yuezhou; Cang, Yong

    2014-05-21

    Ion channels regulate membrane excitation, and mutations of ion channels often cause serious neurological disorders including epilepsy. Compared with extensive analyses of channel protein structure and function, much less is known about the fine tuning of channel activity by post-translational modification. Here we report that the large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels are targeted by the E3 ubiquitin ligase CRL4A(CRBN) for polyubiquitination and retained in the endoplasmic reticulum (ER). Inactivation of CRL4A(CRBN) releases deubiquitinated BK channels from the ER to the plasma membrane, leading to markedly enhanced channel activity. Mice with CRL4A(CRBN) mutation in the brain or treated with a CRL4A(CRBN) inhibitor are very sensitive to seizure induction, which can be attenuated by blocking BK channels. Finally, the mutant mice develop spontaneous epilepsy when aged. Therefore, ubiquitination of BK channels before their cell surface expression is an important step to prevent systemic neuronal excitability and epileptogenesis.

  5. The HECTD3 E3 ubiquitin ligase suppresses cisplatin-induced apoptosis via stabilizing MALT1.

    Science.gov (United States)

    Li, Yi; Chen, Xi; Wang, Zehua; Zhao, Dong; Chen, Hui; Chen, Wenlin; Zhou, Zhongmei; Zhang, Junran; Zhang, Jing; Li, Hongmin; Chen, Ceshi

    2013-01-01

    Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) is an E3 ubiquitin ligase with unknown functions. Here, we show that HECTD3 confers cancer cell resistance to cisplatin. To understand the molecular mechanisms, we performed a yeast two-hybrid analysis and identified mucosa-associated lymphoid tissue 1 (MALT1) as an HECTD3-interacting protein. HECTD3 promotes MALT1 ubiquitination with nondegradative polyubiquitin chains by direct interacting with the MALT1 through its N-terminal destruction of cyclin domain. HECTD3 does not target MALT1 for degradation but stabilize it. HECTD3 depletion dramatically decreases the levels of MALT1 in MCF7 and HeLa cells treated with cisplatin, which is correlated to an increase in apoptosis. Knockdown of MALT1 likewise increases cisplatin-induced apoptosis in these cancer cells. However, HECTD3 over-expression leads to a decreased cisplatin-induced apoptosis, whereas overexpression of MALT1 partially rescues HECTD3 depletion-induced apoptosis. These findings suggest that HECTD3 promotes cell survival through stabilizing MALT1. Our data have important implications in cancer therapy by providing novel molecular targets.

  6. The HECTD3 E3 Ubiquitin Ligase Suppresses Cisplatin-Induced Apoptosis via Stabilizing MALT1

    Directory of Open Access Journals (Sweden)

    Yi Li

    2013-01-01

    Full Text Available Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3 is an E3 ubiquitin ligase with unknown functions. Here, we show that HECTD3 confers cancer cell resistance to cisplatin. To understand the molecular mechanisms, we performed a yeast two-hybrid analysis and identified mucosa-associated lymphoid tissue 1 (MALT1 as an HECTD3-interacting protein. HECTD3 promotes MALT1 ubiquitination with non-degradative polyubiquitin chains by direct interacting with the MALT1 through its N-terminal destruction of cyclin domain. HECTD3 does not target MALT1 for degradation but stabilize it. HECTD3 depletion dramatically decreases the levels of MALT1 in MCF7 and HeLa cells treated with cisplatin, which is correlated to an increase in apoptosis. Knockdown of MALT1 likewise increases cisplatin-induced apoptosis in these cancer cells. However, HECTD3 overexpression leads to a decreased cisplatin-induced apoptosis, whereas overexpression of MALT1 partially rescues HECTD3 depletion–induced apoptosis. These findings suggest that HECTD3 promotes cell survival through stabilizing MALT1. Our data have important implications in cancer therapy by providing novel molecular targets.

  7. Lafora disease E3-ubiquitin ligase malin is related to TRIM32 at both the phylogenetic and functional level

    Directory of Open Access Journals (Sweden)

    Gentry Matthew S

    2011-07-01

    Full Text Available Abstract Background Malin is an E3-ubiquitin ligase that is mutated in Lafora disease, a fatal form of progressive myoclonus epilepsy. In order to perform its function, malin forms a functional complex with laforin, a glucan phosphatase that facilitates targeting of malin to its corresponding substrates. While laforin phylogeny has been studied, there are no data on the evolutionary lineage of malin. Results After an extensive search for malin orthologs, we found that malin is present in all vertebrate species and a cephalochordate, in contrast with the broader species distribution previously reported for laforin. These data suggest that in addition to forming a functional complex, laforin and perhaps malin may also have independent functions. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32, which belongs to the tripartite-motif containing family of proteins. We present experimental evidence that both malin and TRIM32 share some substrates for ubiquitination, although they produce ubiquitin chains with different topologies. However, TRIM32-specific substrates were not reciprocally ubiquitinated by the laforin-malin complex. Conclusions We found that malin and laforin are not conserved in the same genomes. In addition, we found that malin shares significant identity with the E3-ubiquitin ligase TRIM32. The latter result suggests a common origin for malin and TRIM32 and provides insights into possible functional relationships between both proteins.

  8. CHK2 stability is regulated by the E3 ubiquitin ligase SIAH2.

    Science.gov (United States)

    García-Limones, C; Lara-Chica, M; Jiménez-Jiménez, C; Pérez, M; Moreno, P; Muñoz, E; Calzado, M A

    2016-08-18

    The serine threonine checkpoint kinase 2 (CHK2) is a critical protein involved in the DNA damage-response pathway, which is activated by phosphorylation inducing cellular response such as DNA repair, cell-cycle regulation or apoptosis. Although CHK2 activation mechanisms have been amply described, very little is known about degradation control processes. In the present study, we identify the ubiquitin E3 ligase SIAH2 as an interaction partner of CHK2, which mediates its ubiquitination and proteasomal degradation. CHK2 degradation is independent of both its activation and its kinase activity, but also of the phosphorylation in S456. We show that SIAH2-deficient cells present CHK2 accumulation together with lower ubiquitination levels. Accordingly, SIAH2 depletion by siRNA increases CHK2 levels. In response to DNA damage induced by etoposide, interaction between both proteins is disrupted, thus avoiding CHK2 degradation and promoting its stabilization. We also found that CHK2 phosphorylates SIAH2 at three residues (Thr26, Ser28 and Thr119), modifying its ability to regulate certain substrates. Cellular arrest in the G2/M phase induced by DNA damage is reverted by SIAH2 expression through the control of CHK2 levels. We observed that hypoxia decreases CHK2 levels in parallel to SIAH2 induction. Similarly, we provide evidence suggesting that resistance to apoptosis induced by genotoxic agents in cells subjected to hypoxia could be partly explained by the mutual regulation between both proteins. These results indicate that SIAH2 regulates CHK2 basal turnover, with important consequences on cell-cycle control and on the ability of hypoxia to alter the DNA damage-response pathway in cancer cells.

  9. Cyclin F: A component of an E3 ubiquitin ligase complex with roles in neurodegeneration and cancer.

    Science.gov (United States)

    Galper, Jasmin; Rayner, Stephanie L; Hogan, Alison L; Fifita, Jennifer A; Lee, Albert; Chung, Roger S; Blair, Ian P; Yang, Shu

    2017-08-01

    Cyclin F, encoded by CCNF, is the substrate recognition component of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF(cyclin F). E3 ubiquitin ligases play a key role in ubiquitin-proteasome mediated protein degradation, an essential component of protein homeostatic mechanisms within the cell. By recognising and regulating the availability of several protein substrates, SCF(cyclin F) plays a role in regulating various cellular processes including replication and repair of DNA and cell cycle checkpoint control. Cyclin F dysfunction has been implicated in various forms of cancer and CCNF mutations were recently linked to familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia, offering a new lead to understanding the pathogenic mechanisms underlying neurodegeneration. In this review, we evaluate the current literature on the function of cyclin F with an emphasis on its roles in cancer and neurodegeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

    Institute of Scientific and Technical Information of China (English)

    WANG Pingzhang; WANG Xin; WANG Feng; CAI Tianjing; LUO Ying

    2006-01-01

    MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (eIF4E), although the role of eIF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for eIF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hippel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.

  11. The evolutionarily conserved E3 ubiquitin ligase AtCHIP contributes to plant immunity

    Directory of Open Access Journals (Sweden)

    Xin eLi

    2016-03-01

    Full Text Available Plants possess a sophisticated immune system to recognize and respond to microbial threats in their environment. The level of immune signaling must be tightly regulated so that immune responses can be quickly activated in the presence of pathogens, while avoiding autoimmunity. HSP90s, along with their diverse array of co-chaperones, forms chaperone complexes that have been shown to play both positive and negative roles in regulating the accumulation of immune receptors and regulators. In this study, we examined the role of AtCHIP, an evolutionarily conserved E3 ligase that was known to interact with chaperones including HSP90s in multicellular organisms including fruit fly, C. elegans, plants and human. Atchip knockout mutants display enhanced disease susceptibility to a virulent oomycete pathogen, and overexpression of AtCHIP causes enhanced disease resistance at low temperature. Although CHIP was reported to target HSP90 for ubiquitination and degradation, accumulation of HSP90.3 was not affected in Atchip plants. In addition, protein accumulation of nucleotide-binding, leucine-rich repeat domain immune receptor (NLR SNC1 is not altered in Atchip mutant. Thus, while AtCHIP plays a role in immunity, it does not seem to regulate the turnover of HSP90 or SNC1. Further investigation is needed in order to determine the exact mechanism behind AtCHIP’s role in regulating plant immune responses.

  12. A family of Salmonella virulence factors functions as a distinct class of autoregulated E3 ubiquitin ligases

    Science.gov (United States)

    Quezada, Cindy M.; Hicks, Stuart W.; Galán, Jorge E.; Stebbins, C. Erec

    2009-01-01

    Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function. PMID:19273841

  13. E3 Ubiquitin Ligase Cbl-b Regulates Pten via Nedd4 in T Cells Independently of Its Ubiquitin Ligase Activity

    Directory of Open Access Journals (Sweden)

    Hui Guo

    2012-05-01

    Full Text Available E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here, we report that Cbl-b does not inhibit PI3K but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b−/− T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b−/− mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.

  14. Small, N-terminal tags activate Parkin E3 ubiquitin ligase activity by disrupting its autoinhibited conformation.

    Directory of Open Access Journals (Sweden)

    Lynn Burchell

    Full Text Available Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.

  15. Allosteric Interactions by p53 mRNA Govern HDM2 E3 Ubiquitin Ligase Specificity under Different Conditions.

    Science.gov (United States)

    Medina-Medina, Ixaura; García-Beltrán, Paola; de la Mora-de la Mora, Ignacio; Oria-Hernández, Jesús; Millot, Guy; Fahraeus, Robin; Reyes-Vivas, Horacio; Sampedro, José G; Olivares-Illana, Vanesa

    2016-08-15

    HDM2 and HDMX are key negative regulatory factors of the p53 tumor suppressor under normal conditions by promoting its degradation or preventing its trans activity, respectively. It has more recently been shown that both proteins can also act as positive regulators of p53 after DNA damage. This involves phosphorylation by ATM on serine residues HDM2(S395) and HDMX(S403), promoting their respective interaction with the p53 mRNA. However, the underlying molecular mechanisms of how these phosphorylation events switch HDM2 and HDMX from negative to positive regulators of p53 is not known. Our results show that these phosphorylation events reside within intrinsically disordered domains and change the conformation of the proteins. The modifications promote the exposition of N-terminal interfaces that support the formation of a new HDMX-HDM2 heterodimer independent of the C-terminal RING-RING interaction. The E3 ubiquitin ligase activity of this complex toward p53 is prevented by the p53 mRNA ligand but, interestingly, does not affect the capacity to ubiquitinate HDMX and HDM2. These results show how ATM-mediated modifications of HDMX and HDM2 switch HDM2 E3 ubiquitin ligase activity away from p53 but toward HDMX and itself and illustrate how the substrate specificity of HDM2 E3 ligase activity is regulated.

  16. E3 ubiquitin ligase UBR5 drives the growth and metastasis of triple negative breast cancer.

    Science.gov (United States)

    Liao, Liqiu; Song, Mei; Li, Xin; Tang, Lili; Zhang, Tuo; Zhang, Lixing; Pan, Yihang; Chouchane, Lotfi; Ma, Xiaojing

    2017-03-22

    Patients with triple negative breast cancers (TNBC) are at high risk for recurrence and metastasis at an early time despite standard treatment, underscoring the need for novel therapeutic modalities. Here we report for the first time a distinctive and profound role of the E3 ubiquitin ligase UBR5 in the growth and metastasis of TNBC. An analysis of primary TNBC specimen by whole exon sequencing revealed strong gene amplifications of UBR5 associated with the disease. UBR5 overexpression in TNBC tissues was confirmed at mRNA and protein levels. CRISPR/Cas9-mediated deletion of ubr5 in an experimental murine mammary carcinoma model of TNBC dramatically abrogated tumor growth and metastasis in vivo, which could be reversed completely via reconstitution with wild type UBR5 but not a catalytically inactive mutant. Loss of UBR5 caused an impairment in angiogenesis within the tumor, associated with increased apoptosis, necrosis, and growth arrest. Absence of UBR5 in the tumor triggered aberrant epithelial to mesenchymal transition (EMT), principally via abrogated expression of E-cadherin, which resulted in severely reduced tumor metastasis to secondary organs. Use of NOD/SCID mice revealed that tumor-derived UBR5 facilitated tumor growth in a manner completely dependent upon immune cells in the microenvironment, whereas it promoted metastasis in a tumor cell-autonomous fashion. Our findings unveil UBR5 as a novel and critical regulator of tumor growth, metastasis, and immune response, and highlight the potential for UBR5 as an effective therapeutic target for the treatment of highly aggressive breast and ovarian cancers that fail conventional therapy.

  17. E3 Ubiquitin Ligase NEDD4 Promotes Influenza Virus Infection by Decreasing Levels of the Antiviral Protein IFITM3.

    Directory of Open Access Journals (Sweden)

    Nicholas M Chesarino

    2015-08-01

    Full Text Available Interferon (IFN-induced transmembrane protein 3 (IFITM3 is a cell-intrinsic factor that limits influenza virus infections. We previously showed that IFITM3 degradation is increased by its ubiquitination, though the ubiquitin ligase responsible for this modification remained elusive. Here, we demonstrate that the E3 ubiquitin ligase NEDD4 ubiquitinates IFITM3 in cells and in vitro. This IFITM3 ubiquitination is dependent upon the presence of a PPxY motif within IFITM3 and the WW domain-containing region of NEDD4. In NEDD4 knockout mouse embryonic fibroblasts, we observed defective IFITM3 ubiquitination and accumulation of high levels of basal IFITM3 as compared to wild type cells. Heightened IFITM3 levels significantly protected NEDD4 knockout cells from infection by influenza A and B viruses. Similarly, knockdown of NEDD4 in human lung cells resulted in an increase in steady state IFITM3 and a decrease in influenza virus infection, demonstrating a conservation of this NEDD4-dependent IFITM3 regulatory mechanism in mouse and human cells. Consistent with the known association of NEDD4 with lysosomes, we demonstrate for the first time that steady state turnover of IFITM3 occurs through the lysosomal degradation pathway. Overall, this work identifies the enzyme NEDD4 as a new therapeutic target for the prevention of influenza virus infections, and introduces a new paradigm for up-regulating cellular levels of IFITM3 independently of IFN or infection.

  18. Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

    DEFF Research Database (Denmark)

    Boomsma, Wouter Krogh; Nielsen, Sofie Vincents; Lindorff-Larsen, Kresten;

    2016-01-01

    conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology...

  19. The Blue Light-Dependent Polyubiquitination and Degradation of Arabidopsis Cryptochrome2 Requires Multiple E3 Ubiquitin Ligases.

    Science.gov (United States)

    Liu, Qing; Wang, Qin; Liu, Bin; Wang, Wei; Wang, Xu; Park, Joon; Yang, Zhenming; Du, Xinglin; Bian, Mingdi; Lin, Chentao

    2016-10-01

    Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1-SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

  20. Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human osteosarcoma cells.

    Science.gov (United States)

    Chen, Xi; Shen, Jianjun; Li, Xingyu; Wang, Xi; Long, Min; Lin, Fang; Wei, Junxia; Yang, Longfei; Yang, Chinglai; Dong, Ke; Zhang, Huizhong

    2014-07-01

    Stathmin is an oncoprotein and is expressed at high levels in a wide variety of human malignancies, which plays important roles in maintenance of malignant phenotypes. The regulation of Stathmin gene overexpression has been wildly explored, but the exact mechanism still needs to be elucidated. It is believed that regulation of an oncogene protein abundance through post-translational modifications is essential for maintenance of malignant phenotypes. Here we identified the Rlim, a Ring H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a Stathmin-interacting protein that could increase Stathmin turnover through binding with this targeted protein and then induce its degradation by proteasome in a ubiquitin-dependent manner. Inhibition of endogenous Rlim expression by siRNA could increase the level of Stathmin protein, which further led to cell proliferation and cell cycle changes in human osteosarcoma cell lines. On the other hand, forced overexpression of Rlim could decrease the level of Stathmin protein. These results demonstrate that Rlim is involved in the negative regulation of Stathmin protein level through physical interaction and ubiquitin-mediated proteolysis. Hence, Rlim is a novel regulator of Stathmin protein in a ubiquitin-dependent manner, and represents a new pathway for malignant phenotype turnover by modulating the level of Stathmin protein in human osteosarcomas.

  1. Inactivation of the putative ubiquitin-E3 ligase PDLIM2 in classical Hodgkin and anaplastic large cell lymphoma

    Science.gov (United States)

    Wurster, K D; Hummel, F; Richter, J; Giefing, M; Hartmann, S; Hansmann, M-L; Kreher, S; Köchert, K; Krappmann, D; Klapper, W; Hummel, M; Wenzel, S-S; Lenz, G; Janz, M; Dörken, B; Siebert, R; Mathas, S

    2017-01-01

    Apart from its unique histopathological appearance with rare tumor cells embedded in an inflammatory background of bystander cells, classical Hodgkin lymphoma (cHL) is characterized by an unusual activation of a broad range of signaling pathways involved in cellular activation. This includes constitutive high-level activity of nuclear factor-κB (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), activator protein-1 (AP-1) and interferon regulatory factor (IRF) transcription factors (TFs) that are physiologically only transiently activated. Here, we demonstrate that inactivation of the putative ubiquitin E3-ligase PDLIM2 contributes to this TF activation. PDLIM2 expression is lost at the mRNA and protein levels in the majority of cHL cell lines and Hodgkin and Reed–Sternberg (HRS) cells of nearly all cHL primary samples. This loss is associated with PDLIM2 genomic alterations, promoter methylation and altered splicing. Reconstitution of PDLIM2 in HRS cell lines inhibits proliferation, blocks NF-κB transcriptional activity and contributes to cHL-specific gene expression. In non-Hodgkin B-cell lines, small interfering RNA-mediated PDLIM2 knockdown results in superactivation of TFs NF-κB and AP-1 following phorbol 12-myristate 13-acetate (PMA) stimulation. Furthermore, expression of PDLIM2 is lost in anaplastic large cell lymphoma (ALCL) that shares key biological aspects with cHL. We conclude that inactivation of PDLIM2 is a recurrent finding in cHL and ALCL, promotes activation of inflammatory signaling pathways and thereby contributes to their pathogenesis. PMID:27538486

  2. The activities of the E3 ubiquitin ligase COP1/SPA, a key repressor in light signaling.

    Science.gov (United States)

    Hoecker, Ute

    2017-06-01

    Light is a critical signal to integrate plant growth and development with the environment. Downstream of photoreceptors, the E3 ubiquitin ligase COP1/SPA is a key repressor of photomorphogenesis which targets many positive regulators of light signaling, mainly transcription factors, for degradation in darkness. In light-grown plants COP1/SPA activity is repressed, allowing light responses to occur. This review provides an overview on our current knowledge on COP1/SPA repressor function, focusing in particular on the roles of the respective protein domains and the mechanisms of light-induced inactivation of COP1/SPA. Moreover, we summarize how COP1 activity is regulated by other interacting proteins, such as a SUMO E3 ligase and Phytochrome-Interacting Factors (PIFs), as well as by hormones. At last, several novel functions of COP1 that were recently revealed are included. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Protein microarrays for the identification of praja1 e3 ubiquitin ligase substrates.

    Science.gov (United States)

    Loch, Christian M; Eddins, Michael J; Strickler, James E

    2011-06-01

    Although they are the primary determinants of substrate specificity, few E3-substrate pairs have been positively identified, and few E3's profiled in a proteomic fashion. Praja1 is an E3 implicated in bone development and highly expressed in brain. Although it has been well studied relative to the majority of E3's, little is known concerning the repertoire of proteins it ubiquitylates. We sought to identify high confidence substrates for Praja1 from an unbiased proteomic profile of thousands of human proteins using protein microarrays. We first profiled Praja1 activity against a panel of E2's to identify its optimal partner in vitro. We then ubiquitylated multiple, identical protein arrays and detected putative substrates with reagents that vary in ubiquitin recognition according to the extent of chain formation. Gene ontology clustering identified putative substrates consistent with information previously known about Praja1 function, and provides clues into novel aspects of this enzyme's function.

  4. A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A*

    OpenAIRE

    Fishman-Jacob, Tali; Reznichenko, Lydia; Youdim, Moussa B H; Mandel, Silvia A.

    2009-01-01

    The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled v...

  5. Overexpression of denticleless E3 ubiquitin protein ligase homolog (DTL) is related to poor outcome in gastric carcinoma

    OpenAIRE

    Kobayashi, Hiroki; Komatsu, Shuhei; Ichikawa, Daisuke; Kawaguchi, Tsutomu; Hirajima, Shoji; Miyamae, Mahito; Okajima, Wataru; Ohashi, Takuma; Kosuga, Toshiyuki; Konishi, Hirotaka; Shiozaki, Atsushi; FUJIWARA, Hitoshi; Okamoto, Kazuma; Tsuda, Hitoshi; Otsuji, Eigo

    2015-01-01

    Background Denticleless E3 ubiquitin protein ligase homolog (DTL) has been identified in amplified region (1q32) of several cancers and has an oncogenic function. In this study, we tested whether DTL acts as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC). Methods We analyzed 7 GC cell lines and 100 primary tumors that were curatively resected in our hospital between 2001 and 2003. Results Overexpression of the DTL protein was detected in GC cell lines (4/...

  6. Inactivation of Sag/Rbx2/Roc2 E3 Ubiquitin Ligase Triggers Senescence and Inhibits Kras-Induced Immortalization

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    2015-01-01

    Full Text Available Our recent study showed that SAG/RBX2 E3 ubiquitin ligase regulates apoptosis and vasculogenesis by promoting degradation of NOXA and NF1, and co-operates with Kras to promote lung tumorigenesis by activating NFκB and mTOR pathways via targeted degradation of tumor suppressive substrates including IκB, DEPTOR, p21 and p27. Here we investigated the role of Sag/Rbx2 E3 ligase in cellular senescence and immortalization of mouse embryonic fibroblasts (MEFs and report that Sag is required for proper cell proliferation and KrasG12D-induced immortalization. Sag inactivation by genetic deletion remarkably suppresses cell proliferation by inducing senescence, which is associated with accumulation of p16, but not p53. Mechanistically, Sag deletion caused accumulation of Jun-B, a substrate of Sag-Fbxw7 E3 ligase and a transcription factor that drives p16 transcription. Importantly, senescence triggered by Sag deletion can be largely rescued by simultaneous deletion of Cdkn2a, the p16 encoding gene, indicating its causal role. Furthermore, KrasG12D-induced immortalization can also be abrogated by Sag deletion via senescence induction, which is again rescued by simultaneous deletion of Cdkn2a. Finally, we found that Sag deletion inactivates KrasG12D activity and block the MAPK signaling pathway, together with accumulated p16, to induce senescence. Taken together, our results demonstrated that Sag is a KrasG12D-cooperating oncogene required for KrasG12D-induced immortalization and transformation, and targeting SAG-SCF E3 ligase may, therefore, have therapeutic value for senescence-based cancer treatment.

  7. Ezrin ubiquitylation by the E3 ubiquitin ligase, WWP1, and consequent regulation of hepatocyte growth factor receptor activity.

    Directory of Open Access Journals (Sweden)

    Rania F Zaarour

    Full Text Available The membrane cytoskeleton linker ezrin participates in several functions downstream of the receptor Met in response to Hepatocyte Growth Factor (HGF stimulation. Here we report a novel interaction of ezrin with a HECT E3 ubiquitin ligase, WWP1/Aip5/Tiul1, a potential oncogene that undergoes genomic amplification and overexpression in human breast and prostate cancers. We show that ezrin binds to the WW domains of WWP1 via the consensus motif PPVY(477 present in ezrin's C-terminus. This association results in the ubiquitylation of ezrin, a process that requires an intact PPVY(477 motif. Interestingly ezrin ubiquitylation does not target the protein for degradation by the proteasome. We find that ezrin ubiquitylation by WWP1 in epithelial cells leads to the upregulation of Met level in absence of HGF stimulation and increases the response of Met to HGF stimulation as measured by the ability of the cells to heal a wound. Interestingly this effect requires ubiquitylated ezrin since it can be rescued, after depletion of endogenous ezrin, by wild type ezrin but not by a mutant of ezrin that cannot be ubiquitylated. Taken together our data reveal a new role for ezrin in Met receptor stability and activity through its association with the E3 ubiquitin ligase WWP1. Given the role of Met in cell proliferation and tumorigenesis, our results may provide a mechanistic basis for understanding the role of ezrin in tumor progression.

  8. CUL4-DDB1-CDT2 E3 Ligase Regulates the Molecular Clock Activity by Promoting Ubiquitination-Dependent Degradation of the Mammalian CRY1.

    Science.gov (United States)

    Tong, Xin; Zhang, Deqiang; Guha, Anirvan; Arthurs, Blake; Cazares, Victor; Gupta, Neil; Yin, Lei

    2015-01-01

    The CUL4-DDB1 E3 ligase complex serves as a critical regulator in various cellular processes, including cell proliferation, DNA damage repair, and cell cycle progression. However, whether this E3 ligase complex regulates clock protein turnover and the molecular clock activity in mammalian cells is unknown. Here we show that CUL4-DDB1-CDT2 E3 ligase ubiquitinates CRY1 and promotes its degradation both in vitro and in vivo. Depletion of the major components of this E3 ligase complex, including Ddb1, Cdt2, and Cdt2-cofactor Pcna, leads to CRY1 stabilization in cultured cells or in the mouse liver. CUL4A-DDB1-CDT2 E3 ligase targets lysine 585 within the C-terminal region of CRY1 protein, shown by the CRY1 585KA mutant's resistance to ubiquitination and degradation mediated by the CUL4A-DDB1 complex. Surprisingly, both depletion of Ddb1 and over-expression of Cry1-585KA mutant enhance the oscillatory amplitude of the Bmal1 promoter activity without altering its period length, suggesting that CUL4A-DDB1-CDT2 E3 targets CRY1 for degradation and reduces the circadian amplitude. All together, we uncovered a novel biological role for CUL4A-DDB1-CDT2 E3 ligase that regulates molecular circadian behaviors via promoting ubiquitination-dependent degradation of CRY1.

  9. Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.

    Science.gov (United States)

    Cheng, Jie; Guggino, William

    2013-01-01

    Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.

  10. MARCH1 E3 Ubiquitin Ligase Dampens the Innate Inflammatory Response by Modulating Monocyte Functions in Mice.

    Science.gov (United States)

    Galbas, Tristan; Raymond, Maxime; Sabourin, Antoine; Bourgeois-Daigneault, Marie-Claude; Guimont-Desrochers, Fanny; Yun, Tae Jin; Cailhier, Jean-François; Ishido, Satoshi; Lesage, Sylvie; Cheong, Cheolho; Thibodeau, Jacques

    2017-01-15

    Ubiquitination was recently identified as a central process in the pathogenesis and development of numerous inflammatory diseases, such as obesity, atherosclerosis, and asthma. Treatment with proteasomal inhibitors led to severe side effects because ubiquitination is heavily involved in a plethora of cellular functions. Thus, new players regulating ubiquitination processes must be identified to improve therapies for inflammatory diseases. In addition to their role in adaptive immunity, endosomal MHC class II (MHCII) molecules were shown to modulate innate immune responses by fine tuning the TLR4 signaling pathway. However, the role of MHCII ubiquitination by membrane associated ring-CH-type finger 1 (MARCH1) E3 ubiquitin ligase in this process remains to be assessed. In this article, we demonstrate that MARCH1 is a key inhibitor of innate inflammation in response to bacterial endotoxins. The higher mortality of March1(-/-) mice challenged with a lethal dose of LPS was associated with significantly stronger systemic production of proinflammatory cytokines and splenic NK cell activation; however, we did not find evidence that MARCH1 modulates LPS or IL-10 signaling pathways. Instead, the mechanism by which MARCH1 protects against endotoxic shock rests on its capacity to promote the transition of monocytes from Ly6C(Hi) to Ly6C(+/-) Moreover, in competitive bone marrow chimeras, March1(-/-) monocytes and polymorphonuclear neutrophils outcompeted wild-type cells with regard to bone marrow egress and homing to peripheral organs. We conclude that MARCH1 exerts MHCII-independent effects that regulate the innate arm of immunity. Thus, MARCH1 might represent a potential new target for emerging therapies based on ubiquitination reactions in inflammatory diseases.

  11. HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Belzile

    2007-07-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 viral protein R (Vpr has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1, viral protein R binding protein (VPRBP, and cullin 4A (CUL4A--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

  12. Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages

    Science.gov (United States)

    Suzuki, Shiho; Mimuro, Hitomi; Kim, Minsoo; Ogawa, Michinaga; Ashida, Hiroshi; Toyotome, Takahito; Franchi, Luigi; Suzuki, Masato; Sanada, Takahito; Suzuki, Toshihiko; Tsutsui, Hiroko; Núñez, Gabriel; Sasakawa, Chihiro

    2014-01-01

    When nucleotide-binding oligomerization domain–like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN+/− mice were more responsive to inflammasome activation than those from GLMN+/+ mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via

  13. Inactivation of SAG E3 ubiquitin ligase blocks embryonic stem cell differentiation and sensitizes leukemia cells to retinoid acid.

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    Full Text Available Sensitive to Apoptosis Gene (SAG, also known as RBX2 (RING box protein-2, is the RING component of SCF (SKP1, Cullin, and F-box protein E3 ubiquitin ligase. Our previous studies have demonstrated that SAG is an anti-apoptotic protein and an attractive anti-cancer target. We also found recently that Sag knockout sensitized mouse embryonic stem cells (mES to radiation and blocked mES cells to undergo endothelial differentiation. Here, we reported that compared to wild-type mES cells, the Sag(-/- mES cells were much more sensitive to all-trans retinoic acid (RA-induced suppression of cell proliferation and survival. While wild-type mES cells underwent differentiation upon exposure to RA, Sag(-/- mES cells were induced to death via apoptosis instead. The cell fate change, reflected by cellular stiffness, can be detected as early as 12 hrs post RA exposure by AFM (Atomic Force Microscopy. We then extended this novel finding to RA differentiation therapy of leukemia, in which the resistance often develops, by testing our hypothesis that SAG inhibition would sensitize leukemia to RA. Indeed, we found a direct correlation between SAG overexpression and RA resistance in multiple leukemia lines. By using MLN4924, a small molecule inhibitor of NEDD8-Activating Enzyme (NAE, that inactivates SAG-SCF E3 ligase by blocking cullin neddylation, we were able to sensitize two otherwise resistant leukemia cell lines, HL-60 and KG-1 to RA. Mechanistically, RA sensitization by MLN4924 was mediated via enhanced apoptosis, likely through accumulation of pro-apoptotic proteins NOXA and c-JUN, two well-known substrates of SAG-SCF E3 ligase. Taken together, our study provides the proof-of-concept evidence for effective treatment of leukemia patients by RA-MLN4924 combination.

  14. Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Specificity of protein ubiquitylation is conferred by E3 ubiquitin (Ub ligases. We have annotated approximately 617 putative E3s and substrate-recognition subunits of E3 complexes encoded in the human genome. The limited knowledge of the function of members of the large E3 superfamily prompted us to generate genome-wide E3 cDNA and RNAi expression libraries designed for functional screening. An imaging-based screen using these libraries to identify E3s that regulate mitochondrial dynamics uncovered MULAN/FLJ12875, a RING finger protein whose ectopic expression and knockdown both interfered with mitochondrial trafficking and morphology. We found that MULAN is a mitochondrial protein - two transmembrane domains mediate its localization to the organelle's outer membrane. MULAN is oriented such that its E3-active, C-terminal RING finger is exposed to the cytosol, where it has access to other components of the Ub system. Both an intact RING finger and the correct subcellular localization were required for regulation of mitochondrial dynamics, suggesting that MULAN's downstream effectors are proteins that are either integral to, or associated with, mitochondria and that become modified with Ub. Interestingly, MULAN had previously been identified as an activator of NF-kappaB, thus providing a link between mitochondrial dynamics and mitochondria-to-nucleus signaling. These findings suggest the existence of a new, Ub-mediated mechanism responsible for integration of mitochondria into the cellular environment.

  15. The E3 Ubiquitin Ligase SCF(Cyclin F) Transmits AKT Signaling to the Cell-Cycle Machinery.

    Science.gov (United States)

    Choudhury, Rajarshi; Bonacci, Thomas; Wang, Xianxi; Truong, Andrew; Arceci, Anthony; Zhang, Yanqiong; Mills, Christine A; Kernan, Jennifer L; Liu, Pengda; Emanuele, Michael J

    2017-09-26

    The oncogenic AKT kinase is a key regulator of apoptosis, cell growth, and cell-cycle progression. Despite its important role in proliferation, it remains largely unknown how AKT is mechanistically linked to the cell cycle. We show here that cyclin F, a substrate receptor F-box protein for the SCF (Skp1/Cul1/F-box) family of E3 ubiquitin ligases, is a bona fide AKT substrate. Cyclin F expression oscillates throughout the cell cycle, a rare feature among the 69 human F-box proteins, and all of its known substrates are involved in proliferation. AKT phosphorylation of cyclin F enhances its stability and promotes assembly into productive E3 ligase complexes. Importantly, expression of mutant versions of cyclin F that cannot be phosphorylated by AKT impair cell-cycle entry. Our data suggest that cyclin F transmits mitogen signaling through AKT to the core cell-cycle machinery. This discovery has potential implications for proliferative control in malignancies where AKT is activated. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1.

    Science.gov (United States)

    Bodine, Sue C; Baehr, Leslie M

    2014-09-15

    Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered.

  17. Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1

    Science.gov (United States)

    Baehr, Leslie M.

    2014-01-01

    Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered. PMID:25096180

  18. A sporadic Parkinson disease model via silencing of the ubiquitin-proteasome/E3 ligase component SKP1A.

    Science.gov (United States)

    Fishman-Jacob, Tali; Reznichenko, Lydia; Youdim, Moussa B H; Mandel, Silvia A

    2009-11-20

    The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G(0)/G(1) phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for gamma-tubulin, alpha-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration.

  19. A Sporadic Parkinson Disease Model via Silencing of the Ubiquitin-Proteasome/E3 Ligase Component SKP1A*

    Science.gov (United States)

    Fishman-Jacob, Tali; Reznichenko, Lydia; Youdim, Moussa B. H.; Mandel, Silvia A.

    2009-01-01

    The aim of this study was to develop a new model of sporadic Parkinson disease (PD) based on silencing of the SKP1A gene, a component of the ubiquitin-proteasome/E3 ligase complex, Skp1, Cullin 1, F-box protein, which was found to be highly decreased in the substantia nigra of sporadic PD patients. Initially, an embryonic mouse substantia nigra-derived cell line (SN4741 cells) was infected with short hairpin RNA lentiviruses encoding the murine transcript of the SKP1A gene or with scrambled vector. SKP1A silencing resulted in increased susceptibility to neuronal damages induced by the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium ion and serum starvation, in parallel with a decline in the expression of the dopaminergic markers, dopamine transporter and vesicular monoamine transporter-2. SKP1A-deficient cells presented a delay in completion of the cell cycle and the inability to arrest at the G0/G1 phase when induced to differentiate. Instead, the cells progressed through S phase, developing rounded aggregates with characteristics of aggresomes including immunoreactivity for γ-tubulin, α-synuclein, ubiquitin, tyrosine hydroxylase, Hsc-70 (70-kDa heat shock cognate protein), and proteasome subunit, and culminating in a lethal phenotype. Conversely, stably enforced expression of wild type SKP1A duplicated the survival index of naïve SN4741 cells under proteasomal inhibition injury, suggesting a new structural role of SKP1 in dopaminergic neuronal function, besides its E3 ligase activity. These results link, for the first time, SKP1 to dopamine neuronal function and survival, suggesting an essential role in sporadic PD. In summary, this new model has reproduced to a significant extent the molecular alterations described in sporadic PD at the cellular level, implicating Skp1 as a potential modifier in sporadic PD neurodegeneration. PMID:19748892

  20. HDAC7 Ubiquitination by the E3 Ligase CBX4 Is Involved in Contextual Fear Conditioning Memory Formation.

    Science.gov (United States)

    Jing, Xu; Sui, Wen-Hai; Wang, Shuai; Xu, Xu-Feng; Yuan, Rong-Rong; Chen, Xiao-Rong; Ma, Hui-Xian; Zhu, Ying-Xiao; Sun, Jin-Kai; Yi, Fan; Chen, Zhe-Yu; Wang, Yue

    2017-04-05

    Histone acetylation, an epigenetic modification, plays an important role in long-term memory formation. Recently, histone deacetylase (HDAC) inhibitors were demonstrated to promote memory formation, which raises the intriguing possibility that they may be used to rescue memory deficits. However, additional research is necessary to clarify the roles of individual HDACs in memory. In this study, we demonstrated that HDAC7, within the dorsal hippocampus of C57BL6J mice, had a late and persistent decrease after contextual fear conditioning (CFC) training (4-24 h), which was involved in long-term CFC memory formation. We also showed that HDAC7 decreased via ubiquitin-dependent degradation. CBX4 was one of the HDAC7 E3 ligases involved in this process. Nur77, as one of the target genes of HDAC7, increased 6-24 h after CFC training and, accordingly, modulated the formation of CFC memory. Finally, HDAC7 was involved in the formation of other hippocampal-dependent memories, including the Morris water maze and object location test. The current findings facilitate an understanding of the molecular and cellular mechanisms of HDAC7 in the regulation of hippocampal-dependent memory.SIGNIFICANCE STATEMENT The current findings demonstrated the effects of histone deacetylase 7 (HDAC7) on hippocampal-dependent memories. Moreover, we determined the mechanism of decreased HDAC7 in contextual fear conditioning (CFC) through ubiquitin-dependent protein degradation. We also verified that CBX4 was one of the HDAC7 E3 ligases. Finally, we demonstrated that Nur77, as one of the important targets for HDAC7, was involved in CFC memory formation. All of these proteins, including HDAC7, CBX4, and Nur77, could be potential therapeutic targets for preventing memory deficits in aging and neurological diseases. Copyright © 2017 the authors 0270-6474/17/373848-16$15.00/0.

  1. Reconstruction of an active SOCS3-based E3 ubiquitin ligase complex in vitro: identification of the active components and JAK2 and gp130 as substrates.

    Science.gov (United States)

    Kershaw, Nadia J; Laktyushin, Artem; Nicola, Nicos A; Babon, Jeffrey J

    2014-02-01

    SOCS3 (suppressor of cytokine signaling 3) inhibits the intracellular signaling cascade initiated by exposure of cells to cytokines. SOCS3 regulates signaling via two distinct mechanisms: directly inhibiting the catalytic activity of Janus kinases (JAKs) that initiate the intracellular signaling cascade and catalysing the ubiquitination of signaling components by recruiting components of an E3 ubiquitin ligase complex. Here we investigate the latter mode-of-action biochemically by reconstructing a SOCS3-based E3 ubiquitin ligase complex in vitro using fully purified, recombinant components and examining its ability to promote the ubiquitination of molecules involved in the cytokine signaling cascade. We show that SOCS3 is an active substrate recruitment module for a Cullin5-based E3 ligase and have defined the core protein components required for ubiquitination. SOCS3-induced polyubiquitination was rapid and could proceed through a number of different ubiquitin lysines. SOCS3 catalyzed the ubiquitination of both the IL-6 receptor common chain (gp130) and JAK2.

  2. Genetically engineered mouse models for functional studies of SKP1-CUL1-F-box-protein (SCF) E3 ubiquitin ligases

    Institute of Scientific and Technical Information of China (English)

    Weihua Zhou; Wenyi Wei; Yi Sun

    2013-01-01

    The SCF (SKP1 (S-phase-kinase-associated protein 1),Cullin-1,F-box protein) E3 ubiquitin ligases,the founding member of Cullin-RING ligases (CRLs),are the largest family of E3 ubiquitin ligases in mammals.Each individual SCF E3 ligase consists of one adaptor protein SKP1,one scaffold protein cullin-1 (the first family member of the eight cullins),one F-box protein out of 69 family members,and one out of two RING (Really Interesting New Gene) family proteins RBX1/ROC1 or RBX2/ROC2/SAG/RNF7.Various combinations of these four components construct a large number of SCF E3s that promote the degradation of many key regulatory proteins in cell-context,temporally,and spatially dependent manners,thus controlling precisely numerous important cellular processes,including cell cycle progression,apoptosis,gene transcription,signal transduction,DNA replication,maintenance of genome integrity,and tumorigenesis.To understand how the SCF E3 ligases regulate these cellular processes and embryonic development under in vivo physiological conditions,a number of mouse models with transgenic (Tg) expression or targeted deletion of components of SCF have been established and characterized.In this review,we will provide a brief introduction to the ubiquitin-proteasome system (UPS) and the SCF E3 ubiquitin ligases,followed by a comprehensive overview on the existing Tg and knockout (KO) mouse models of the SCF E3s,and discuss the role of each component in mouse embryogenesis,cell proliferation,apoptosis,carcinogenesis,as well as other pathogenic processes associated with human diseases.We will end with a brief discussion on the future directions of this research area and the potential applications of the knowledge gained to more effective therapeutic interventions of human diseases.

  3. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

    Science.gov (United States)

    Aghajan, Mariam; Jonai, Nao; Flick, Karin; Fu, Fei; Luo, Manlin; Cai, Xiaolu; Ouni, Ikram; Pierce, Nathan; Tang, Xiaobo; Lomenick, Brett; Damoiseaux, Robert; Hao, Rui; Del Moral, Pierre M; Verma, Rati; Li, Ying; Li, Cheng; Houk, Kendall N; Jung, Michael E; Zheng, Ning; Huang, Lan; Deshaies, Raymond J; Kaiser, Peter; Huang, Jing

    2010-07-01

    The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

  4. Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2.

    Science.gov (United States)

    Juanes, M Angeles; Piatti, Simonetta

    2016-09-01

    Formins are widespread actin-polymerizing proteins that play pivotal roles in a number of processes, such as cell polarity, morphogenesis, cytokinesis, and cell migration. In agreement with their crucial function, formins are prone to a variety of regulatory mechanisms that include autoinhibition, post-translational modifications, and interaction with formin modulators. Furthermore, activation and function of formins is intimately linked to their ability to interact with membranes. In the budding yeast Saccharomyces cerevisiae, the two formins Bni1 and Bnr1 play both separate and overlapping functions in the organization of the actin cytoskeleton. In addition, they are controlled by both common and different regulatory mechanisms. Here we show that proper localization of both formins requires the redundant E3 ubiquitin ligases Dma1 and Dma2, which were previously involved in spindle positioning and septin organization. In dma1 dma2 double mutants, formin distribution at polarity sites is impaired, thus causing defects in the organization of the actin cable network and hypersensitivity to the actin depolymerizer latrunculin B. Expression of a hyperactive variant of Bni1 (Bni1-V360D) rescues these defects and partially restores proper spindle positioning in the mutant, suggesting that the failure of dma1 dma2 mutant cells to position the spindle is partly due to faulty formin activity. Strikingly, Dma1/2 interact physically with both formins, while their ubiquitin-ligase activity is required for formin function and polarized localization. Thus, ubiquitylation of formin or a formin interactor(s) could promote formin binding to membrane and its ability to nucleate actin. Altogether, our data highlight a novel level of formin regulation that further expands our knowledge of the complex and multilayered controls of these key cytoskeleton organizers.

  5. Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling.

    Science.gov (United States)

    Berndt, Jason D; Aoyagi, Atsushi; Yang, Peitzu; Anastas, Jamie N; Tang, Lan; Moon, Randall T

    2011-09-05

    Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steady-state levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/β-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A-mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling.

  6. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

    NARCIS (Netherlands)

    Luijsterburg, Martijn S.; Goedhart, Joachim; Moser, Jill; Kool, Hanneke; Geverts, Bart; Houtsmuller, Adriaan B.; Mullenders, Leon H. F.; Vermeulen, Wim; van Driel, Roel

    2007-01-01

    Damage DNA binding protein 2 ( DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and

  7. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC.

    NARCIS (Netherlands)

    Luijsterburg, M.S.; Goedhart, J.; Moser, J.; Kool, H.; Geverts, B.; Houtsmuller, A.B.; Mullenders, L.H.; Vermeulen, W.; van Driel, R.

    2007-01-01

    Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A

  8. Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase

    DEFF Research Database (Denmark)

    Hernández-Muñoz, Inmaculada; Lund, Anders H; van der Stoop, Petra

    2005-01-01

    . This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group...... inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3/SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing....

  9. UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans

    Science.gov (United States)

    Safdar, Komal; Gu, Anniya; Xu, Xia; Au, Vinci; Taylor, Jon; Flibotte, Stephane; Moerman, Donald G.; Maine, Eleanor M.

    2016-01-01

    Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells) to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components. PMID:27185398

  10. UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Komal Safdar

    2016-07-01

    Full Text Available Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components.

  11. PEX2 is the E3 ubiquitin ligase required for pexophagy during starvation

    NARCIS (Netherlands)

    Sargent, Graeme; van Zutphen, Tim; Shatseva, Tatiana; Zhang, Ling; Di Giovanni, Valeria; Bandsma, Robert; Kim, Peter Kijun

    2016-01-01

    Peroxisomes are metabolic organelles necessary for anabolic and catabolic lipid reactions whose numbers are highly dynamic based on the metabolic need of the cells. One mechanism to regulate peroxisome numbers is through an autophagic process called pexophagy. In mammalian cells, ubiquitination of

  12. Heterologous expression of the gourd E3 ubiquitin ligase gene LsRZF1 compromises the drought stress tolerance in Arabidopsis thaliana.

    Science.gov (United States)

    Min, Ji-Hee; Ju, Hyun-Woo; Yang, Kwang-Yeol; Chung, Jung-Sung; Cho, Baik-Ho; Kim, Cheol Soo

    2014-04-01

    Protein ubiquitination is one of the major regulatory processes used by eukaryotic cells. The ubiquitin E3 ligase acts as a main determinant of substrate specificity. However, the precise roles of E3 ligase in plants to drought stress are poorly understood. In this study, a gourd family (Lagenaria siceraria) ortholog of Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1) gene, designated LsRZF1, was identified and characterized. LsRZF1 was reduced by abscisic acid (ABA), osmotic stress, and drought conditions. Compared to wild type, transgenic Arabidopsis plants ectopic expressing LsRZF1 were hypersensitive to ABA and osmotic stress during early seedling development, indicating that LsRZF1 negatively regulates drought-mediated control of early seedling development. Moreover, the ectopic expression of the LsRZF1 gene was very influential in drought sensitive parameters including proline content, water loss, and the expression of dehydration stress-related genes. Furthermore, ubiquitin E3 ligase activity and genetic data indicate that AtRZF1 and LsRZF1 function in similar pathway to control proline metabolism in Arabidopsis under drought condition. Together, these results suggest that the E3 ligase LsRZF1 is an important regulator of water deficit stress during early seedling development.

  13. Regulation of Chloroplast Protein Import by the Ubiquitin E3 Ligase SP1 Is Important for Stress Tolerance in Plants.

    Science.gov (United States)

    Ling, Qihua; Jarvis, Paul

    2015-10-05

    Chloroplasts are the organelles responsible for photosynthesis in plants [1, 2]. The chloroplast proteome comprises ∼3,000 different proteins, including components of the photosynthetic apparatus, which are highly abundant. Most chloroplast proteins are nucleus-encoded and imported following synthesis in the cytosol. Such import is mediated by multiprotein complexes in the envelope membranes that surround each organelle [3, 4]. The translocon at the outer envelope membrane of chloroplasts (TOC) mediates client protein recognition and early stages of import. The TOC apparatus is regulated by the ubiquitin-proteasome system (UPS) in a process controlled by the envelope-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) [5, 6]. Previous work showed that SP1-mediated regulation of chloroplast protein import contributes to the organellar proteome changes that occur during plant development (e.g., during de-etiolation). Here, we reveal a critical role for SP1 in plant responses to abiotic stress, which is a major and increasing cause of agricultural yield losses globally [7]. Arabidopsis plants lacking SP1 are hypersensitive to salt, osmotic, and oxidative stresses, whereas plants overexpressing SP1 are considerably more stress tolerant than wild-type. We present evidence that SP1 acts to deplete the TOC apparatus under stress conditions to limit the import of photosynthetic apparatus components, which may attenuate photosynthetic activity and reduce the potential for reactive oxygen species production and photo-oxidative damage. Our results indicate that chloroplast protein import is responsive to environmental cues, enabling dynamic regulation of the organellar proteome, and suggest new approaches for improving stress tolerance in crops.

  14. A non-proteolytic role for ubiquitin in deadenylation of MHC-I mRNA by the RNA-binding E3-ligase MEX-3C

    Science.gov (United States)

    Cano, Florencia; Rapiteanu, Radu; Sebastiaan Winkler, G.; Lehner, Paul J.

    2015-01-01

    The regulation of protein and mRNA turnover is essential for many cellular processes. We recently showed that ubiquitin—traditionally linked to protein degradation—directly regulates the degradation of mRNAs through the action of a newly identified family of RNA-binding E3 ubiquitin ligases. How ubiquitin regulates mRNA decay remains unclear. Here, we identify a new role for ubiquitin in regulating deadenylation, the initial and often rate-limiting step in mRNA degradation. MEX-3C, a canonical member of this family of RNA-binding ubiquitin ligases, associates with the cytoplasmic deadenylation complexes and ubiquitinates CNOT7(Caf1), the main catalytic subunit of the CCR4-NOT deadenylation machinery. We establish a new role for ubiquitin in regulating MHC-I mRNA deadenylation as ubiquitination of CNOT7 by MEX-3C regulates its deadenylation activity and is required for MHC-I mRNA degradation. Since neither proteasome nor lysosome inhibitors rescued MEX-3C-mediated MHC-I mRNA degradation, our findings suggest a new non-proteolytic function for ubiquitin in the regulation of mRNA decay. PMID:26471122

  15. Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila.

    Directory of Open Access Journals (Sweden)

    Roland Le Borgne

    2005-04-01

    Full Text Available Signaling by the Notch ligands Delta (Dl and Serrate (Ser regulates a wide variety of essential cell-fate decisions during animal development. Two distinct E3 ubiquitin ligases, Neuralized (Neur and Mind bomb (Mib, have been shown to regulate Dl signaling in Drosophila melanogaster and Danio rerio, respectively. While the neur and mib genes are evolutionarily conserved, their respective roles in the context of a single organism have not yet been examined. We show here that the Drosophila mind bomb (D-mib gene regulates a subset of Notch signaling events, including wing margin specification, leg segmentation, and vein determination, that are distinct from those events requiring neur activity. D-mib also modulates lateral inhibition, a neur- and Dl-dependent signaling event, suggesting that D-mib regulates Dl signaling. During wing development, expression of D-mib in dorsal cells appears to be necessary and sufficient for wing margin specification, indicating that D-mib also regulates Ser signaling. Moreover, the activity of the D-mib gene is required for the endocytosis of Ser in wing imaginal disc cells. Finally, ectopic expression of neur in D-mib mutant larvae rescues the wing D-mib phenotype, indicating that Neur can compensate for the lack of D-mib activity. We conclude that D-mib and Neur are two structurally distinct proteins that have similar molecular activities but distinct developmental functions in Drosophila.

  16. The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A.

    Science.gov (United States)

    Deyter, Gary M R; Biggins, Sue

    2014-08-15

    Centromere identity and its epigenetic maintenance require the incorporation of a histone H3 variant called CENP-A at centromeres. CENP-A mislocalization to ectopic sites may disrupt chromatin-based processes and chromosome segregation, so it is important to uncover the mechanisms by which this variant is exclusively localized to centromeres. Here, we identify a role for the conserved chromatin-modifying complex FACT (facilitates chromatin transcription/transactions) in preventing budding yeast CENP-A(Cse4) mislocalization to euchromatin by mediating its proteolysis. The Spt16 subunit of the FACT complex binds to Psh1 (Pob3/Spt16/histone), an E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. The interaction between Psh1 and Spt16 is critical for both CENP-A(Cse4) ubiquitylation and its exclusion from euchromatin. We found that Psh1 cannot efficiently ubiquitylate CENP-A(Cse4) nucleosomes in vitro, suggesting that additional factors must facilitate CENP-A(Cse4) removal from chromatin in vivo. Consistent with this, a Psh1 mutant that cannot associate with FACT has a reduced interaction with CENP-A(Cse4) in vivo. Together, our data identify a previously unknown mechanism to maintain centromere identity and genomic stability through the FACT-mediated degradation of ectopically localized CENP-A(Cse4). © 2014 Deyter and Biggins; Published by Cold Spring Harbor Laboratory Press.

  17. E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells

    Science.gov (United States)

    Xu, Qingqiang; Zhu, Naiwei; Chen, Shenglin; Zhao, Ping; Ren, Hao; Zhu, Shiying; Tang, Hailin; Zhu, Yongzhe; Qi, Zhongtian

    2017-01-01

    Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection. PMID:28349961

  18. Su(dx) E3 ubiquitin ligase-dependent and -independent functions of polychaetoid, the Drosophila ZO-1 homologue.

    Science.gov (United States)

    Djiane, Alexandre; Shimizu, Hideyuki; Wilkin, Marian; Mazleyrat, Sabine; Jennings, Martin D; Avis, Johanna; Bray, Sarah; Baron, Martin

    2011-01-10

    Zona occludens (ZO) proteins are molecular scaffolds localized to cell junctions, which regulate epithelial integrity in mammals. Using newly generated null alleles, we demonstrate that polychaetoid (pyd), the unique Drosophila melanogaster ZO homologue, regulates accumulation of adherens junction-localized receptors, such as Notch, although it is dispensable for epithelial polarization. Pyd positively regulates Notch signaling during sensory organ development but acts negatively on Notch to restrict the ovary germline stem cell niche. In both contexts, we identify a core antagonistic interaction between Pyd and the WW domain E3 ubiquitin ligase Su(dx). Pyd binds Su(dx) directly, in part through a noncanonical WW-binding motif. Pyd also restricts epithelial wing cell numbers to control adult wing shape, a function associated with the FERM protein Expanded and independent of Su(dx). As both Su(dx) and Expanded regulate trafficking, we propose that a conserved role of ZO proteins is to coordinate receptor trafficking and signaling with junctional organization.

  19. Notch-induced Asb2 expression promotes protein ubiquitination by forming non-canonical E3 ligase complexes

    Institute of Scientific and Technical Information of China (English)

    Lei Nie; Ying Zhao; Wei Wu; Yuan-Zheng Yang; Hong-Cheng Wang; Xiao-Hong Sun

    2011-01-01

    Notch signaling controls multiple developmental processes, thus demanding versatile functions. We have previously shown that this may be partly achieved by accelerating ubiquitin-mediated degradation of important regulators of differentiation. However, the underlying mechanism was unknown. We now find that Notch signaling transcriptionally activates the gene encoding ankyrin-repeat SOCS box-containing protein 2(Asb2). Asb2 promotes the ubiquidnation of Notch targets such as E2A and Janus kinase(Jak)2, and a dominant-negative(DN)mutant of Asb2blocks Notch-induced degradation of these proteins. Asb2 likely binds Jak2 directly but associates with E2A through Skp2. We next provide evidence to suggest that Asb2 bridges the formation of non-canonical cullin-based complexes through interaction with not only ElonginB/C and Cullin(Cul)5, but also the F-box-containing protein, Skp2, which is known to associate with Skpl and Cull. Consistently, ablating the function of Cull or Cu15 using DN mutants or siRNAs protected both E2A and Jak2 from Asb2-mediated or Notch-induced degradation. By shifting monomeric E3ligase complexes to dimeric forms through activation of Asb2 transcription, Notch could effectively control the turnover of a variety of substrates and it exerts diverse effects on cell proliferation and differentiation.

  20. Effects of ageing on expression of the muscle-specific E3 ubiquitin ligases and Akt-dependent regulation of Foxo transcription factors in skeletal muscle.

    Science.gov (United States)

    Wagatsuma, Akira; Shiozuka, Masataka; Takayama, Yuzo; Hoshino, Takayuki; Mabuchi, Kunihiko; Matsuda, Ryoichi

    2016-01-01

    Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.

  1. MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades

    Science.gov (United States)

    Liu, Siqi; Chen, Jueqi; Cai, Xin; Wu, Jiaxi; Chen, Xiang; Wu, You-Tong; Sun, Lijun; Chen, Zhijian J

    2013-01-01

    RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses. DOI: http://dx.doi.org/10.7554/eLife.00785.001 PMID:23951545

  2. A Family of Salmonella Virulence Factors Functions as a Distinct Class of Autoregulated E3 Ubiquitin Ligases

    Energy Technology Data Exchange (ETDEWEB)

    Quezada, C.; Hicks, S; Galan, J; Stebbins, C

    2009-01-01

    Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E{sub 3} ligase [which we have termed NEL for Novel E{sub 3} Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E{sub 3} ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.

  3. Distinct Functional Domains Contribute to Degradation of the Low Density Lipoprotein Receptor (LDLR) by the E3 Ubiquitin Ligase Inducible Degrader of the LDLR (IDOL)

    Science.gov (United States)

    Sorrentino, Vincenzo; Scheer, Lilith; Santos, Ana; Reits, Eric; Bleijlevens, Boris; Zelcer, Noam

    2011-01-01

    We recently identified the liver X receptor-regulated E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) as a modulator of lipoprotein metabolism. Acting as an E3 ubiquitin ligase, IDOL triggers ubiquitination and subsequent degradation of the low density lipoprotein receptor (LDLR). We demonstrate here that this outcome requires the conserved FERM and RING domains present in IDOL. The RING domain promotes ubiquitination in vitro and Lys-63-specific ubiquitination of the LDLR in vivo in response to IDOL or liver X receptor activation. We further identify RING residues that differentially influence ubiquitination of the LDLR or stability of IDOL. The FERM domain interacts with the LDLR and in living cells co-localizes with the receptor at the plasma membrane. Homology modeling revealed a phosphotyrosine-binding element embedded in the FERM domain. Mutating residues within this region or residues in the LDLR preceding the NPVY endocytosis motif abrogate LDLR degradation by IDOL. Collectively, our results indicate that both the FERM and RING domains are required for promoting lysosomal degradation of the LDLR by IDOL. Our findings may facilitate development of structure-based IDOL inhibitors aimed at increasing LDLR abundance in therapeutic strategies to treat cardiovascular disease. PMID:21734303

  4. E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

    Science.gov (United States)

    Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

    2016-10-01

    C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response.

  5. PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Cho, Seok Keun; Bae, Hansol; Ryu, Moonyoung

    2015-01-01

    and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only......, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress....

  6. The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death[W

    Science.gov (United States)

    Serrano, Irene; Gu, Yangnan; Qi, Dong; Dubiella, Ullrich

    2014-01-01

    Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events. PMID:25398498

  7. Inhibition of xanthine oxidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Frederic Derbre

    Full Text Available Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO. The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1 and Muscle RING (Really Interesting New Gene Finger-1 (MuRF-1. We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ~20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.

  8. A novel missense mutation of the ubiquitin protein ligase E3A gene in a patient with Angelman syndrome

    Institute of Scientific and Technical Information of China (English)

    BAI Jin-li; QU Yu-jin; ZOU Li-ping; YANG Xin-ying; LIU Li-jun; SONG Fang

    2011-01-01

    Background Angelman syndrome (AS) is a neurogenetic disorder caused by an expression defect of the maternally inherited copy of ubiquitin protein ligase E3A (UBE3A) gene from chromosome 15. Although the most common genetic defects include maternal deletions of chromosome 15q11-13, paternal uniparental disomy and imprinting defect,mutations in the UBE3A gene have been identified in approximately 10% of AS patients.Methods A Chinese girl of 28 months presented clinical manifestation of AS. Genetic diagnosis and molecular genetic defects were studied by methylation-specific PCR (MS-PCR) and linkage analysis by short tandem repeat (STR). We further performed sequence analysis of all the coding exons and flanking sequences of the UBE3A gene. The novel mutation screening was also performed in 100 unrelated healthy individuals to exclude the possibility of identifying a polymorphism variation.Results The MS-PCR analysis of the patient showed biparental inheritance of chromosome 15 with a normal methylation pattern in the 15q11-q13 region. And STR analysis revealed that the patient also inherited biparental alleles for six microsatellites. A novel mutation, cDNA1199 C>A (p. P400H), in exon 9 of the maternal UBE3A gene, was identified in the patient. Meanwhile, the mutation was observed in the patient's mother who had a normal phenotype.Conclusions It is necessary to perform the UBE3A gene mutation analysis in non-deletion/non-UPD/non-ID patients with AS. The clinical picture of the patient is concordant with that observed in previously reported AS patients with UBE3A mutation.

  9. Centromere architecture breakdown induced by the viral E3 ubiquitin ligase ICP0 protein of herpes simplex virus type 1.

    Directory of Open Access Journals (Sweden)

    Sylvain Gross

    Full Text Available The viral E3 ubiquitin ligase ICP0 protein has the unique property to temporarily localize at interphase and mitotic centromeres early after infection of cells by the herpes simplex virus type 1 (HSV-1. As a consequence ICP0 induces the proteasomal degradation of several centromeric proteins (CENPs, namely CENP-A, the centromeric histone H3 variant, CENP-B and CENP-C. Following ICP0-induced centromere modification cells trigger a specific response to centromeres called interphase Centromere Damage Response (iCDR. The biological significance of the iCDR is unknown; so is the degree of centromere structural damage induced by ICP0. Interphase centromeres are complex structures made of proximal and distal protein layers closely associated to CENP-A-containing centromeric chromatin. Using several cell lines constitutively expressing GFP-tagged CENPs, we investigated the extent of the centromere destabilization induced by ICP0. We show that ICP0 provokes the disappearance from centromeres, and the proteasomal degradation of several CENPs from the NAC (CENP-A nucleosome associated and CAD (CENP-A Distal complexes. We then investigated the nucleosomal occupancy of the centromeric chromatin in ICP0-expressing cells by micrococcal nuclease (MNase digestion analysis. ICP0 expression either following infection or in cell lines constitutively expressing ICP0 provokes significant modifications of the centromeric chromatin structure resulting in higher MNase accessibility. Finally, using human artificial chromosomes (HACs, we established that ICP0-induced iCDR could also target exogenous centromeres. These results demonstrate that, in addition to the protein complexes, ICP0 also destabilizes the centromeric chromatin resulting in the complete breakdown of the centromere architecture, which consequently induces iCDR.

  10. Muscle-specific E3 ubiquitin ligases are involved in muscle atrophy of cancer cachexia: an in vitro and in vivo study.

    Science.gov (United States)

    Yuan, Lei; Han, Jun; Meng, Qingyang; Xi, Qiulei; Zhuang, Qiulin; Jiang, Yi; Han, Yusong; Zhang, Bo; Fang, Jing; Wu, Guohao

    2015-05-01

    Muscle atrophy F-Box (MAFbx)/atrogin-1 and muscle ring-finger-1 (MuRF-1) have been identified as two muscle-specific E3 ubiquitin ligases that are highly expressed in skeletal muscle during muscle atrophy. However, the role of muscle-specific E3 ubiquitin ligases during the process of muscle atrophy of cancer cachexia remains largely unknown. In the present study, we analyzed the expression of atrogin-1 and MuRF-1 in the skeletal muscle of patients with malignant and benign disease. The possible mechanisms were studied both in a colon 26-induced cancer cachexia mouse model and in tumor necrosis factor-α (TNF-α) induced atrophy C2C12 cells. Our results demonstrated that atrogin-1 and MuRF-1 tended to be increased in the skeletal muscle of patients with malignant disease even before weight loss. Non-tumor body weights and gastrocnemius weights were significantly decreased while expression levels of ubiquitin proteasome pathway associated genes (atrogin-1, MuRF-1, ubiquitin and E2-14K) were upregulated in cancer cachexia mice. Significant myotube atrophy with atrogin-1 overexpression was observed in the C2C12 cells treated with TNF-α. Meanwhile, knockdown of atrogin-1 by small interfering RNA (siRNA) protected C2C12 cells from the adverse effect of TNF-α. In conclusion, muscle-specific E3 ubiquitin ligases were upregulated during cancer cachexia, and atrogin-1 may be a potential molecular target for treating muscle atrophy induced by cancer cachexia.

  11. The E3 ubiquitin-ligase Bmi1/Ring1A controls the proteasomal degradation of Top2alpha cleavage complex - a potentially new drug target.

    Directory of Open Access Journals (Sweden)

    Iris Alchanati

    Full Text Available BACKGROUND: The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha. METHODOLOGY/PRINCIPAL FINDINGS: We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner. CONCLUSIONS/SIGNIFICANCE: The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.

  12. Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection.

    Directory of Open Access Journals (Sweden)

    Smita Srivastava

    2008-05-01

    Full Text Available Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4-based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

  13. WW domain containing E3 ubiquitin protein ligase 1 targets the full-length ErbB4 for ubiquitin-mediated degradation in breast cancer.

    Science.gov (United States)

    Li, Y; Zhou, Z; Alimandi, M; Chen, C

    2009-08-20

    ErbB4, a member of the epidermal growth factor receptor family, plays a role in normal breast and breast cancer development by regulating mammary epithelial cell proliferation, survival and differentiation. In this study, we show that WWP1, a C2-WW-HECT type E3 ubiquitin ligase, binds, ubiquitinates and destructs ErbB4-CYT1, but much less efficiently for CYT2, isoforms (both JMa and JMb). The protein-protein interaction occurs primarily between the first and third WW domains of WWP1 and the second PY motif of ErbB4. Knockdown of WWP1 by two different small interfering RNAs increases the endogenous ErbB4 protein levels in both MCF7 and T47D breast cancer cell lines. In addition, overexpression of the wild type, but not the catalytic inactive WWP1, dramatically decreases the endogenous ErbB4 protein levels in MCF7. Importantly, we found that WWP1 negatively regulates the heregulin-beta1-stimulated ErbB4 activity as measured by the serum response element report assay and the BRCA1 mRNA expression. After a systematic screening of all WWP1 family members by small interfering RNA, we found that AIP4/Itch and HECW1/NEDL1 also negatively regulate the ErbB4 protein expression in T47D. Interestingly, the protein expression levels of both WWP1 and ErbB4 are higher in estrogen receptor-alpha-positive than in estrogen receptor-alpha-negative breast cancer cell lines. These data suggest that WWP1 and its family members suppress the ErbB4 expression and function in breast cancer.

  14. The Arabidopsis U-box E3 Ubiquitin ligase PUB30 Negatively Regulates Salt Tolerance by Facilitating BRI1 KINASE INHIBITOR 1 (BKI1) Degradation.

    Science.gov (United States)

    Zhang, Ming; Zhao, Jinfeng; Li, Long; Gao, Yanan; Zhao, Linlin; Patil, Suyash Bhimgonda; Fang, Jingjing; Zhang, Wenhui; Yang, Yuhong; Li, Ming; Li, Xueyong

    2017-09-02

    The Arabidopsis U-box E3 ubiquitin (Ub) ligases play an important role in the ubiquitin/26S proteasome-mediated protein degradation pathway. Recently PUB30 has been reported to participate in the salt stress response during seed germination stage in ABA-independent manner, but the molecular mechanism remains to be elucidated. Here we displayed that the pub30 mutant was more tolerant to salt stress during seed germination, whereas the mutant of its closest homologue PUB31 showed mild sensitivity to salt stress. PUB30 exhibited E3 ubiquitin ligase activity in vitro. PUB30 specifically interacted with BRI1 KINASE INHIBITOR 1 (BKI1), a regulator playing dual roles in brassinosteroids (BRs) signaling, in vitro and in vivo. We found that BKI1 protein was ubiquitinated and degraded by the 26S proteasome. The degradation of BKI1 was slowed down in the pub30-1 mutant compared with that in the wild-type. The bki1 mutant was sensitive to salt whereas the transgenic plants overexpressing BKI1 showed salt tolerant phenotype. All these results indicate that PUB30 negatively regulates salt tolerance probably through regulating the degradation of BKI1 and BRs signaling in Arabidopsis. This article is protected by copyright. All rights reserved.

  15. The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels.

    Science.gov (United States)

    Chen, Yi-An; Peng, Yi-Jheng; Hu, Meng-Chun; Huang, Jing-Jia; Chien, Yun-Chia; Wu, June-Tai; Chen, Tsung-Yu; Tang, Chih-Yung

    2015-05-29

    Voltage-gated CLC-1 chloride channels play a critical role in controlling the membrane excitability of skeletal muscles. Mutations in human CLC-1 channels have been linked to the hereditary muscle disorder myotonia congenita. We have previously demonstrated that disease-associated CLC-1 A531V mutant protein may fail to pass the endoplasmic reticulum quality control system and display enhanced protein degradation as well as defective membrane trafficking. Currently the molecular basis of protein degradation for CLC-1 channels is virtually unknown. Here we aim to identify the E3 ubiquitin ligase of CLC-1 channels. The protein abundance of CLC-1 was notably enhanced in the presence of MLN4924, a specific inhibitor of cullin-RING E3 ligases. Subsequent investigation with dominant-negative constructs against specific subtypes of cullin-RING E3 ligases suggested that CLC-1 seemed to serve as the substrate for cullin 4A (CUL4A) and 4B (CUL4B). Biochemical examinations further indicated that CUL4A/B, damage-specific DNA binding protein 1 (DDB1), and cereblon (CRBN) appeared to co-exist in the same protein complex with CLC-1. Moreover, suppression of CUL4A/B E3 ligase activity significantly enhanced the functional expression of the A531V mutant. Our data are consistent with the idea that the CUL4A/B-DDB1-CRBN complex catalyses the polyubiquitination and thus controls the degradation of CLC-1 channels.

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

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    Isabelle Bergiers

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

  17. MALT1 cleaves the E3 ubiquitin ligase HOIL-1 in activated T cells, generating a dominant negative inhibitor of LUBAC-induced NF-κB signaling.

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    Elton, Lynn; Carpentier, Isabelle; Staal, Jens; Driege, Yasmine; Haegman, Mira; Beyaert, Rudi

    2016-02-01

    Human paracaspase 1 (PCASP1), better known as mucosa associated lymphoid tissue lymphoma translocation 1 (MALT1), plays a key role in immunity and inflammation by regulating gene expression in lymphocytes and other immune cell types. Deregulated MALT1 activity has been implicated in autoimmunity, immunodeficiency and certain types of lymphoma. As a scaffold MALT1 assembles downstream signaling proteins for nuclear factor-κB (NF-κB) activation, while its proteolytic activity further enhances NF-κB activation by cleaving NF-κB inhibitory proteins. MALT1 also processes and inactivates a number of mRNA destabilizing proteins, which further fine-tunes gene expression. MALT1 protease inhibitors are currently developed for therapeutic targeting. Here we show that T cell activation, as well as overexpression of the oncogenic fusion protein API2-MALT1, induces the MALT1-mediated cleavage of haem-oxidized IRP2 ubiquitin ligase 1 (HOIL-1). In addition, to acting as a K48-polyubiquitin specific E3 ubiquitin ligase for different substrates, HOIL-1 co-operates in a catalytic-independent manner with the E3 ubiquitin ligase HOIL-1L interacting protein (HOIP) as part of the linear ubiquitin chain assembly complex (LUBAC). Intriguingly, cleavage of HOIL-1 does not directly abolish its ability to support HOIP-induced NF-κB signaling, which is still mediated by the N-terminal cleavage fragment, but generates a C-terminal fragment with LUBAC inhibitory properties. We propose that MALT1-mediated HOIL-1 cleavage provides a gain-of-function mechanism that is involved in the negative feedback regulation of NF-κB signaling.

  18. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule pathway, stabilizes Tex19.1 during spermatogenesis.

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    Fang Yang

    Full Text Available Ubiquitin E3 ligases target their substrates for ubiquitination, leading to proteasome-mediated degradation or altered biochemical properties. The ubiquitin ligase Ubr2, a recognition E3 component of the N-end rule proteolytic pathway, recognizes proteins with N-terminal destabilizing residues and plays an important role in spermatogenesis. Tex19.1 (also known as Tex19 has been previously identified as a germ cell-specific protein in mouse testis. Here we report that Tex19.1 forms a stable protein complex with Ubr2 in mouse testes. The binding of Tex19.1 to Ubr2 is independent of the second position cysteine of Tex19.1, a putative target for arginylation by the N-end rule pathway R-transferase. The Tex19.1-null mouse mutant phenocopies the Ubr2-deficient mutant in three aspects: heterogeneity of spermatogenic defects, meiotic chromosomal asynapsis, and embryonic lethality preferentially affecting females. In Ubr2-deficient germ cells, Tex19.1 is transcribed, but Tex19.1 protein is absent. Our results suggest that the binding of Ubr2 to Tex19.1 metabolically stabilizes Tex19.1 during spermatogenesis, revealing a new function for Ubr2 outside the conventional N-end rule pathway.

  19. Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL) domain effector of Rhizobium sp. strain NGR234.

    Science.gov (United States)

    Xin, Da-Wei; Liao, Sha; Xie, Zhi-Ping; Hann, Dagmar R; Steinle, Lea; Boller, Thomas; Staehelin, Christian

    2012-01-01

    Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.

  20. Time-of-day- and light-dependent expression of ubiquitin protein ligase E3 component N-recognin 4 (UBR4 in the suprachiasmatic nucleus circadian clock.

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    Harrod H Ling

    Full Text Available Circadian rhythms of behavior and physiology are driven by the biological clock that operates endogenously but can also be entrained to the light-dark cycle of the environment. In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN, which is composed of individual cellular oscillators that are driven by a set of core clock genes interacting in transcriptional/translational feedback loops. Light signals can trigger molecular events in the SCN that ultimately impact on the phase of expression of core clock genes to reset the master pacemaker. While transcriptional regulation has received much attention in the field of circadian biology in the past, other mechanisms including targeted protein degradation likely contribute to the clock timing and entrainment process. In the present study, proteome-wide screens of the murine SCN led to the identification of ubiquitin protein ligase E3 component N-recognin 4 (UBR4, a novel E3 ubiquitin ligase component of the N-end rule pathway, as a time-of-day-dependent and light-inducible protein. The spatial and temporal expression pattern of UBR4 in the SCN was subsequently characterized by immunofluorescence microscopy. UBR4 is expressed across the entire rostrocaudal extent of the SCN in a time-of-day-dependent fashion. UBR4 is localized exclusively to arginine vasopressin (AVP-expressing neurons of the SCN shell. Upon photic stimulation in the early subjective night, the number of UBR4-expressing cells within the SCN increases. This study is the first to identify a novel E3 ubiquitin ligase component, UBR4, in the murine SCN and to implicate the N-end rule degradation pathway as a potential player in regulating core clock mechanisms and photic entrainment.

  1. Functional analysis of NopM, a novel E3 ubiquitin ligase (NEL domain effector of Rhizobium sp. strain NGR234.

    Directory of Open Access Journals (Sweden)

    Da-Wei Xin

    Full Text Available Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.

  2. Skeletal muscle 11beta-HSD1 controls glucocorticoid-induced proteolysis and expression of E3 ubiquitin ligases atrogin-1 and MuRF-1.

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    Katrin Biedasek

    Full Text Available Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1 in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx and MuRF-1 (Muscle RING-Finger-1. We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [(3H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [(3H]-cortisone to [(3H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12 as well as in primary human myotubes. 11Beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p < 0.01, suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p < 0.001, which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p < 0.01, respectively. Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1.

  3. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

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    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  4. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  5. Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

    Science.gov (United States)

    Ashida, Hiroshi; Nakano, Hiroyasu; Sasakawa, Chihiro

    2013-01-01

    NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation. PMID:23754945

  6. The Arabidopsis paralogs, PUB46 and PUB48, encoding U-box E3 ubiquitin ligases, are essential for plant response to drought stress.

    Science.gov (United States)

    Adler, Guy; Konrad, Zvia; Zamir, Lyad; Mishra, Amit Kumar; Raveh, Dina; Bar-Zvi, Dudy

    2017-01-11

    Plants respond to abiotic stress on physiological, biochemical and molecular levels. This includes a global change in their cellular proteome achieved by changes in the pattern of their protein synthesis and degradation. The ubiquitin-proteasome system (UPS) is a key player in protein degradation in eukaryotes. Proteins are marked for degradation by the proteasome by coupling short chains of ubiquitin polypeptides in a three-step pathway. The last and regulatory stage is catalyzed by a member of a large family of substrate-specific ubiquitin ligases. We have identified AtPUB46 and AtPUB48-two paralogous genes that encode ubiquitin ligases (E3s)-to have a role in the plant environmental response. The AtPUB46, -47, and -48 appear as tandem gene copies on chromosome 5, and we present a phylogenetic analysis that traces their evolution from an ancestral PUB-ARM gene. Single homozygous T-DNA insertion mutants of AtPUB46 and AtPUB48 displayed hypersensitivity to water stress; this was not observed for similar mutants of AtPUB47. Although the three genes show a similar spatial expression pattern, the steady state levels of their transcripts are differentially affected by abiotic stresses and plant hormones. AtPUB46 and AtPUB48 encode plant U-Box E3s and are involved in the response to water stress. Our data suggest that despite encoding highly homologous proteins, AtPUB46 and AtPUB48 biological activity does not fully overlap.

  7. E3 ubiquitin ligase gene CMPG1-V from Haynaldia villosa L. contributes to powdery mildew resistance in common wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhu, Yanfei; Li, Yingbo; Fei, Fei; Wang, Zongkuan; Wang, Wei; Cao, Aizhong; Liu, Yuan; Han, Shuang; Xing, Liping; Wang, Haiyan; Chen, Wei; Tang, Sanyuan; Huang, Xiahe; Shen, Qianhua; Xie, Qi; Wang, Xiue

    2015-10-01

    Powdery mildew is one of the most devastating wheat fungal diseases. A diploid wheat relative, Haynaldia villosa L., is highly resistant to powdery mildew, and its genetic resource of resistances, such as the Pm21 locus, is now widely used in wheat breeding. Here we report the cloning of a resistance gene from H. villosa, designated CMPG1-V, that encodes a U-box E3 ubiquitin ligase. Expression of the CMPG1-V gene was induced in the leaf and stem of H. villosa upon inoculation with Blumeria graminis f. sp. tritici (Bgt) fungus, and the presence of Pm21 is essential for its rapid induction of expression. CMPG1-V has conserved key residues for E3 ligase, and possesses E3 ligase activity in vitro and in vivo. CMPG1-V is localized in the nucleus, endoplasmic reticulum, plasma membrane and partially in trans-Golgi network/early endosome vesicles. Transgenic wheat over-expressing CMPG1-V showed improved broad-spectrum powdery mildew resistance at seedling and adult stages, associated with an increase in expression of salicylic acid-responsive genes, H2 O2 accumulation, and cell-wall protein cross-linking at the Bgt infection sites, and the expression of CMPG1-V in H. villosa was increased when treated with salicylic acid, abscisic acid and H2 O2 . These results indicate the involvement of E3 ligase in defense responses to Bgt fungus in wheat, particularly in broad-spectrum disease resistance, and suggest association of reactive oxidative species and the phytohormone pathway with CMPG1-V-mediated powdery mildew resistance.

  8. Involvement of NF-κB and muscle specific E3 ubiquitin ligase MuRF1 in cigarette smoke-induced catabolism in C2 myotubes.

    Science.gov (United States)

    Kaisari, Sharon; Rom, Oren; Aizenbud, Dror; Reznick, Abraham Z

    2013-01-01

    Cigarette smoking has been identified as a risk factor for muscular damage and sarcopenia, the age-related loss of muscle mass and strength in old age. Cigarette smoke (CS)-induced oxidative stress and p38 MAPK activation have been shown to be the main cellular mechanisms leading to skeletal muscle catabolism. In order to investigate the involvement of NF-κB as another possible cellular mechanism by which CS promotes muscle catabolism, C2 myotubes, from an in vitro skeletal muscle cell line, were exposed to different time periods of whole vapor phase CS in the presence or absence of NF-κB inhibitor, IMD-0354. The CS-induced reduction in diameter of myotubes and time-dependent degradation of the main contractile protein myosin heavy chain were abolished by NF-κB inhibition. Also, C2 exposure to CS resulted in IκB-α degradation and NF-κB activation, which led to upregulation of the muscle specific E3 ubiquitin ligase MuRF1, but not MAFbx/atrogin-1. In conclusion, our results demonstrate that vapor phase CS exposure to skeletal myotubes triggers NF-κB activation leading to skeletal muscle cell damage and breakdown of muscle proteins mediated by muscle specific E3 ubiquitin ligase MuRF1. Our findings provide another possible molecular mechanism for the catabolic effects of CS in skeletal muscle.

  9. The E3 Ubiquitin Ligase IDOL Induces the Degradation of the Low Density Lipoprotein Receptor Family Members VLDLR and ApoER2*

    Science.gov (United States)

    Hong, Cynthia; Duit, Sarah; Jalonen, Pilvi; Out, Ruud; Scheer, Lilith; Sorrentino, Vincenzo; Boyadjian, Rima; Rodenburg, Kees W.; Foley, Edan; Korhonen, Laura; Lindholm, Dan; Nimpf, Johannes; van Berkel, Theo J. C.; Tontonoz, Peter; Zelcer, Noam

    2010-01-01

    We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism. PMID:20427281

  10. AtPUB 19, a U-Box E3 Ubiquitin Ligase, Negatively Regulates Abscisic Acid and Drought Responses in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang Liu; Yao-Rong Wu; Xia-He Huang; Jie Sun; Qi Xie

    2011-01-01

    Ubiquitination is an important protein post-translational modification,which is involved in various cellular processes in higher plants,and U-box E3 ligases play important roles in diverse functions in eukaryotes.Here,we describe the functions of Arabidopsis thaliana PUB19 (AtPUB19),which we demonstrated in an in vitro assay to encode a U-box type E3 ubiquitin ligase.AtPUB19 was up-regulated by drought,salt,cold,and abscisic acid (ABA).Down-regulation of AtPUB19led to hypersensitivity to ABA,enhanced ABA-induced stomatal closing,and enhanced drought tolerance,while AtPUB 19overexpression resulted in the reverse phenotypes.Molecular analysis showed that the expression levels of a number of ABA and stress marker genes were altered in both AtPUB 19 overexpressing and atpub 19-1 mutant plants.In summary,our data show that AtPUB19 negatively regulates ABA and drought responses in A.thaliana.

  11. The Medicago truncatula E3 Ubiquitin Ligase PUB1 Interacts with the LYK3 Symbiotic Receptor and Negatively Regulates Infection and Nodulation[W][OA

    Science.gov (United States)

    Mbengue, Malick; Camut, Sylvie; de Carvalho-Niebel, Fernanda; Deslandes, Laurent; Froidure, Solène; Klaus-Heisen, Dörte; Moreau, Sandra; Rivas, Susana; Timmers, Ton; Hervé, Christine; Cullimore, Julie; Lefebvre, Benoit

    2010-01-01

    LYK3 is a lysin motif receptor-like kinase of Medicago truncatula, which is essential for the establishment of the nitrogen-fixing, root nodule symbiosis with Sinorhizobium meliloti. LYK3 is a putative receptor of S. meliloti Nod factor signals, but little is known of how it is regulated and how it transduces these symbiotic signals. In a screen for LYK3-interacting proteins, we identified M. truncatula Plant U-box protein 1 (PUB1) as an interactor of the kinase domain. In planta, both proteins are localized and interact in the plasma membrane. In M. truncatula, PUB1 is expressed specifically in symbiotic conditions, is induced by Nod factors, and shows an overlapping expression pattern with LYK3 during nodulation. Biochemical studies show that PUB1 has a U-box–dependent E3 ubiquitin ligase activity and is phosphorylated by the LYK3 kinase domain. Overexpression and RNA interference studies in M. truncatula show that PUB1 is a negative regulator of the LYK3 signaling pathway leading to infection and nodulation and is important for the discrimination of rhizobia strains producing variant Nod factors. The potential role of PUB E3 ubiquitin ligases in controlling plant–microbe interactions and development through interacting with receptor-like kinases is discussed. PMID:20971894

  12. Overexpression of E3 Ubiquitin Ligase Gene AdBiL Contributes to Resistance against Chilling Stress and Leaf Mold Disease in Tomato

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    Shuangchen Chen

    2017-06-01

    Full Text Available Ubiquitination is a common regulatory mechanism, playing a critical role in diverse cellular and developmental processes in eukaryotes. However, a few reports on the functional correlation between E3 ubiquitin ligases and reactive oxygen species (ROS or reactive nitrogen species (RNS metabolism in response to stress are currently available in plants. In the present study, the E3 ubiquitin ligase gene AdBiL (Adi3 Binding E3 Ligase was introduced into tomato line Ailsa Craig via Agrobacterium-mediated method. Transgenic lines were confirmed for integration into the tomato genome using PCR. Transcription of AdBiL in various transgenic lines was determined using real-time PCR. Evaluation of stress tolerance showed that T1 generation of transgenic tomato lines showed only mild symptoms of chilling injury as evident by higher biomass accumulation and chlorophyll content than those of non-transformed plants. Compared with wild-type plants, the contents of AsA, AsA/DHA, GSH and the activity of GaILDH, γ-GCS and GSNOR were increased, while H2O2, O2.−, MDA, NO, SNOs, and GSNO accumulations were significantly decreased in AdBiL overexpressing plants in response to chilling stress. Furthermore, transgenic tomato plants overexpressing AdBiL showed higher activities of enzymes such as G6PDH, 6PGDH, NADP-ICDH, and NADP-ME involved in pentose phosphate pathway (PPP. The transgenic tomato plants also exhibited an enhanced tolerance against the necrotrophic fungus Cladosporium fulvum. Tyrosine nitration protein was activated in the plants infected with leaf mold disease, while the inhibition could be recovered in AdBiL gene overexpressing lines. Taken together, our results revealed a possible physiological role of AdBiL in the activation of the key enzymes of AsA–GSH cycle, PPP and down-regulation of GSNO reductase, thereby reducing oxidative and nitrosative stress in plants. This study demonstrates an optimized transgenic strategy using AdBiL gene for crop

  13. Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration.

    Science.gov (United States)

    Del Prete, Dolores; Rice, Richard C; Rajadhyaksha, Anjali M; D'Adamio, Luciano

    2016-08-12

    The amyloid precursor protein (APP), whose mutations cause Alzheimer disease, plays an important in vivo role and facilitates transmitter release. Because the APP cytosolic region (ACR) is essential for these functions, we have characterized its brain interactome. We found that the ACR interacts with proteins that regulate the ubiquitin-proteasome system, predominantly with the E3 ubiquitin-protein ligases Stub1, which binds the NH2 terminus of the ACR, and CRL4(CRBN), which is formed by Cul4a/b, Ddb1, and Crbn, and interacts with the COOH terminus of the ACR via Crbn. APP shares essential functions with APP-like protein-2 (APLP2) but not APP-like protein-1 (APLP1). Noteworthy, APLP2, but not APLP1, interacts with Stub1 and CRL4(CRBN), pointing to a functional pathway shared only by APP and APLP2. In vitro ubiquitination/ubiquitome analysis indicates that these E3 ligases are enzymatically active and ubiquitinate the ACR residues Lys(649/650/651/676/688) Deletion of Crbn reduces ubiquitination of Lys(676) suggesting that Lys(676) is physiologically ubiquitinated by CRL4(CRBN) The ACR facilitated in vitro ubiquitination of presynaptic proteins that regulate exocytosis, suggesting a mechanism by which APP tunes transmitter release. Other dementia-related proteins, namely Tau and apoE, interact with and are ubiquitinated via the ACR in vitro This, and the evidence that CRBN and CUL4B are linked to intellectual disability, prompts us to hypothesize a pathogenic mechanism, in which APP acts as a modulator of E3 ubiquitin-protein ligase(s), shared by distinct neuronal disorders. The well described accumulation of ubiquitinated protein inclusions in neurodegenerative diseases and the link between the ubiquitin-proteasome system and neurodegeneration make this concept plausible.

  14. Gp78, an E3 ubiquitin ligase acts as a gatekeeper suppressing nonalcoholic steatohepatitis (NASH and liver cancer.

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    Tianpeng Zhang

    Full Text Available Nonalcoholic steatohepatitis (NASH is related to metabolic dysregulation and the perturbation of endoplasmic reticulum (ER homeostasis that frequently develops into hepatocellular carcinoma (HCC. Gp78 is E3 ligase, which regulates endoplasmic reticulum-associated degradation (ERAD by ubiquitinylation of misfolded ER proteins. Here, we report that upon ageing (12 months, gp78-/- mice developed obesity, recapitulating age-related human NASH. Liver histology of gp78-/- mice revealed typical steatosis, hepatic inflammation and fibrosis, followed by progression to hepatocellular tumors. Acute ER stress revealed that loss of gp78 results in up regulation of unfolded protein response (UPR pathways and SREBP-1 regulating de novo lipogenesis, responsible for fatty liver. Tissue array of human hepatocellular carcinoma (HCC demonstrated that the expression of gp78 was inversely correlated with clinical grades of cancer. Here, we have described the generation of the first preclinical experimental model system which spontaneously develops age-related NASH and HCC, linking ERAD to hepatosteatosis, cirrhosis, and cancer. It suggests that gp78 is a regulator of normal liver homeostasis and a tumor suppressor in human liver.

  15. The Role of E3 Ubiquitin Ligase Cbl Proteins in β-Elemene Reversing Multi-Drug Resistance of Human Gastric Adenocarcinoma Cells

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    Xue-Jun Hu

    2013-05-01

    Full Text Available Recent studies indicate that β-elemene, a compound isolated from the Chinese herbal medicine Curcuma wenyujin, is capable of reversing tumor MDR, although the mechanism remains elusive. In this study, β-Elemene treatment markedly increased the intracellular accumulation of doxorubicin (DOX and rhodamine 123 in both K562/DNR and SGC7901/ADR cells and significantly inhibited the expression of P-gp. Treatment of SGC7901/ADR cells with β-elemene led to downregulation of Akt phosphorylation and significant upregulation of the E3 ubiquitin ligases, c-Cbl and Cbl-b. Importantly, β-elemene significantly enhanced the anti-tumor activity of DOX in nude mice bearing SGC7901/ADR xenografts. Taken together, our results suggest that β-elemene may target P-gp-overexpressing leukemia and gastric cancer cells to enhance the efficacy of DOX treatment.

  16. Iron-induced skeletal muscle atrophy involves an Akt-forkhead box O3-E3 ubiquitin ligase-dependent pathway.

    Science.gov (United States)

    Ikeda, Yasumasa; Imao, Mizuki; Satoh, Akiho; Watanabe, Hiroaki; Hamano, Hirofumi; Horinouchi, Yuya; Izawa-Ishizawa, Yuki; Kihira, Yoshitaka; Miyamoto, Licht; Ishizawa, Keisuke; Tsuchiya, Koichiro; Tamaki, Toshiaki

    2016-05-01

    Skeletal muscle wasting or sarcopenia is a critical health problem. Skeletal muscle atrophy is induced by an excess of iron, which is an essential trace metal for all living organisms. Excessive amounts of iron catalyze the formation of highly toxic hydroxyl radicals via the Fenton reaction. However, the molecular mechanism of iron-induced skeletal muscle atrophy has remained unclear. In this study, 8-weeks-old C57BL6/J mice were divided into 2 groups: vehicle-treated group and the iron-injected group (10 mg iron day(-1)mouse(-1)) during 2 weeks. Mice in the iron-injected group showed an increase in the iron content of the skeletal muscle and serum and ferritin levels in the muscle, along with reduced skeletal muscle mass. The skeletal muscle showed elevated mRNA expression of the muscle atrophy-related E3 ubiquitin ligases, atrogin-1 and muscle ring finger-1(MuRF1), on days 7 and 14 of iron treatment. Moreover, iron-treated mice showed reduced phosphorylation of Akt and forkhead box O3 (FOXO3a) in skeletal muscles. Inhibition of FOXO3a using siRNA in vitro in C2C12 myotube cells inhibited iron-induced upregulation of atrogin-1 and MuRF1 and reversed the reduction in myotube diameters. Iron-load caused oxidative stress, and an oxidative stress inhibitor abrogated iron-induced muscle atrophy by reactivating the Akt-FOXO3a pathway. Iron-induced skeletal muscle atrophy is suggested to involve the E3 ubiquitin ligase mediated by the reduction of Akt-FOXO3a signaling by oxidative stress.

  17. HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3 Transcriptional Activity.

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    Gary T ZeRuth

    Full Text Available The transcription factor Gli-similar 3 (Glis3 plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

  18. Hepatitis B Virus X Protein Sensitizes TRAIL-Induced Hepatocyte Apoptosis by Inhibiting the E3 Ubiquitin Ligase A20.

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    Hang Zhang

    Full Text Available Hepatitis B virus (HBV infection causes hepatocyte death and liver damage, which may eventually lead to cirrhosis and liver cancer. Hepatitis B virus X protein (HBx is a key antigen that is critically involved in HBV-associated liver diseases. However, the molecular basis for its pathogenesis, particularly in liver damage, has not been well defined. Herein, we report that HBx was able to enhance the susceptibility of hepatocytes to TNF-related apoptosis-inducing ligand (TRAIL-induced apoptosis. Increased sensitivity to TRAIL was associated with HBx-induced upregulation of miR-125a, which, in turn, suppressed the expression of its putative target gene, A20 E3 ligase. Importantly, we demonstrate that the defective expression of A20 impaired the K63-linked polyubiquitination of caspase-8, which reciprocally enhanced the activation of caspase-8, the recruitment of Fas-associated death domain (FADD, and the formation of death-inducing signaling complex (DISC, thereby promoting HBx-mediated apoptotic signaling. Accordingly, antagonizing miR-125a or ectopically expressing A20 in hepatocytes abolished the pro-apoptotic effect of HBx. Conversely, the overexpression of miR-125a or knockdown of A20 mimicked HBx to enhance TRAIL susceptibility in hepatocytes. Thus, we establish, for the first time, a miR-125a/A20-initiated and caspase-8-targeted mechanism by which HBx modulates apoptotic signaling and increases hepatic susceptibility to the damaging agent, which might provide novel insight into HBV-related liver pathology.

  19. The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters*

    Science.gov (United States)

    Aleidi, Shereen M.; Howe, Vicky; Sharpe, Laura J.; Yang, Alryel; Rao, Geetha; Brown, Andrew J.; Gelissen, Ingrid C.

    2015-01-01

    The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity. PMID:26296893

  20. A Tale of Two PMLs: Elements Regulating a Differential Substrate Recognition by the ICP0 E3 Ubiquitin Ligase of Herpes Simplex Virus 1.

    Science.gov (United States)

    Zheng, Yi; Samrat, Subodh Kumar; Gu, Haidong

    2016-12-01

    Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an α gene product required for viral replication at low multiplicities of infection. Upon entry, nuclear domain 10 (ND10) converges at the incoming DNA and represses viral gene expression. ICP0 contains a RING-type E3 ubiquitin ligase that degrades the ND10 organizer PML and disperses ND10 to alleviate the repression. In the present study, we focused on understanding the regulation of ICP0 E3 ligase activity in the degradation of different ICP0 substrates. We report the following. (i) A SUMO interaction motif located at ICP0 residues 362 to 364 is required for the degradation of PML isoforms II, IV, and VI but not isoform I. This differentiation mechanism exists in both HEp-2 and U2OS cells, regardless of the cell's permissiveness to the ICP0-null virus. (ii) Physical interaction between SIM362-364 and PML II is necessary but not sufficient for PML II degradation. Both proximal sequences surrounding SIM362-364 and distal sequences located at the ICP0 C terminus enhance the degradation of PML II. (iii) The ICP0 C terminus is dispensable for PML I degradation. Instead, bipartite PML I binding domains located in the N-terminal half of ICP0 coordinate to promote the degradation of PML I. (iv) The stability of ICP0, but not its ND10 fusion ability, affects the rate of PML I degradation. Taken together, our results show that ICP0 uses at least two regulatory mechanisms to differentiate its substrates. The disparate recognition of the ICP0 E3 substrates may be related to the different roles these substrates may play in HSV-1 infection.

  1. Progressive Purkinje cell degeneration in tambaleante mutant mice is a consequence of a missense mutation in HERC1 E3 ubiquitin ligase.

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    Tomoji Mashimo

    2009-12-01

    Full Text Available The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domains have been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2 and small (HERC3-6. The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu in the highly conserved N-terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein

  2. U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans.

    Science.gov (United States)

    He, Qin; McLellan, Hazel; Boevink, Petra C; Sadanandom, Ari; Xie, Conghua; Birch, Paul R J; Tian, Zhendong

    2015-06-01

    Ubiquitination regulates many processes in plants, including immunity. The E3 ubiquitin ligase PUB17 is a positive regulator of programmed cell death (PCD) triggered by resistance proteins CF4/9 in tomato. Its role in immunity to the potato late blight pathogen, Phytophthora infestans, was investigated here. Silencing StPUB17 in potato by RNAi and NbPUB17 in Nicotiana benthamiana by virus-induced gene silencing (VIGS) each enhanced P. infestans leaf colonization. PAMP-triggered immunity (PTI) transcriptional responses activated by flg22, and CF4/Avr4-mediated PCD were attenuated by silencing PUB17. However, silencing PUB17 did not compromise PCD triggered by P. infestans PAMP INF1, or co-expression of R3a/AVR3a, demonstrating that not all PTI- and PCD-associated responses require PUB17. PUB17 localizes to the plant nucleus and especially in the nucleolus. Transient over-expression of a dominant-negative StPUB17(V314I,V316I) mutant, which retained nucleolar localization, suppressed CF4-mediated cell death and enhanced P. infestans colonization. Exclusion of the StPUB17(V314I,V316I) mutant from the nucleus abolished its dominant-negative activity, demonstrating that StPUB17 functions in the nucleus. PUB17 is a positive regulator of immunity to late blight that acts in the nucleus to promote specific PTI and PCD pathways.

  3. Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

    Science.gov (United States)

    Benvegnù, Stefano; Mateo, María Inés; Palomer, Ernest; Jurado-Arjona, Jerónimo; Dotti, Carlos G

    2017-05-04

    A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. HTLV-1 Tax Functions as a Ubiquitin E3 Ligase for Direct IKK Activation via Synthesis of Mixed-Linkage Polyubiquitin Chains.

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    Wang, Chong; Long, Wenying; Peng, Chao; Hu, Lin; Zhang, Qiong; Wu, Ailing; Zhang, Xiaoqing; Duan, Xiaotao; Wong, Catherine C L; Tanaka, Yuetsu; Xia, Zongping

    2016-04-01

    The HTLV-1 oncoprotein Tax plays a key role in CD4+ T cell transformation by promoting cell proliferation and survival, mainly through permanent activation of the NK-κB pathway and induction of many NF-κB target genes. Elucidating the underlying molecular mechanism is therefore critical in understanding HTLV-1-mediated transformation. Current studies have suggested multiple but controversial mechanisms regarding Tax-induced IKK activation mainly due to blending of primary Tax-induced IKK activation events and secondary IKK activation events induced by cytokines secreted by the primary Tax-induced IKK-NF-κB activation events. We reconstituted Tax-stimulated IKK activation in a cell-free system to dissect the essential cellular components for primary IKK activation by Tax and studied the underlying biochemical mechanism. We found that Tax is a putative E3 ubiquitin ligase, which, together with UbcH2, UhcH5c, or UbcH7, catalyzes the assembly of free mixed-linkage polyubiquitin chains. These free mixed-linkage polyubiquitin chains are then responsible for direct IKK activation by binding to the NEMO subunit of IKK. Our studies revealed the biochemical function of Tax in the process of IKK activation, which utilizes the minimal cellular ubiquitination components for NF-κB activation.

  5. Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.

    Science.gov (United States)

    Pettersson, Susanne; Sczaniecka, Matylda; McLaren, Lorna; Russell, Fiona; Gladstone, Karen; Hupp, Ted; Wallace, Maura

    2013-03-15

    The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.

  6. Haploinsufficiency of the E3 ubiquitin ligase C-terminus of heat shock cognate 70 interacting protein (CHIP) produces specific behavioral impairments.

    Science.gov (United States)

    McLaughlin, Bethann; Buendia, Matthew A; Saborido, Tommy P; Palubinsky, Amy M; Stankowski, Jeannette N; Stanwood, Gregg D

    2012-01-01

    The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET) mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and cellular stress.

  7. Haploinsufficiency of the E3 ubiquitin ligase C-terminus of heat shock cognate 70 interacting protein (CHIP produces specific behavioral impairments.

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    Bethann McLaughlin

    Full Text Available The multifunctional E3 ubiquitin ligase CHIP is an essential interacting partner of HSP70, which together promote the proteasomal degradation of client proteins. Acute CHIP overexpression provides neuroprotection against neurotoxic mitochondrial stress, glucocorticoids, and accumulation of toxic amyloid fragments, as well as genetic mutations in other E3 ligases, which have been shown to result in familial Parkinson's disease. These studies have created a great deal of interest in understanding CHIP activity, expression and modulation. While CHIP knockout mice have the potential to provide essential insights into the molecular control of cell fate and survival, the animals have been difficult to characterize in vivo due to severe phenotypic and behavioral dysfunction, which have thus far been poorly characterized. Therefore, in the present study we conducted a battery of neurobehavioral and physiological assays of adult CHIP heterozygotic (HET mutant mice to provide a better understanding of the functional consequence of CHIP deficiency. We found that CHIP HET mice had normal body and brain weight, body temperature, muscle tone and breathing patterns, but do have a significant elevation in baseline heart rate. Meanwhile basic behavioral screens of sensory, motor, emotional and cognitive functions were normative. We observed no alterations in performance in the elevated plus maze, light-dark preference and tail suspension assays, or two simple cognitive tasks: novel object recognition and spontaneous alternation in a Y maze. Significant deficits were found, however, when CHIP HET mice performed wire hang, inverted screen, wire maneuver, and open field tasks. Taken together, our data indicate a clear subset of behaviors that are altered at baseline in CHIP deficient animals, which will further guide whole animal studies of the effects of CHIP dysregulation on cardiac function, brain circuitry and function, and responsiveness to environmental and

  8. Arabidopsis RING E3 ubiquitin ligase AtATL80 is negatively involved in phosphate mobilization and cold stress response in sufficient phosphate growth conditions.

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    Suh, Ji Yeon; Kim, Woo Taek

    2015-08-07

    Phosphate (Pi) remobilization in plants is critical to continuous growth and development. AtATL80 is a plasma membrane (PM)-localized RING E3 ubiquitin (Ub) ligase that belongs to the Arabidopsis Tóxicos en Levadura (ATL) family. AtATL80 was upregulated by long-term low Pi (0-0.02 mM KH2PO4) conditions in Arabidopsis seedlings. AtATL80-overexpressing transgenic Arabidopsis plants (35S:AtATL80-sGFP) displayed increased phosphorus (P) accumulation in the shoots and lower biomass, as well as reduced P-utilization efficiency (PUE) under high Pi (1 mM KH2PO4) conditions compared to wild-type plants. The loss-of-function atatl80 mutant line exhibited opposite phenotypic traits. The atatl80 mutant line bolted earlier than wild-type plants, whereas AtATL80-overexpressors bloomed significantly later and produced lower seed yields than wild-type plants under high Pi conditions. Thus, AtATL80 is negatively correlated not only with P content and PUE, but also with biomass and seed yield in Arabidopsis. In addition, AtATL80-overexpressors were significantly more sensitive to cold stress than wild-type plants, while the atatl80 mutant line exhibited an increased tolerance to cold stress. Taken together, our results suggest that AtATL80, a PM-localized ATL-type RING E3 Ub ligase, participates in the Pi mobilization and cold stress response as a negative factor in Arabidopsis.

  9. Yeast sterol regulatory element-binding protein (SREBP) cleavage requires Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing subunit of the Golgi Dsc E3 ligase.

    Science.gov (United States)

    Stewart, Emerson V; Lloyd, S Julie-Ann; Burg, John S; Nwosu, Christine C; Lintner, Robert E; Daza, Riza; Russ, Carsten; Ponchner, Karen; Nusbaum, Chad; Espenshade, Peter J

    2012-01-01

    Schizosaccharomyces pombe Sre1 is a membrane-bound transcription factor that controls adaptation to hypoxia. Like its mammalian homolog, sterol regulatory element-binding protein (SREBP), Sre1 activation requires release from the membrane. However, in fission yeast, this release occurs through a strikingly different mechanism that requires the Golgi Dsc E3 ubiquitin ligase complex and the proteasome. The mechanistic details of Sre1 cleavage, including the link between the Dsc E3 ligase complex and proteasome, are not well understood. Here, we present results of a genetic selection designed to identify additional components required for Sre1 cleavage. From the selection, we identified two new components of the fission yeast SREBP pathway: Dsc5 and Cdc48. The AAA (ATPase associated with diverse cellular activities) ATPase Cdc48 and Dsc5, a ubiquitin regulatory X domain-containing protein, interact with known Dsc complex components and are required for SREBP cleavage. These findings provide a mechanistic link between the Dsc E3 ligase complex and the proteasome in SREBP cleavage and add to a growing list of similarities between the Dsc E3 ligase and membrane E3 ligases involved in endoplasmic reticulum-associated degradation.

  10. Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease

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    Laurent P. Bogdanik

    2013-05-01

    Charcot-Marie-Tooth disease (CMT is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P. Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.

  11. Unique Pattern of Component Gene Disruption in the NRF2 Inhibitor KEAP1/CUL3/RBX1 E3-Ubiquitin Ligase Complex in Serous Ovarian Cancer

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    Victor D. Martinez

    2014-01-01

    Full Text Available The NFE2-related factor 2 (NRF2 pathway is critical to initiate responses to oxidative stress; however, constitutive activation occurs in different cancer types, including serous ovarian carcinomas (OVCA. The KEAP1/CUL3/RBX1 E3-ubiquitin ligase complex is a regulator of NRF2 levels. Hence, we investigated the DNA-level mechanisms affecting these genes in OVCA. DNA copy-number loss (CNL, promoter hypermethylation, mRNA expression, and sequence mutation for KEAP1, CUL3, and RBX1 were assessed in a cohort of 568 OVCA from The Cancer Genome Atlas. Almost 90% of cases exhibited loss-of-function alterations in any components of the NRF2 inhibitory complex. CNL is the most prominent mechanism of component disruption, with RBX1 being the most frequently disrupted component. These alterations were associated with reduced mRNA expression of complex components, and NRF2 target gene expression was positively enriched in 90% of samples harboring altered complex components. Disruption occurs through a unique DNA-level alteration pattern in OVCA. We conclude that a remarkably high frequency of DNA and mRNA alterations affects components of the KEAP1/CUL3/RBX1 complex, through a unique pattern of genetic mechanisms. Together, these results suggest a key role for the KEAP1/CUL3/RBX1 complex and NRF2 pathway deregulation in OVCA.

  12. CDK1-dependent inhibition of the E3 ubiquitin ligase CRL4CDT2 ensures robust transition from S Phase to Mitosis.

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    Rizzardi, Lindsay F; Coleman, Kate E; Varma, Dileep; Matson, Jacob P; Oh, Seeun; Cook, Jeanette Gowen

    2015-01-02

    Replication-coupled destruction of a cohort of cell cycle proteins ensures efficient and precise genome duplication. Three proteins destroyed during replication via the CRL4(CDT2) ubiquitin E3 ligase, CDT1, p21, and SET8 (PR-SET7), are also essential or important during mitosis, making their reaccumulation after S phase a critical cell cycle event. During early and mid-S phase and during DNA repair, proliferating cell nuclear antigen (PCNA) loading onto DNA (PCNA(DNA)) triggers the interaction between CRL4(CDT2) and its substrates, resulting in their degradation. We have discovered that, beginning in late S phase, PCNA(DNA) is no longer sufficient to trigger CRL4(CDT2)-mediated degradation. A CDK1-dependent mechanism that blocks CRL4(CDT2) activity by interfering with CDT2 recruitment to chromatin actively protects CRL4(CDT2) substrates. We postulate that deliberate override of replication-coupled destruction allows anticipatory accumulation in late S phase. We further show that (as for CDT1) de novo SET8 reaccumulation is important for normal mitotic progression. In this manner, CDK1-dependent CRL4(CDT2) inactivation contributes to efficient transition from S phase to mitosis.

  13. The crystal structure of the Sgt1-Skp1 complex: the link between Hsp90 and both SCF E3 ubiquitin ligases and kinetochores

    Science.gov (United States)

    Willhoft, Oliver; Kerr, Richard; Patel, Dipali; Zhang, Wenjuan; Al-Jassar, Caezar; Daviter, Tina; Millson, Stefan H.; Thalassinos, Konstantinos; Vaughan, Cara K.

    2017-01-01

    The essential cochaperone Sgt1 recruits Hsp90 chaperone activity to a range of cellular factors including SCF E3 ubiquitin ligases and the kinetochore in eukaryotes. In these pathways Sgt1 interacts with Skp1, a small protein that heterodimerizes with proteins containing the F-box motif. We have determined the crystal structure of the interacting domains of Saccharomyces cerevisiae Sgt1 and Skp1 at 2.8 Å resolution and validated the interface in the context of the full-length proteins in solution. The BTB/POZ domain of Skp1 associates with Sgt1 via the concave surface of its TPR domain using residues that are conserved in humans. Dimerization of yeast Sgt1 occurs via an insertion that is absent from monomeric human Sgt1. We identify point mutations that disrupt dimerization and Skp1 binding in vitro and find that the interaction with Skp1 is an essential function of Sgt1 in yeast. Our data provide a structural rationale for understanding the phenotypes of temperature-sensitive Sgt1 mutants and for linking Skp1-associated proteins to Hsp90-dependent pathways. PMID:28139700

  14. Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia-reoxygenation injury in pheochromocytoma (PC12) cells

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    Tan, Can [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Zhang, Li-Yang [Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Cancer Research Institute, Central South University, 110 Xiang Ya Road, Changsha 410078 (China); Chen, Hong [Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Xiao, Ling [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Liu, Xian-Peng, E-mail: xliu@lsuhsc.edu [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130-3932 (United States); Zhang, Jian-Xiang, E-mail: jianxiangzhang@yahoo.cn [Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, 172 Tong Zipo Road, Changsha 410013 (China); Department of Developmental Biology, School of Biological Science and Technology, Central South University, 172 Tong Zipo Road, Changsha 410013 (China)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Overexpression of human CUL4A (hCUL4A) in PC12 cells. Black-Right-Pointing-Pointer The effects of hCUL4A on hypoxia-reoxygenation injury were investigated. Black-Right-Pointing-Pointer hCUL4A suppresses apoptosis and DNA damage and thus promotes cell survival. Black-Right-Pointing-Pointer hCUL4A regulates apoptosis-related proteins and cell cycle regulators. -- Abstract: The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia-reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia-reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia-reoxygenation injury.

  15. Submicroscopic duplications of the hydroxysteroid dehydrogenase HSD17B10 and the E3 ubiquitin ligase HUWE1 are associated with mental retardation.

    Science.gov (United States)

    Froyen, Guy; Corbett, Mark; Vandewalle, Joke; Jarvela, Irma; Lawrence, Owen; Meldrum, Cliff; Bauters, Marijke; Govaerts, Karen; Vandeleur, Lucianne; Van Esch, Hilde; Chelly, Jamel; Sanlaville, Damien; van Bokhoven, Hans; Ropers, Hans-Hilger; Laumonnier, Frederic; Ranieri, Enzo; Schwartz, Charles E; Abidi, Fatima; Tarpey, Patrick S; Futreal, P Andrew; Whibley, Annabel; Raymond, F Lucy; Stratton, Michael R; Fryns, Jean-Pierre; Scott, Rodney; Peippo, Maarit; Sipponen, Marjatta; Partington, Michael; Mowat, David; Field, Michael; Hackett, Anna; Marynen, Peter; Turner, Gillian; Gécz, Jozef

    2008-02-01

    Submicroscopic copy-number imbalances contribute significantly to the genetic etiology of human disease. Here, we report a novel microduplication hot spot at Xp11.22 identified in six unrelated families with predominantly nonsyndromic XLMR. All duplications segregate with the disease, including the large families MRX17 and MRX31. The minimal, commonly duplicated region contains three genes: RIBC1, HSD17B10, and HUWE1. RIBC1 could be excluded on the basis of its absence of expression in the brain and because it escapes X inactivation in females. For the other genes, expression array and quantitative PCR analysis in patient cell lines compared to controls showed a significant upregulation of HSD17B10 and HUWE1 as well as several important genes in their molecular pathways. Loss-of-function mutations of HSD17B10 have previously been associated with progressive neurological disease and XLMR. The E3 ubiquitin ligase HUWE1 has been implicated in TP53-associated regulation of the neuronal cell cycle. Here, we also report segregating sequence changes of highly conserved residues in HUWE1 in three XLMR families; these changes are possibly associated with the phenotype. Our findings demonstrate that an increased gene dosage of HSD17B10, HUWE1, or both contribute to the etiology of XLMR and suggest that point mutations in HUWE1 are associated with this disease too.

  16. Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes.

    Science.gov (United States)

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J; Liu, Yi; Burlingame, A L; Correia, Maria Almira

    2015-02-06

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  17. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development*♦

    Science.gov (United States)

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J.; West, Christopher M.

    2016-01-01

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. PMID:26719340

  18. Phosphorylation of the E3 ubiquitin ligase RNF41 by the kinase Par-1b is required for epithelial cell polarity.

    Science.gov (United States)

    Lewandowski, Katherine T; Piwnica-Worms, Helen

    2014-01-15

    The establishment and maintenance of cell polarity is an essential property governing organismal homeostasis, and loss of polarity is a common feature of cancer cells. The ability of epithelial cells to establish apical-basal polarity depends on intracellular signals generated from polarity proteins, such as the Par-1 family of proteins, as well as extracellular signals generated through cell contacts with the extracellular matrix (ECM). The Par-1 family has a well-established role in regulating cell-cell contacts in the form of tight junctions by phosphorylating Par-3. In addition, Par-1 has been shown to impact on cell-ECM interactions by regulating laminin receptor localization and laminin deposition on the basal surface of epithelial cells. Laminins are major structural and signaling components of basement membrane (BM), a sheet of specialized ECM underlying epithelia. In this study, we identify RNF41, an E3 ubiquitin ligase, as a novel Par-1b (also known as MARK2) effector in the cell-ECM pathway. Par-1b binds to and phosphorylates RNF41 on serine 254. Phosphorylation of RNF41 by Par-1b is required for epithelial cells to localize laminin-111 receptors to their basolateral surfaces and to properly anchor to laminin-111. In addition, phosphorylation of RNF41 is required for epithelial cells to establish apical-basal polarity. Our data suggests that phosphorylation of RNF41 by Par-1b regulates basolateral membrane targeting of laminin-111 receptors, thereby facilitating cell anchorage to laminin-111 and ultimately forming the cell-ECM contacts required for epithelial cells to establish apical-basal cell polarity.

  19. The putative E3 ubiquitin ligase ECERIFERUM9 regulates abscisic acid biosynthesis and response during seed germination and postgermination growth in arabidopsis

    KAUST Repository

    Zhao, Huayan

    2014-05-08

    The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination. © 2014 American Society of Plant Biologists. All Rights Reserved.

  20. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    Science.gov (United States)

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

  1. O2 sensing associated glycosylation exposes the F-box combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases.

    Science.gov (United States)

    Sheikh, M Osman; Thieker, David; Chalmers, Gordon; Schafer, Christopher M; Ishihara, Mayumi; Azadi, Parastoo; Woods, Robert J; Glushka, John N; Bendiak, Brad; Prestegard, James H; West, Christopher M

    2017-09-19

    Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1-Cullin1-F-box) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box binding region in Skp1, and aids in Skp1's association with F-box proteins. Here, nuclear magnetic resonance and mass spectrometry approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box binding domain of Skp1. MD trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two alpha-helices of the F-box combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  2. miR-135A regulates preimplantation embryo development through down-regulation of E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (SIAH1A expression.

    Directory of Open Access Journals (Sweden)

    Ronald T K Pang

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are small non-coding RNA molecules capable of regulating transcription and translation. Previously, a cluster of miRNAs that are specifically expressed in mouse zygotes but not in oocytes or other preimplantation stages embryos are identified by multiplex real-time polymerase chain reaction-based miRNA profiling. The functional role of one of these zygote-specific miRNAs, miR-135a, in preimplantation embryo development was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Microinjection of miR-135a inhibitor suppressed first cell cleavage in more than 30% of the zygotes. Bioinformatics analysis identified E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (Siah1a as a predicted target of miR-135a. Western blotting and 3'UTR luciferase functional assays demonstrated that miR-135a down-regulated the expression of Siah1 in HeLa cells and in mouse zygotes. Siah1a was expressed in preimplantation embryos and its expression pattern negatively correlated with that of miR-135a. Co-injection of Siah1a-specific antibody with miR-135a inhibitor partially nullified the effect of miR-135a inhibition. Proteasome inhibition by MG-132 revealed that miR-135a regulated proteasomal degradation and potentially controlled the expression of chemokinesin DNA binding protein (Kid. CONCLUSIONS/SIGNIFICANCE: The present study demonstrated for the first time that zygotic specific miRNA modulates the first cell cleavage through regulating expression of Siah1a.

  3. Tyrosine phosphorylation of the E3 ubiquitin ligase TRIM21 positively regulates interaction with IRF3 and hence TRIM21 activity.

    Directory of Open Access Journals (Sweden)

    Kevin B Stacey

    Full Text Available Patients suffering from Systemic Lupus Erythematous (SLE have elevated type I interferon (IFN levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs. However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F. We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFN-β promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-β promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics.

  4. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

    Science.gov (United States)

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Hein, Marco Y; Müller, Marcel A; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-08-30

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

  5. Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

    Science.gov (United States)

    Ranaweera, Ruchira S.; Yang, Xiaolu

    2013-01-01

    The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

  6. Fission yeast arrestin-related trafficking adaptor, Arn1/Any1, is ubiquitinated by Pub1 E3 ligase and regulates endocytosis of Cat1 amino acid transporter

    Directory of Open Access Journals (Sweden)

    Akio Nakashima

    2014-05-01

    Full Text Available The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1. Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1+ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1+ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.

  7. Genome-Wide Identification of Soybean U-Box E3 Ubiquitin Ligases and Roles of GmPUB8 in Negative Regulation of Drought Stress Response in Arabidopsis.

    Science.gov (United States)

    Wang, Ning; Liu, Yaping; Cong, Yahui; Wang, Tingting; Zhong, Xiujuan; Yang, Shouping; Li, Yan; Gai, Junyi

    2016-06-01

    Plant U-box (PUB) E3 ubiquitin ligases play important roles in hormone signaling pathways and response to abiotic stresses, but little is known about them in soybean, Glycine max. Here, we identified and characterized 125 PUB genes from the soybean genome, which were classified into eight groups according to their protein domains. Soybean PUB genes (GmPUB genes) are broadly expressed in many tissues and are a little more abundant in the roots than in the other tissues. Nine GmPUB genes, GmPUB1-GmPUB9, showed induced expression patterns by drought, and the expression of GmPUB8 was also induced by exogenous ABA and NaCl. GmPUB8 was localized to post-Golgi compartments, interacting with GmE2 protein as demonstrated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments, and showed E3 ubiquitin ligase activity by in vitro ubiquitination assay. Heterogeneous overexpression of GmPUB8 in Arabidopsis showed decreased drought tolerance, enhanced sensitivity with respect to osmotic and salt stress inhibition of seed germination and seedling growth, and inhibited ABA- and mannitol-mediated stomatal closure. Eight drought stress-related genes were less induced in GmPUB8-overexpressing Arabidopsis after drought treatment compared with the wild type and the pub23 mutant. Taken together, our results suggested that GmPUB8 might negatively regulate plant response to drought stress. In addition, Y2H and BiFC showed that GmPUB8 interacted with soybean COL (CONSTANS LIKE) protein. GmPUB8-overexpressing Arabidopsis flowered earlier under middle- and short-day conditions but later under long-day conditions, indicating that GmPUB8 might regulate flowering time in the photoperiod pathway. This study helps us to understand the functions of PUB E3 ubiquitin ligases in soybean.

  8. p90RSK targets the ERK5-CHIP ubiquitin E3 ligase activity in diabetic hearts and promotes cardiac apoptosis and dysfunction.

    Science.gov (United States)

    Le, Nhat-Tu; Takei, Yuichiro; Shishido, Tetsuro; Woo, Chang-Hoon; Chang, Eugene; Heo, Kyung-Sun; Lee, Hakjoo; Lu, Yan; Morrell, Craig; Oikawa, Masayoshi; McClain, Carolyn; Wang, Xin; Tournier, Cathy; Molina, Carlos A; Taunton, Jack; Yan, Chen; Fujiwara, Keigi; Patterson, Cam; Yang, Jay; Abe, Jun-ichi

    2012-02-17

    Cardiomyocyte apoptosis is one of the key events in the development and progression of heart failure, and a crucial role for ICER (inducible cAMP early repressor) in this process has been previously reported. ERK5 is known to inhibit cardiac apoptosis after myocardial infarction (MI), especially in hyperglycemic states, via association with CHIP ubiquitin (Ub) ligase and subsequent upregulation of CHIP ligase activity, which induces ICER ubiquitination and subsequent protein degradation. The regulatory mechanism governing ERK5/CHIP interaction is unknown. We previously demonstrated increased p90RSK activation in the diabetic heart. As a logical extension of this work, we now investigate whether p90RSK activation inhibits ERK5-mediated CHIP activation, and subsequently increases ICER levels and apoptosis. p90RSK activation inhibits ERK5/CHIP association and CHIP Ub ligase activity. p90RSK and CHIP share a common binding site in the ERK5 C-terminal domain (aa571-807). Overexpression of either p90RSK or an ERK5 fragment (aa571-807) inhibits ERK5/CHIP association, suggesting that p90RSK and CHIP competes for ERK5 binding and that p90RSK activation is critical for inhibiting ERK5/CHIP interaction. We also identified ERK5-S496 as being directly phosphorylated by p90RSK and demonstrated that an ERK5-S496A mutant significantly impairs Angiotensin II-mediated inhibition of CHIP activity and subsequent increase in ICER levels. In vivo, either cardiac-specific depletion of ERK5 or overexpression of p90RSK inhibits CHIP activity and accelerates cardiac apoptosis after MI-a phenomenon fully reversible by activating ERK5. These data suggest a role for p90RSK in inhibiting CHIP activity and promoting cardiac apoptosis through binding to and phosphorylation of ERK5-S496.

  9. BRCA1/BARD1 E3 ubiquitin ligase can modify histones H2A and H2B in the nucleosome particle.

    Science.gov (United States)

    Thakar, Amit; Parvin, Jeffrey D; Zlatanova, Jordanka

    2010-02-01

    BRCA1, the protein product of the Breast Cancer Susceptibility Gene (BRCA1) has been implicated in multiple pathways that preserve genome stability, including cell cycle control, DNA repair, transcription, and chromatin remodeling. BRCA1, in complex with another RING-domain protein BARD1, possesses ubiquitin-ligase activity. Only a few targets for this activity have been identified in vivo. Nucleosomal histones may also be targets in vivo since they can be modified by the BRCA1/BARD1 complex in vitro. Here we demonstrate that the BRCA1/BARD1 complex can ubiquitylate both free H2A and H2B histones and histones in the context of nucleosomal particles. These results raise the possibility that BRCA1/BARD1 can directly affect nucleosomal structure, dynamics, and function through its ability to modify nucleosomal histones.

  10. In silico analysis identifies a C3HC4-RING finger domain of a putative E3 ubiquitin-protein ligase located at the C-terminus of a polyglutamine-containing protein

    Directory of Open Access Journals (Sweden)

    T. Scior

    2007-03-01

    Full Text Available Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229. We infer that an identical new gene (RING finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3 as a binding site for ubiquitin-conjugating enzyme (E2. Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4 may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744. Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734. The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ.

  11. The E3 Ubiquitin Ligase Activity Associated with the Adenoviral E1B-55K–E4orf6 Complex Does Not Require CRM1-Dependent Export ▿

    OpenAIRE

    Schmid, Melanie; Kindsmüller, Kathrin; Wimmer, Peter; Groitl, Peter; Gonzalez, Ramon A.; Dobner, Thomas

    2011-01-01

    The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via l...

  12. BTB-BACK Domain Protein POB1 Suppresses Immune Cell Death by Targeting Ubiquitin E3 ligase PUB17 for Degradation

    Science.gov (United States)

    Mesmar, Joelle; McLellan, Hazel; Yang, Chengwei; Craig, Adam; Zhang, Cunjin; Moore, Jonathan David; Tian, Zhendong; Birch, Paul R. J.; Sadanandom, Ari

    2017-01-01

    Hypersensitive response programmed cell death (HR-PCD) is a critical feature in plant immunity required for pathogen restriction and prevention of disease development. The precise control of this process is paramount to cell survival and an effective immune response. The discovery of new components that function to suppress HR-PCD will be instrumental in understanding the regulation of this fundamental mechanism. Here we report the identification and characterisation of a BTB domain E3 ligase protein, POB1, that functions to suppress HR-PCD triggered by evolutionarily diverse pathogens. Nicotiana benthamiana and tobacco plants with reduced POB1 activity show accelerated HR-PCD whilst those with increased POB1 levels show attenuated HR-PCD. We demonstrate that POB1 dimerization and nuclear localization are vital for its function in HR-PCD suppression. Using protein-protein interaction assays, we identify the Plant U-Box E3 ligase PUB17, a well established positive regulator of plant innate immunity, as a target for POB1-mediated proteasomal degradation. Using confocal imaging and in planta immunoprecipitation assays we show that POB1 interacts with PUB17 in the nucleus and stimulates its degradation. Mutated versions of POB1 that show reduced interaction with PUB17 fail to suppress HR-PCD, indicating that POB1-mediated degradation of PUB17 U-box E3 ligase is an important step for negative regulation of specific immune pathways in plants. Our data reveals a new mechanism for BTB domain proteins in suppressing HR-PCD in plant innate immune responses. PMID:28056034

  13. Structure of the HHARI catalytic domain shows glimpses of a HECT E3 ligase.

    Directory of Open Access Journals (Sweden)

    Donald E Spratt

    Full Text Available The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING, or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT. The RING-inBetweenRING-RING (RBR proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI. These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.

  14. Overview of the membrane-associated RING-CH (MARCH) E3 ligase family.

    Science.gov (United States)

    Bauer, Johannes; Bakke, Oddmund; Morth, J Preben

    2016-12-14

    E3 ligases are critical checkpoints for protein ubiquitination, a signal that often results in protein sorting and degradation but has also been linked to regulation of transcription and DNA repair. In line with their key role in cellular trafficking and cell-cycle control, malfunction of E3 ligases is often linked to human disease. Thus, they have emerged as prime drug targets. However, the molecular basis of action of membrane-bound E3 ligases is still unknown. Here, we review the current knowledge on the membrane-embedded MARCH E3 ligases (MARCH-1-6,7,8,11) with a focus on how the transmembrane regions can contribute via GxxxG-motifs to the selection and recognition of other membrane proteins as substrates for ubiquitination. Further understanding of the molecular parameters that govern target protein recognition of MARCH E3 ligases will contribute to development of strategies for therapeutic regulation of MARCH-induced ubiquitination.

  15. Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy

    Directory of Open Access Journals (Sweden)

    Zhang Hui

    2007-02-01

    Full Text Available Abstract Recent investigation of Cullin 4 (CUL4 has ushered this class of multiprotein ubiquitin E3 ligases to center stage as critical regulators of diverse processes including cell cycle regulation, developmental patterning, DNA replication, DNA damage and repair, and epigenetic control of gene expression. CUL4 associates with DNA Damage Binding protein 1 (DDB1 to assemble an ubiquitin E3 ligase that targets protein substrates for ubiquitin-dependent proteolysis. CUL4 ligase activity is also regulated by the covalent attachment of the ubiquitin-like protein NEDD8 to CUL4, or neddylation, and the COP9 signalosome complex (CSN that removes this important modification. Recently, multiple WD40-repeat proteins (WDR were found to interact with DDB1 and serve as the substrate-recognition subunits of the CUL4-DDB1 ubiquitin ligase. As more than 150–300 WDR proteins exist in the human genome, these findings impact a wide array of biological processes through CUL4 ligase-mediated proteolysis. Here, we review the recent progress in understanding the mechanism of CUL4 ubiquitin E3 ligase and discuss the architecture of CUL4-assembled E3 ubiquitin ligase complexes by comparison to CUL1-based E3s (SCF. Then, we will review several examples to highlight the critical roles of CUL4 ubiquitin ligase in genome stability, cell cycle regulation, and histone lysine methylation. Together, these studies provide insights into the mechanism of this novel ubiquitin ligase in the regulation of important biological processes.

  16. Akt is negatively regulated by the MULAN E3 ligase

    Institute of Scientific and Technical Information of China (English)

    Seunghee Bae; Jongdoo Kim; Hong-Duck Um; In-Chul Park; Su-Jae Lee; Seon Young Nam; Young-Woo Jin; Jae Ho Lee; Sungkwan An; Sun-Yong Kim; Jin Hyuk Jung; Yeongmin Yoon; Hwa Jun Cha; Hyunjin Lee; Karam Kim; Jongran Kim; In-Sook An

    2012-01-01

    The serine/threonine kinase Akt functions in multiple cellular processes,including cell survival and tumor development.Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation.In this study,we identified a negative regulator of Akt,MULAN,which possesses both a RING finger domain and E3 ubiquitin ligase activity.Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo.Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN.The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability.These data provide insight into the Akt ubiquitination signaling network.

  17. Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity

    Directory of Open Access Journals (Sweden)

    Joshua R. Kane

    2015-05-01

    Full Text Available HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac] and non-primate (FIV, BIV, and MVV viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA, for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

  18. Lineage-Specific Viral Hijacking of Non-canonical E3 Ubiquitin Ligase Cofactors in the Evolution of Vif Anti-APOBEC3 Activity.

    Science.gov (United States)

    Kane, Joshua R; Stanley, David J; Hultquist, Judd F; Johnson, Jeffrey R; Mietrach, Nicole; Binning, Jennifer M; Jónsson, Stefán R; Barelier, Sarah; Newton, Billy W; Johnson, Tasha L; Franks-Skiba, Kathleen E; Li, Ming; Brown, William L; Gunnarsson, Hörður I; Adalbjornsdóttir, Adalbjorg; Fraser, James S; Harris, Reuben S; Andrésdóttir, Valgerður; Gross, John D; Krogan, Nevan J

    2015-05-26

    HIV-1 encodes the accessory protein Vif, which hijacks a host Cullin-RING ubiquitin ligase (CRL) complex as well as the non-canonical cofactor CBFβ, to antagonize APOBEC3 antiviral proteins. Non-canonical cofactor recruitment to CRL complexes by viral factors, to date, has only been attributed to HIV-1 Vif. To further study this phenomenon, we employed a comparative approach combining proteomic, biochemical, structural, and virological techniques to investigate Vif complexes across the lentivirus genus, including primate (HIV-1 and simian immunodeficiency virus macaque [SIVmac]) and non-primate (FIV, BIV, and MVV) viruses. We find that CBFβ is completely dispensable for the activity of non-primate lentiviral Vif proteins. Furthermore, we find that BIV Vif requires no cofactor and that MVV Vif requires a novel cofactor, cyclophilin A (CYPA), for stable CRL complex formation and anti-APOBEC3 activity. We propose modular conservation of Vif complexes allows for potential exaptation of functions through the acquisition of non-CRL-associated host cofactors while preserving anti-APOBEC3 activity.

  19. A novel brain-enriched E3 ubiquitin ligase RNF182 is up regulated in the brains of Alzheimer's patients and targets ATP6V0C for degradation

    Directory of Open Access Journals (Sweden)

    Sikorska Marianna

    2008-02-01

    Full Text Available Abstract Background Alterations in multiple cellular pathways contribute to the development of chronic neurodegeneration such as a sporadic Alzheimer's disease (AD. These, in turn, involve changes in gene expression, amongst which are genes regulating protein processing and turnover such as the components of the ubiquitin-proteosome system. Recently, we have identified a cDNA whose expression was altered in AD brains. It contained an open reading frame of 247 amino acids and represented a novel RING finger protein, RNF182. Here we examined its biochemical properties and putative role in brain cells. Results RNF182 is a low abundance cytoplasmic protein expressed preferentially in the brain. Its expression was elevated in post-mortem AD brain tissue and the gene could be up regulated in vitro in cultured neurons subjected to cell death-inducing injuries. Subsequently, we have established that RNF182 protein possessed an E3 ubiquitin ligase activity and stimulated the E2-dependent polyubiquitination in vitro. Yeast two-hybrid screening, overexpression and co-precipitation approaches revealed, both in vitro and in vivo, an interaction between RNF182 and ATP6V0C, known for its role in the formation of gap junction complexes and neurotransmitter release channels. The data indicated that RNF182 targeted ATP6V0C for degradation by the ubiquitin-proteosome pathway. Overexpression of RNF182 reduced cell viability and it would appear that by itself the gene can disrupt cellular homeostasis. Conclusion Taken together, we have identified a novel brain-enriched RING finger E3 ligase, which was up regulated in AD brains and neuronal cells exposed to injurious insults. It interacted with ATP6V0C protein suggesting that it may play a very specific role in controlling the turnover of an essential component of neurotransmitter release machinery.

  20. Cloning and Expression Analysis of E3 Ubiquitin Ligase Nrdp1 from Grass Carp Ctenopharyngodon idella%草鱼E3泛素连接酶Nrdp1基因cDNA克隆和表达分析

    Institute of Scientific and Technical Information of China (English)

    隗黎丽; 杨竹青; 王自蕊; 熊六凤; 周秋白

    2013-01-01

    神经调节素受体降解蛋白1(neuregulin receptor degradation protein-1,Nrdpl)是一种新的E3泛素连接酶,可调节细胞增殖和凋亡等.研究采用RACE方法从草鱼(Ctenopharyngodon idella)肝脏中克隆了Nrdpl的全长cDNA序列;采用半定量RT-PCR方法分析该基因在草鱼不同组织中表达情况.结果表明:该序列全长cDNA大小为1 865 bp(GenBank登录号:JX864048),其中开放阅读框为954 bp,编码318个氨基酸,预测分子量大小为36.09 ku,pH为7.0时理论等电点为5.83;5’和3’非翻译区的长度分别为291 bp和620 bp,在3’非翻译区发现2个mRNA不稳定信号(ATTTA)和1个多聚腺苷酸加尾信号(ATTAAA).该蛋白序列没有信号肽和跨膜结构,其二级结构主要以α-螺旋为主,不含β-折叠.氨基酸序列保守性分析表明,草鱼Nrdp1与斑马鱼、虹鳟及人的同源性分别为98%、94%和89%;该基因在心脏和脑中的表达量较多,在肌肉和血液中表达很少.%Nrdpl ( neuregulin receptor degradation protein - 1) is a newly defined E3 ubiquitin ligase and regulates the cell proliferation and apoptosis. In the present study, the cDNA sequence of grass carp Ctenopha-ryngodon idella Nrdp1 (gcNrdpl) consists of 1865 bp with a 291 bp 5' untranslated region (UTR) and a 620 bp 3' UTR, with an open reading frame of 954 bp encoding the 318 amino acids which is predicted to have no signal petptide and transmembrane helices. The deduced amino acid sequence of gcNrdpl showed 98% , 94% and 89% of identity to zebra fish Danio rerio, rainbow trout Oncorhynchus mykiss and human Homo sapiens Nrdpl, respectively. The domain search revealed that gcNrdpl contains a RING domain (at amino acids 18 -56) and Pfam domain (at amino acids 137 -315). Phylogenetic analysis demonstrated that gcNrdpl is clustered in a same clade with zebra fish and rainbow trout Nrdpl. RT-PCR demonstrated that the mRNA is con-stitutively expressed in all the examined organs examined of healthy fish

  1. Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U

    DEFF Research Database (Denmark)

    Davis, Matti; Hatzubai, Ada; Andersen, Jens S

    2002-01-01

    beta-TrCP/E3RS (E3RS) is the F-box protein that functions as the receptor subunit of the SCF(beta-TrCP) ubiquitin ligase (E3). Surprisingly, although its two recognized substrates, IkappaB(alpha) and beta-catenin, are present in the cytoplasm, we have found that E3RS is located predominantly......, and efficacy of a specific protein-ubiquitin ligase....

  2. Selectivity of E2-E3 interactions in the human ubiquitin system

    NARCIS (Netherlands)

    van Wijk, S.J.L.

    2010-01-01

    Highly selective interactions between ubiquitin-conjugating enzymes and RING-type E3 ligases are crucial for the adequate and efficient action of ubiquitin and ubiquitin-like pathways. Within these cascades, E2 enzymes provide a connecting link between activation and the final covalent conjugation,

  3. Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

    OpenAIRE

    Xiaolian Zhang; Ning Wang; Pei Chen; Mengmeng Gao; Juge Liu; Yufeng Wang; Tuanjie Zhao; Yan Li; Junyi Gai

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine...

  4. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.).

    Science.gov (United States)

    Gandhi, Anita K; Kang, Jian; Havens, Courtney G; Conklin, Thomas; Ning, Yuhong; Wu, Lei; Ito, Takumi; Ando, Hideki; Waldman, Michelle F; Thakurta, Anjan; Klippel, Anke; Handa, Hiroshi; Daniel, Thomas O; Schafer, Peter H; Chopra, Rajesh

    2014-03-01

    Cereblon (CRBN), the molecular target of lenalidomide and pomalidomide, is a substrate receptor of the cullin ring E3 ubiquitin ligase complex, CRL4(CRBN) . T cell co-stimulation by lenalidomide or pomalidomide is cereblon dependent: however, the CRL4(CRBN) substrates responsible for T cell co-stimulation have yet to be identified. Here we demonstrate that interaction of the transcription factors Ikaros (IKZF1, encoded by the IKZF1 gene) and Aiolos (IKZF3, encoded by the IKZF3 gene) with CRL4(CRBN) is induced by lenalidomide or pomalidomide. Each agent promotes Aiolos and Ikaros binding to CRL4(CRBN) with enhanced ubiquitination leading to cereblon-dependent proteosomal degradation in T lymphocytes. We confirm that Aiolos and Ikaros are transcriptional repressors of interleukin-2 expression. The findings link lenalidomide- or pomalidomide-induced degradation of these transcriptional suppressors to well documented T cell activation. Importantly, Aiolos could serve as a proximal pharmacodynamic marker for lenalidomide and pomalidomide, as healthy human subjects administered lenalidomide demonstrated Aiolos degradation in their peripheral T cells. In conclusion, we present a molecular model in which drug binding to cereblon results in the interaction of Ikaros and Aiolos to CRL4(CRBN) , leading to their ubiquitination, subsequent proteasomal degradation and T cell activation.

  5. Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

    Science.gov (United States)

    Kumazoe, Motofumi; Nakamura, Yuki; Yamashita, Mai; Suzuki, Takashi; Takamatsu, Kanako; Huang, Yuhui; Bae, Jaehoon; Yamashita, Shuya; Murata, Motoki; Yamada, Shuhei; Shinoda, Yuki; Yamaguchi, Wataru; Toyoda, Yui; Tachibana, Hirofumi

    2017-03-10

    Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Cyclin F/FBXO1 interacts with HIV-1 Vif and restricts progeny virion infectivity by ubiquitination and proteasomal degradation of Vif through SCF (Cyclin F) E3 ligase machinery.

    Science.gov (United States)

    Augustine, Tracy; Chaudhary, Priyanka; Gupta, Kailash; Islam, Sehbanul; Ghosh, Payel; Santra, Manas Kumar; Mitra, Debashis

    2017-02-09

    Cyclin F, also known as FBXO1, is the largest among all cyclins which oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, Cyclin F functions as the substrate binding subunit of SCFCyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of Cyclin F (CCNF) gene. Later, using gene over expression and knockdown studies, we identified that Cyclin F negatively influences HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that Cyclin F negatively regulates the expression of viral protein, Vif (Viral infectivity factor), at the protein level. We also identified a novel host-pathogen interaction between Cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a Cyclin F-specific amino acid motif in the C-terminal region of Vif shows rescue of the protein from Cyclin F-mediated down-regulation. Subsequently, we have shown that Vif is a novel substrate of the SCFCyclin F E3 ligase, where Cyclin F mediates ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we have shown that Cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate Cyclin F as a novel F-box protein which functions as an intrinsic cellular regulator of HIV-1 Vif and imparts a negative regulatory effect on maintenance of viral infectivity by restoring APOBEC3G expression.

  7. The Role of Ubiquitin Ligases in Cardiac Disease

    Science.gov (United States)

    Willis, Monte S.; Bevilacqua, Ariana; Pulinilkunnil, Thomas; Kienesberger, Petra; Tannu, Manasi; Patterson, Cam

    2014-01-01

    Rigorous surveillance of protein quality control is essential for the maintenance of normal cardiac function, while the dysregulation of protein turnover is present in a diverse array of common cardiac diseases. Central to the protein quality control found in all cells is the ubiquitin proteasome system (UPS). The UPS plays a critical role in protein trafficking, cellular signaling, and most prominently, protein degradation. As ubiquitin ligases (E3s) control the specificity of the UPS, their description in the cardiomyocyte has highlighted how ubiquitin ligases are critical to the turnover and function of the sarcomere complex, responsible for the heart’s required continuous contraction. In this review, we provide an overview of the UPS, highlighting a comprehensive overview of the cardiac ubiquitin ligases identified to date. We then focus on recent studies of new cardiac ubiquitin ligases outlining their novel roles in protein turnover, cellular signaling, and the regulation of mitochondrial dynamics and receptor turnover in the pathophysiology of cardiac hypertrophy, cardiac atrophy, myocardial infarction, and heart failure. PMID:24262338

  8. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function

    OpenAIRE

    Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R.; Xu, Guoqiang

    2015-01-01

    The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains a...

  9. The role of HERC2 and RNF8 ubiquitin E3 ligases in the promotion of translesion DNA synthesis in the chicken DT40 cell line

    DEFF Research Database (Denmark)

    Mohiuddin, Mohammed; Kobayashi, Shunsuke; Keka, Islam Shamima

    2016-01-01

    immediately after exposure to UV while retaining proficient post-replicative gap filling. These mutants are both proficient in mono-ubiquitination of PCNA. Taken together, these results suggest that HERC2 and RNF8 promote TLS past abasic sites and UV-lesions at or very close to stalled replication forks....

  10. Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

    Energy Technology Data Exchange (ETDEWEB)

    Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; Tang, Xiaojing; Marcon, Edyta; Kurinov, Igor; Greenblatt, Jack F.; Tyers, Mike; Moffat, Jason; Sicheri, Frank; Sidhu, Sachdev S.

    2016-03-14

    The ubiquitin proteasome components are often misregulated in numerous diseases, encouraging the search for drug targets and inhibitors. E3 ligases that specify ubiquitination targets are of particular interest. Multimeric Skp1–Cul1–F-box (SCF) E3 ligases constitute one of the largest E3 families connected to every cellular process and multiple diseases; however, their characterization as therapeutic targets is impeded by functional diversity and poor characterization of its members. Herein we describe a strategy to inhibit SCF E3 ligases using engineered ubiquitin-based binders. We identify a previously uncharacterized inhibitory site and design ubiquitin-based libraries targeting this site. Our strategy to target SCF E3 ligases with small-molecule–like agents will have broad applications for basic research and drug development relating to SCF E3 ligase function.

  11. Endoplasmic Reticulum Exit of Golgi-resident Defective for SREBP Cleavage (Dsc) E3 Ligase Complex Requires Its Activity.

    Science.gov (United States)

    Raychaudhuri, Sumana; Espenshade, Peter J

    2015-06-01

    Layers of quality control ensure proper protein folding and complex formation prior to exit from the endoplasmic reticulum. The fission yeast Dsc E3 ligase is a Golgi-localized complex required for sterol regulatory element-binding protein (SREBP) transcription factor activation that shows architectural similarity to endoplasmic reticulum-associated degradation E3 ligases. The Dsc E3 ligase consists of five integral membrane proteins (Dsc1-Dsc5) and functionally interacts with the conserved AAA-ATPase Cdc48. Utilizing an in vitro ubiquitination assay, we demonstrated that Dsc1 has ubiquitin E3 ligase activity that requires the E2 ubiquitin-conjugating enzyme Ubc4. Mutations that specifically block Dsc1-Ubc4 interaction prevent SREBP cleavage, indicating that SREBP activation requires Dsc E3 ligase activity. Surprisingly, Golgi localization of the Dsc E3 ligase complex also requires Dsc1 E3 ligase activity. Analysis of Dsc E3 ligase complex formation, glycosylation, and localization indicated that Dsc1 E3 ligase activity is specifically required for endoplasmic reticulum exit of the complex. These results define enzyme activity-dependent sorting as an autoregulatory mechanism for protein trafficking.

  12. A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation.

    Science.gov (United States)

    Hong, Eun-Jin Erica; Villén, Judit; Gerace, Erica L; Gygi, Steven P; Moazed, Danesh

    2005-01-01

    The assembly of heterochromatin in fission yeast and metazoans requires histone H3-lysine 9 (-K9) methylation by the conserved Clr4/Suv39h methyltransferase. In fission yeast, H3-K9 methylation requires components of the RNAi machinery and is initiated by the RNA-Induced Transcriptional Silencing (RITS) complex. Here we report the purification of a novel complex that associates with the Clr4 methyltransferase, termed the CLRC (CLr4-Rik1-Cul4) complex. By affinity purification of the Clr4-associated protein Rik1, we show that, in addition to Clr4, Rik1 is associated with the fission yeast E3 ubiquitin ligase Cullin4 (Cul4, encoded by cul4(+)), the ubiquitin-like protein, Ned8, and two previously uncharacterized proteins, designated Cmc1 and Cmc2. In addition, the complex contains substochiometric amounts of histones H2B and H4, and the 14-3-3 protein, Rad24. Deletion of cul4(+), cmc1(+), cmc2(+) and rad24(+) results in a complete loss of silencing of a ura4(+) reporter gene inserted within centromeric DNA repeats or the silent mating type locus. Each of the above deletions also results in accumulation of noncoding RNAs transcribed from centromeric repeats and telomeric DNA regions, and a corresponding loss of small RNAs that are homologous to centromeric repeats, suggesting a defect in the processing of noncoding RNA to small RNA. Based on these results, we propose that the components of the Clr4-Rik1-Cul4 complex act concertedly at an early step in heterochromatin formation.

  13. The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

    Science.gov (United States)

    Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

    2013-01-01

    The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

  14. A novel effect of thalidomide and its analogs: suppression of cereblon ubiquitination enhances ubiquitin ligase function.

    Science.gov (United States)

    Liu, Yaobin; Huang, Xiangao; He, Xian; Zhou, Yanqing; Jiang, Xiaogang; Chen-Kiang, Selina; Jaffrey, Samie R; Xu, Guoqiang

    2015-12-01

    The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide are highly effective in treating clinical indications. Thalidomide binds to cereblon (CRBN), a substrate receptor of the cullin-4 really interesting new gene (RING) E3 ligase complex. Here, we examine the effect of thalidomide and its analogs on CRBN ubiquitination and its functions in human cell lines. We find that the ubiquitin modification of CRBN includes K48-linked polyubiquitin chains and that thalidomide blocks the formation of CRBN-ubiquitin conjugates. Furthermore, we show that ubiquitinated CRBN is targeted for proteasomal degradation. Treatment of human myeloma cell lines such as MM1.S, OPM2, and U266 with thalidomide (100 μM) and its structural analog lenalidomide (10 μM) results in stabilization of CRBN and elevation of CRBN protein levels. This in turn leads to the reduced level of CRBN target proteins and enhances the sensitivity of human multiple myeloma cells to IMiDs. Our results reveal a novel mechanism by which thalidomide and its analogs modulate the CRBN function in cells. Through inhibition of CRBN ubiquitination, thalidomide and its analogs allow CRBN to accumulate, leading to the increased cullin-4 RING E3 ligase-mediated degradation of target proteins.

  15. Defining the interactions and role of DCAF1/VPRBP in the DDB1-cullin4A E3 ubiquitin ligase complex engaged by HIV-1 Vpr to induce a G2 cell cycle arrest.

    Directory of Open Access Journals (Sweden)

    Francine C A Gérard

    Full Text Available HIV viral protein R (Vpr induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD. Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

  16. Investigation of the Expression of Myogenic Transcription Factors, microRNAs and Muscle-Specific E3 Ubiquitin Ligases in the Medial Gastrocnemius and Soleus Muscles following Peripheral Nerve Injury.

    Directory of Open Access Journals (Sweden)

    Rebecca Wiberg

    Full Text Available Despite surgical innovation, the sensory and motor outcome after a peripheral nerve injury remains incomplete. One contributing factor to the poor outcome is prolonged denervation of the target organ, leading to apoptosis of both mature myofibres and satellite cells with subsequent replacement of the muscle tissue with fibrotic scar and adipose tissue. In this study, we investigated the expression of myogenic transcription factors, muscle specific microRNAs and muscle-specific E3 ubiquitin ligases at several time points following denervation in two different muscles, the gastrocnemius (containing predominantly fast type fibres and soleus (slow type muscles, since these molecules may influence the degree of atrophy following denervation. Both muscles exhibited significant atrophy (compared with the contra-lateral sides at 7 days following either a nerve transection or crush injury. In the crush model, the soleus muscle showed significantly increased muscle weights at days 14 and 28 which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. This study provides further insights regarding the intracellular regulatory molecules that generate and maintain distinct patterns of gene expression in different fibre types following peripheral nerve injury.

  17. Origin and diversification of TRIM ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Ignacio Marín

    Full Text Available Most proteins of the TRIM family (also known as RBCC family are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.

  18. KF-1 ubiquitin ligase: an anxiety suppressor

    Directory of Open Access Journals (Sweden)

    Tamotsu Hashimoto-Gotoh

    2009-05-01

    Full Text Available Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located to the endoplasmic reticulum (ER, may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior against light or heights. Thus, KF-1 may degrade some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson's disease (PD. Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice, be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds.

  19. The auto-ubiquitylation of E3 ubiquitin-protein ligase Chfr at G2 phase is required for accumulation of polo-like kinase 1 and mitotic entry in mammalian cells.

    Science.gov (United States)

    Kim, Jo-Sun; Park, Yong-Yea; Park, Sun-Yi; Cho, Hyeseon; Kang, Dongmin; Cho, Hyeseong

    2011-09-01

    The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.

  20. Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8

    OpenAIRE

    Yu, Clinton; Mao, Haibin; Novitsky, Eric J.; Tang, Xiaobo; Rychnovsky, Scott D.; Zheng, Ning; Huang, Lan

    2015-01-01

    The full enzymatic activity of the cullin-RING ubiquitin ligases (CRLs) requires a ubiquitin-like protein (that is, Nedd8) modification. By deamidating Gln40 of Nedd8 to glutamate (Q40E), the bacterial cycle-inhibiting factor (Cif) family is able to inhibit CRL E3 activities, thereby interfering with cellular functions. Despite extensive structural studies on CRLs, the molecular mechanism by which Nedd8 Gln40 deamidation affects CRL functions remains unclear. We apply a new quantitative cross...

  1. MDM2 is a novel E3 ligase for HIV-1 Vif

    Directory of Open Access Journals (Sweden)

    Tomonaga Mitsunori

    2009-01-01

    Full Text Available Abstract The human immunodeficiency virus type 1 (HIV-1 Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G. Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3 complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

  2. (1)H, (15)N and (13)C resonance assignments for free and IEEVD peptide-bound forms of the tetratricopeptide repeat domain from the human E3 ubiquitin ligase CHIP.

    Science.gov (United States)

    Zhang, Huaqun; McGlone, Cameron; Mannion, Matthew M; Page, Richard C

    2017-04-01

    The ubiquitin ligase CHIP catalyzes covalent attachment of ubiquitin to unfolded proteins chaperoned by the heat shock proteins Hsp70/Hsc70 and Hsp90. CHIP interacts with Hsp70/Hsc70 and Hsp90 by binding of a C-terminal IEEVD motif found in Hsp70/Hsc70 and Hsp90 to the tetratricopeptide repeat (TPR) domain of CHIP. Although recruitment of heat shock proteins to CHIP via interaction with the CHIP-TPR domain is well established, alterations in structure and dynamics of CHIP upon binding are not well understood. In particular, the absence of a structure for CHIP-TPR in the free form presents a significant limitation upon studies seeking to rationally design inhibitors that may disrupt interactions between CHIP and heat shock proteins. Here we report the (1)H, (13)C, and (15)N backbone and side chain chemical shift assignments for CHIP-TPR in the free form, and backbone chemical shift assignments for CHIP-TPR in the IEEVD-bound form. The NMR resonance assignments will enable further studies examining the roles of dynamics and structure in regulating interactions between CHIP and the heat shock proteins Hsp70/Hsc70 and Hsp90.

  3. Identification of a ubiquitin-protein ligase subunit within the CCR4-NOT transcription repressor complex

    NARCIS (Netherlands)

    Albert, TK; Hanzawa, H; Legtenberg, YIA; de Ruwe, MJ; van den Heuvel, FAJ; Collart, MA; Boelens, R; Timmers, HTM

    2002-01-01

    The RING finger protein CNOT4 is a component of the CCR4-NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin-protein ligase (E3). We show that the unique C4C4 RING domain of CNOT4 interacts with a subset of

  4. RNF38 encodes a nuclear ubiquitin protein ligase that modifies p53

    Energy Technology Data Exchange (ETDEWEB)

    Sheren, Jamie E. [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Kassenbrock, C. Kenneth, E-mail: ken.kassenbrock@ucdenver.edu [Department of Pathology, The University of Colorado Anschutz Medical Campus, Aurora, CO 80045 (United States); Department of Biology, Colorado State University, Fort Collins, CO 80523-1878 (United States)

    2013-11-01

    Highlights: •RNF38 is shown to be a nuclear protein with a bipartite nuclear localization signal. •RNF38 protein is purified and shown to have ubiquitin protein ligase (E3) activity. •We show that RNF38 binds p53 and can ubiquitinate p53 in vitro. •Overexpression of RNF38 increases p53 ubiquitination in HEK293T cells. •Overexpression of RNF38 in HEK293T cells alters p53 localization. -- Abstract: The RNF38 gene encodes a RING finger protein of unknown function. Here we demonstrate that RNF38 is a functional ubiquitin protein ligase (E3). We show that RNF38 isoform 1 is localized to the nucleus by a bipartite nuclear localization sequence (NLS). We confirm that RNF38 is a binding partner of p53 and demonstrate that RNF38 can ubiquitinate p53 in vitro and in vivo. Finally, we show that overexpression of RNF38 in HEK293T cells results in relocalization of p53 to discrete foci associated with PML nuclear bodies. These results suggest RNF38 is an E3 ubiquitin ligase that may play a role in regulating p53.

  5. Cullin-RING Ubiquitin Ligase Family in Plant Abiotic Stress Pathways

    Institute of Scientific and Technical Information of China (English)

    Liquan Guo; Cynthia D.Nezames; Lianxi Sheng; Xingwang Deng; Ning Wei

    2013-01-01

    The ubiquitin-proteasome system is a key mechanism that plants use to generate adaptive responses in coping with various environmental stresses.Cullin-RING (CRL) complexes represent a predominant group of ubiquitin E3 ligases in this system.In this review,we focus on the CRL E3s that have been implicated in abiotic stress signaling pathways in Arabidopsis.By comparing and analyzing these cases,we hope to gain a better understanding on how CRL complexes work under various settings in an attempt to decipher the clues about the regulatory mechanism of CRL E3s.

  6. The ubiquitin ligase HectH9 regulates transcriptional activation by Myc and is essential for tumor cell proliferation

    DEFF Research Database (Denmark)

    Adhikary, Sovana; Marinoni, Federica; Hock, Andreas

    2005-01-01

    The Myc oncoprotein forms a binary activating complex with its partner protein, Max, and a ternary repressive complex that, in addition to Max, contains the zinc finger protein Miz1. Here we show that the E3 ubiquitin ligase HectH9 ubiquitinates Myc in vivo and in vitro, forming a lysine 63-linked...

  7. TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

    DEFF Research Database (Denmark)

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke

    2016-01-01

    , allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes...... ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced...... chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication....

  8. RNF13: a novel RING-type ubiquitin ligase over-expressed in pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Qiang Zhang; Yunxiao Meng; Lei Zhang; Jie Chen; Dahai Zhu

    2009-01-01

    Protein ubiquitination by E3 ubiquitin ligases plays an important role in cancer development. In this study, we provide experimental evidence that a RING-finger-containing protein RNF13 is an ER/Golgi membrane-associated E3 ubiquitin ligase and its RING finger domain is required for the ubiquitin iigase activity, lmmunohistochemical analysis of pancreatic ductal adenocarcinoma (PDAC) and paracancerous normal tissues from 72 patients documented RNF13 over-expression in 30 tumor samples (41.7%, 30/72), and its expression was significantly associated with histological grading (P= 0.024). In addition, RNFI3 was detected in precancerous lesions: tubular complexes in chronic pancreatitis (CP) and pancreatic intraepithelial neoplasia (PanlN) (79.3%, 23/29 and 62.8%, 22/35, respectively). Moreover, RNF13 staining was significantly correlated with Tenascin-C expression (P = 0.004) in PDAC samples, further supporting the role of RNF13 in cancer progression. Over-expression of wild type but not RING domain-mutant RNF13 in pancreatic MiaPaca-2 cancer cells increased invasive potential and gelatinolytic activity by matrix metalloproteinase-9. Taken together, these findings reveal that RNF13 is a novel E3 ubiquitin ligase involved in pancreatic carcinogenesis; ubiqui-tin-mediated modification of proteins by RNF13 may participate in pancreatic cancer development.

  9. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

    Science.gov (United States)

    Stewart, Emerson V; Nwosu, Christine C; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W; Krogan, Nevan J; Espenshade, Peter J

    2011-04-22

    Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

  10. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors

    NARCIS (Netherlands)

    Koo, B.K.; Spit, M.; Jordens, I.; Low, T.Y.; Stange, D.E.; van de Wetering, M.; van Es, J.H.; Mohammed, S.; Heck, A.J.R.; Maurice, M.M.; Clevers, H.

    2012-01-01

    LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes en

  11. Ancient origin of animal U-box ubiquitin ligases

    Directory of Open Access Journals (Sweden)

    Marín Ignacio

    2010-10-01

    Full Text Available Abstract Background The patterns of emergence and diversification of the families of ubiquitin ligases provide insights about the evolution of the eukaryotic ubiquitination system. U-box ubiquitin ligases (UULs are proteins characterized by containing a peculiar protein domain known as U box. In this study, the origin of the animal UUL genes is described. Results Phylogenetic and structural data indicate that six of the seven main UUL-encoding genes found in humans (UBE4A, UBE4B, UIP5, PRP19, CHIP and CYC4 were already present in the ancestor of all current metazoans and the seventh (WDSUB1 is found in placozoans, cnidarians and bilaterians. The fact that only 4 - 5 genes orthologous to the human ones are present in the choanoflagellate Monosiga brevicollis suggests that several animal-specific cooptions of the U box to generate new genes occurred. Significantly, Monosiga contains five additional UUL genes that are not present in animals. One of them is also present in distantly-related protozoans. Along animal evolution, losses of UUL-encoding genes are rare, except in nematodes, which lack three of them. These general patterns are highly congruent with those found for other two families (RBR, HECT of ubiquitin ligases. Conclusions Finding that the patterns of emergence, diversification and loss of three unrelated families of ubiquitin ligases (RBR, HECT and U-box are parallel indicates that there are underlying, linage-specific evolutionary forces shaping the complexity of the animal ubiquitin system.

  12. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis.

    Science.gov (United States)

    Maier, Alexander; Schrader, Andrea; Kokkelink, Leonie; Falke, Christian; Welter, Bastian; Iniesto, Elisa; Rubio, Vicente; Uhrig, Joachim F; Hülskamp, Martin; Hoecker, Ute

    2013-05-01

    Anthocyanins are natural pigments that accumulate only in light-grown and not in dark-grown Arabidopsis plants. Repression of anthocyanin accumulation in darkness requires the CONSTITUTIVELY PHOTOMORPHOGENIC1/SUPPRESSOR OF PHYA-105 (COP1/SPA) ubiquitin ligase, as cop1 and spa mutants produce anthocyanins also in the dark. Here, we show that COP1 and SPA proteins interact with the myeloblastosis (MYB) transcription factors PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP)1 and PAP2, two members of a small protein family that is required for anthocyanin accumulation and for the expression of structural genes in the anthocyanin biosynthesis pathway. The increased anthocyanin levels in cop1 mutants requires the PAP1 gene family, indicating that COP1 functions upstream of the PAP1 gene family. PAP1 and PAP2 proteins are degraded in the dark and this degradation is dependent on the proteasome and on COP1. Hence, the light requirement for anthocyanin biosynthesis results, at least in part, from the light-mediated stabilization of PAP1 and PAP2. Consistent with this conclusion, moderate overexpression of PAP1 leads to an increase in anthocyanin levels only in the light and not in darkness. Here we show that SPA genes are also required for reducing PAP1 and PAP2 transcript levels in dark-grown seedlings. Taken together, these results indicate that the COP1/SPA complex affects PAP1 and PAP2 both transcriptionally and post-translationally. Thus, our findings have identified mechanisms via which the COP1/SPA complex controls anthocyanin levels in Arabidopsis that may be useful for applications in biotechnology directed towards increasing anthocyanin content in plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  13. Self-clearance mechanism of mitochondrial E3 ligase MARCH5 contributes to mitochondria quality control.

    Science.gov (United States)

    Kim, Song-Hee; Park, Yong-Yea; Yoo, Young-Suk; Cho, Hyeseong

    2016-01-01

    MARCH5, a mitochondrial E3 ubiquitin ligase, controls mitochondrial dynamics proteins and misfolded proteins, and has been proposed to play a role in mitochondria quality control. However, it remains unclear how mutant MARCH5 found in cancer tissues is removed from cells. Here, we show that mutation in the MARCH5 ligase domain increased its half-life fourfold, resulting in a drastic increase in its protein level. Abnormal accumulation of the E3 ligase-defective MARCH5 mutants MARCH5(H43W) and MARCH5(C65/68S) was diminished by overexpression of active MARCH5(WT) ; the mutant proteins were degraded through the ubiquitin-proteasome pathway. Coimmunoprecipitation revealed that MARCH5 forms homodimers, and that substitution of Gly to Leu at the first putative GxxxG dimerization motif, but not the second, resulted in a loss of dimeric interaction. Moreover, overexpression of the dimerization-defective mutant MARCH5(4GL) could not decrease the level of accumulated MARCH5(H43W) , suggesting that dimerization of MARCH5 is necessary for self-clearance. Abnormal accumulation of MARCH5(H43W) and mitochondrial hyperfusion led to NF-ĸB activation, which was suppressed by overexpression of MARCH5(WT) . Together, the data reveal a self-protective mechanism involving MARCH5, which can target its own dysfunctional mutant for degradation in order to maintain mitochondrial homeostasis.

  14. An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

    KAUST Repository

    Lin, Xiao-Li

    2016-04-29

    COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.

  15. The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

    Science.gov (United States)

    Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

    2017-06-01

    Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene (CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer.Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR. ©2017 American Association for Cancer Research.

  16. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Xiaolian Zhang

    Full Text Available Ariadne (ARI subfamily of RBR (Ring Between Ring fingers proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L. Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2-4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.

  17. Overexpression of a soybean ariadne-like ubiquitin ligase gene GmARI1 enhances aluminum tolerance in Arabidopsis.

    Science.gov (United States)

    Zhang, Xiaolian; Wang, Ning; Chen, Pei; Gao, Mengmeng; Liu, Juge; Wang, Yufeng; Zhao, Tuanjie; Li, Yan; Gai, Junyi

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2-4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.

  18. The prolific ATL family of RING-H2 ubiquitin ligases.

    Science.gov (United States)

    Guzmán, Plinio

    2012-08-01

    An abundant class of E3 ubiquitin ligases encodes the RING-finger domain. The RING finger binds to the E2 ubiquitin-conjugating enzyme and brings together both the E2 and substrate. It is predicted that 477 RING finger E3 ligases exist in Arabidopsis thaliana. A particular family among them, named Arabidopsis Tóxicos en Levadura (ATL), consists of 91 members that contain the RING-H2 variation and a hydrophobic domain located at the N-terminal end. Transmembrane E3 ligases are important in several biological processes. For instance, some transmembrane RING finger E3 ligases are main participants in the endoplasmic reticulum-associated degradation pathway that targets misfolded proteins. Functional analysis of a number of ATLs has shown that some of them regulate distinct pathways in plants. Several ATLs have been shown to participate in defense responses, while others play a role in the regulation of the carbon/nitrogen response during post-germinative seedling growth transition, in the regulation of cell death during root development, in endosperm development, or in the transition to flowering under short day conditions. The ATL family has also been instrumental in evolution studies for showing how gene families are expanded in plant genomes.

  19. Post-translational control of IL-1β via the human papillomavirus type 16 E6 oncoprotein: a novel mechanism of innate immune escape mediated by the E3-ubiquitin ligase E6-AP and p53.

    Directory of Open Access Journals (Sweden)

    Martina Niebler

    Full Text Available Infections with high-risk human papillomaviruses (HPVs are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1

  20. Post-Translational Control of IL-1β via the Human Papillomavirus Type 16 E6 Oncoprotein: A Novel Mechanism of Innate Immune Escape Mediated by the E3-Ubiquitin Ligase E6-AP and p53

    Science.gov (United States)

    Niebler, Martina; Qian, Xu; Höfler, Daniela; Kogosov, Vlada; Kaewprag, Jittranan; Kaufmann, Andreas M.; Ly, Regina; Böhmer, Gerd; Zawatzky, Rainer; Rösl, Frank; Rincon-Orozco, Bladimiro

    2013-01-01

    Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1β towards cervical

  1. The APC/C Ubiquitin Ligase: from Cell Biology to Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Clara ePenas

    2012-01-01

    Full Text Available The ubiquitin proteasome system (UPS is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5 kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1-Cullin-F-box proteins (SCF ubiquitin ligases and the Anaphase Promoting Complex/cyclosome (APC/C are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, thus underscoring its possible contribution to transformation. We will also put forth the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

  2. A palmitoylated RING finger ubiquitin ligase and its homologue in the brain membranes.

    Science.gov (United States)

    Araki, Kazuaki; Kawamura, Meiko; Suzuki, Toshiaki; Matsuda, Noriyuki; Kanbe, Daiji; Ishii, Kyoko; Ichikawa, Tomio; Kumanishi, Toshiro; Chiba, Tomoki; Tanaka, Keiji; Nawa, Hiroyuki

    2003-08-01

    Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).

  3. Tyrosine phosphorylation of NEDD4 activates its ubiquitin ligase activity.

    Science.gov (United States)

    Persaud, Avinash; Alberts, Philipp; Mari, Sara; Tong, Jiefei; Murchie, Ryan; Maspero, Elena; Safi, Frozan; Moran, Michael F; Polo, Simona; Rotin, Daniela

    2014-10-07

    Ligand binding to the receptor tyrosine kinase fibroblast growth factor (FGF) receptor 1 (FGFR1) causes dimerization and activation by transphosphorylation of tyrosine residues in the kinase domain. FGFR1 is ubiquitylated by the E3 ligase NEDD4 (also known as NEDD4-1), which promotes FGFR1 internalization and degradation. Although phosphorylation of FGFR1 is required for NEDD4-dependent endocytosis, NEDD4 directly binds to a nonphosphorylated region of FGFR1. We found that activation of FGFR1 led to activation of c-Src kinase-dependent tyrosine phosphorylation of NEDD4, enhancing the ubiquitin ligase activity of NEDD4. Using mass spectrometry, we identified several FGF-dependent phosphorylated tyrosines in NEDD4, including Tyr(43) in the C2 domain and Tyr(585) in the HECT domain. Mutating these tyrosines to phenylalanine to prevent phosphorylation inhibited FGF-dependent NEDD4 activity and FGFR1 endocytosis and enhanced cell proliferation. Mutating the tyrosines to glutamic acid to mimic phosphorylation enhanced NEDD4 activity. Moreover, the NEDD4 C2 domain bound the HECT domain, and the presence of phosphomimetic mutations inhibited this interaction, suggesting that phosphorylation of NEDD4 relieves an inhibitory intra- or intermolecular interaction. Accordingly, activation of FGFR1 was not required for activation of NEDD4 that lacked its C2 domain. Activation of c-Src by epidermal growth factor (EGF) also promoted tyrosine phosphorylation and enhanced the activity of NEDD4. Thus, we identified a feedback mechanism by which receptor tyrosine kinases promote catalytic activation of NEDD4 and that may represent a mechanism of receptor crosstalk.

  4. Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

    Science.gov (United States)

    Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

    2015-02-01

    Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  5. Ubiquitin ligase gene neurl3 plays a role in spermatogenesis of half-smooth tongue sole (Cynoglossus semilaevis) by regulating testis protein ubiquitination.

    Science.gov (United States)

    Xu, Wenteng; Li, Hailong; Dong, Zhongdian; Cui, Zhongkai; Zhang, Ning; Meng, Liang; Zhu, Ying; Liu, Yang; Li, Yangzhen; Guo, Hua; Ma, Jialu; Wei, Zhanfei; Zhang, Nianwei; Yang, Yingming; Chen, Songlin

    2016-10-30

    E3 ubiquitin ligases are a large gene family that plays a diversity of roles in spermatogenesis. In this study, the functional characterization of a neuralized E3 ubiquitin protein ligase 3 (neurl3) revealed its potential participation in spermatogenesis. Firstly, we found that neurl3 exhibited male-biased transcription and that its translation was predominant in testis germ cells. The knockdown of neurl3 by RNA interference caused increased transcription of spermatogenesis-related genes. These results corroborate previous studies indicating a role for neurl3 in spermatogenesis. Moreover, the levels of neurl3 transcription and testis protein ubiquitination were closely correlated. Based on these findings, we speculate that neurl3 modulates testis protein ubiquitination in a dosage-dependent manner and that this influences spermatogenesis.

  6. Novel roles of Skp2 E3 ligase in cellular senescence, cancer progression, and metastasis

    Institute of Scientific and Technical Information of China (English)

    Guocan Wang; Chia-Hsin Chan; Yuan Gao; Hui-Kuan Lin

    2012-01-01

    S-phase kinase-associated protein 2 (Skp2) belongs to the F-box protein family.It is a component of the SCF E3 ubiquitin ligase complex.Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation,including cyclin-dependent kinase inhibitor p27.Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers.This review discusses the recent discoveries on the novel roles of Skp2 in regulating cellular senescence,cancer progression,and metastasis,as well as the therapeutic potential of targeting Skp2 for human cancer treatment.

  7. The pineal gland: A model for adrenergic modulation of ubiquitin ligases

    Science.gov (United States)

    Liu, Wenjun; Reiter, Russel J.

    2017-01-01

    Introduction A recent study of the pineal gland of the rat found that the expression of more than 3000 genes showed significant day/night variations (The Hartley dataset). The investigators of this report made available a supplemental table in which they tabulated the expression of many genes that they did not discuss, including those coding for components of the ubiquitin proteasome system. Herein we identify the genes of the ubiquitin proteasome system whose expression were significantly influenced by environmental lighting in the Hartley dataset, those that were stimulated by DBcAMP in pineal glands in culture, and those that were stimulated by norepinephrine. Purpose Using the Ubiquitin and Ubiquitin-like Conjugation Database (UUCA) we identified ubiquitin ligases and conjugases, and deubiquitinases in the Hartley dataset for the purpose of determining whether expression of genes of the ubiquitin proteasome pathway were significantly influenced by day/night variations and if these variations were regulated by autonomic innervation of the pineal gland from the superior cervical ganglia. Methods In the Hartley experiments pineal glands groups of rats sacrificed during the day and groups sacrificed during the night were examined for gene expression. Additional groups of rats had their superior cervical ganglia removed surgically or surgically decentralized and the pineal glands likewise examined for gene expression. Results The genes with at least a 2-fold day/night significant difference in expression included genes for 5 ubiquitin conjugating enzymes, genes for 58 ubiquitin E3 ligases and genes for 6 deubiquitinases. A 35-fold day/night difference was noted in the expression of the gene Sik1, which codes for a protein containing both an ubiquitin binding domain (UBD) and an ubiquitin-associated (UBA) domain. Most of the significant differences in these genes were prevented by surgical removal, or disconnection, of the superior cervical ganglia, and most were

  8. Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation.

    Science.gov (United States)

    Stolz, Alexandra; Besser, Stefanie; Hottmann, Heike; Wolf, Dieter H

    2013-09-17

    Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.

  9. Deficiency for the ubiquitin ligase UBE3B in a blepharophimosis-ptosis-intellectual-disability syndrome.

    Science.gov (United States)

    Basel-Vanagaite, Lina; Dallapiccola, Bruno; Ramirez-Solis, Ramiro; Segref, Alexandra; Thiele, Holger; Edwards, Andrew; Arends, Mark J; Miró, Xavier; White, Jacqueline K; Désir, Julie; Abramowicz, Marc; Dentici, Maria Lisa; Lepri, Francesca; Hofmann, Kay; Har-Zahav, Adi; Ryder, Edward; Karp, Natasha A; Estabel, Jeanne; Gerdin, Anna-Karin B; Podrini, Christine; Ingham, Neil J; Altmüller, Janine; Nürnberg, Gudrun; Frommolt, Peter; Abdelhak, Sonia; Pasmanik-Chor, Metsada; Konen, Osnat; Kelley, Richard I; Shohat, Mordechai; Nürnberg, Peter; Flint, Jonathan; Steel, Karen P; Hoppe, Thorsten; Kubisch, Christian; Adams, David J; Borck, Guntram

    2012-12-07

    Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.

  10. Structural basis for c-KIT inhibition by the suppressor of cytokine signaling 6 (SOCS6) ubiquitin ligase

    DEFF Research Database (Denmark)

    Zadjali, Fahad; Pike, Ashley C W; Vesterlund, Mattias

    2011-01-01

    of cytokine signaling 6 (SOCS6) is a member of the SOCS family of E3 ubiquitin ligases that can interact with c-KIT and suppress c-KIT-dependent pathways. Here, we analyzed the molecular mechanisms that determine SOCS6 substrate recognition. Our results show that the SH2 domain of SOCS6 is essential for its...... to substrate residue position pY+6 and envelopes the c-KIT phosphopeptide with a large BG loop insertion that contributes significantly to substrate interaction. We demonstrate that SOCS6 has ubiquitin ligase activity toward c-KIT and regulates c-KIT protein turnover in cells. Our data support a role of SOCS6...... as a feedback inhibitor of SCF-dependent signaling and provides molecular data to account for target specificity within the SOCS family of ubiquitin ligases....

  11. Classificati,Expression Patter,and E3 Ligase Activity Assay of Rice U-Box-Containing Proteins

    Institute of Scientific and Technical Information of China (English)

    Li-Rong Zeng; Chan Ho Park; R.C.Venu; Julian Gough; Guo-Liang Wang

    2008-01-01

    Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification.The U-box is a recently identified,ubiquitin ligase activityrelated protein domain that shows greater presence in plants than in other organisms.In this study,we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms.Most of the U-box protein genes are expressed,as supported by the identification of their corresponding expressed sequence tags (ESTs),full-length cDNAs,or massively parallel signature sequencing(MPSS)tags.Using the same algorithms,we identified 61 U-box proteins from the Arabidopsis genome.The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions.Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations.The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins.Cell death assay using the rice protoplast system suggests that one rice U-box gene,OsPU851,might act as a negative regulator of cell death signaling.In addition,the selected U-box proteins were found to be functional E3 ubiquitin ligases.The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

  12. The MLLE domain of the ubiquitin ligase UBR5 binds to its catalytic domain to regulate substrate binding.

    Science.gov (United States)

    Muñoz-Escobar, Juliana; Matta-Camacho, Edna; Kozlov, Guennadi; Gehring, Kalle

    2015-09-11

    E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity.

  13. TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

    DEFF Research Database (Denmark)

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke;

    2016-01-01

    , allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes......Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks...... ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced...

  14. Human DNA Ligase I Interacts with and Is Targeted for Degradation by the DCAF7 Specificity Factor of the Cul4-DDB1 Ubiquitin Ligase Complex.

    Science.gov (United States)

    Peng, Zhimin; Liao, Zhongping; Matsumoto, Yoshihiro; Yang, Austin; Tomkinson, Alan E

    2016-10-14

    The synthesis, processing, and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and coordinates the activity of several DNA replication proteins, including the enzymes flap endonuclease 1 (FEN-1) and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. Although it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Because DNA ligase I has been reported to be ubiquitylated, we used a proteomic approach to map ubiquitylation sites and screen for DNA ligase I-associated E3 ubiquitin ligases. We identified three ubiquitylated lysine residues and showed that DNA ligase I interacts with and is targeted for ubiquitylation by DCAF7, a specificity factor for the Cul4-DDB1 complex. Notably, knockdown of DCAF7 reduced the degradation of DNA ligase I in response to inhibition of proliferation and replacement of ubiquitylated lysine residues reduced the in vitro ubiquitylation of DNA ligase I by Cul4-DDB1 and DCAF7. In contrast, a different E3 ubiquitin ligase regulates FEN-1 turnover. Thus, although the expression of many of the genes encoding DNA replication proteins is coordinately regulated, our studies reveal that different mechanisms are involved in the turnover of these proteins.

  15. RING finger E3 ligase PPP1R11 regulates TLR2 signaling and innate immunity

    Science.gov (United States)

    McKelvey, Alison C; Lear, Travis B; Dunn, Sarah R; Evankovich, John; Londino, James D; Bednash, Joseph S; Zhang, Yingze; McVerry, Bryan J; Liu, Yuan; Chen, Bill B

    2016-01-01

    Toll-like receptor 2 (TLR2) is a pattern recognition receptor that recognizes many types of PAMPs that originate from gram-positive bacteria. Here we describe a novel mechanism regulating TLR2 protein expression and subsequent cytokine release through the ubiquitination and degradation of the receptor in response to ligand stimulation. We show a new mechanism in which an uncharacterized RING finger E3 ligase, PPP1R11, directly ubiquitinates TLR2 both in vitro and in vivo, which leads to TLR2 degradation and disruption of the signaling cascade. Lentiviral gene transfer or knockdown of PPP1R11 in mouse lungs significantly affects lung inflammation and the clearance of Staphylococcus aureus. There is a negative correlation between PPP1R11 and TLR2 levels in white blood cell samples isolated from patients with Staphylococcus aureus infections. These results suggest that PPP1R11 plays an important role in regulating innate immunity and gram-positive bacterial clearance by functioning, in part, through the ubiquitination and degradation of TLR2. DOI: http://dx.doi.org/10.7554/eLife.18496.001 PMID:27805901

  16. Crystal structure of the substrate-recognition domain of the Shigella E3 ligase IpaH9.8.

    Science.gov (United States)

    Takagi, Kenji; Kim, Minsoo; Sasakawa, Chihiro; Mizushima, Tsunehiro

    2016-04-01

    Infectious diseases caused by bacteria have significant impacts on global public health. During infection, pathogenic bacteria deliver a variety of virulence factors, called effectors, into host cells. The Shigella effector IpaH9.8 functions as an ubiquitin ligase, ubiquitinating the NF-κB essential modulator (NEMO)/IKK-γ to inhibit host inflammatory responses. IpaH9.8 contains leucine-rich repeats (LRRs) involved in substrate recognition and an E3 ligase domain. To elucidate the structural basis of the function of IpaH9.8, the crystal structure of the LRR domain of Shigella IpaH9.8 was determined and this structure was compared with the known structures of other IpaH family members. This model provides insights into the structural features involved in substrate specificity.

  17. The RING finger domain E3 ubiquitin ligases BRCA1 and the RNF20/RNF40 complex in global loss of the chromatin mark histone H2B monoubiquitination (H2Bub1) in cell line models and primary high-grade serous ovarian cancer.

    Science.gov (United States)

    Dickson, Kristie-Ann; Cole, Alexander J; Gill, Anthony J; Clarkson, Adele; Gard, Gregory B; Chou, Angela; Kennedy, Catherine J; Henderson, Beric R; Fereday, Sian; Traficante, Nadia; Alsop, Kathryn; Bowtell, David D; deFazio, Anna; Clifton-Bligh, Roderick; Marsh, Deborah J

    2016-12-15

    Enzymatic factors driving cancer-associated chromatin remodelling are of increasing interest as the role of the cancer epigenome in gene expression and DNA repair processes becomes elucidated. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) is a central histone modification that functions in histone cross-talk, transcriptional elongation, DNA repair, maintaining centromeric chromatin and replication-dependent histone mRNA 3'-end processing, as well as being required for the differentiation of stem cells. The loss of global H2Bub1 is seen in a number of aggressive malignancies and has been linked to tumour progression and/or a poorer prognosis in some cancers. Here, we analyse a large cohort of high-grade serous ovarian cancers (HGSOC) and show loss of global H2Bub1 in 77% (313 of 407) of tumours. Loss of H2Bub1 was seen at all stages (I-IV) of HGSOC, indicating it is a relatively early epigenomic event in this aggressive malignancy. Manipulation of key H2Bub1 E3 ubiquitin ligases, RNF20, RNF40 and BRCA1, in ovarian cancer cell line models modulated H2Bub1 levels, indicative of the role of these RING finger ligases in monoubiquitination of H2Bub1 in vitro. However, in primary HGSOC, loss of RNF20 protein expression was identified in just 6% of tumours (26 of 424) and did not correlate with global H2Bub1 loss. Similarly, germline mutation of BRCA1 did not show a correlation with the global H2Bub1 loss. We conclude that the regulation of tumour-associated H2Bub1 levels is complex. Aberrant expression of alternative histone-associated 'writer' or 'eraser' enzymes are likely responsible for the global loss of H2Bub1 seen in HGSOC. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. MdCOP1 Ubiquitin E3 Ligases Interact with MdMYB1 to Regulate Light-Induced Anthocyanin Biosynthesis and Red Fruit Coloration in Apple1[W][OA

    Science.gov (United States)

    Li, Yuan-Yuan; Mao, Ke; Zhao, Cheng; Zhao, Xian-Yan; Zhang, Hua-Lei; Shu, Huai-Rui; Hao, Yu-Jin

    2012-01-01

    MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYB1 interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vector-based transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species. PMID:22855936

  19. Blocking an N-terminal acetylation–dependent protein interaction inhibits an E3 ligase

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Daniel C.; Hammill, Jared T.; Min, Jaeki; Rhee, David Y.; Connelly, Michele; Sviderskiy, Vladislav O.; Bhasin, Deepak; Chen, Yizhe; Ong, Su-Sien; Chai, Sergio C.; Goktug, Asli N.; Huang, Guochang; Monda, Julie K.; Low, Jonathan; Kim, Ho Shin; Paulo, Joao A.; Cannon, Joe R.; Shelat, Anang A.; Chen, Taosheng; Kelsall, Ian R.; Alpi, Arno F.; Pagala, Vishwajeeth; Wang, Xusheng; Peng, Junmin; Singh , Bhuvanesh; Harper, J. Wade; Schulman, Brenda A.; Guy, R. Kip (MSKCC); (Dundee); (SJCH); (Harvard-Med); (MXPL)

    2017-06-05

    N-terminal acetylation is an abundant modification influencing protein functions. Because ~80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.

  20. DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact.

    Science.gov (United States)

    Cao, Zhifang; Huett, Alan; Kuballa, Petric; Giallourakis, Cosmas; Xavier, Ramnik J

    2008-01-01

    PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.

  1. The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated β-catenin by sequestering TCF4 to the nuclear membrane

    NARCIS (Netherlands)

    Loregger, Anke; Grandl, Martina; Mejías-Luque, Raquel; Allgäuer, Michael; Degenhart, Kathrin; Haselmann, Verena; Oikonomou, Christina; Hatzis, Pantelis; Janssen, Klaus Peter; Nitsche, Ulrich; Gradl, Dietmar; Van Den Broek, Olaf; Destree, Olivier; Ulm, Kurt; Neumaier, Michael; Kalali, Behnam; Jung, Andreas; Varela, Ignacio; Schmid, Roland M.; Rad, Roland; Busch, Dirk H.; Gerhard, Markus

    2015-01-01

    Given its fundamental role in development and cancer, the Wnt-β-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of t

  2. Expansion and diversification of BTL ring-H2 ubiquitin ligases in angiosperms: putative Rabring7/BCA2 orthologs.

    Science.gov (United States)

    Aguilar-Hernández, Victor; Medina, Juliana; Aguilar-Henonin, Laura; Guzmán, Plinio

    2013-01-01

    RING finger E3 ligases are components of the ubiquitin proteasome system (UPS) that mediate the transfer of ubiquitin to substrates. Single-subunit RING finger E3s binds the E2 ubiquitin-conjugating enzyme and contains recognition sequences for the substrate within the same polypeptide. Here we describe the characterization of a class of RING finger E3 ligases that is conserved among eukaryotes. This class encodes a RING-H2 domain related in sequence to the ATL RING-H2 domain, another class of E3 ligases, and a C2/C2 zing finger at the amino-terminus, formerly described as BZF. In viridiplantae (green algae and land plants), we designed this family as BTL for BZF ATLs. BTLs are putative orthologs of the mammalian Rabring7/BCA2 RING-H2 E3s that have expanded in angiosperms. They are found in numbers ranging from three to thirty-one, which is in contrast to the one to three members normally found in animals, fungi, and protists. Furthermore, the number of sequence LOGOs generated in angiosperms is four times greater than that in other eukaryotes. In contrast to ATLs, which show expansion by tandem duplication, tandemly duplicated BTLs are scarce. The mode of action of Rabring7/BCA2 and BTLs may be similar since both the Rabring7/BCA2 BZF and the ath|BTL4 BZF are likely to mediate the binding of ubiquitin. This study introduces valuable information on the evolution and domain structure of the Rabring7/BCA2/BTL class of E3 ligases which may be important for core eukaryotic genes.

  3. 骨形成蛋白7干预肝纤维化小鼠肝组织E3泛素连接酶Arkadia表达的实验研究%Effects of bone morphogenetic protein-7 therapy on E3 ubiquitin ligase expression in mouse liver with experimentally induced fibrosis

    Institute of Scientific and Technical Information of China (English)

    申春燕; 陈永平; 阳韬; 陆小蒟; 李春艳; 林镯; 宋梅

    2012-01-01

    均<0.01);与12周模型组相比,BMP-7干预组Arkadia、Smad7、TGF β1的蛋白质表达量明显降低(t值分别为23.438、11.667和42.889,P值均<0.01).结论 Arkadia在肝纤维化进展过程中呈上升趋势,BMP-7具有抗肝纤维化作用并可以抑制纤维化过程中Arkadia的表达.%Objective This study explored the dynamic expression of the E3 ubiquitin-protein ligase gene,Arkadia,in response to carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model and investigated the differential expression that occurs following treatment with the anti-fibrotic bone morphogenetic protein-7 (BMP-7).Methods Thirty healthy male imprinting control region (ICR) mice were randomly assigned to three groups:normal (control; n =6),CCl4-induced model group (model; n =18),and CCl4-induced model with BMP-7 treatment group (treatment; n =6).The model group was further divided into three subgroups (n =6 each) for analysis at 4,8 and 12 weeks after fibrosis induction.Liver fibrosis was induced by hypodermic injections of 60% CCl4/peanut oil (5 mL/kg)to the hind legs of mice two-times per week in alternating legs for a period of 12 weeks.At week 9,the treatment group of CC14-induced mice were given an intraperitoneal injection of BMP-7 (300 pg/g) simultaneously with that day's hypodermic injection of 60% CCl4/peanut oil,and then every other day for a period of four weeks.The pathological changes in liver tissues were observed after staining with hematoxylin-eosin (HE) and Masson's trichrome.Messenger RNA (mRNA) and protein expression of Arkadia in liver were evaluated using reverse transcription-polymerase chain reaction and immunohistochemistry and Western blotting,respectively.Results Mouse models of liver fibrosis were successfully established by CCl4 exposure.Arkadia,Stmad7 and TGF-β1 mRNA levels were up-regulated in the model group in a time-dependent manner (vs.control group),and BMP-7 treatment led to significant down-regulation of the CCl4-induced

  4. The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis.

    Science.gov (United States)

    Li, Wei; Ahn, Il-Pyung; Ning, Yuese; Park, Chan-Ho; Zeng, Lirong; Whitehill, Justin G A; Lu, Haibin; Zhao, Qingzhen; Ding, Bo; Xie, Qi; Zhou, Jian-Min; Dai, Liangying; Wang, Guo-Liang

    2012-05-01

    The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

  5. TMEM129 is a Derlin-1 associated ERAD E3 ligase essential for virus-induced degradation of MHC-I

    DEFF Research Database (Denmark)

    van den Boomen, Dick J H; Timms, Richard T; Grice, Guinevere L

    2014-01-01

    role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11......-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation......, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129-dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for "free" US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component...

  6. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA.

    Science.gov (United States)

    Zhong, Bo; Zhang, Lu; Lei, Caoqi; Li, Ying; Mao, Ai-Ping; Yang, Yan; Wang, Yan-Yi; Zhang, Xiao-Lian; Shu, Hong-Bing

    2009-03-20

    Viral infection activates transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and elicit innate antiviral response. MITA (also known as STING) has recently been identified as an adaptor that links virus-sensing receptors to IRF3 activation. Here, we showed that the E3 ubiquitin ligase RNF5 interacted with MITA in a viral-infection-dependent manner. Overexpression of RNF5 inhibited virus-triggered IRF3 activation, IFNB1 expression, and cellular antiviral response, whereas knockdown of RNF5 had opposite effects. RNF5 targeted MITA at Lys150 for ubiquitination and degradation after viral infection. Both MITA and RNF5 were located at the mitochondria and endoplasmic reticulum (ER) and viral infection caused their redistribution to the ER and mitochondria, respectively. We further found that virus-induced ubiquitination and degradation of MITA by RNF5 occurred at the mitochondria. These findings suggest that RNF5 negatively regulates virus-triggered signaling by targeting MITA for ubiquitination and degradation at the mitochondria.

  7. Antagonistic roles of ubiquitin ligase HEI10 and SUMO ligase RNF212 regulate meiotic recombination.

    Science.gov (United States)

    Qiao, Huanyu; Prasada Rao, H B D; Yang, Ye; Fong, Jared H; Cloutier, Jeffrey M; Deacon, Dekker C; Nagel, Kathryn E; Swartz, Rebecca K; Strong, Edward; Holloway, J Kim; Cohen, Paula E; Schimenti, John; Ward, Jeremy; Hunter, Neil

    2014-02-01

    Crossover recombination facilitates the accurate segregation of homologous chromosomes during meiosis. In mammals, poorly characterized regulatory processes ensure that every pair of chromosomes obtains at least one crossover, even though most recombination sites yield non-crossovers. Designation of crossovers involves selective localization of the SUMO ligase RNF212 to a minority of recombination sites, where it stabilizes pertinent factors such as MutSγ (ref. 4). Here we show that the ubiquitin ligase HEI10 (also called CCNB1IP1) is essential for this crossover/non-crossover differentiation process. In HEI10-deficient mice, RNF212 localizes to most recombination sites, and dissociation of both RNF212 and MutSγ from chromosomes is blocked. Consequently, recombination is impeded, and crossing over fails. In wild-type mice, HEI10 accumulates at designated crossover sites, suggesting that it also has a late role in implementing crossing over. As with RNF212, dosage sensitivity for HEI10 indicates that it is a limiting factor for crossing over. We suggest that SUMO and ubiquitin have antagonistic roles during meiotic recombination that are balanced to effect differential stabilization of recombination factors at crossover and non-crossover sites.

  8. FERM-dependent E3 ligase recognition is a conserved mechanism for targeted degradation of lipoprotein receptors.

    Science.gov (United States)

    Calkin, Anna C; Goult, Benjamin T; Zhang, Li; Fairall, Louise; Hong, Cynthia; Schwabe, John W R; Tontonoz, Peter

    2011-12-13

    The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.

  9. The Anaphase-Promoting Complex (APC ubiquitin ligase affects chemosensory behavior in C. elegans

    Directory of Open Access Journals (Sweden)

    Julia Wang

    2016-05-01

    Full Text Available The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS, which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143 of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and

  10. PUB13, a U-box/ARM E3 ligase, regulates plant defense, cell death, and flowering time.

    Science.gov (United States)

    Li, Wei; Dai, Liangying; Wang, Guo-Liang

    2012-08-01

    The ubiquitination pathway is involved in a variety of cellular processes in plant growth, development, and immune responses. However, the function of this pathway in connecting plant development and innate immunity is still largely unknown. Recently, we characterized the U-box/ARM E3 ubiquitin ligase PUB13, which regulates both immune responses and flowering time in Arabidopsis. Here, we show that the rice Spl11 gene can complement the cell death and flowering functions of PUB13 in the pub13 mutant. In addition, HFR1, which functions mainly in photomorphogenesis, was identified as one of the PUB13-interacting proteins through yeast two-hybrid screening and pull-down assays. Because the flowering phenotype of pub13 depends on photoperiod, we propose that PUB13 may regulate HFR1 to fine-tune photomorphogenesis and flowering time in Arabidopsis.

  11. Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1.

    Science.gov (United States)

    Blees, Johanna S; Bokesch, Heidi R; Rübsamen, Daniela; Schulz, Kathrin; Milke, Larissa; Bajer, Magdalena M; Gustafson, Kirk R; Henrich, Curtis J; McMahon, James B; Colburn, Nancy H; Schmid, Tobias; Brüne, Bernhard

    2012-01-01

    Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70(S6K1)-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.

  12. Erioflorin stabilizes the tumor suppressor Pdcd4 by inhibiting its interaction with the E3-ligase β-TrCP1.

    Directory of Open Access Journals (Sweden)

    Johanna S Blees

    Full Text Available Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70(S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin, it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target and HIF-1α (a pVHL-target, implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.

  13. Natural polymorphisms in C. elegans HECW-1 E3 ligase affect pathogen avoidance behaviour.

    Science.gov (United States)

    Chang, Howard C; Paek, Jennifer; Kim, Dennis H

    2011-11-16

    Heritable variation in behavioural traits generally has a complex genetic basis, and thus naturally occurring polymorphisms that influence behaviour have been defined only in rare instances. The isolation of wild strains of Caenorhabditis elegans has facilitated the study of natural genetic variation in this species and provided insights into its diverse microbial ecology. C. elegans responds to bacterial infection with conserved innate immune responses and, although lacking the immunological memory of vertebrate adaptive immunity, shows an aversive learning response to pathogenic bacteria. Here, we report the molecular characterization of naturally occurring coding polymorphisms in a C. elegans gene encoding a conserved HECT domain-containing E3 ubiquitin ligase, HECW-1. We show that two distinct polymorphisms in neighbouring residues of HECW-1 each affect C. elegans behavioural avoidance of a lawn of Pseudomonas aeruginosa. Neuron-specific rescue and ablation experiments and genetic interaction analysis indicate that HECW-1 functions in a pair of sensory neurons to inhibit P. aeruginosa lawn avoidance behaviour through inhibition of the neuropeptide receptor NPR-1 (ref. 10), which we have previously shown promotes P. aeruginosa lawn avoidance behaviour. Our data establish a molecular basis for natural variation in a C. elegans behaviour that may undergo adaptive changes in response to microbial pathogens.

  14. Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination

    Science.gov (United States)

    Ziolkowski, Piotr A.; Underwood, Charles J.; Lambing, Christophe; Martinez-Garcia, Marina; Lawrence, Emma J.; Ziolkowska, Liliana; Griffin, Catherine; Choi, Kyuha; Franklin, F. Chris H.; Martienssen, Robert A.; Henderson, Ian R.

    2017-01-01

    During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection. PMID:28223312

  15. Myc protein is stabilized by suppression of a novel E3 ligase complex in cancer cells

    Science.gov (United States)

    Choi, Seung H.; Wright, Jason B.; Gerber, Scott A.; Cole, Michael D.

    2010-01-01

    Rapid Myc protein turnover is critical for maintaining basal levels of Myc activity in normal cells and a prompt response to changing growth signals. We characterize a new Myc-interacting factor, TRPC4AP (transient receptor potential cation channel, subfamily C, member 4-associated protein)/TRUSS (tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein), which is the receptor for a DDB1 (damage-specific DNA-binding protein 1)–CUL4 (Cullin 4) E3 ligase complex for selective Myc degradation through the proteasome. TRPC4AP/TRUSS binds specifically to the Myc C terminus and promotes its ubiquitination and destruction through the recognition of evolutionarily conserved domains in the Myc N terminus. TRPC4AP/TRUSS suppresses Myc-mediated transactivation and transformation in a dose-dependent manner. Finally, we found that TRPC4AP/TRUSS expression is strongly down-regulated in most cancer cell lines, leading to Myc protein stabilization. These studies identify a novel pathway targeting Myc degradation that is suppressed in cancer cells. PMID:20551172

  16. Mutation in SUMO E3 ligase, SIZ1, disrupts the mature female gametophyte in Arabidopsis

    KAUST Repository

    Ling, Yu

    2012-01-09

    Female gametophyte is the multicellular haploid structure that can produce embryo and endosperm after fertilization, which has become an attractive model system for investigating molecular mechanisms in nuclei migration, cell specification, cell-to-cell communication and many other processes. Previous reports found that the small ubiquitin-like modifier (SUMO) E3 ligase, SIZ1, participated in many processes depending on particular target substrates and suppression of salicylic acid (SA) accumulation. Here, we report that SIZ1 mediates the reproductive process. SIZ1 showed enhanced expression in female organs, but was not detected in the anther or pollen. A defect in the siz1-2 maternal source resulted in reduced seed-set regardless of high SA concentration within the plant. Moreover, aniline blue staining and scanning electron microscopy revealed that funicular and micropylar pollen tube guidance was arrested in siz1-2 plants. Some of the embryo sacs of ovules in siz1-2 were also disrupted quickly after stage FG7. There was no significant affects of the siz1-2 mutation on expression of genes involved in female gametophyte development- or pollen tube guidance in ovaries. Together, our results suggest that SIZ1 sustains the stability and normal function of the mature female gametophyte which is necessary for pollen tube guidance. © 2012 Ling et al.

  17. Ubiquitin Ligase HUWE1 Regulates Axon Branching through the Wnt/beta-Catenin Pathway in a Drosophila Model for Intellectual Disability

    NARCIS (Netherlands)

    Vandewalle, J.; Langen, M.; Zschaetzsch, M.; Nijhof, B.; Kramer, J.M.; Brems, H.; Bauters, M.; Lauwers, E.; Srahna, M.; Marynen, P.; Verstreken, P.; Schenck, A.; Hassan, B.A.; Froyen, G.

    2013-01-01

    We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developi

  18. The ubiquitin ligase STUB1 regulates stability and activity of RUNX1 and RUNX1-RUNX1T1.

    Science.gov (United States)

    Yonezawa, Taishi; Takahashi, Hirotaka; Shikata, Shiori; Liu, Xiaoxiao; Tamura, Moe; Asada, Shuhei; Fukushima, Tsuyoshi; Fukuyama, Tomofusa; Tanaka, Yosuke; Sawasaki, Tatsuya; Kitamura, Toshio; Goyama, Susumu

    2017-07-28

    RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. Disruption of RUNX1 activity has been implicated in the development of hematopoietic neoplasms. Chromosomal translocations involving the RUNX1 gene are associated with several types of leukemia, including acute myeloid leukemia driven by a leukemogenic fusion protein RUNX1-RUNX1T1. Previous studies have shown that RUNX1 is an unstable protein and is subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. However, the precise mechanisms of RUNX1 ubiquitination have not been fully understood. Furthermore, much less is known about the mechanisms to regulate the stability of RUNX1-RUNX1T1. In this study, we identified several RUNX1-interacting E3 ubiquitin ligases using a novel high-throughput binding assay. Among them, we found that STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus. Immunofluorescence analyses revealed that the STUB1-induced ubiquitination also promoted nuclear export of RUNX1, which probably contributes to the reduced transcriptional activity of RUNX1 in STUB1-overexpressing cells. STUB1 also induced ubiquitination of RUNX1-RUNX1T1 and down-regulated its expression. Importantly, STUB1 overexpression showed a substantial growth-inhibitory effect in myeloid leukemia cells that harbor RUNX1-RUNX1T1, whereas it showed only a marginal effect in other non-RUNX1-RUNX1T1 leukemia cells and normal human cord blood cells. Taken together, these data suggest that the E3 ubiquitin ligase STUB1 is a negative regulator of both RUNX1 and RUNX1-RUNX1T1. Activation of STUB1 could be a promising therapeutic strategy for RUNX1-RUNX1T1 leukemia. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Subunit architecture of the Golgi Dsc E3 ligase required for sterol regulatory element-binding protein (SREBP) cleavage in fission yeast.

    Science.gov (United States)

    Lloyd, S Julie-Ann; Raychaudhuri, Sumana; Espenshade, Peter J

    2013-07-19

    The membrane-bound sterol regulatory element-binding protein (SREBP) transcription factors regulate lipogenesis in mammalian cells and are activated through sequential cleavage by the Golgi-localized Site-1 and Site-2 proteases. The mechanism of fission yeast SREBP cleavage is less well defined and, in contrast, requires the Golgi-localized Dsc E3 ligase complex. The Dsc E3 ligase consists of five integral membrane subunits, Dsc1 through Dsc5, and resembles membrane E3 ligases that function in endoplasmic reticulum-associated degradation. Using immunoprecipitation assays and blue native electrophoresis, we determined the subunit architecture for the complex of Dsc1 through Dsc5, showing that the Dsc proteins form subcomplexes and display defined connectivity. Dsc2 is a rhomboid pseudoprotease family member homologous to mammalian UBAC2 and a central component of the Dsc E3 ligase. We identified conservation in the architecture of the Dsc E3 ligase and the multisubunit E3 ligase gp78 in mammals. Specifically, Dsc1-Dsc2-Dsc5 forms a complex resembling gp78-UBAC2-UBXD8. Further characterization of Dsc2 revealed that its C-terminal UBA domain can bind to ubiquitin chains but that the Dsc2 UBA domain is not essential for yeast SREBP cleavage. Based on the ability of rhomboid superfamily members to bind transmembrane proteins, we speculate that Dsc2 functions in SREBP recognition and binding. Homologs of Dsc1 through Dsc4 are required for SREBP cleavage and virulence in the human opportunistic pathogen Aspergillus fumigatus. Thus, these studies advance our organizational understanding of multisubunit E3 ligases involved in endoplasmic reticulum-associated degradation and fungal pathogenesis.

  20. Structural Basis of Dimerization-dependent Ubiquitination by the SCFFbx4 Ubiquitin Ligase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Y.; Hao, B

    2010-01-01

    The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F-box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF{sup Fbx4} (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Fbx4 in complex with an adaptor protein Skp1 reveals an antiparallel dimer configuration in which the linker domain of Fbx4 interacts with the C-terminal substrate-binding domain of the other protomer, whereas the C-terminal domain of the protein adopts a compact {alpha}/{beta} fold distinct from those of known F-box proteins. Biochemical studies indicate that both the N-terminal domain and a loop connecting the linker and C-terminal domain of Fbx4 are critical for the dimerization and activation of the protein. Our findings provide a framework for understanding the role of F-box dimerization in the SCF-mediated ubiquitination reaction.

  1. PARAQUAT TOLERANCE3 Is an E3 Ligase That Switches off Activated Oxidative Response by Targeting Histone-Modifying PROTEIN METHYLTRANSFERASE4b.

    Directory of Open Access Journals (Sweden)

    Chao Luo

    2016-09-01

    Full Text Available Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3 as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance.

  2. Roles of the TRAF6 and Pellino E3 ligases in MyD88 and RANKL signaling.

    Science.gov (United States)

    Strickson, Sam; Emmerich, Christoph H; Goh, Eddy T H; Zhang, Jiazhen; Kelsall, Ian R; Macartney, Thomas; Hastie, C James; Knebel, Axel; Peggie, Mark; Marchesi, Francesco; Arthur, J Simon C; Cohen, Philip

    2017-04-25

    It is widely accepted that the essential role of TRAF6 in vivo is to generate the Lys63-linked ubiquitin (K63-Ub) chains needed to activate the "master" protein kinase TAK1. Here, we report that TRAF6 E3 ligase activity contributes to but is not essential for the IL-1-dependent formation of K63-Ub chains, TAK1 activation, or IL-8 production in human cells, because Pellino1 and Pellino2 generate the K63-Ub chains required for signaling in cells expressing E3 ligase-inactive TRAF6 mutants. The IL-1-induced formation of K63-Ub chains and ubiquitylation of IRAK1, IRAK4, and MyD88 was abolished in TRAF6/Pellino1/Pellino2 triple-knockout (KO) cells, but not in TRAF6 KO or Pellino1/2 double-KO cells. The reexpression of E3 ligase-inactive TRAF6 mutants partially restored IL-1 signaling in TRAF6 KO cells, but not in TRAF6/Pellino1/Pellino2 triple-KO cells. Pellino1-generated K63-Ub chains activated the TAK1 complex in vitro with similar efficiently to TRAF6-generated K63-Ub chains. The early phase of TLR signaling and the TLR-dependent secretion of IL-10 (controlled by IRAKs 1 and 2) was only reduced modestly in primary macrophages from knockin mice expressing the E3 ligase-inactive TRAF6[L74H] mutant, but the late-phase production of IL-6, IL-12, and TNFα (controlled only by the pseudokinase IRAK2) was abolished. RANKL-induced signaling in macrophages and the differentiation of bone marrow to osteoclasts was similar in TRAF6[L74H] and wild-type cells, explaining why the bone structure and teeth of the TRAF6[L74H] mice was normal, unlike TRAF6 KO mice. We identify two essential roles of TRAF6 that are independent of its E3 ligase activity.

  3. Actin Cytoskeletal Organization in Drosophila Germline Ring Canals Depends on Kelch Function in a Cullin-RING E3 Ligase.

    Science.gov (United States)

    Hudson, Andrew M; Mannix, Katelynn M; Cooley, Lynn

    2015-11-01

    The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and cross-links F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. We used targeted mutagenesis to investigate the mechanism of Kelch function. We tested a model in which Cul3-dependent degradation of Kelch is required for its function, but we found no evidence to support this hypothesis. However, we found that mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. We also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome.

  4. Zn-binding AZUL domain of human ubiquitin protein ligase Ube3A

    Energy Technology Data Exchange (ETDEWEB)

    Lemak, Alexander; Yee, Adelinda [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health Center, Department of Molecular Microbial and Structural Biology (United States); Dhe-Paganon, Sirano, E-mail: sirano.dhepaganon@utoronto.ca [University of Toronto, Structural Genomics Consortium (Canada); Arrowsmith, Cheryl H., E-mail: carrow@uhnresearch.ca [University of Toronto, and Northeast Structural Genomics Consortium, Ontario Cancer Institute, Campbell Family Cancer Research Institute and Department of Medical Biophysics (Canada)

    2011-09-15

    Ube3A (also referred to as E6AP for E6 Associated Protein) is a E3 ubiquitin-protein ligase implicated in the development of Angelman syndrome by controlling degradation of synaptic protein Arc and oncogenic papilloma virus infection by controlling degradation of p53. This article describe the solution NMR structure of the conserved N-terminal domain of human Ube3A (residues 24-87) that contains two residues (Cys44 and Arg62) found to be mutated in patients with Angelman syndrome. The structure of this domain adopts a novel Zn-binding fold we called AZUL (Amino-terminal Zn-finger of Ube3a Ligase). The AZUL domain has a helix-loop-helix architecture with a Zn ion coordinated by four Cys residues arranged in Cys-X{sub 4}-Cys-X{sub 4}-Cys-X{sub 28}-Cys motif. Three of the Zn-bound residues are located in a 23-residue long and well structured loop that connects two {alpha}-helicies.

  5. SUMO E3 Ligase AtMMS21 Regulates Drought Tolerance in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Shengchun Zhang; Yanli Qi; Ming Liu; ChengweiYang

    2013-01-01

    Post-translational modifications of proteins by small ubiquitin-like modifiers (SUMOs) play crucial roles in plant growth and development,and in stress responses.The MMS21 is a newly-identified Arabidopsis thaliana L.SUMO E3 ligase gene aside from the SIZ1,and its function requires further elucidation.Here,we show that MMS21 deficient plants display improved drought tolerance,and constitutive expression of MMS21 reduces drought tolerance.The expression of MMS21 was reduced by abscisic acid (ABA),polyethylene glycol (PEG) or drought stress.Under drought conditions,mms21 mutants showed the highest survival rate and the slowest water loss,and accumulated a higher level of free proline compared to wild-type (WT) and MMS21 over-expression plants.Stomatal aperture,seed germination and cotyledon greening analysis indicated that mms21 was hypersensitive to ABA.Molecular genetic analysis revealed that MMS21 deficiency led to elevated expression of a series of ABA-mediated stress-responsive genes,including COR15A,RD22,and P5CS1 The ABA and drought-induced stress-responsive genes,including RAB18,RD29A and RD29B,were inhibited by constitutive expression of MMS21.Moreover,ABA-induced accumulation of SUMO-protein conjugates was blocked in the mms21 mutant.We thus conclude that MMS21 plays a role in the drought stress response,likely through regulation of gene expression in an ABA-dependent pathway.

  6. The role of the e3 ligase cbl-B in murine dendritic cells.

    Directory of Open Access Journals (Sweden)

    Stephanie Wallner

    Full Text Available Dendritic cells (DCs are potent antigen-presenting cells with a promising potential in cancer immunotherapy. Cbl proteins are E3 ubiquitin ligases and have been implicated in regulating the functional activity of various immune cells. As an example, c-Cbl negatively affects DC activation. We here describe that another member of the Cbl-protein family (i.e. Cbl-b is highly expressed in murine bone-marrow-derived DCs (BMDCs. Differentiation of cblb-/- bone marrow mononuclear cells into classical BMDCs is unaltered, except enhanced induction of DEC-205 (CD205 expression. When tested in mixed-lymphocyte reaction (MLR, cblb-/- BMDCs exhibit increased allo-stimulatory capacity in vitro. BMDCs were next in vitro stimulated by various toll like receptor (TLR-agonists (LPS, Poly(I:C, CpG and exposed to FITC-labeled dextran. Upon TLR-stimulation, cblb-/- BMDCs produce higher levels of proinflammatory cytokines (IL-1α, IL-6 and TNF-α and exhibit a slightly higher level of FITC-dextran uptake. To further characterize the functional significance of cblb-/- BMDCs we tested them in antigen-specific T cell responses against ovalbumin (OVA protein and peptides, activating either CD8(+ OT-I or CD4(+ OT-II transgenic T cells. However, cblb-/- BMDCs are equally effective in inducing antigen-specific T cell responses when compared to wildtype BMDCs both in vitro and in vivo. The migratory capacity into lymph nodes during inflammation was similarly not affected by the absence of Cbl-b. In line with these observations, cblb-/- peptide-pulsed BMDCs are equally effective vaccines against OVA-expressing B16 tumors in vivo when compared to wildtype BMDCs. We conclude that in contrast to c-Cbl, Cbl-b plays only a limited role in the induction of Ag-specific T cell responses by murine BMDCs in vitro and in vivo.

  7. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    Science.gov (United States)

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  8. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.

    Science.gov (United States)

    Matyskiela, Mary E; Lu, Gang; Ito, Takumi; Pagarigan, Barbra; Lu, Chin-Chun; Miller, Karen; Fang, Wei; Wang, Nai-Yu; Nguyen, Derek; Houston, Jack; Carmel, Gilles; Tran, Tam; Riley, Mariko; Nosaka, Lyn'Al; Lander, Gabriel C; Gaidarova, Svetlana; Xu, Shuichan; Ruchelman, Alexander L; Handa, Hiroshi; Carmichael, James; Daniel, Thomas O; Cathers, Brian E; Lopez-Girona, Antonia; Chamberlain, Philip P

    2016-07-14

    Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.

  9. Iron-binding E3 ligase mediates iron response in plants by targeting basic helix-loop-helix transcription factors.

    Science.gov (United States)

    Selote, Devarshi; Samira, Rozalynne; Matthiadis, Anna; Gillikin, Jeffrey W; Long, Terri A

    2015-01-01

    Iron uptake and metabolism are tightly regulated in both plants and animals. In Arabidopsis (Arabidopsis thaliana), BRUTUS (BTS), which contains three hemerythrin (HHE) domains and a Really Interesting New Gene (RING) domain, interacts with basic helix-loop-helix transcription factors that are capable of forming heterodimers with POPEYE (PYE), a positive regulator of the iron deficiency response. BTS has been shown to have E3 ligase capacity and to play a role in root growth, rhizosphere acidification, and iron reductase activity in response to iron deprivation. To further characterize the function of this protein, we examined the expression pattern of recombinant ProBTS::β-GLUCURONIDASE and found that it is expressed in developing embryos and other reproductive tissues, corresponding with its apparent role in reproductive growth and development. Our findings also indicate that the interactions between BTS and PYE-like (PYEL) basic helix-loop-helix transcription factors occur within the nucleus and are dependent on the presence of the RING domain. We provide evidence that BTS facilitates 26S proteasome-mediated degradation of PYEL proteins in the absence of iron. We also determined that, upon binding iron at the HHE domains, BTS is destabilized and that this destabilization relies on specific residues within the HHE domains. This study reveals an important and unique mechanism for plant iron homeostasis whereby an E3 ubiquitin ligase may posttranslationally control components of the transcriptional regulatory network involved in the iron deficiency response.

  10. The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members.

    Directory of Open Access Journals (Sweden)

    Jana Kamanova

    2016-04-01

    Full Text Available Salmonella Typhimurium stimulates inflammatory responses in the intestinal epithelium, which are essential for its ability to replicate within the intestinal tract. Stimulation of these responses is strictly dependent on the activity of a type III secretion system encoded within its pathogenicity island 1, which through the delivery of effector proteins, triggers signaling pathways leading to inflammation. One of these effectors is SopA, a HECT-type E3 ligase, which is required for the efficient stimulation of inflammation in an animal model of Salmonella Typhimurium infection. We show here that SopA contributes to the stimulation of innate immune responses by targeting two host E3 ubiquitin ligases, TRIM56 and TRIM65. We also found that TRIM65 interacts with the innate immune receptor MDA5 enhancing its ability to stimulate interferon-β signaling. Therefore, by targeting TRIM56 and TRIM65, SopA can stimulate signaling through two innate immune receptors, RIG-I and MDA5. These findings describe a Salmonella mechanism to modulate inflammatory responses by directly targeting innate immune signaling mechanisms.

  11. Targeted ubiquitination and degradation of G-protein-coupled receptor kinase 5 by the DDB1-CUL4 ubiquitin ligase complex.

    Directory of Open Access Journals (Sweden)

    Ziyan Wu

    Full Text Available The G protein-coupled receptor kinases (GRKs phosphorylate agonist occupied G protein-coupled receptors (GPCRs and desensitize GPCR-mediated signaling. Recent studies indicate they also function non-catalytically via interaction with other proteins. In this study, a proteomic approach was used to screen interacting proteins of GRK5 in MDA-MB-231 cells and HUVEC cells. Mass spectrometry analysis reveals several proteins in the GRK5 immunocomplex including damaged DNA-binding protein 1 (DDB1, an adaptor subunit of the CUL4-ROC1 E3 ubiquitin ligase complex. Co-immunoprecipitation experiments confirmed the association of GRK5 with DDB1-CUL4 complex, and reveal that DDB1 acts as an adapter to link GRK5 to CUL4 to form the complex. Overexpression of DDB1 promoted, whereas knockdown of DDB1 inhibited the ubiquitination of GRK5, and the degradation of GRK5 was reduced in cells deficient of DDB1. Furthermore, the depletion of DDB1 decreased Hsp90 inhibitor-induced GRK5 destabilization and UV irradiation-induced GRK5 degradation. Thus, our study identified potential GRK5 interacting proteins, and reveals the association of GRK5 with DDB1 in cell and the regulation of GRK5 level by DDB1-CUL4 ubiquitin ligase complex-dependent proteolysis pathway.

  12. Ubiquitylation of FACT by the cullin-E3 ligase Rtt101 connects FACT to DNA replication.

    Science.gov (United States)

    Han, Junhong; Li, Qing; McCullough, Laura; Kettelkamp, Charisse; Formosa, Tim; Zhang, Zhiguo

    2010-07-15

    FACT plays important roles in both gene transcription and DNA replication. However, how this protein complex is targeted to these two distinct cellular processes remains largely unknown. Here we show that ubiquitylation of the Spt16 subunit of FACT by Rtt101, the cullin subunit of an E3 ubiquitin ligase in Saccharomyces cerevisiae, links FACT to DNA replication. We find Rtt101 interacts with and ubiquitylates Spt16 in vitro and in vivo. Deletion of RTT101 leads to reduced association of both FACT and the replicative helicase MCM with replication origins. Loss of Rtt101 also reduces binding of FACT to MCM, but not the association of FACT with Leo1 and Spt5, two proteins involved in transcription. Origin function is compromised in cells lacking Rtt101 or with an Spt16 mutation. These findings identify Spt16 as an Rtt101 substrate, and suggest that Spt16 ubiquitylation is important for FACT to function during DNA replication.

  13. Ubiquitin ligase UBE3C promotes melanoma progression by increasing epithelial-mesenchymal transition in melanoma cells

    OpenAIRE

    TANG, Li; Yi, Xue-Mei; Chen, Jia; Chen, Fu-Juan; Lou, Wei; Gao, Yun-Lu; Zhou, Jing; Su, Li-Na; Xu, Xin; Lu, Jia-Qing; Ma, Jun; Yu, Ning; Ding, Yang-Feng

    2016-01-01

    Melanoma is the most aggressive type of skin cancer, exhibiting extensive local invasion and early distant metastasis. Aberrant expression of ubiquitin-protein ligase E3C (UBE3C) plays a key role in tumor development and progression. In the present study, we analyzed UBE3C expression in samples of cancerous and normal skin tissue. Levels of UBE3C expression were much higher in primary and metastatic melanoma tissues than in normal skin, cutaneous squamous cell carcinoma or basal cell carcinom...

  14. Transactivation of Schizosaccharomyces pombe cdt2+ stimulates a Pcu4-Ddb1-CSN ubiquitin ligase

    DEFF Research Database (Denmark)

    Liu, C.; Poitelea, M.; Watson, A.

    2005-01-01

    Cullin-4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe, the Cullin-4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S...... phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4-Ddb1-CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from...... degradation. We propose that Cdt2 incorporation into the Pcu4-Ddb1-CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin-4-based ubiquitin ligase....

  15. TRIM4; a novel mitochondrial interacting RING E3 ligase, sensitizes the cells to hydrogen peroxide (H2O2) induced cell death.

    Science.gov (United States)

    Tomar, Dhanendra; Prajapati, Paresh; Lavie, Julie; Singh, Kritarth; Lakshmi, Sripada; Bhatelia, Khyati; Roy, Milton; Singh, Rochika; Bénard, Giovanni; Singh, Rajesh

    2015-12-01

    The emerging evidences suggest that posttranslational modification of target protein by ubiquitin (Ub) not only regulate its turnover through ubiquitin proteasome system (UPS) but is a critical regulator of various signaling pathways. During ubiquitination, E3 ligase recognizes the target protein and determines the topology of ubiquitin chains. In current study, we studied the role of TRIM4, a member of the TRIM/RBCC protein family of RING E3 ligase, in regulation of hydrogen peroxide (H2O2) induced cell death. TRIM4 is expressed differentially in human tissues and expressed in most of the analyzed human cancer cell lines. The subcellular localization studies showed that TRIM4 forms distinct cytoplasmic speckle like structures which transiently interacts with mitochondria. The expression of TRIM4 induces mitochondrial aggregation and increased level of mitochondrial ROS in the presence of H2O2. It sensitizes the cells to H2O2 induced death whereas knockdown reversed the effect. TRIM4 potentiates the loss of mitochondrial transmembrane potential and cytochrome c release in the presence of H2O2. The analysis of TRIM4 interacting proteins showed its interaction with peroxiredoxin 1 (PRX1), including other proteins involved in regulation of mitochondrial and redox homeostasis. TRIM4 interaction with PRX1 is critical for the regulation of H2O2 induced cell death. Collectively, the evidences in the current study suggest the role of TRIM4 in regulation of oxidative stress induced cell death.

  16. The SOCS2 Ubiquitin Ligase Complex Regulates Growth Hormone Receptor Levels

    DEFF Research Database (Denmark)

    Vesterlund, Mattias; Zadjali, Fahad; Persson, Torbjörn

    2011-01-01

    that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2(-/-) mice and in the absence...... to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions....

  17. Degradation of phosphorylated p53 by viral protein-ECS E3 ligase complex.

    Directory of Open Access Journals (Sweden)

    Yoshitaka Sato

    2009-07-01

    Full Text Available p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.

  18. BAG-2 Acts as an Inhibitor of the Chaperone-associated Ubiquitin Ligase CHIP

    OpenAIRE

    Arndt, Verena; Daniel, Christina; Nastainczyk, Wolfgang; Alberti, Simon; Höhfeld, Jörg

    2005-01-01

    Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a u...

  19. Study the effect of kidney stones on serum xanthine oxidase, ecto-5ʹ-nucleotidase activity and E3 SUMO-protein ligase NSE2 (NSMCE2 in Malaysian individuals

    Directory of Open Access Journals (Sweden)

    Faridah Yusof

    2015-08-01

    Conclusions: The present study suggests that the increase in serum of xanthine oxidase,ecto-5ʹ-nucleotidase activities E3 small ubiquitin-like modifier-protein ligase NSE2 concentration can be used as biomarkers for diagnosis of kidney damage in patients with kidney stone, also in developments of change DNA damage and inflammation disorders in these patients.

  20. Rice RING protein OSBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

    Institute of Scientific and Technical Information of China (English)

    Wei Li; Zuhua He; Sihui Zhong; Guojun Li; Qun Li; Bizeng Mao; Yiwen Deng; Huijuan Zhang; Longjun Zeng; Fengming Song

    2011-01-01

    Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear.We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OSBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resistance to multiple races of M.oryzae. This indicates that OsBBI1 modulates broad-spectrum resistance against the blast fungus. The OsBBII-overexpressing plants showed higher levels of H,O, accumulation in cells and higher levels of phenolic compounds and cross-linking of proteins in cell walls at infection sites by M. Oryzae compared with wild-type(WT)plants. The cell walls were thicker in the OsBB11-overexpressing plants and thinner in the mutant plants than in the WT plants. Our results suggest that OsBBH modulates broad-spectrum resistance to blast fungus by modifying cell wall defence responses. The functional characterization of OsBBI1 provides insight into the E3 ligase-mediated innate immunity, and a practical tool for constructing broad-spectrum resistance against the most destructive disease in rice.

  1. CRL2(LRR-1 E3-ligase regulates proliferation and progression through meiosis in the Caenorhabditis elegans germline.

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    Julien Burger

    2013-03-01

    Full Text Available The ubiquitin-proteolytic system controls the stability of proteins in space and time. In this study, using a temperature-sensitive mutant allele of the cul-2 gene, we show that CRL2(LRR-1 (CUL-2 RING E3 ubiquitin-ligase and the Leucine Rich Repeat 1 substrate recognition subunit acts at multiple levels to control germline development. CRL2(LRR-1 promotes germ cell proliferation by counteracting the DNA replication ATL-1 checkpoint pathway. CRL2(LRR-1 also participates in the mitotic proliferation/meiotic entry decision, presumably controlling the stability of meiotic promoting factors in the mitotic zone of the germline. Finally, CRL2(LRR-1 inhibits the first steps of meiotic prophase by targeting in mitotic germ cells degradation of the HORMA domain-containing protein HTP-3, required for loading synaptonemal complex components onto meiotic chromosomes. Given its widespread evolutionary conservation, CUL-2 may similarly regulate germline development in other organisms as well.

  2. E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

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    Bill B. Chen

    2014-04-01

    Full Text Available Acute lung injury (ALI is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1. Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN-induced putative kinase 1 (PINK1, which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

  3. NEDD4 E3 ligase inhibits the activity of the Hippo pathway by targeting LATS1 for degradation.

    Science.gov (United States)

    Salah, Zaidoun; Cohen, Sherri; Itzhaki, Ella; Aqeilan, Rami I

    2013-12-15

    Proper regulation of cell proliferation, cell apoptosis, and cell death are vital for the development and survival of living organisms. Failure or dysfunction of any of these processes can have devastating effects, including cancer. The Hippo pathway, first discovered in Drosophila, has been found to be a major growth-regulatory signaling pathway that controls these crucial processes and has been implicated in cell-progress regulation and organ size determination. Abnormal regulation of this pathway has been found in several cancer types. However, the mechanisms that regulate the pathway and its core members yet have to be elucidated. One of the main core components of this pathway is LATS1, a serine/threonine kinase. Therefore, understanding how LATS1 activity is regulated is expected to shed light on new mechanisms that regulate the Hippo pathway. In the current work, we identified several potential LATS1 regulators and proved that NEDD4 E3 ubiquitin ligase controls LATS1 stability. We demonstrate that NEDD4 directly interacts with LATS1, leading to ubiquitination and decreased levels of LATS1 and, thus, increased YAP localization in the nucleus, which subsequently increases the transcriptional activity of YAP. As such, we show that NEDD4 acts as an additional regulator of the Hippo pathway on the protein level via interactions between WW domain-containing and PPxY motif-containing proteins. These findings might be applied in the development of new therapeutic approaches through the activation of LATS1.

  4. E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

    Science.gov (United States)

    Li, Peili; Kurata, Yasutaka; Maharani, Nani; Mahati, Endang; Higaki, Katsumi; Hasegawa, Akira; Shirayoshi, Yasuaki; Yoshida, Akio; Kondo, Tatehito; Kurozawa, Youichi; Yamamoto, Kazuhiro; Ninomiya, Haruaki; Hisatome, Ichiro

    2015-09-01

    Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

  5. RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

    Science.gov (United States)

    Brown, Nicholas G.; VanderLinden, Ryan; Watson, Edmond R.; Qiao, Renping; Grace, Christy R. R.; Yamaguchi, Masaya; Weissmann, Florian; Frye, Jeremiah J.; Dube, Prakash; Ei Cho, Shein; Actis, Marcelo L.; Rodrigues, Patrick; Fujii, Naoaki; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A.

    2015-01-01

    For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2∼Ub catalytic modules such as APC11–UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APCCDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APCCDH1–UBCH10∼Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation. PMID:25825779

  6. Arabidopsis HIGH PLOIDY2 sumoylates and stabilizes flowering locus C through its E3 ligase activity

    Directory of Open Access Journals (Sweden)

    Jun Soo eKwak

    2016-04-01

    Full Text Available Flowering Locus C (FLC, a floral repressor, plays an important role in flowering. The mechanisms regulating FLC gene expression and protein function have been studied extensively; however, post-translational regulation of FLC remains unclear. Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2 as an E3 SUMO ligase for FLC. In vitro and vivo pull-down assays showed that FLC physically interacts with HPY2. In vitro assays showed that the stimulation of FLC sumoylation by HPY2 was dependent on SUMO-activating enzyme E1 and -conjugating enzyme E2, indicating that HPY2 was an E3 SUMO ligase for FLC. In transgenic plants, inducible HPY2 overexpression increased the concentration of FLC, indicating that HPY2 stabilized FLC through direct sumoylation. Flowering time in hpy2-2 mutants was shorter than in wild-type plants under long- and short-day conditions, with a greater effect under short-day conditions, and FLC was downregulated in hpy2-2 mutants. These data indicate that HPY2 regulates FLC function and stability at both the transcriptional and post-translational levels through its E3 SUMO ligase activity.

  7. XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms,Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature

    Institute of Scientific and Technical Information of China (English)

    Sofia D.Carvalho; Rita Saraiva; Teresa M.Maia; Isabel A.Abreu; Paula Duque

    2012-01-01

    The Arabidopsis XBAT35 is one of five structurally related ankyrin repeat-containing Really interesting New Gene (RING) E3 ligases involved in ubiquitin-mediated protein degradation,which plays key roles in a wide range of cellular processes.Here,we show that the XBAT35 gene undergoes alternative splicing,generating two transcripts that are constitutively expressed in all plant tissues.The two splice variants derive from an exon skipping event that excludes an in-frame segment from the XBAT35 precursor mRNA,giving rise to two protein isoforms that differ solely in the presence of a nuclear localization signal (NLS).Transient expression assays indicate that the isoform lacking the NLS localizes in the cytoplasm of plant cells,whereas the other is targeted to the nucleus,accumulating in nuclear speckles.Both isoforms are functional E3 ligases,as assessed by in vitro ubiquitination assays.Two insertion mutant alleles and RNA-interference (RNAi) silencing lines for XBAT35 display no evident phenotypes under normal growth conditions,but exhibit hypersensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) during apical hook exaggeration in the dark,which is rescued by an inhibitor of ethylene perception.Independent expression of each XBAT35 splice variant in the mutant background indicates that the two isoforms may differentially contribute to apical hook formation but are both functional in this ethylene-mediated response.Thus,XBAT35 defines a novel player in ethylene signaling involved in negatively regulating apical hook curvature,with alternative splicing controlling dual targeting of this E3 ubiquitin ligase to the nuclear and cytoplasmic compartments.

  8. The Highwire ubiquitin ligase promotes axonal degeneration by tuning levels of Nmnat protein.

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    Xin Xiong

    Full Text Available Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw, as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat, which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury.

  9. Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling.

    Science.gov (United States)

    Barsi, Julius C; Rajendra, Rashmi; Wu, Jiang I; Artzt, Karen

    2005-10-01

    The Notch-Delta signaling pathway controls many conserved cell determination events. While the Notch end is fairly well characterized, the Delta end remains poorly understood. Mind bomb1 (MIB1) is one of two E3 ligases known to ubiquitinate Delta. We report here that a targeted mutation of Mib1 in mice results in embryonic lethality by E10.5. Mutants exhibit multiple defects due to their inability to modulate Notch signaling. As histopathology revealed a strong neurogenic phenotype, this study concentrates on characterizing the Mib1 mutant by analyzing Notch pathway components in embryonic neuroepithelium prior to developmental arrest. Premature neurons were observed to undergo apoptosis soon after differentiation. Aberrant neurogenesis is a direct consequence of lowered Hes1 and Hes5 expression resulting from the inability to generate Notch1 intracellular domain (NICD1). We conclude that MIB1 activity is required for S3 cleavage of the Notch1 receptor. These results have direct implications for manipulating the differentiation of neuronal stem cells and provide a putative target for the modulation of specific tumors.

  10. RNF185 is a novel E3 ligase of endoplasmic reticulum-associated degradation (ERAD) that targets cystic fibrosis transmembrane conductance regulator (CFTR).

    Science.gov (United States)

    El Khouri, Elma; Le Pavec, Gwenaëlle; Toledano, Michel B; Delaunay-Moisan, Agnès

    2013-10-25

    In the endoplasmic reticulum (ER), misfolded or improperly assembled proteins are exported to the cytoplasm and degraded by the ubiquitin-proteasome pathway through a process called ER-associated degradation (ERAD). ER-associated E3 ligases, which coordinate substrate recognition, export, and proteasome targeting, are key components of ERAD. Cystic fibrosis transmembrane conductance regulator (CFTR) is one ERAD substrate targeted to co-translational degradation by the E3 ligase RNF5/RMA1. RNF185 is a RING domain-containing polypeptide homologous to RNF5. We show that RNF185 controls the stability of CFTR and of the CFTRΔF508 mutant in a RING- and proteasome-dependent manner but does not control that of other classical ERAD model substrates. Reciprocally, its silencing stabilizes CFTR proteins. Turnover analyses indicate that, as RNF5, RNF185 targets CFTR to co-translational degradation. Importantly, however, simultaneous depletion of RNF5 and RNF185 profoundly blocks CFTRΔF508 degradation not only during translation but also after synthesis is complete. Our data thus identify RNF185 and RNF5 as a novel E3 ligase module that is central to the control of CFTR degradation.

  11. Both Rbx1 and Rbx2 exhibit a functional role in the HIV-1 Vif-Cullin5 E3 ligase complex in vitro.

    Science.gov (United States)

    Wang, Xiaodan; Wang, Xiaoying; Wang, Weiran; Zhang, Jingyao; Wang, Jiawen; Wang, Chu; Lv, Mingyu; Zuo, Tao; Liu, Donglai; Zhang, Haihong; Wu, Jiaxin; Yu, Bin; Kong, Wei; Wu, Hui; Yu, Xianghui

    2015-06-12

    Rbx1 and Rbx2 are essential components of Cullin-RING E3 Ligases. Vif is generally believed to preferentially recruit the Cul5-Rbx2 module to induce proteasomal degradation of antiretroviral enzyme APOBEC3G, although some investigators have found that the Cul5-Rbx1 module is recruited. Here, to investigate the function of the two Rbx proteins in the Vif-Cul5 complex, we analyzed the performance of Cul5-Rbx1/Cul5-Rbx2 module in the activity of Vif E3 ligase and evaluated the interactions between Rbx1/Rbx2 and Cul5. We found that either Rbx1 or Rbx2 could promote ubiquitination of APOBE3G (A3G) in vitro. We also found that both Rbx1 and Rbx2 could bind Cul5 in cells and Rbx2 could dose-dependently inhibit the interaction of Rbx1 with Cul5. Furthermore, only the decrease of endogenous Rbx2 but not Rbx1 could impair the Vif-induced A3G degradation in cells. These findings indicate that Rbx1 and Rbx2 can both activate Cul5-Vif E3 ligase in vitro, but they may undergo a more delicate selection mechanism in vivo.

  12. Deficiency in ubiquitin ligase TRIM2 causes accumulation of neurofilament light chain and neurodegeneration

    OpenAIRE

    Balastik, M.; Ferraguti, F.; A. Pires da Silva; Lee, T; Alvarez-Bolado, G.; Lu, K.; Gruss, P

    2008-01-01

    TRIM RING finger proteins have been shown to play an important role in cancerogenesis, in the pathogenesis of some human hereditary disorders, and in the defense against viral infection, but the function of the majority of TRIM proteins remains unknown. Here, we show that TRIM RING finger protein TRIM2, highly expressed in the nervous system, is an UbcH5a-dependent ubiquitin ligase. We further demonstrate that TRIM2 binds to neurofilament light subunit (NF-L) and regulates NF-L ubiquitination...

  13. The Ubiquitin Ligase Siah2 Regulates PPARγ Activity in Adipocytes

    OpenAIRE

    Kilroy, Gail; Kirk-Ballard, Heather; Carter, Lauren E.; Floyd, Z. Elizabeth

    2012-01-01

    Moderate reductions in peroxisome proliferator-activated receptor (PPAR)γ levels control insulin sensitivity as effectively as activation of PPARγ in adipocytes by the thiazolidinediones. That observation suggests that PPARγ activity can be regulated by modulating the amount of PPARγ protein in adipocytes. Activation of PPARγ in adipocytes is linked to changes in PPARγ protein levels via increased degradation of PPARγ proteins by the ubiquitin proteasome system. Identification of the ubiquiti...

  14. The fate of tandemly duplicated genes assessed by the expression analysis of a group of Arabidopsis thaliana RING-H2 ubiquitin ligase genes of the ATL family.

    Science.gov (United States)

    Aguilar-Hernández, Victor; Guzmán, Plinio

    2014-03-01

    Gene duplication events exert key functions on gene innovations during the evolution of the eukaryotic genomes. A large portion of the total gene content in plants arose from tandem duplications events, which often result in paralog genes with high sequence identity. Ubiquitin ligases or E3 enzymes are components of the ubiquitin proteasome system that function during the transfer of the ubiquitin molecule to the substrate. In plants, several E3s have expanded in their genomes as multigene families. To gain insight into the consequences of gene duplications on the expansion and diversification of E3s, we examined the evolutionary basis of a cluster of six genes, duplC-ATLs, which arose from segmental and tandem duplication events in Brassicaceae. The assessment of the expression suggested two patterns that are supported by lineage. While retention of expression domains was observed, an apparent absence or reduction of expression was also inferred. We found that two duplC-ATL genes underwent pseudogenization and that, in one case, gene expression is probably regained. Our findings provide insights into the evolution of gene families in plants, defining key events on the expansion of the Arabidopsis Tóxicos en Levadura family of E3 ligases.

  15. The Arabidopsis MIEL1 E3 ligase negatively regulates ABA signalling by promoting protein turnover of MYB96

    OpenAIRE

    Lee, Hong Gil; Seo, Pil Joon

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant responses to various environmental challenges. Controlled protein turnover is an important component of ABA signalling. Here we show that the RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1) regulates ABA sensitivity by promoting MYB96 turnover in Arabidopsis. Germination of MIEL1-deficient mutant seeds is hypersensitive to ABA, whereas MIEL1-overexpressing transgenic seeds are less sensitive. MIEL1 can interact with MYB96, a regul...

  16. Datasets from an interaction proteomics screen for substrates of the SCFβTrCP ubiquitin ligase

    NARCIS (Netherlands)

    Magliozzi, Roberto; Peng, Mao; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J R; Low, Teck Yew

    2015-01-01

    An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCFβTrCP ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCFβTrCP, was used as bait. βTrCP2 wild type and the two mutants

  17. Distinct and overlapping functions of the cullin E3 ligase scaffolding proteins CUL4A and CUL4B

    Science.gov (United States)

    Hannah, Jeffrey

    2016-01-01

    The cullin 4 subfamily of genes includes CUL4A and CUL4B, which share a mostly identical amino acid sequence aside from the elongated N-terminal region in CUL4B. Both act as scaffolding proteins for modular cullin RING ligase 4 (CRL4) complexes which promote the ubiquitination of a variety of substrates. CRL4 function is vital to cells as loss of both genes or their shared substrate adaptor protein DDB1 halts proliferation and eventually leads to cell death. Due to their high structural similarity, CUL4A and CUL4B share a substantial overlap in function. However, in some cases, differences in subcellular localization, spatiotemporal expression patterns and stress-inducibility preclude functional compensation. In this review, we highlight the most essential functions of the CUL4 genes in: DNA repair and replication, chromatin-remodeling, cell cycle regulation, embryogenesis, hematopoiesis and spermatogenesis. CUL4 genes are also clinically relevant as dysregulation can contribute to the onset of cancer and CRL4 complexes are often hijacked by certain viruses to promote viral replication and survival. Also, mutations in CUL4B have been implicated in a subset of patients suffering from syndromic X-linked intellectual disability (AKA mental retardation). Interestingly, the antitumor effects of immunomodulatory drugs are caused by their binding to the CRL4CRBN complex and re-directing the E3 ligase towards the Ikaros transcription factors IKZF1 and IKZF3. Because of their influence over key cellular functions and relevance to human disease, CRL4s are considered promising targets for therapeutic intervention. PMID:26344709

  18. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase.

    Science.gov (United States)

    Petzold, Georg; Fischer, Eric S; Thomä, Nicolas H

    2016-04-07

    Thalidomide and its derivatives, lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of haematologic malignancies. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4(CRBN)) E3 ubiquitin ligase, and inhibit ubiquitination of endogenous CRL4(CRBN) substrates. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degradation. Lenalidomide induces degradation of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3), and casein kinase 1α (CK1α), which contributes to its clinical efficacy in the treatment of multiple myeloma and 5q-deletion associated myelodysplastic syndrome (del(5q) MDS), respectively. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α β-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4(CRBN) is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compound and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy. We anticipate that high-affinity protein-protein interactions induced by small molecules will provide opportunities for drug development, particularly for targeted protein degradation.

  19. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

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    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

  20. Clomipramine causes osteoporosis by promoting osteoclastogenesis via E3 ligase Itch, which is prevented by Zoledronic acid

    Science.gov (United States)

    Li, Xing; Sun, Wen; Li, Jinbo; Wang, Mengmeng; Zhang, Hengwei; Pei, Lingpeng; Boyce, Brendan F.; Wang, Zhiyu; Xing, Lianping

    2017-01-01

    Patients taking antidepressants, including Clomipramine (CLP), have an increased risk of osteoporotic fracture. However, the effects of CLP on bone metabolism are unknown. Here, we demonstrate that WT mice treated with CLP for 2 weeks had significantly reduced trabecular bone volume and cortical bone thickness, associated with increased osteoclast (OC) numbers, but had no change in osteoblast numbers or bone formation rate. Bone marrow cells from CLP-treated mice had normal OC precursor frequency, but formed significantly more OCs when they were cultured with RANKL and M-CSF. CLP promoted OC formation and bone resorption and expression of OC-associated genes. CLP-induced bone loss was prevented by Zoledronic acid. At the molecular level, CLP inhibited the activity of the ubiquitin E3 ligase Itch. CLP did not promote OC formation from bone marrow cells of Itch−/− mice in vitro nor induce bone loss in Itch−/− mice. Our findings indicate that CLP causes bone loss by enhancing Itch-mediated osteoclastogenesis, which was prevented by Zoledronic acid. Thus, anti-resorptive therapy could be used to prevent bone loss in patients taking antidepressants, such as CLP. PMID:28145497

  1. Identification of the ubiquitin ligase Triad1 as a regulator of endosomal transport

    Directory of Open Access Journals (Sweden)

    Gerco Hassink

    2012-05-01

    The ubiquitin system plays an important role in trafficking of signaling receptors from the plasma membrane to lysosomes. Triad1 is a ubiquitin ligase that catalyzes the formation of poly-ubiquitin chains linked via lysine-48 as well as lysine-63 residues. We show that depletion of Triad1 affects the sorting of both growth hormone and epidermal growth factor. Triad1-depleted cells accumulate both ligands in endosomes. While fluid phase transport to the lysosomes is reduced in the absence of Triad1, growth hormone receptor can recycle back to the plasma membrane together with transferrin. Using immune electron microscopy we show that Triad1 depletion results in enlarged endosomes with enlarged and irregular shaped intraluminal vesicles. The endosomes display prominent clathrin coats and show increased levels of growth hormone label. We conclude that Triad1 is required for the proper function of multivesicular bodies.

  2. Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

    Science.gov (United States)

    Won, Minho; Ro, Hyunju; Dawid, Igor B

    2015-10-06

    The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

  3. Deficiency of UBE2T, the E2 Ubiquitin Ligase Necessary for FANCD2 and FANCI Ubiquitination, Causes FA-T Subtype of Fanconi Anemia

    Directory of Open Access Journals (Sweden)

    Kimberly A. Rickman

    2015-07-01

    Full Text Available Fanconi anemia (FA is a rare bone marrow failure and cancer predisposition syndrome resulting from pathogenic mutations in genes encoding proteins participating in the repair of DNA interstrand crosslinks (ICLs. Mutations in 17 genes (FANCA-FANCS have been identified in FA patients, defining 17 complementation groups. Here, we describe an individual presenting with typical FA features who is deficient for the ubiquitin-conjugating enzyme (E2, UBE2T. UBE2T is known to interact with FANCL, the E3 ubiquitin-ligase component of the multiprotein FA core complex, and is necessary for the monoubiquitination of FANCD2 and FANCI. Proband fibroblasts do not display FANCD2 and FANCI monoubiquitination, do not form FANCD2 foci following treatment with mitomycin C, and are hypersensitive to crosslinking agents. These cellular defects are complemented by expression of wild-type UBE2T, demonstrating that deficiency of the protein UBE2T can lead to Fanconi anemia. UBE2T gene gains an alias of FANCT.

  4. A lysine-to-arginine mutation on NEDD8 markedly reduces the activity of cullin RING E3 ligase through the impairment of neddylation cascades

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    Sui, Yiyan; Liu, Yaobin; Xu, Guoqiang, E-mail: gux2002@suda.edu.cn

    2015-06-12

    Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions. - Highlights: • Lys27 in NEDD8 is critical for protein neddylation. • NEDD8 K27R mutant impairs the NEDD8 conjugation. • NEDD8 K27R mutant significantly reduces the activity of cullin-RING E3 ligases.

  5. Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels.

    Science.gov (United States)

    Hantouche, Christine; Williamson, Brittany; Valinsky, William C; Solomon, Joshua; Shrier, Alvin; Young, Jason C

    2017-02-10

    Cardiac long QT syndrome type 2 is caused by mutations in the human ether a go-go-related gene (hERG) potassium channel, many of which cause misfolding and degradation at the endoplasmic reticulum instead of normal trafficking to the cell surface. The Hsc70/Hsp70 chaperones assist the folding of the hERG cytosolic domains. Here, we demonstrate that the Hsp70 nucleotide exchange factor Bag1 promotes hERG degradation by the ubiquitin-proteasome system at the endoplasmic reticulum to regulate hERG levels and channel activity. Dissociation of hERG complexes containing Hsp70 and the E3 ubiquitin ligase CHIP requires the interaction of Bag1 with Hsp70, but this does not involve the Bag1 ubiquitin-like domain. The interaction with Bag1 then shifts hERG degradation to the membrane-anchored E3 ligase TRC8 and its E2-conjugating enzyme Ube2g2, as determined by siRNA screening. TRC8 interacts through the transmembrane region with hERG and decreases hERG functional expression. TRC8 also mediates degradation of the misfolded hERG-G601S disease mutant, but pharmacological stabilization of the mutant structure prevents degradation. Our results identify TRC8 as a previously unknown Hsp70-independent quality control E3 ligase for hERG. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Ubiquitin ligase MARCH 8 cooperates with CD83 to control surface MHC II expression in thymic epithelium and CD4 T cell selection.

    Science.gov (United States)

    Liu, Haiyin; Jain, Reema; Guan, Jing; Vuong, Vivian; Ishido, Satoshi; La Gruta, Nicole L; Gray, Daniel H; Villadangos, Jose A; Mintern, Justine D

    2016-08-22

    Major histocompatibility complex class II (MHC II) expression is tightly regulated, being subjected to cell type-specific mechanisms that closely control its levels at the cell surface. Ubiquitination by the E3 ubiquitin ligase MARCH 1 regulates MHC II expression in dendritic cells and B cells. In this study, we demonstrate that the related ligase MARCH 8 is responsible for regulating surface MHC II in thymic epithelial cells (TECs). March8(-/-) mice have elevated MHC II at the surface of cortical TECs and autoimmune regulator (AIRE)(-) medullary TECs (mTECs), but not AIRE(+) mTECs. Despite this, thymic and splenic CD4(+) T cell numbers and repertoires remained unaltered in March8(-/-) mice. Notably, the ubiquitination of MHC II by MARCH 8 is controlled by CD83. Mice expressing a mutated form of CD83 (Cd83(anu/anu) mice) have impaired CD4(+) T cell selection, but deleting March8 in Cd83(anu/anu) mice restored CD4(+) T cell selection to normal levels. Therefore, orchestrated regulation of MHC II surface expression in TECs by MARCH 8 and CD83 plays a major role in CD4(+) T cell selection. Our results also highlight the specialized use of ubiquitinating machinery in distinct antigen-presenting cell types, with important functional consequences and implications for therapeutic manipulation.

  7. The bacterial effector Cif interferes with SCF ubiquitin ligase function by inhibiting deneddylation of Cullin1.

    Science.gov (United States)

    Morikawa, Hanako; Kim, Minsoo; Mimuro, Hitomi; Punginelli, Claire; Koyama, Tomohiro; Nagai, Shinya; Miyawaki, Atsushi; Iwai, Kazuhiro; Sasakawa, Chihiro

    2010-10-15

    Cycle inhibiting factor (Cif) is one of the effectors delivered into epithelial cells by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) via the type III secretion system (TTSS). Cif family proteins, which inhibit host cell-cycle progression via mechanisms not yet precisely understood, are highly conserved among EPEC, EHEC, Yersinia pseudotuberculosis, Photorhabdus luminescens and Burkholderia pseudomallei. Levels of several proteins relevant to cell-cycle progression are modulated by Cullin-RING ligases (CRLs), which in turn are activated by conjugation and deconjugation of NEDD8 to Cullins. Here we show that Cif interacts with NEDD8 and interferes with SCF (Skp1-Cullin1-F-box protein) complex ubiquitin ligase function. We found that neddylated Cullin family proteins accumulated and ubiquitination of p27 decreased in cells infected with EPEC. Consequently, Cif stabilized SCF substrates such as CyclinD1, Cdt1, and p27, and caused G1 cell-cycle arrest. Using time-lapse-imaging of fluorescent ubiquitination-based cell-cycle indicator (Fucci)-expressing cells, we were able to monitor cell-cycle progression during EPEC infection and confirmed the arrest of infected cells at G1. Our in vitro and in vivo data show that Cif-NEDD8 interaction inhibits deneddylation of Cullins, suppresses CRL activity and induces G1 arrest. We thus conclude that the bacterial effector Cif interferes with neddylation-mediated cell-cycle control.

  8. TRIM E3 ligases interfere with early and late stages of the retroviral life cycle.

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    Pradeep D Uchil

    2008-02-01

    Full Text Available Members of the TRIpartite interaction Motif (TRIM family of E3 ligases have been shown to exhibit antiviral activities. Here we report a near comprehensive screen for antiretroviral activities of 55 TRIM proteins (36 human, 19 mouse. We identified approximately 20 TRIM proteins that, when transiently expressed in HEK293 cells, affect the entry or release of human immunodeficiency virus 1 (HIV, murine leukemia virus (MLV, or avian leukosis virus (ALV. While TRIM11 and 31 inhibited HIV entry, TRIM11 enhanced N-MLV entry by interfering with Ref1 restriction. Strikingly, many TRIM proteins affected late stages of the viral life cycle. Gene silencing of endogenously expressed TRIM 25, 31, and 62 inhibited viral release indicating that they play an important role at late stages of the viral life cycle. In contrast, downregulation of TRIM11 and 15 enhanced virus release suggesting that these proteins contribute to the endogenous restriction of retroviruses in cells.

  9. The HERC2 ubiquitin ligase is essential for embryonic development and regulates motor coordination

    Science.gov (United States)

    Cubillos-Rojas, Monica; Schneider, Taiane; Hadjebi, Ouadah; Pedrazza, Leonardo; de Oliveira, Jarbas Rodrigues; Langa, Francina; Guénet, Jean-Louis; Duran, Joan; de Anta, Josep Maria; Alcántara, Soledad; Ruiz, Rocio; Pérez-Villegas, Eva María; Aguilar, Francisco J.; Carrión, Ángel M.; Armengol, Jose Angel; Baple, Emma; Crosby, Andrew H.; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-01-01

    A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed. Our findings establish a crucial role of HERC2 in embryonic development and motor coordination. PMID:27528230

  10. Pathogenic role of the CRL4 ubiquitin ligase in human disease

    Directory of Open Access Journals (Sweden)

    Pengbo eZhou

    2012-03-01

    Full Text Available The cullin 4-RING ubiquitin ligase (CRL4 family employs multiple DCAF (DDB1-CUL4 associated factors substrate receptors to direct the degradation of proteins involved in a wide spectrum of cellular functions. Aberrant expression of the cullin 4A (CUL4A gene is found in many tumor types, while mutations of the cullin 4B (CUL4B gene are causally associated with human X-linked mental retardation. This focused review will summarize our current knowledge of the two CUL4 family members in the pathogenesis of human malignancy and neuronal disease, and discuss their potential as new targets for cancer prevention and therapeutic intervention.

  11. Poxvirus targeting of E3 ligase β-TrCP by molecular mimicry: a mechanism to inhibit NF-κB activation and promote immune evasion and virulence.

    Science.gov (United States)

    Mansur, Daniel S; Maluquer de Motes, Carlos; Unterholzner, Leonie; Sumner, Rebecca P; Ferguson, Brian J; Ren, Hongwei; Strnadova, Pavla; Bowie, Andrew G; Smith, Geoffrey L

    2013-02-01

    The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKβ causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase β-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished β-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.

  12. Four Closely-related RING-type E3 Ligases, APD1-4,are Involved in Pollen Mitosis Ⅱ Regulation in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Guo Luo; Hongya Gu; Jingjing Liu; Li-Jia Qu

    2012-01-01

    Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes.In higher plants,protein ubiquitination plays an essential role in many biological processes,including hormone signaling,photomorphogenesis,and pathogen defense.However,the roles of protein ubiquitination in the reproductive process are not clear.In this study,we identified four plant-specific RING-finger genes designated (A)berrant (P)ollen (D)evelopment (1) (APD1) to APD4,as regulators of pollen mitosis Ⅱ (PMII) in Arabidopsis thaliana (L.).The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage.Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2,suggesting that cell division was defective in male gametogenesis.All of the four genes were expressed in multiple stages at different levels during male gametophyte development.Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes.Furthermore,APD2 had E2-dependent E3 ligase activity in vitro,and five APD2-interacting proteins were identified.Our results suggest that these four genes may be involved,redundantly,in regulating the PMII process during male gametogenesis.

  13. The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

    Science.gov (United States)

    Miao, Min; Niu, Xiangli; Kud, Joanna; Du, Xinran; Avila, Julian; Devarenne, Timothy P; Kuhl, Joseph C; Liu, Yongsheng; Xiao, Fangming

    2016-07-01

    We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling.

  14. Ubiquitin ligase gp78 targets unglycosylated prion protein PrP for ubiquitylation and degradation.

    Science.gov (United States)

    Shao, Jia; Choe, Vitnary; Cheng, Haili; Tsai, Yien Che; Weissman, Allan M; Luo, Shiwen; Rao, Hai

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.

  15. The ubiquitin ligase Siah2 regulates obesity-induced adipose tissue inflammation.

    Science.gov (United States)

    Kilroy, Gail; Carter, Lauren E; Newman, Susan; Burk, David H; Manuel, Justin; Möller, Andreas; Bowtell, David D; Mynatt, Randall L; Ghosh, Sujoy; Floyd, Z Elizabeth

    2015-11-01

    Chronic, low-grade adipose tissue inflammation associated with adipocyte hypertrophy is an important link in the relationship between obesity and insulin resistance. Although ubiquitin ligases regulate inflammatory processes, the role of these enzymes in metabolically driven adipose tissue inflammation is relatively unexplored. Herein, the effect of the ubiquitin ligase Siah2 on obesity-related adipose tissue inflammation was examined. Wild-type and Siah2KO mice were fed a low- or high-fat diet for 16 weeks. Indirect calorimetry, body composition, and glucose and insulin tolerance were assayed along with glucose and insulin levels. Gene and protein expression, immunohistochemistry, adipocyte size distribution, and lipolysis were also analyzed. Enlarged adipocytes in obese Siah2KO mice were not associated with obesity-induced insulin resistance. Proinflammatory gene expression, stress kinase signaling, fibrosis, and crown-like structures were reduced in the Siah2KO adipose tissue, and Siah2KO adipocytes were more responsive to insulin-dependent inhibition of lipolysis. Loss of Siah2 increased expression of PPARγ target genes involved in lipid metabolism and decreased expression of proinflammatory adipokines regulated by PPARγ. Siah2 links adipocyte hypertrophy with adipocyte dysfunction and recruitment of proinflammatory immune cells to adipose tissue. Selective regulation of PPARγ activity is a Siah2-mediated mechanism contributing to obesity-induced adipose tissue inflammation. © 2015 The Obesity Society.

  16. The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus.

    Science.gov (United States)

    Lee, Wan Yin; Goh, Germaine; Chia, Joanne; Boey, Adrian; Gunko, Natalia V; Bard, Frederic

    2015-01-01

    The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.

  17. The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus.

    Directory of Open Access Journals (Sweden)

    Wan Yin Lee

    Full Text Available The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC's regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC's action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.

  18. PJA1, encoding a RING-H2 finger ubiquitin ligase, is a novel human X chromosome gene abundantly expressed in brain.

    Science.gov (United States)

    Yu, Ping; Chen, Yiwang; Tagle, Danilo A; Cai, Tao

    2002-06-01

    RING-finger proteins contain cysteine-rich, zinc-binding domains and are involved in the formation of macromolecular scaffolds important for transcriptional repression and ubiquitination. In this study, we have identified a RING-H2 finger gene, PJA1 (for praja-1), from a human brain cDNA library and mapped it to human chromosome Xq12 between markers DXS983 and DXS1216, a region implicated in X-linked mental retardation (MRX). Northern blot analysis indicated a 2.7-kb transcript that was abundantly expressed in the brain, including regions of the cerebellum, cerebral cortex, medulla, occipital pole, frontal lobe, temporal lobe, and putamen. Amino acid sequence analysis of the 71-kDa protein PJA1 showed 52.3% identity to human PJA2 (for praja-2, also known as NEURODAP1/KIAA0438) and also a significant identity to its homologs in rat, mouse, and zebrafish. In vitro binding and immunoprecipitation assays demonstrated that both PJA1 and PJA2 are able to bind the ubiquitin-conjugating enzyme UbcH5B. Moreover, the ubiquitination assay indicated that PJA1 and PJA2 have an E2-dependent E3 ubiquitin ligase activity. Thus our findings demonstrate that PJA1 can be involved in protein ubiquitination in the brain and is a suitable candidate gene for MRX.

  19. Insights into Ubiquitination from the Unique Clamp-like Binding of the RING E3 AO7 to the E2 UbcH5B*

    Science.gov (United States)

    Li, Shengjian; Liang, Yu-He; Mariano, Jennifer; Metzger, Meredith B.; Stringer, Daniel K.; Hristova, Ventzislava A.; Li, Jess; Randazzo, Paul A.; Tsai, Yien Che; Ji, Xinhua; Weissman, Allan M.

    2015-01-01

    RING proteins constitute the largest class of E3 ubiquitin ligases. Unlike most RINGs, AO7 (RNF25) binds the E2 ubiquitin-conjugating enzyme, UbcH5B (UBE2D2), with strikingly high affinity. We have defined, by co-crystallization, the distinctive means by which AO7 binds UbcH5B. AO7 contains a structurally unique UbcH5B binding region (U5BR) that is connected by an 11-amino acid linker to its RING domain, forming a clamp surrounding the E2. The U5BR interacts extensively with a region of UbcH5B that is distinct from both the active site and the RING-interacting region, referred to as the backside of the E2. An apparent paradox is that the high-affinity binding of the AO7 clamp to UbcH5B, which is dependent on the U5BR, decreases the rate of ubiquitination. We establish that this is a consequence of blocking the stimulatory, non-covalent, binding of ubiquitin to the backside of UbcH5B. Interestingly, when non-covalent backside ubiquitin binding cannot occur, the AO7 clamp now enhances the rate of ubiquitination. The high-affinity binding of the AO7 clamp to UbcH5B has also allowed for the co-crystallization of previously described and functionally important RING mutants at the RING-E2 interface. We show that mutations having marked effects on function only minimally affect the intermolecular interactions between the AO7 RING and UbcH5B, establishing a high degree of complexity in activation through the RING-E2 interface. PMID:26475854

  20. The CUL4A ubiquitin ligase is a potential therapeutic target in skin cancer and other malignancies

    Institute of Scientific and Technical Information of China (English)

    Jeffrey Hannah; Peng-Bo Zhou

    2013-01-01

    Cullin 4A (CUL4A) is an E3 ubiquitin ligase that directly affects DNA repair and cel cycle progression by targeting substrates including damage-specific DNA-binding protein 2 (DDB2), xeroderma pigmentosum complementation group C (XPC), chromatin licensing and DNA replication factor 1 (Cdt1), and p21. Recent work from our laboratory has shown that Cul4a-deficient mice have greatly reduced rates of ultraviolet-induced skin carcinomas. On a cel ular level, Cul4a-deficient cel s have great capacity for DNA repair and demonstrate a slow rate of proliferation due primarily to increased expression of DDB2 and p21, respectively. This suggests that CUL4A promotes tumorigenesis (as well as accumulation of skin damage and subsequent premature aging) by limiting DNA repair activity and expediting S phase entry. In addition, CUL4A has been found to be up-regulated via gene amplification or overexpression in breast cancers, hepatocellular carcinomas, squamous cell carcinomas, adrenocortical carcinomas, childhood medulloblastomas, and malignant pleural mesotheliomas. Because of its oncogenic activity in skin cancer and up-regulation in other malignancies, CUL4A has arisen as a potential candidate for targeted therapeutic approaches. In this review, we outline the established functions of CUL4A and discuss the E3 ligase’s emergence as a potential driver of tumorigenesis.

  1. The SOCS2 ubiquitin ligase complex regulates growth hormone receptor levels.

    Directory of Open Access Journals (Sweden)

    Mattias Vesterlund

    Full Text Available Growth Hormone is essential for the regulation of growth and the homeostatic control of intermediary metabolism. GH actions are mediated by the Growth Hormone Receptor; a member of the cytokine receptor super family that signals chiefly through the JAK2/STAT5 pathway. Target tissue responsiveness to GH is under regulatory control to avoid excessive and off-target effects upon GHR activation. The suppressor of cytokine signalling 2 (SOCS is a key regulator of GHR sensitivity. This is clearly shown in mice where the SOCS2 gene has been inactivated, which show 30-40% increase in body length, a phenotype that is dependent on endogenous GH secretion. SOCS2 is a GH-stimulated, STAT5b-regulated gene that acts in a negative feedback loop to downregulate GHR signalling. Since the biochemical basis for these actions is poorly understood, we studied the molecular function of SOCS2. We demonstrated that SOCS2 is part of a multimeric complex with intrinsic ubiquitin ligase activity. Mutational analysis shows that the interaction with Elongin B/C controls SOCS2 protein turnover and affects its molecular activity. Increased GHR levels were observed in livers from SOCS2⁻/⁻ mice and in the absence of SOCS2 in in vitro experiments. We showed that SOCS2 regulates cellular GHR levels through direct ubiquitination and in a proteasomally dependent manner. We also confirmed the importance of the SOCS-box for the proper function of SOCS2. Finally, we identified two phosphotyrosine residues in the GHR to be responsible for the interaction with SOCS2, but only Y487 to account for the effects of SOCS2. The demonstration that SOCS2 is an ubiquitin ligase for the GHR unveils the molecular basis for its physiological actions.

  2. Regulation of EGFR protein stability by the HECT-type ubiquitin ligase SMURF2.

    Science.gov (United States)

    Ray, Dipankar; Ahsan, Aarif; Helman, Abigail; Chen, Guoan; Hegde, Ashok; Gurjar, Susmita Ramanand; Zhao, Lili; Kiyokawa, Hiroaki; Beer, David G; Lawrence, Theodore S; Nyati, Mukesh K

    2011-07-01

    Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA)-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nude mice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expression microarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.

  3. Regulation of EGFR Protein Stability by the HECT-type Ubiquitin Ligase SMURF2

    Directory of Open Access Journals (Sweden)

    Dipankar Ray

    2011-07-01

    Full Text Available Epidermal growth factor receptor (EGFR is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nudemice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expressionmicroarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.

  4. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase

    Energy Technology Data Exchange (ETDEWEB)

    Orlicky, Steve; Tang, Xiaojing; Willems, Andrew; Tyers, Mike; Sicheri, Frank

    2010-12-01

    Cell cycle progression depends on precise elimination of cyclins and cyclin-dependent kinase (CDK) inhibitors by the ubiquitin system. Elimination of the CDK inhibitor Sic1 by the SCF{sup Cdc4} ubiquitin ligase at the onset of S phase requires phosphorylation of Sic1 on at least six of its nine Cdc4-phosphodegron (CPD) sites. A 2.7 {angstrom} X-ray crystal structure of a Skp1-Cdc4 complex bound to a high-affinity CPD phosphopeptide from human cyclin E reveals a core CPD motif, Leu-Leu-pThr-Pro, bound to an eight-bladed WD40 propeller domain in Cdc4. The low affinity of each CPD motif in Sic1 reflects structural discordance with one or more elements of the Cdc4 binding site. Reengineering of Cdc4 to reduce selection against Sic1 sequences allows ubiquitination of lower phosphorylated forms of Sic1. These features account for the observed phosphorylation threshold in Sic1 recognition and suggest an equilibrium binding mode between a single receptor site in Cdc4 and multiple low-affinity CPD sites in Sic1.

  5. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase.

    Science.gov (United States)

    Kolas, Nadine K; Chapman, J Ross; Nakada, Shinichiro; Ylanko, Jarkko; Chahwan, Richard; Sweeney, Frédéric D; Panier, Stephanie; Mendez, Megan; Wildenhain, Jan; Thomson, Timothy M; Pelletier, Laurence; Jackson, Stephen P; Durocher, Daniel

    2007-12-07

    Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.

  6. Hijacking of the host SCF ubiquitin ligase machinery by plant pathogens

    Directory of Open Access Journals (Sweden)

    Shimpei eMagori

    2011-11-01

    Full Text Available The SCF (SKP1-CUL1-F-box protein ubiquitin ligase complex mediates polyubiquitination of proteins targeted for degradation, thereby controlling a plethora of biological processes in eukaryotic cells. Although this ubiquitination machinery is found and functional only in eukaryotes, many non-eukaryotic pathogens also encode F-box proteins, the critical subunits of the SCF complex. Increasing evidence indicates that such non-eukaryotic F-box proteins play an essential role in subverting or exploiting the host ubiquitin/proteasome system for efficient pathogen infection. A recent bioinformatic analysis has identified more than 70 F-box proteins in 22 different bacterial species, suggesting that use of pathogen-encoded F-box effectors in the host cell may be a widespread infection strategy. In this review, we focus on plant pathogen-encoded F-box effectors, such as VirF of Agrobacterium tumefaciens, GALAs of Ralstonia solanacearum, and P0 of Poleroviruses, and discuss the molecular mechanism by which plant pathogens use these factors to manipulate the host cell for their own benefit.

  7. Modulation of immune cell functions by the E3 ligase CBL-b

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    Christina eLutz-Nicoladoni

    2015-03-01

    Full Text Available Maintenance of immunological tolerance is a critical hallmark of the immune system. Several signaling checkpoints necessary to balance activating and inhibitory input to immune cells have been described so far, among which the E3 ligase Cbl-b appears to be a central player. Cbl-b is expressed in all leukocyte subsets and regulates several signaling pathways in T cells, NK cells, B cells and different types of myeloid cells. In most cases Cbl-b negatively regulates activation signals through antigen or pattern recognition receptors and co-stimulatory molecules. In line with this function, cblb-deficient immune cells display lower activation thresholds and cblb knockout mice spontaneously develop autoimmunity and are highly susceptible to experimental autoimmunity. Interestingly, genetic association studies link cblb-polymorphisms with autoimmunity also in humans. Vice versa, the increased activation potential of cblb-deficient cells renders them more potent to fight against malignancies or infections. Accordingly, several reports have shown that cblb knockout mice reject tumors, which mainly depends on cytotoxic T and NK cells. Thus targeting Cbl-b may be an interesting strategy to enhance anti-cancer immunity. In this review we summarize the findings on the molecular function of Cbl-b in different cell types and illustrate the potential of Cbl-b as target for immunomodulatory therapies.

  8. SCF Ubiquitin Ligase F-box Protein Fbx15 Controls Nuclear Co-repressor Localization, Stress Response and Virulence of the Human Pathogen Aspergillus fumigatus

    Science.gov (United States)

    Jöhnk, Bastian; Bayram, Özgür; Heinekamp, Thorsten; Mattern, Derek J.; Brakhage, Axel A.; Jacobsen, Ilse D.; Valerius, Oliver; Braus, Gerhard H.

    2016-01-01

    F-box proteins share the F-box domain to connect substrates of E3 SCF ubiquitin RING ligases through the adaptor Skp1/A to Cul1/A scaffolds. F-box protein Fbx15 is part of the general stress response of the human pathogenic mold Aspergillus fumigatus. Oxidative stress induces a transient peak of fbx15 expression, resulting in 3x elevated Fbx15 protein levels. During non-stress conditions Fbx15 is phosphorylated and F-box mediated interaction with SkpA preferentially happens in smaller subpopulations in the cytoplasm. The F-box of Fbx15 is required for an appropriate oxidative stress response, which results in rapid dephosphorylation of Fbx15 and a shift of the cellular interaction with SkpA to the nucleus. Fbx15 binds SsnF/Ssn6 as part of the RcoA/Tup1-SsnF/Ssn6 co-repressor and is required for its correct nuclear localization. Dephosphorylated Fbx15 prevents SsnF/Ssn6 nuclear localization and results in the derepression of gliotoxin gene expression. fbx15 deletion mutants are unable to infect immunocompromised mice in a model for invasive aspergillosis. Fbx15 has a novel dual molecular function by controlling transcriptional repression and being part of SCF E3 ubiquitin ligases, which is essential for stress response, gliotoxin production and virulence in the opportunistic human pathogen A. fumigatus. PMID:27649508

  9. A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

    DEFF Research Database (Denmark)

    Luijsterburg, Martijn S; Acs, Klara; Ackermann, Leena

    2012-01-01

    . Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks......, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.......The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation...

  10. Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

    Science.gov (United States)

    Lazarou, Michael; Jin, Seok Min; Kane, Lesley A; Youle, Richard J

    2012-02-14

    Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. The ubiquitin ligase HUWE1 regulates hematopoietic stem cell maintenance and lymphoid commitment

    Science.gov (United States)

    King, Bryan; Boccalatte, Francesco; Moran-Crusio, Kelly; Wolf, Elmar; Wang, Jingjing; Kayembe, Clarisse; Lazaris, Charalampos; Yu, Xiaofeng; Aranda-Orgilles, Beatriz; Lasorella, Anna; Aifantis, Iannis

    2016-01-01

    Hematopoietic stem cells (HSCs) are dormant in the bone marrow and can be activated in response to diverse stresses to replenish all blood cell types. Here we identify the ubiquitin ligase Huwe1 as a crucial regulator of HSC functions via its post-translational control of N-myc. We found Huwe1 to be essential for HSC self-renewal, quiescence and lymphoid fate specification. Using a novel fluorescent fusion allele (MycnM), we observed that N-myc expression was restricted to the most immature, multipotent stem and progenitor populations. N-myc was upregulated in response to stress or upon loss of Huwe1, leading to increased proliferation and stem cell exhaustion. Mycn depletion reversed most of these phenotypes in vivo, suggesting that the attenuation of N-myc by Huwe1 is essential to reestablish homeostasis following stress. PMID:27668798

  12. Control of mRNA Export by Adenovirus E4orf6 and E1B55K Proteins during Productive Infection Requires E4orf6 Ubiquitin Ligase Activity▿

    OpenAIRE

    Blanchette, Paola; Kindsmüller, Kathrin; Groitl, Peter; Dallaire, Frédéric; Speiseder, Thomas; Branton, Philip E.; Dobner, Thomas

    2008-01-01

    During the adenovirus infectious cycle, the early proteins E4orf6 and E1B55K are known to perform several functions. These include nuclear export of late viral mRNAs, a block of nuclear export of the bulk of cellular mRNAs, and the ubiquitin-mediated degradation of selected proteins, including p53 and Mre11. Degradation of these proteins occurs via a cellular E3 ubiquitin ligase complex that is assembled through interactions between elongins B and C and BC boxes present in E4orf6 to form a cu...

  13. An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.

    Directory of Open Access Journals (Sweden)

    Nadia Litterman

    2011-05-01

    Full Text Available The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8 localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8 by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8 is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8 also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8 in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8 in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8 ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

  14. p21-Activated kinase 6 (PAK6) inhibits prostate cancer growth via phosphorylation of androgen receptor and tumorigenic E3 ligase murine double minute-2 (Mdm2).

    Science.gov (United States)

    Liu, Tong; Li, Yang; Gu, Hui; Zhu, Ge; Li, Jiabin; Cao, Liu; Li, Feng

    2013-02-01

    The androgen receptor (AR) signaling pathway plays a crucial role in the development and growth of prostate malignancies. Regulation of AR homeostasis in prostate tumorigenesis has not yet been fully characterized. In this study, we demonstrate that p21-activated kinase 6 (PAK6) inhibits prostate tumorigenesis by regulating AR homeostasis. First, we demonstrated that in normal prostate epithelium, AR co-localizes with PAK6 in the cytoplasm and translocates into the nucleus in malignant prostate. Furthermore, AR phosphorylation at Ser-578 by PAK6 promotes AR-E3 ligase murine double minute-2 (Mdm2) association, causing AR degradation upon androgen stimuli. We also showed that PAK6 phosphorylates Mdm2 on Thr-158 and Ser-186, which is critical for AR ubiquitin-mediated degradation. Moreover, we found that Thr-158 collaborates with Ser-186 for AR-Mdm2 association and AR ubiquitin-mediated degradation as it facilitates PAK6-mediated AR homeostasis. PAK6 knockdown promotes prostate tumor growth in vivo. Interestingly, we found a strong inverse correlation between PAK6 and AR expression in the cytoplasm of prostate cancer cells. These observations indicate that PAK6 may be important for the maintenance of androgen-induced AR signaling homeostasis and in prostate malignancy, as well as being a possible new therapeutic target for AR-positive and hormone-sensitive prostate cancer.

  15. The E3 ligase Ubr3 regulates Usher syndrome and MYH9 disorder proteins in the auditory organs of Drosophila and mammals

    Science.gov (United States)

    Li, Tongchao; Giagtzoglou, Nikolaos; Eberl, Daniel F; Jaiswal, Sonal Nagarkar; Cai, Tiantian; Godt, Dorothea; Groves, Andrew K; Bellen, Hugo J

    2016-01-01

    Myosins play essential roles in the development and function of auditory organs and multiple myosin genes are associated with hereditary forms of deafness. Using a forward genetic screen in Drosophila, we identified an E3 ligase, Ubr3, as an essential gene for auditory organ development. Ubr3 negatively regulates the mono-ubiquitination of non-muscle Myosin II, a protein associated with hearing loss in humans. The mono-ubiquitination of Myosin II promotes its physical interaction with Myosin VIIa, a protein responsible for Usher syndrome type IB. We show that ubr3 mutants phenocopy pathogenic variants of Myosin II and that Ubr3 interacts genetically and physically with three Usher syndrome proteins. The interactions between Myosin VIIa and Myosin IIa are conserved in the mammalian cochlea and in human retinal pigment epithelium cells. Our work reveals a novel mechanism that regulates protein complexes affected in two forms of syndromic deafness and suggests a molecular function for Myosin IIa in auditory organs. DOI: http://dx.doi.org/10.7554/eLife.15258.001 PMID:27331610

  16. New Functions of APC/C Ubiquitin Ligase in the Nervous System and Its Role in Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Tanja Fuchsberger

    2017-05-01

    Full Text Available The E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C regulates important processes in cells, such as the cell cycle, by targeting a set of substrates for degradation. In the last decade, APC/C has been related to several major functions in the nervous system, including axon guidance, synaptic plasticity, neurogenesis, and neuronal survival. Interestingly, some of the identified APC/C substrates have been related to neurodegenerative diseases. There is an accumulation of some degradation targets of APC/C in Alzheimer’s disease (AD brains, which suggests a dysregulation of the protein complex in the disorder. Moreover, recently evidence has been provided for an inactivation of APC/C in AD. It has been shown that oligomers of the AD-related peptide, Aβ, induce degradation of the APC/C activator subunit cdh1, in vitro in neurons in culture and in vivo in the mouse hippocampus. Furthermore, in the AD mouse model APP/PS1, lower cdh1 levels were observed in pyramidal neurons in CA1 when compared to age-matched wildtype mice. In this review, we provide a complete list of APC/C substrates that are involved in the nervous system and we discuss their functions. We also summarize recent studies that show neurobiological effects in cdh1 knockout mouse models. Finally, we discuss the role of APC/C in the pathophysiology of AD.

  17. The CUL4-DDB1 ubiquitin ligase complex controls adult and embryonic stem cell differentiation and homeostasis.

    Science.gov (United States)

    Gao, Jie; Buckley, Shannon M; Cimmino, Luisa; Guillamot, Maria; Strikoudis, Alexandros; Cang, Yong; Goff, Stephen P; Aifantis, Iannis

    2015-11-27

    Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation.

  18. An Atypical SCF-like Ubiquitin Ligase Complex Promotes Wallerian Degeneration through Regulation of Axonal Nmnat2

    Directory of Open Access Journals (Sweden)

    Yuya Yamagishi

    2016-10-01

    Full Text Available Axon degeneration is a tightly regulated, self-destructive program that is a critical feature of many neurodegenerative diseases, but the molecular mechanisms regulating this program remain poorly understood. Here, we identify S-phase kinase-associated protein 1A (Skp1a, a core component of a Skp/Cullin/F-box (SCF-type E3 ubiquitin ligase complex, as a critical regulator of axon degeneration after injury in mammalian neurons. Depletion of Skp1a prolongs survival of injured axons in vitro and in the optic nerve in vivo. We demonstrate that Skp1a regulates the protein level of the nicotinamide adenine dinucleotide (NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2 in axons. Loss of axonal Nmnat2 contributes to a local ATP deficit that triggers axon degeneration. Knockdown of Skp1a elevates basal levels of axonal Nmnat2, thereby delaying axon degeneration through prolonged maintenance of axonal ATP. Consistent with Skp1a functioning through regulation of Nmnat2, Skp1a knockdown fails to protect axons from Nmnat2 knockdown. These results illuminate the molecular mechanism underlying Skp1a-dependent axonal destruction.

  19. Centromere binding and a conserved role in chromosome stability for SUMO-dependent ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Loes A L van de Pasch

    Full Text Available The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs. Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis.

  20. Both ubiquitin ligases FBXW8 and PARK2 are sequestrated into insolubility by ATXN2 PolyQ expansions, but only FBXW8 expression is dysregulated.

    Directory of Open Access Journals (Sweden)

    Melanie Vanessa Halbach

    Full Text Available The involvement of the ubiquitin-proteasome system (UPS in the course of various age-associated neurodegenerative diseases is well established. The single RING finger type E3 ubiquitin-protein ligase PARK2 is mutated in a Parkinson's disease (PD variant and was found to interact with ATXN2, a protein where polyglutamine expansions cause Spinocerebellar ataxia type 2 (SCA2 or increase the risk for Levodopa-responsive PD and for the motor neuron disease Amyotrophic lateral sclerosis (ALS. We previously reported evidence for a transcriptional induction of the multi-subunit RING finger Skp1/Cul/F-box (SCF type E3 ubiquitin-protein ligase complex component FBXW8 in global microarray profiling of ATXN2-expansion mouse cerebellum and demonstrated its role for ATXN2 degradation in vitro. Now, we documented co-localization in vitro and co-immunoprecipitations both in vitro and in vivo, which indicate associations of FBXW8 with ATXN2 and PARK2. Both FBXW8 and PARK2 proteins are driven into insolubility by expanded ATXN2. Whereas the FBXW8 transcript upregulation by ATXN2- expansion was confirmed also in qPCR of skin fibroblasts and blood samples of SCA2 patients, a FBXW8 expression dysregulation was not observed in ATXN2-deficient mice, nor was a PARK2 transcript dysregulation observed in any samples. Jointly, all available data suggest that the degradation of wildtype and mutant ATXN2 is dependent on FBXW8, and that ATXN2 accumulation selectively modulates FBXW8 levels, while PARK2 might act indirectly through FBXW8. The effects of ATXN2-expansions on FBXW8 expression in peripheral tissues like blood may become useful for clinical diagnostics.

  1. The ubiquitin ligase tripartite-motif-protein 32 is induced in Duchenne muscular dystrophy.

    Science.gov (United States)

    Assereto, Stefania; Piccirillo, Rosanna; Baratto, Serena; Scudieri, Paolo; Fiorillo, Chiara; Massacesi, Manuela; Traverso, Monica; Galietta, Luis J; Bruno, Claudio; Minetti, Carlo; Zara, Federico; Gazzerro, Elisabetta

    2016-08-01

    Activation of the proteasome pathway is one of the secondary processes of cell damage, which ultimately lead to muscle degeneration and necrosis in Duchenne muscular dystrophy (DMD). In mdx mice, the proteasome inhibitor bortezomib up-regulates the membrane expression of members of the dystrophin complex and reduces the inflammatory reaction. However, chronic inhibition of the 26S proteasome may be toxic, as indicated by the systemic side-effects caused by this drug. Therefore, we sought to determine the components of the ubiquitin-proteasome pathway that are specifically activated in human dystrophin-deficient muscles. The analysis of a cohort of patients with genetically determined DMD or Becker muscular dystrophy (BMD) unveiled a selective up-regulation of the ubiquitin ligase tripartite motif-containing protein 32 (TRIM32). The induction of TRIM32 was due to a transcriptional effect and it correlated with disease severity in BMD patients. In contrast, atrogin1 and muscle RING-finger protein-1 (MuRF-1), which are strongly increased in distinct types of muscular atrophy, were not affected by the DMD dystrophic process. Knock-out models showed that TRIM32 is involved in ubiquitination of muscle cytoskeletal proteins as well as of protein inhibitor of activated STAT protein gamma (Piasγ) and N-myc downstream-regulated gene, two inhibitors of satellite cell proliferation and differentiation. Accordingly, we showed that in DMD/BMD muscle tissue, TRIM32 induction was more pronounced in regenerating myofibers rather than in necrotic muscle cells, thus pointing out a role of this protein in the regulation of human myoblast cell fate. This finding highlights TRIM32 as a possible therapeutic target to favor skeletal muscle regeneration in DMD patients.

  2. Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b

    Directory of Open Access Journals (Sweden)

    Tomoki Abe

    2013-01-01

    Full Text Available Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA, IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.

  3. Endoplasmic reticulum protein quality control is determined by cooperative interactions between Hsp/c70 protein and the CHIP E3 ligase.

    Science.gov (United States)

    Matsumura, Yoshihiro; Sakai, Juro; Skach, William R

    2013-10-25

    The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.

  4. The Pepper RING Finger E3 Ligase, CaDIR1, Regulates the Drought Stress Response via ABA-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Sang-Wook Han

    2017-04-01

    Full Text Available Drought stress from soil or air limits plant growth and development, leading to a reduction in crop productivity. Several E3 ligases positively or negatively regulate the drought stress response. In the present study, we show that the pepper (Capsicum annuum Drought Induced RING type E3 ligase 1, CaDIR1, regulates the drought stress response via abscisic acid (ABA-mediated signaling. CaDIR1 contains a C3HC4-type RING finger domain in the N-terminal region; this domain functions during protein degradation via attachment of ubiquitins to the substrate target proteins. The expression levels of the CaDIR1 gene were suppressed and induced by ABA and drought treatments, respectively. We conducted loss-of-function and gain-of function genetic studies to examine the in vivo function of CaDIR1 in response to ABA and drought stress. CaDIR1-silenced pepper plants displayed a drought-tolerant phenotype characterized by a low level of transpirational water loss via increased stomatal closure and elevated leaf temperatures. CaDIR1-overexpressing (OX Arabidopsis plants exhibited an ABA-hypersensitive phenotype during the germination stage, but an ABA-hyposensitive phenotype—characterized by decreased stomatal closure and reduced leaf temperatures—at the adult stage. Moreover, adult CaDIR1-OX plants exhibited a drought-sensitive phenotype characterized by high levels of transpirational water loss. Our results indicate that CaDIR1 functions as a negative regulator of the drought stress response via ABA-mediated signaling. Our findings provide a valuable insight into the plant defense mechanism that operates during drought stress.

  5. Datasets from an interaction proteomics screen for substrates of the SCF(βTrCP) ubiquitin ligase

    NARCIS (Netherlands)

    Magliozzi, Roberto; Peng, Mao; Mohammed, Shabaz; Guardavaccaro, Daniele; Heck, Albert J R; Low, Teck Yew

    An affinity purification-mass spectrometry (AP-MS) method was employed to identify novel substrates of the SCF(βTrCP) ubiquitin ligase. A FLAG-HA tagged version of the F-box protein βTrCP2, the substrate recognition subunit of SCF(βTrCP), was used as bait. βTrCP2 wild type and the two mutants

  6. Structure of the Siz/PIAS SUMO E3 Ligase Siz1 and Determinants Required for SUMO Modification of PCNA

    Energy Technology Data Exchange (ETDEWEB)

    Yunus, Ali A.; Lima, Christopher D.; (SKI)

    2010-01-12

    Siz1 is a founding member of the Siz/PIAS RING family of SUMO E3 ligases. The X-ray structure of an active Siz1 ligase revealed an elongated tripartite architecture comprised of an N-terminal PINIT domain, a central zinc-containing RING-like SP-RING domain, and a C-terminal domain we term the SP-CTD. Structure-based mutational analysis and biochemical studies show that the SP-RING and SP-CTD are required for activation of the E2SUMO thioester, while the PINIT domain is essential for redirecting SUMO conjugation to the proliferating cell nuclear antigen (PCNA) at lysine 164, a nonconsensus lysine residue that is not modified by the SUMO E2 in the absence of Siz1. Mutational analysis of Siz1 and PCNA revealed surfaces on both proteins that are required for efficient SUMO modification of PCNA in vitro and in vivo.

  7. Identification of the receptor component of the IkappaBalpha-ubiquitin ligase

    DEFF Research Database (Denmark)

    Yaron, A; Hatzubai, A; Davis, M;

    1998-01-01

    NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade......-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS...

  8. Systematic In Vivo RNAi Analysis Identifies IAPs as NEDD8-E3 Ligases

    DEFF Research Database (Denmark)

    Broemer, Meike; Tenev, Tencho; Rigbolt, Kristoffer T G;

    2010-01-01

    The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-li...

  9. The ubiquitin ligase Cullin5(SOCS2) regulates NDR1/STK38 stability and NF-κB transactivation

    DEFF Research Database (Denmark)

    Paul, Indranil; Batth, Tanveer S; Iglesias-Gato, Diego

    2017-01-01

    SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic...... show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results...

  10. Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

    Science.gov (United States)

    Dantuma, Nico P; Pfeiffer, Annika

    2016-01-01

    Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

  11. SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

    Science.gov (United States)

    Buster, Daniel W.; Daniel, Scott G.; Nguyen, Huy Q.; Windler, Sarah L.; Skwarek, Lara C.; Peterson, Maureen; Roberts, Meredith; Meserve, Joy H.; Hartl, Tom; Klebba, Joseph E.; Bilder, David; Bosco, Giovanni

    2013-01-01

    Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCFSlimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCFSlimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCFSlimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus. PMID:23530065

  12. An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

    Science.gov (United States)

    Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

    2017-01-01

    The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

  13. Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.

    Science.gov (United States)

    Faresse, Nourdine; Ruffieux-Daidie, Dorothée; Salamin, Mélanie; Gomez-Sanchez, Celso E; Staub, Olivier

    2010-12-01

    The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

  14. Membrane-Associated Ubiquitin Ligase SAUL1 Suppresses Temperature- and Humidity-Dependent Autoimmunity in Arabidopsis.

    Science.gov (United States)

    Disch, Eva-Maria; Tong, Meixuezi; Kotur, Tanja; Koch, Gerald; Wolf, Carl-Asmus; Li, Xin; Hoth, Stefan

    2016-01-01

    Plants have evolved elaborate mechanisms to regulate pathogen defense. Imbalances in this regulation may result in autoimmune responses that are affecting plant growth and development. In Arabidopsis, SAUL1 encodes a plant U-box ubiquitin ligase and regulates senescence and cell death. Here, we show that saul1-1 plants exhibit characteristics of an autoimmune mutant. A decrease in relative humidity or temperature resulted in reduced growth and systemic lesioning of saul1-1 rosettes. These physiological changes are associated with increased expression of salicylic acid-dependent and pathogenesis-related (PR) genes. Consistently, resistance of saul1-1 plants against Pseudomonas syringae pv. maculicola ES4326, P. syringae pv. tomato DC3000, or Hyaloperonospora arabidopsidis Noco2 was enhanced. Transmission electron microscopy revealed alterations in saul1-1 chloroplast ultrastructure and cell-wall depositions. Confocal analysis on aniline blue-stained leaf sections and cellular universal micro spectrophotometry further showed that these cell-wall depositions contain callose and lignin. To analyze signaling downstream of SAUL1, we performed epistasis analyses between saul1-1 and mutants in the EDS1/PAD4/SAG101 hub. All phenotypes observed in saul1-1 plants at low temperature were dependent on EDS1 and PAD4 but not SAG101. Taken together, SAUL1 negatively regulates immunity upstream of EDS1/PAD4, likely through the degradation of an unknown activator of the pathway.

  15. The Oncogenic Role of WWP1 E3 Ubiquitin Ligase in Prostate Cancer Development

    Science.gov (United States)

    2011-05-01

    and suppresses its ligand-independent activation. Current Biology, 14, 2228–2236. 42. Chastagner, P., Israel , A., & Brou, C. (2006). Itch/AIP4...proteasome destruction of activated RhoA is impaired in Smurf1−/− cells. Molecular Biology of the Cell, 17, 2489– 2497. 145. Sahai, E., Garcia -Medina...Haga K, Asaka M, Ramirez F, Yoshida T (2003) Downregulation and growth inhibitory effect of epithelial-type Kruppel-like tran- scription factor KLF4

  16. The Role of Ubiquitin E3 Ligase SCF-SKP2 in Prostate Cancer Development

    Science.gov (United States)

    2007-02-01

    that CUL4 was also present in the H3 methylated nucleo - some fractions. Interestingly, CUL4A, but not its paralogue CUL4B, is enriched in methylated...2, e51 (2006). 23. Zou, L., Cortez , D. & Elledge, S. J. Regulation of ATR substrate selection by Rad17- dependent loading of Rad9 complexes onto

  17. Interplay between the myc oncoprotein, cyclin-dependent kinases and e3 ubiquitin ligases

    OpenAIRE

    Sharifi, Hamid Reza

    2013-01-01

    Mammalian cells grow, divide, and die in a precise and orderly fashion. For cells to grow and maintain their integrity they need to communicate at both intracellular and extracellular level. This communicative circuit is maintained by means of many different factors functioning at different level to for example receive, transmit and respond to the delivered message. Among these factors are transcription factors whose function is to regulate expr...

  18. Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase

    Indian Academy of Sciences (India)

    Rachana Tripathi; K Seetharama; Satya Keerthi Kota; Usha K Srinivas

    2005-06-01

    Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.

  19. Characterization of an E3 Ubiquitin Ligase that Degrades Neurofibromin in Vitro and Vivo

    Science.gov (United States)

    2012-04-01

    cystic embryoid bodies (cEBs) (Figure 2A) with blood island structures, lined with endothelial cells (Figure 2B, arrows). In contrast, Sag/ ES cells... embryoid bodies: ES cells underwent endothelial differentiation for indicated periods and photographed. Arrows indicate cystic EBs that occurred in...attached onto the Petri dish during suspension culture for differentiation, leading to much fewer numbers of embryoid bodies, although we did observe

  20. The Expression of the Ubiquitin Ligase SIAH2 (Seven In Absentia Homolog 2 Is Increased in Human Lung Cancer.

    Directory of Open Access Journals (Sweden)

    Paula Moreno

    Full Text Available Lung cancer is the leading cause of cancer-related deaths worldwide. Overall 5-year survival has shown little improvement over the last decades. Seven in absentia homolog (SIAH proteins are E3 ubiquitin ligases that mediate proteasomal protein degradation by poly-ubiquitination. Even though SIAH proteins play a key role in several biological processes, their role in human cancer remains controversial. The aim of the study was to document SIAH2 expression pattern at different levels (mRNA, protein level and immunohistochemistry in human non-small cell lung cancer (NSCLC samples compared to surrounding healthy tissue from the same patient, and to analyse the association with clinicopathological features.One hundred and fifty-two samples from a patient cohort treated surgically for primary lung cancer were obtained for the study. Genic and protein expression levels of SIAH2 were analysed and compared with clinic-pathologic variables.The present study is the first to analyze the SIAH2 expression pattern at different levels (RNA, protein expression and immunohistochemistry in non-small cell lung cancer (NSCLC. We found that SIAH2 protein expression is significantly enhanced in human lung adenocarcinoma (ADC and squamous cell lung cancer (SCC. Paradoxically, non-significant changes at RNA level were found, suggesting a post-traductional regulatory mechanism. More importantly, an increased correlation between SIAH2 expression and tumor grade was detected, suggesting that this protein could be used as a prognostic biomarker to predict lung cancer progression. Likewise, SIAH2 protein expression showed a strong positive correlation with fluorodeoxyglucose (2-deoxy-2(18Ffluoro-D-glucose uptake in primary NSCLC, which may assist clinicians in stratifying patients at increased overall risk of poor survival. Additionally, we described an inverse correlation between the expression of SIAH2 and the levels of one of its substrates, the serine/threonine kinase

  1. The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection.

    Science.gov (United States)

    Indriolo, Emily; Tharmapalan, Pirashaanthy; Wright, Stephen I; Goring, Daphne R

    2012-11-01

    Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.

  2. SCF(JFK) is a bona fide E3 ligase for ING4 and a potent promoter of the angiogenesis and metastasis of breast cancer.

    Science.gov (United States)

    Yan, Ruorong; He, Lin; Li, Zhongwu; Han, Xiao; Liang, Jing; Si, Wenzhe; Chen, Zhe; Li, Lei; Xie, Guojia; Li, Wanjin; Wang, Peiyan; Lei, Liandi; Zhang, Hongquan; Pei, Fei; Cao, Dengfeng; Sun, Luyang; Shang, Yongfeng

    2015-03-15

    Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1-Cul1-F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCF(JFK) as a bona fide E3 ligase for ING4 and unraveled the JFK-ING4-NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention.

  3. Ubiquitin ligases of the N-end rule pathway: assessment of mutations in UBR1 that cause the Johanson-Blizzard syndrome.

    Directory of Open Access Journals (Sweden)

    Cheol-Sang Hwang

    Full Text Available BACKGROUND: Johanson-Blizzard syndrome (JBS; OMIM 243800 is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons recognized by UBR1 are destabilizing N-terminal residues of protein substrates. METHODOLOGY/PRINCIPAL FINDINGS: Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R was inactive in yeast-based activity assays, the other one (Q1224E had a detectable but weak activity, and the third one (V146L exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients. CONCLUSIONS/SIGNIFICANCE: These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.

  4. The ubiquitin ligase TRIM27 functions as a host restriction factor antagonized by Mycobacterium tuberculosis PtpA during mycobacterial infection

    Science.gov (United States)

    Wang, Jing; Teng, Jade L. L.; Zhao, Dongdong; Ge, Pupu; Li, Bingxi; Woo, Patrick C. Y.; Liu, Cui Hua

    2016-01-01

    Macrophage-mediated innate immune responses play crucial roles in host defense against pathogens. Recent years have seen an explosion of host proteins that act as restriction factors blocking viral replication in infected cells. However, the essential factors restricting Mycobacterium tuberculosis (Mtb) and their regulatory roles during mycobacterial infection remain largely unknown. We previously reported that Mtb tyrosine phosphatase PtpA, a secreted effector protein required for intracellular survival of Mtb, inhibits innate immunity by co-opting the host ubiquitin system. Here, we identified a new PtpA-interacting host protein TRIM27, which is reported to possess a conserved RING domain and usually acts as an E3 ubiquitin ligase that interferes with various cellular processes. We further demonstrated that TRIM27 restricts survival of mycobacteria in macrophages by promoting innate immune responses and cell apoptosis. Interestingly, Mtb PtpA could antagonize TRIM27-promoted JNK/p38 MAPK pathway activation and cell apoptosis through competitively binding to the RING domain of TRIM27. TRIM27 probably works as a potential restriction factor for Mtb and its function is counteracted by Mtb effector proteins such as PtpA. Our study suggests a potential tuberculosis treatment via targeting of the TRIM27-PtpA interfaces. PMID:27698396

  5. Down-regulation of ubiquitin ligase Cbl induced by twist haploinsufficiency in Saethre-Chotzen syndrome results in increased PI3K/Akt signaling and osteoblast proliferation.

    Science.gov (United States)

    Guenou, Hind; Kaabeche, Karim; Dufour, Cécilie; Miraoui, Hichem; Marie, Pierre J

    2006-10-01

    Genetic mutations of Twist, a basic helix-loop-helix transcription factor, induce premature fusion of cranial sutures in Saethre-Chotzen syndrome (SCS). We report here a previously undescribed mechanism involved in the altered osteoblastogenesis in SCS. Cranial osteoblasts from an SCS patient with a Twist mutation causing basic helix-loop-helix deletion exhibited decreased expression of E3 ubiquitin ligase Cbl compared with wild-type osteoblasts. This was associated with decreased ubiquitin-mediated degradation of phosphatidyl inositol 3 kinase (PI3K) and increased PI3K expression and PI3K/Akt signaling. Increased PI3K immunoreactivity was also found in osteoblasts in histological sections of affected cranial sutures from SCS patients. Transfection with Twist or Cbl abolished the increased PI3K/Akt signaling in Twist mutant osteoblasts. Forced overexpression of Cbl did not correct the altered expression of osteoblast differentiation markers in Twist mutant cells. In contrast, pharmacological inhibition of PI3K/Akt, but not ERK signaling, corrected the increased cell growth in Twist mutant osteoblasts. The results show that Twist haploinsufficiency results in decreased Cbl-mediated PI3K degradation in osteoblasts, causing PI3K accumulation and activation of PI3K/Akt-dependent osteoblast growth. This provides genetic and biochemical evidence for a role for Cbl-mediated PI3K signaling in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS.

  6. Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

    Science.gov (United States)

    Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

    2017-03-01

    Voltage-gated CaV2.1 channels comprise a pore-forming α1A subunit with auxiliary α2δ and β subunits. CaV2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the CaV2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of CaV2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human CaV2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel CaV2.1-binding partner. In neurons, RNF138 and CaV2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of CaV2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the CaV2.1 protein level and enhances CaV2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of CaV2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on CaV2.1 WT functional expression, which can be attributed to defective membrane trafficking of CaV2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of CaV2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human CaV2.1 subunits.SIGNIFICANCE STATEMENT Loss-of-function mutations in the human CaV2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and

  7. Defects in DNA Ligase I Trigger PCNA Ubiquitination at Lysine 107

    OpenAIRE

    Das-Bradoo, Sapna; Nguyen, Hai Dang; Wood, Jamie L; Ricke, Robin M; Haworth, Justin C.; Bielinsky, Anja-Katrin

    2009-01-01

    In all eukaryotes, the ligation of newly synthesized DNA, also known as Okazaki fragments, is catalyzed by DNA ligase I1. An individual with a DNA ligase I deficiency exhibited growth retardation, sunlight sensitivity and severe immunosuppression2, likely due to accumulation of DNA damage. Surprisingly, not much is known about the DNA damage response (DDR) in DNA ligase I-deficient cells. Because DNA replication and DDR pathways are highly conserved in eukaryotes, we utilized Saccharomyces ce...

  8. Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

    DEFF Research Database (Denmark)

    Moss, Jennifer; Tinline-Purvis, Helen; Walker, Carol A

    2010-01-01

    Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found...... the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed...... through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair....

  9. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    Science.gov (United States)

    Kondapalli, Chandana; Kazlauskaite, Agne; Zhang, Ning; Woodroof, Helen I.; Campbell, David G.; Gourlay, Robert; Burchell, Lynn; Walden, Helen; Macartney, Thomas J.; Deak, Maria; Knebel, Axel; Alessi, Dario R.; Muqit, Miratul M. K.

    2012-01-01

    Summary Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD. PMID:22724072

  10. The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin.

    Directory of Open Access Journals (Sweden)

    Claire Pardieu

    2010-04-01

    Full Text Available Tetherin (CD317/BST2 is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18 in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.

  11. A HECT ubiquitin-protein ligase as a novel candidate gene for altered quinine and quinidine responses in Plasmodium falciparum.

    Science.gov (United States)

    Sanchez, Cecilia P; Liu, Chia-Hao; Mayer, Sybille; Nurhasanah, Astutiati; Cyrklaff, Marek; Mu, Jianbing; Ferdig, Michael T; Stein, Wilfred D; Lanzer, Michael

    2014-05-01

    The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors.

  12. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    Science.gov (United States)

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  13. Non-canonical NF-κB activation and abnormal B cell accumulation in mice expressing ubiquitin protein ligase-inactive c-IAP2.

    Directory of Open Access Journals (Sweden)

    Dietrich B Conze

    Full Text Available Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion proteins lack the c-IAP2 RING domain, the region responsible for its ubiquitin protein ligase (E3 activity. Ectopic expression of the fusion protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.

  14. Endocytosis of the Aspartic Acid/Glutamic Acid Transporter Dip5 Is Triggered by Substrate-Dependent Recruitment of the Rsp5 Ubiquitin Ligase via the Arrestin-Like Protein Aly2 ▿

    Science.gov (United States)

    Hatakeyama, Riko; Kamiya, Masao; Takahara, Terunao; Maeda, Tatsuya

    2010-01-01

    Endocytosis of nutrient transporters is stimulated under various conditions, such as elevated nutrient availability. In Saccharomyces cerevisiae, endocytosis is triggered by ubiquitination of transporters catalyzed by the E3 ubiquitin ligase Rsp5. However, how the ubiquitination is accelerated under certain conditions remains obscure. Here we demonstrate that closely related proteins Aly2/Art3 and Aly1/Art6, which are poorly characterized members of the arrestin-like protein family, mediate endocytosis of the aspartic acid/glutamic acid transporter Dip5. In aly2Δ cells, Dip5 is stabilized at the plasma membrane and is not endocytosed efficiently. Efficient ubiquitination of Dip5 is dependent on Aly2. aly1Δ cells also show deficiency in Dip5 endocytosis, although less remarkably than aly2Δ cells. Aly2 physically interacts in vivo with Rsp5 at its PY motif and also with Dip5, thus serving as an adaptor linking Rsp5 with Dip5 to achieve Dip5 ubiquitination. Importantly, the interaction between Aly2 and Dip5 is accelerated in response to elevated aspartic acid availability. This result indicates that the regulation of Dip5 endocytosis is accomplished by dynamic recruitment of Rsp5 via Aly2. PMID:20956561

  15. Functional characterization of the SIZ/PIAS-type SUMO E3 ligases, OsSIZ1 and OsSIZ2 in rice

    KAUST Repository

    Park, Hyeongcheol

    2010-06-18

    Sumoylation is a post-translational regulatory process in diverse cellular processes in eukaryotes, involving conjugation/deconjugation of small ubiquitin-like modifier (SUMO) proteins to other proteins thus modifying their function. The PIAS [protein inhibitor of activated signal transducers and activators of transcription (STAT)] and SAP (scaffold attachment factor A/B/acinus/PIAS)/MIZ (SIZ) proteins exhibit SUMO E3-ligase activity that facilitates the conjugation of SUMO proteins to target substrates. Here, we report the isolation and molecular characterization of Oryza sativa SIZ1 (OsSIZ1) and SIZ2 (OsSIZ2), rice homologs of Arabidopsis SIZ1. The rice SIZ proteins are localized to the nucleus and showed sumoylation activities in a tobacco system. Our analysis showed increased amounts of SUMO conjugates associated with environmental stresses such as high and low temperature, NaCl and abscisic acid (ABA) in rice plants. The expression of OsSIZ1 and OsSIZ2 in siz1-2 Arabidopsis plants partially complemented the morphological mutant phenotype and enhanced levels of SUMO conjugates under heat shock conditions. In addition, ABA-hypersensitivity of siz1-2 seed germination was partially suppressed by OsSIZ1 and OsSIZ2. The results suggest that rice SIZ1 and SIZ2 are able to functionally complement Arabidopsis SIZ1 in the SUMO conjugation pathway. Their effects on the Arabidopsis mutant suggest a function for these genes related to stress responses and stress adaptation. © 2010 Blackwell Publishing Ltd.

  16. Arabidopsis BPM proteins function as substrate adaptors to a cullin3-based E3 ligase to affect fatty acid metabolism in plants.

    Science.gov (United States)

    Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R; Hellmann, Hanjo

    2013-06-01

    Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that cullin3-based E3 ligases have the potential to interact with a broad range of ethylene response factor (ERF)/APETALA2 (AP2) transcription factors, mediated by Math-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the wrinkled1 ERF/AP2 protein. Furthermore, loss of Math-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

  17. Arabidopsis BPM Proteins Function as Substrate Adaptors to a CULLIN3-Based E3 Ligase to Affect Fatty Acid Metabolism in Plants[W

    Science.gov (United States)

    Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R.; Hellmann, Hanjo

    2013-01-01

    Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members. PMID:23792371

  18. The deubiquitylating enzyme USP44 counteracts the DNA double-strand break response mediated by the RNF8 and RNF168 ubiquitin ligases

    DEFF Research Database (Denmark)

    Mosbech, Anna; Lukas, Claudia; Bekker-Jensen, Simon

    2013-01-01

    Protein recruitment to DNA double-strand breaks (DSBs) relies on ubiquitylation of the surrounding chromatin by the RING finger ubiquitin ligases RNF8 and RNF168. Flux through this pathway is opposed by several deubiquitylating enzymes (DUBs), including OTUB1 and USP3. By analyzing the effect...

  19. CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer

    DEFF Research Database (Denmark)

    Cepeda, Diana; Ng, Hwee-Fang; Sharifi, Hamid Reza

    2013-01-01

    SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28...

  20. Structural basis for hijacking CBF-β and CUL5 E3 ligase complex by HIV-1 Vif.

    Science.gov (United States)

    Guo, Yingying; Dong, Liyong; Qiu, Xiaolin; Wang, Yishu; Zhang, Bailing; Liu, Hongnan; Yu, You; Zang, Yi; Yang, Maojun; Huang, Zhiwei

    2014-01-09

    The human immunodeficiency virus (HIV)-1 protein Vif has a central role in the neutralization of host innate defences by hijacking cellular proteasomal degradation pathways to subvert the antiviral activity of host restriction factors; however, the underlying mechanism by which Vif achieves this remains unclear. Here we report a crystal structure of the Vif-CBF-β-CUL5-ELOB-ELOC complex. The structure reveals that Vif, by means of two domains, organizes formation of the pentameric complex by interacting with CBF-β, CUL5 and ELOC. The larger domain (α/β domain) of Vif binds to the same side of CBF-β as RUNX1, indicating that Vif and RUNX1 are exclusive for CBF-β binding. Interactions of the smaller domain (α-domain) of Vif with ELOC and CUL5 are cooperative and mimic those of SOCS2 with the latter two proteins. A unique zinc-finger motif of Vif, which is located between the two Vif domains, makes no contacts with the other proteins but stabilizes the conformation of the α-domain, which may be important for Vif-CUL5 interaction. Together, our data reveal the structural basis for Vif hijacking of the CBF-β and CUL5 E3 ligase complex, laying a foundation for rational design of novel anti-HIV drugs.

  1. The SOCS-box of HIV-1 Vif interacts with ElonginBC by induced-folding to recruit its Cul5-containing ubiquitin ligase complex.

    Directory of Open Access Journals (Sweden)

    Julien R C Bergeron

    Full Text Available The HIV-1 viral infectivity factor (Vif protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC, cullin 5 (Cul5 and RING-box 2 (Rbx2, to the anti-viral proteins APOBEC3G (A3G and APOBEC3F (A3F and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif's BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.

  2. Cullin 3 mediates SRC-3 ubiquitination and degradation to control the retinoic acid response.

    Science.gov (United States)

    Ferry, Christine; Gaouar, Samia; Fischer, Benoit; Boeglin, Marcel; Paul, Nicodeme; Samarut, Eric; Piskunov, Aleksandr; Pankotai-Bodo, Gabriella; Brino, Laurent; Rochette-Egly, Cecile

    2011-12-20

    SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitination and subsequent degradation of SRC-3. We also show that the RA-induced ubiquitination of SRC-3 depends on its prior phosphorylation at serine 860 that promotes binding of the CUL-3-based E3 ligase in the nucleus. Finally, phosphorylation, ubiquitination, and degradation of SRC-3 cooperate to control the dynamics of transcription. In all, this process participates to the antiproliferative effect of RA.

  3. Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members.

    Science.gov (United States)

    Ariani, Pietro; Regaiolo, Alice; Lovato, Arianna; Giorgetti, Alejandro; Porceddu, Andrea; Camiolo, Salvatore; Wong, Darren; Castellarin, Simone; Vandelle, Elodie; Polverari, Annalisa

    2016-12-02

    The Arabidopsis Tóxicos en Levadura (ATL) protein family is a class of E3 ubiquitin ligases with a characteristic RING-H2 Zn-finger structure that mediates diverse physiological processes and stress responses in plants. We carried out a genome-wide survey of grapevine (Vitis vinifera L.) ATL genes and retrieved 96 sequences containing the canonical ATL RING-H2 domain. We analysed their genomic organisation, gene structure and evolution, protein domains and phylogenetic relationships. Clustering revealed several clades, as already reported in Arabidopsis thaliana and rice (Oryza sativa), with an expanded subgroup of grapevine-specific genes. Most of the grapevine ATL genes lacked introns and were scattered among the 19 chromosomes, with a high level of duplication retention. Expression profiling revealed that some ATL genes are expressed specifically during early or late development and may participate in the juvenile to mature plant transition, whereas others may play a role in pathogen and/or abiotic stress responses, making them key candidates for further functional analysis. Our data offer the first genome-wide overview and annotation of the grapevine ATL family, and provide a basis for investigating the roles of specific family members in grapevine physiology and stress responses, as well as potential biotechnological applications.

  4. Ubiquitin ligase HUWE1 regulates axon branching through the Wnt/β-catenin pathway in a Drosophila model for intellectual disability.

    Science.gov (United States)

    Vandewalle, Joke; Langen, Marion; Zschätzsch, Marlen; Zschaetzsch, Marlen; Nijhof, Bonnie; Kramer, Jamie M; Brems, Hilde; Bauters, Marijke; Lauwers, Elsa; Srahna, Mohammed; Marynen, Peter; Verstreken, Patrik; Schenck, Annette; Hassan, Bassem A; Froyen, Guy

    2013-01-01

    We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID) in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN) was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh) by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of β-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/β-catenin pathway possibly by enhancing the degradation of dsh.

  5. Ubiquitin ligase HUWE1 regulates axon branching through the Wnt/β-catenin pathway in a Drosophila model for intellectual disability.

    Directory of Open Access Journals (Sweden)

    Joke Vandewalle

    Full Text Available We recently reported that duplication of the E3 ubiquitin ligase HUWE1 results in intellectual disability (ID in male patients. However, the underlying molecular mechanism remains unknown. We used Drosophila melanogaster as a model to investigate the effect of increased HUWE1 levels on the developing nervous system. Similar to the observed levels in patients we overexpressed the HUWE1 mRNA about 2-fold in the fly. The development of the mushroom body and neuromuscular junctions were not altered, and basal neurotransmission was unaffected. These data are in agreement with normal learning and memory in the courtship conditioning paradigm. However, a disturbed branching phenotype at the axon terminals of the dorsal cluster neurons (DCN was detected. Interestingly, overexpression of HUWE1 was found to decrease the protein levels of dishevelled (dsh by 50%. As dsh as well as Fz2 mutant flies showed the same disturbed DCN branching phenotype, and the constitutive active homolog of β-catenin, armadillo, could partially rescue this phenotype, our data strongly suggest that increased dosage of HUWE1 compromises the Wnt/β-catenin pathway possibly by enhancing the degradation of dsh.

  6. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

    DEFF Research Database (Denmark)

    Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K

    2013-01-01

    ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together...

  7. The vertebrate makorin ubiquitin ligase gene family has been shaped by large-scale duplication and retroposition from an ancestral gonad-specific, maternal-effect gene

    Directory of Open Access Journals (Sweden)

    Volff Jean-Nicolas

    2010-12-01

    fingers and even complete gene elimination from certain groups of vertebrates. Comparative expression analysis strongly suggests that the ancestral E3 ubiquitin ligase function of the single copy mkrn gene before duplication in vertebrates was gonad-specific, with maternal expression in early embryos.

  8. The stress phenotype makes cancer cells addicted to CDT2, a substrate receptor of the CRL4 ubiquitin ligase.

    Science.gov (United States)

    Olivero, Martina; Dettori, Daniela; Arena, Sabrina; Zecchin, Davide; Lantelme, Erica; Di Renzo, Maria Flavia

    2014-08-15

    CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.

  9. Ubiquitin Ligase, MuRF-1 regulates myosin heavy chain type IIa transcripts during muscle atrophy under microgravity conditions

    Science.gov (United States)

    Kagawa, Sachiko

    Skeletal muscles are vulnerable to marked atrophy under microgravity conditions. We previously reported that gastrocnemius muscle atrophy by spaceflight was specifically sensitive to the ubiquitin-proteasome proteolytic pathway. We also screened more over 26,000 skeletal muscle genes in rats exposed to real weightlessness and found that the expression of Ubiquitin Ligase, Muscle specific Ring Finger-1 (MuRF-1) upregulated under microgravity. In the present study, we examined the role of MuRF-1 in microgravity-induced muscle atrophy. The amounts of MuRF-1 transcripts significantly increased in skeletal muscle after denervation, an in vivo model of microgravity-induced unloading. MuRF-1 deficient (MuRF-1-/-) mice significantly inhibited reduction of muscle weight for muscle atrophy, compared with wild type mice. Interestingly, MuRF-1-/- mice significantly inhibited upregulation of myosin heavy chain (MyHC) type IIa transcrips, while wild type mice significantly increased expression of MyHC type IIa transcripts in denervated skeletal muscle. Our present results suggest that MuRF-1 may play an important role in regulation of MyHC type IIa during muscle atrophy under microgravity conditions.

  10. Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2000-01-01

    Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccha......Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which......, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference...... ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells....

  11. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies

    NARCIS (Netherlands)

    Reggiori, Fulvio; Pelham, Hugh R B; Reggiori, Fulvio

    2002-01-01

    Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. W

  12. A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies

    NARCIS (Netherlands)

    Reggiori, Fulvio; Pelham, Hugh R B; Reggiori, Fulvio

    2002-01-01

    Membrane proteins with transmembrane domains (TMDs) that contain polar residues exposed to the lipid bilayer are selectively sorted into multivesicular bodies (MVBs) and delivered to the yeast vacuole. Sorting of some, although not all, proteins into these structures is mediated by ubiquitination. W

  13. Differential gene expression of muscle-specific ubiquitin ligase MAFbx/Atrogin-1 and MuRF1 in response to immobilization-induced atrophy of slow-twitch and fast-twitch muscles.

    Science.gov (United States)

    Okamoto, Takeshi; Torii, Suguru; Machida, Shuichi

    2011-11-01

    We examined muscle-specific ubiquitin ligases MAFbx/Atrogin-1 and MuRF1 gene expression resulting from immobilization-induced skeletal muscle atrophy of slow-twitch soleus and fast-twitch plantaris muscles. Male C57BL/6 mice were subjected to hindlimb immobilization, which induced similar percentage decreases in muscle mass in the soleus and plantaris muscles. Expression of MAFbx/Atrogin-1 and MuRF1 was significantly greater in the plantaris muscle than in the soleus muscle during the early stage of atrophy. After a 3-day period of atrophy, total FOXO3a protein level had increased in both muscles, while phosphorylated FOXO3a protein had decreased in the plantaris muscle, but not in the soleus muscle. PGC-1α protein expression did not change following immobilization in both muscles, but basal PGC-1α protein in the soleus was markedly higher than that in plantaris muscles. These data suggest that although soleus and plantaris muscles atrophied to a similar extent and that muscle-specific ubiquitin protein ligases (E3) may contribute more to the atrophy of fast-twitch muscle than to that of slow-twitch muscle during immobilization.

  14. Regulation of APCCdh1 E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7

    Institute of Scientific and Technical Information of China (English)

    Alan W Lau; Hiroyuki Inuzuka; Hidefumi Fukushima; Lixin Wan; Pengda Liu; Daming Gao; Yi Sun

    2013-01-01

    Fbw7 and Cdh1 are substrate-recognition subunits of the SCF-and APC-type E3 ubiquitin ligases,respectively.There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction.Loss of Fbw7,but not Cdh1,is frequently observed in various human tumors.However,it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1.Here,we report that Fbw7-deficient cells not only display elevated expression levels of SCFFbw7 substrates,including cyclin E,but also have increased expression of various APCdh1 substrates.We further defined cyclin E as the critical signaling link by which Fbw7 governs APCCdh1 activity,as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APCdh1 substrates to levels comparable to those in wildtype (WT) cells.Conversely,ectopic expression of cyclin E recapitulates the aberrant APCdh1 substrate expression observed in Fbw7-deficient cells.More importantly,4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation,but not WT-Cdh1,reversed the elevated expression of various APCCdh1 substrates in Fbw7-deficient cells.Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation.Altogether,we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner.As a result,loss of Fbw7 can lead to aberrant increase in the expression of both SCFFbw7and APCCdh1 substrates.Our study provides a better understanding of the tumor suppressor function of Fbw7,and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.

  15. Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag.

    Directory of Open Access Journals (Sweden)

    Eric R Weiss

    Full Text Available Retroviruses engage the ESCRT pathway through late assembly (L domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA. The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.

  16. Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

    OpenAIRE

    Kazlauskaite, Agne; Kelly MR; Johnson, Clare; Baillie, Carla; Hastie, C James; Peggie, Mark; Macartney, Thomas; Woodroof, Helen I.; Alessi, Dario R; Pedrioli, Patrick G.A.; Muqit, Miratul M.K.

    2014-01-01

    Mutations in PINK1 and Parkin are associated with early-onset Parkinson's disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser65) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser65 phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Mir...

  17. The E3 Ligase APC/C-Cdh1 Is Required for Associative Fear Memory and Long-Term Potentiation in the Amygdala of Adult Mice

    Science.gov (United States)

    Pick, Joseph E.; Malumbres, Marcos; Klann, Eric

    2013-01-01

    The anaphase promoting complex/cyclosome (APC/C) is an E3 ligase regulated by Cdh1. Beyond its role in controlling cell cycle progression, APC/C-Cdh1 has been detected in neurons and plays a role in long-lasting synaptic plasticity and long-term memory. Herein, we further examined the role of Cdh1 in synaptic plasticity and memory by generating…

  18. Protein Interaction Screening for the Ankyrin Repeats and Suppressor of Cytokine Signaling (SOCS) Box (ASB) Family Identify Asb11 as a Novel Endoplasmic Reticulum Resident Ubiquitin Ligase

    DEFF Research Database (Denmark)

    Andresen, Christina Aaen; Smedegaard, Stine; Sylvestersen, Kathrine Beck

    2014-01-01

    remain largely unexplored. To increase our understanding of the ASB proteins function, we conducted a family-wide SILAC (Stable Isotope Labeling by Amino acids in Cell Culture)-based protein-protein interaction analysis. This investigation led to the identification of novel as well as known ASB...... in vivo. In summary, we provide a comprehensive protein-protein interaction data resource that can aid the biological and functional characterization of ASB ubiquitin ligases....

  19. 筛选调控Sonic Hedgehog信号转导的泛素连接酶%Identification of E3 ligases that regulate Sonic Hedgehog signaling transduction

    Institute of Scientific and Technical Information of China (English)

    汤颖; 乐珅; 程雁

    2013-01-01

    Objeetive:Screening for HECT E3 ligases that can regulate Sonic Hedgehog (Shh) signaling pathway.Methods:In the first round of screening,siRNAs of the HECT E3 ligases were tested for Shh pathway transduction by GliBS-luciferase assay.And in the second round,these siRNAs were tested for the localization of Patched1-GFP in primary cilia by immunofluorescence staining.Results:After two rounds of screening,5 HECT E3 ligases were identified to regulate Shh pathway,which are Smurf1,Smurf2,Ube3c,Wwp1 and Wwp2.Conclusion:A screening system of new regulators of Shh signaling was setup,and 5 HECT E3 ligases were found from our preliminary screening.%目的:发现调控Sonic Hedgehog (Shh)信号转导的泛素连接酶.方法:第一轮筛选,利用荧光素酶报告基因(8×GliBS-Luc)检测系统,检测相应HECT家族E3泛素连接酶的siRNA对Shh信号通路活性的影响;第二轮筛选,利用细胞免疫荧光激光共聚焦检测系统,检测上述siRNA对Shh的受体Ptch1蛋白原纤毛定位的影响.综合2轮筛选结果,初步发现影响Shh信号通路活性的HECT家族E3泛素连接酶.结果:通过2轮筛选,发现Smurf1、Smurf2、Ube3c、Wwp1、Wwp2共5个泛素连接酶,不仅能调节Ptch1蛋白的原纤毛定位,也可以增加通路下游转录因子Gli的活性.结论:建立了筛选调控Shh信号通路的泛素连接酶的平台.在初步筛选的27个HECT E3泛素连接酶中,有5个成员参与调控Shh信号通路.

  20. The ubiquitin ligase human TRIM71 regulates let-7 microRNA biogenesis via modulation of Lin28B protein.

    Science.gov (United States)

    Lee, Seo Hyun; Cho, Sungchan; Kim, M Sun; Choi, Kwangman; Cho, Jae Youl; Gwak, Ho-Shin; Kim, Youn-Jae; Yoo, Heon; Lee, Seung-Hoon; Park, Jong Bae; Kim, Jong Heon

    2014-05-01

    let-7 microRNA (miRNA) is implicated in various biological processes, and its downregulation essentially linked to human malignancy. Regulation of gene expression of the let-7 family is critically linked to RNA-binding proteins. For instance, Lin28B and its paralog, Lin28A, inhibit the pre-let-7 precursor from being processed to mature miRNA by recruiting terminal uridyltransferase, TUT4, which adds oligomeric U at the 3' end, suggesting that deregulation of Lin28B, together with Lin28A, may alter various biological processes through modulation of let-7 expression. Here, we showed that the Lin28B protein level is regulated via ubiquitin-mediated proteasomal degradation, and identified the ubiquitin ligase as human TRIM-NHL domain-containing TRIM71. In cells, TRIM71 negatively regulates Lin28B protein stability by catalyzing polyubiquitination. Compared with its paralog, Lin28A, a C-terminal unique ~50 amino acid stretch of Lin28B is essential for TRIM71 interactions and subsequent polyubiquitination. Moreover, the N-terminal RING finger motif of TRIM71 is critical for protein-protein interactions and polyubiquitination of Lin28B, and consequent let-7 expression. Consistent with the let-7 stimulatory role of TRIM71 via Lin28B polyubiquitination, specific knockdown of TRIM71 led to downregulation of let-7 expression. Expression of one of the known let-7 targets, HMGA2, was derepressed after knockdown of TRIM71. We additionally showed that enhanced expression of let-7 is part of a feedback loop that targets TRIM71 3'UTR, which contains two conserved let-7 target sites. Our findings collectively reveal critical aspects of regulatory complexity of let-7 biogenesis at the posttranscriptional level. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

    OpenAIRE

    Jia Shao; Vitnary Choe; Haili Cheng; Yien Che Tsai; Weissman, Allan M.; Shiwen Luo; Hai Rao

    2014-01-01

    Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrPSc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligas...

  2. Prenatal stress increased Snk Polo-like kinase 2, SCF β-TrCP ubiquitin ligase and ubiquitination of SPAR in the hippocampus of the offspring at adulthood.

    Science.gov (United States)

    Chutabhakdikul, Naunchan; Surakul, Pornprom

    2013-11-01

    Exposure to excessive glucocorticoids during fetal development period contributes to later life psychopathology. Prenatal stress decreases dendritic spine density and impair LTP in the hippocampus of rat pups, however, the mechanisms regulating these changes are still unclear. Glutamate receptors are localized in the postsynaptic density. PSD-95 is a postsynaptic scaffolding protein that plays a role in synaptic maturation and regulation of the synaptic strength and plasticity. PSD-95 interacts with other proteins to form the protein networks that promote dendritic spine formation. The present study investigated the effect of prenatal stress on the levels of scaffolding proteins of NMDA receptor in the hippocampus in order to explain how prenatal stress alters the amount of NMDA receptor in the pups' brain. Pregnant rats were randomly assigned to either the prenatal stress (PS) or the control group (C). The pregnant rats in the PS group were restrained in a plexiglas restrainer for 4h/day during the GD 14-21. Control rats were left undisturbed for the duration of their pregnancies. The amount of PSD-95, SPAR, NR2A and NR2B, as well as the levels of Snk Polo-like kinase 2 and the SCF β-TrCP ubiquitin ligase were measured in the hippocampus of the offspring. The results show that prenatal stress induces a reduction in the amount of NR2B and NR2A subunits in the hippocampus of rat pups, parallel to the decrease in PSD-95 and SPAR at P40 and P60. Moreover, prenatal stress increases Snk and β-TrCP in the hippocampus of rat pups, and the timing correlates with the decrease of SPAR and PSD-95. Prenatal stress also induces a significantly increases in the level of ubiquitinated SPAR in the hippocampus of rat pups at adulthood. The results suggest that degradation of SPAR via UPS system may contribute to the loss of PSD-95 and NMDA receptor subunits in the hippocampus of rat pups at adulthood. In conclusion, the present work demonstrates that the developing brain is

  3. The Brain Proteome of the Ubiquitin Ligase Peli1 Knock-Out Mouse during Experimental Autoimmune Encephalomyelitis

    Science.gov (United States)

    Lereim, Ragnhild Reehorst; Oveland, Eystein; Xiao, Yichuan; Torkildsen, Øivind; Wergeland, Stig; Myhr, Kjell-Morten; Sun, Shao-Cong; Berven, Frode S

    2016-01-01

    The ubiquitin ligase Peli1 has previously been suggested as a potential treatment target in multiple sclerosis. In the multiple sclerosis disease model, experimental autoimmune encephalomyelitis, Peli1 knock-out led to less activated microglia and less inflammation in the central nervous system. Despite being important in microglia, Peli1 expression has also been detected in glial and neuronal cells. In the present study the overall brain proteomes of Peli1 knock-out mice and wild-type mice were compared prior to experimental autoimmune encephalomyelitis induction, at onset of the disease and at disease peak. Brain samples from the frontal hemisphere, peripheral from the extensive inflammatory foci, were analyzed using TMT-labeling of sample pools, and the discovered proteins were verified in individual mice using label-free proteomics. The greatest proteomic differences between Peli1 knock-out and wild-type mice were observed at the disease peak. In Peli1 knock-out a higher degree of antigen presentation, increased activity of adaptive and innate immune cells and alterations to proteins involved in iron metabolism were observed during experimental autoimmune encephalomyelitis. These results unravel global effects to the brain proteome when abrogating Peli1 expression, underlining the importance of Peli1 as a regulator of the immune response also peripheral to inflammatory foci during experimental autoimmune encephalomyelitis. The proteomics data is available in PRIDE with accession PXD003710.

  4. SPA Proteins Affect the Subcellular Localization of COP1 in the COP1/SPA Ubiquitin Ligase Complex during Photomorphogenesis.

    Science.gov (United States)

    Balcerowicz, Martin; Kerner, Konstantin; Schenkel, Christian; Hoecker, Ute

    2017-07-01

    The Arabidopsis (Arabidopsis thaliana) COP1/SPA ubiquitin ligase is a central repressor that suppresses light signaling in darkness by targeting positive regulators of the light response, mainly transcription factors, for degradation. Light inactivates COP1/SPA, in part by excluding COP1 from the nucleus. SPA proteins are essential cofactors of COP1, but their exact role in the COP1/SPA complex is thus far unknown. To unravel a potential role of SPA proteins in COP1 nucleocytoplasmic partitioning, we monitored the subcellular localization of COP1 in a spa1234 quadruple mutant (spaQn). We analyzed a YFP-COP1-expressing transgenic line and endogenous COP1 after subcellular fractionation. In dark-grown seedlings, both YFP-COP1 and endogenous COP1 accumulated in the nucleus in the absence and presence of SPA proteins, indicating that SPA proteins are not required for nuclear localization of COP1 in darkness. In contrast, in white light-grown seedlings, spaQn mutants failed to relocalize COP1 from the nucleus to the cytoplasm. Hence, SPA proteins are necessary for the light-controlled change in COP1 subcellular localization. We conclude that SPA proteins have a dual role: (1) they are required for light-responsiveness of COP1 subcellular localization, and (2) they promote COP1 activity in darkness in a fashion that is independent of the nuclear import/nuclear retention of COP1. © 2017 American Society of Plant Biologists. All Rights Reserved.

  5. Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

    Institute of Scientific and Technical Information of China (English)

    Liren Liu; Yan Yin; Yuewei Li; Lisa Prevedel; Elizabeth H Lacy; Liang Ma; Pengbo Zhou

    2012-01-01

    Mutations of the CUL4B ubiquitin ligase gene are causally linked to syndromic X-linked mental retardation (XLMR).However,the pathogenic role of CUL4B mutations in neuronal and developmental defects is not understood.We have generated mice with targeted disruption of Cul4b,and observed embryonic lethality with pronounced growth inhibition and increased apoptosis in extra-embryonic tissues.Cul4b,but not its paralog Cul4a,is expressed at high levels in extra-embryonic tissues post implantation.Silencing of CUL4B expression in an extra-embryonic cell line resulted in the robust accumulation of the CUL4 substrate p21Cip1/WAF and G2/M cell cycle arrest,which could be partially rescued by silencing of p21cip1/WAF.Epiblast-specific deletion of Cul4b prevented embryonic lethality and gave rise to viable Cul4b null mice.Therefore,while dispensable in the embryo proper,Cul4b performs an essential developmental role in the extra-embryonic tissues.Our study offers a strategy to generate viable Cul4b-deficient mice to model the potential neuronal and behavioral deficiencies of human CUL4B XLMR patients.

  6. Overexpressed ubiquitin ligase Cullin7 in breast cancer promotes cell proliferation and invasion via down-regulating p53

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Hongsheng [Department of Histology and Embryology, Guangdong Medical College, Dongguan 523808, Guangdong (China); Wu, Fenping [The 7th People’s Hospital of Chengdu, Chengdu 610041, Sichuan (China); Wang, Yan [The Second School of Clinical Medicine, Guangdong Medical College, Dongguan 523808, Guangdong (China); Yan, Chong [School of Pharmacy, Guangdong Medical College, Dongguan 523808, Guangdong (China); Su, Wenmei, E-mail: wenmeisutg@126.com [Oncology of Affiliated Hospital Guangdong Medical College, Zhanjiang 524000, Guangdong (China)

    2014-08-08

    Highlights: • Cullin7 is overexpressed in human breast cancer samples. • Cullin7 stimulated proliferation and invasion of breast cancer cells. • Inhibition of p53 contributes to Cullin7-induced proliferation and invasion. - Abstract: Ubiquitin ligase Cullin7 has been identified as an oncogene in some malignant diseases such as choriocarcinoma and neuroblastoma. However, the role of Cullin7 in breast cancer carcinogenesis remains unclear. In this study, we compared Cullin7 protein levels in breast cancer tissues with normal breast tissues and identified significantly higher expression of Cullin7 protein in breast cancer specimens. By overexpressing Cullin7 in breast cancer cells HCC1937, we found that Cullin7 could promote cell growth and invasion in vitro. In contrast, the cell growth and invasion was inhibited by silencing Cullin7 in breast cancer cell BT474. Moreover, we demonstrated that Cullin7 promoted breast cancer cell proliferation and invasion via down-regulating p53 expression. Thus, our study provided evidence that Cullin7 functions as a novel oncogene in breast cancer and may be a potential therapeutic target for breast cancer management.

  7. A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells.

    Directory of Open Access Journals (Sweden)

    Andrew L Mammen

    Full Text Available Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish, UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3' introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.

  8. Resistance to UV-induced apoptosis by β-HPV5 E6 involves targeting of activated BAK for proteolysis by recruitment of the HERC1 ubiquitin ligase.

    Science.gov (United States)

    Holloway, Amy; Simmonds, Mark; Azad, Abul; Fox, Joanna L; Storey, Alan

    2015-06-15

    UV exposure is the main etiological agent in the development of non-melanoma skin cancer (NMSC), but mounting evidence suggests a co-factorial role for β-genus HPV types early in tumor initiation or progression. UV damage initiates an apoptotic response, driven at the mitochondrial level by BCL-2 family proteins, that eliminates damaged cells that may accumulate deleterious mutations and acquire tumorigenic properties. BAK is a pro-apoptotic BCL-2 protein that functions ultimately to form pores that permeabilize the mitochondrial outer membrane, thereby committing a cell to death, a process involving changes in BAK phosphorylation and conformation. The E6 protein of β-type HPV5 signals BAK for proteasomal degradation, a function that confers protection from UV-induced apoptosis. We find that HPV5 E6 does not constitutively target BAK for proteolysis, but targets the latter stages of BAK activation, following changes in phosphorylation and conformation. A mutational analysis identified the lysine residue on BAK required for proteolysis, and a functional siRNA screen identified the HECT domain E3 ubiquitin ligase HERC1 as being required for E6-mediated BAK degradation. We show that HERC1 interacts with BAK in E6-expressing cells that have been damaged by UV, and provide evidence that the interaction of HERC1 with BAK requires access to a hydrophobic surface on BAK that binds BH3 domains of BCL-2 proteins. We also show that HERC1 contains a putative BH3 domain that can bind to BAK. These findings reveal a specific and unique mechanism used by the HPV5 E6 protein to target BAK.

  9. Scores of RINGS but no PHDs in ubiquitin signaling.

    Science.gov (United States)

    Aravind, L; Iyer, L M; Koonin, E V

    2003-01-01

    Recently, it has been reported that PHD fingers of MEKK1 kinase and a family of viral and cellular membrane proteins have E3 ubiquitin ligase activity. Here we describe unique sequence and structural signatures that distinguish PHD fingers from RING fingers, which function primarily as E3 ubiquitin ligases, and demonstrate that the Zn-binding modules of the above proteins are distinct versions of the RING domain rather than PHD fingers. Thus, currently available data reveal extreme versatility of RINGs and their derivatives that function as E3 ubiquitin ligases but provide no evidence of this activity among PHD fingers whose principal function appears to involve specific protein-protein and possibly protein-DNA interactions in chromatin.

  10. Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation

    Science.gov (United States)

    Kobayashi, Takanori; Nagasaka, Seiji; Senoura, Takeshi; Itai, Reiko Nakanishi; Nakanishi, Hiromi; Nishizawa, Naoko K.

    2013-01-01

    Iron is essential for most living organisms. Plants transcriptionally induce genes involved in iron acquisition under conditions of low iron availability, but the nature of the deficiency signal and its sensors are unknown. Here we report the identification of new iron regulators in rice, designated Oryza sativa Haemerythrin motif-containing Really Interesting New Gene (RING)- and Zinc-finger protein 1 (OsHRZ1) and OsHRZ2. OsHRZ1, OsHRZ2 and their Arabidopsis homologue BRUTUS bind iron and zinc, and possess ubiquitination activity. OsHRZ1 and OsHRZ2 are susceptible to degradation in roots irrespective of iron conditions. OsHRZ-knockdown plants exhibit substantial tolerance to iron deficiency, and accumulate more iron in their shoots and grains irrespective of soil iron conditions. The expression of iron deficiency-inducible genes involved in iron utilization is enhanced in OsHRZ-knockdown plants, mostly under iron-sufficient conditions. These results suggest that OsHRZ1 and OsHRZ2 are iron-binding sensors that negatively regulate iron acquisition under conditions of iron sufficiency. PMID:24253678

  11. Kaposi's sarcoma-associated herpesvirus K-Rta exhibits SUMO-targeting ubiquitin ligase (STUbL like activity and is essential for viral reactivation.

    Directory of Open Access Journals (Sweden)

    Yoshihiro Izumiya

    Full Text Available The small ubiquitin-like modifier (SUMO is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML and K-bZIP. PML-NBs (nuclear bodies or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate

  12. Regulation of voltage-gated ion channels in excitable cells by the ubiquitin ligases Nedd4 and Nedd4-2.

    Science.gov (United States)

    Bongiorno, Daria; Schuetz, Friderike; Poronnik, Philip; Adams, David J

    2011-01-01

    The electrical excitability of neurons is mediated primarily by voltage-gated ion channels, particularly voltage-gated Na(+) (Na(v)), K(+) (K(v)) and Cl(-) (ClC) channels. Cells regulate their electrical excitability by controlling not only the activity, but also the number of individual ion channels in the plasma membrane. There exist several mechanisms for regulating levels of voltage-gated ion channels: transcription and translation, retention and export from the endoplasmic reticulum as well as insertion and retrieval from the plasma membrane. Alterations in voltage-gated ion channel activity, composition and distribution can contribute to the pathophysiology of epilepsy, hypertension, neuropathic and inflammatory pain. One mechanism for retrieval is ubiquitination. Here specific ubiquitin ligases bind to membrane proteins to modulate and regulate their cellular fate. In this review, we focus on Nedd4 and Nedd4-2 ubiquitin ligases and the mechanisms by which they regulate voltage-gated ion channels and describe a novel paradigm on the mechanisms that underpin aberrant ion channel function in neurological disorders.

  13. BRCC36, a Novel Subunit of a BRCA1 E3 Ubiquitin Ligase Complex: Candidates for BRCA3

    Science.gov (United States)

    2008-06-01

    J, Dahia PL, Wang SI, Zheng Z, Bose S, Call KM, Tsou HC, Peacocke M and others. 1997. Germline mutations of the PTEN gene in Cowden disease, an...Hum Mutat 20:65–73. Arnold N, Peper H, Bandick K, Kreikemeier M, Karow D, Teegen B, Jonat W. 2002. Establishing a control population to screen for

  14. BRCC36, A Novel Subunit of a BRCA1 E3 Ubiquitin Ligase Complex, Candidates for BRCA3

    Science.gov (United States)

    2006-06-01

    Lymphocytes isolated from FRAP blood samples were infected with Epstein-Barr virus ( EBV ) to establish the immortal lymphoblastoid cell lines (LCLs...BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures . Genes Dev 2000;14:927–39. 27...inhibitor, puromycin (Sigma, St. Louis, MO; www.sigmaaldrich.com), was added to the EBV cells at the concentration of 200 mg/ml for 14 hr before total RNAs

  15. The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ

    OpenAIRE

    Watanabe, Masashi; Takahashi, Hidehisa; Saeki, Yasushi; Ozaki, Takashi; Itoh, Shihori; Suzuki, Masanobu; Mizushima, Wataru; Tanaka, Keiji; Hatakeyama, Shigetsugu

    2015-01-01

    eLife digest The world is facing a global epidemic of obesity, which also increases the risk for diabetes and heart disease. Obesity is caused when excess fat is stored in fat cells, and overweight individuals have larger fat cells compared to healthy weight people. Therefore understanding how fat cells are created in the body can provide new ways to combat obesity. Fat cells, also known as adipocytes, arise from precursor cells via a process called adipogenesis. This requires the activity of...

  16. Mouse RING finger protein Rnf133 is a testis-specific endoplasmic reticulum-associated E3 ubiquitin ligase

    Institute of Scientific and Technical Information of China (English)

    Hong Nian; Chunsheng Han; Wei Zhang; Hexin Shi; Qingzhen Zhao; Qi Xie; Shangying Liao; Yan Zhang; Zhuqiang Zhang; Chen Wang

    2008-01-01

    @@ Dear Editor, Spermatogenesis, the process by which sperms are generated within the male gonads, involves a number of events that occur only in the testis. Examples include the nucleus condensation as a result of histone sequential replacement by transition proteins 1, 2 and protamin, the formation of sperm-specific organelles such as acrosomes and sperm tails, and the shedding of majority of the cytoplasm as residual bodies.

  17. Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals

    Institute of Scientific and Technical Information of China (English)

    Faridah Yusof; Atheer Awad Mehde; Wesen Adel Mehdi; Raha Ahmed Raus; Hamid Ghazali; Azlina Abd Rahman

    2015-01-01

    Objective Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. Methods Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. Results The result indicated that mean levels of sera NSMCE2 have a significantly increase (P Conclusion This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

  18. Effects of Nephrolithiasis on Serum DNase (Deoxyribonuclease I and II) Activity and E3 SUMO-Protein Ligase NSE2 (NSMCE2) in Malaysian Individuals.

    Science.gov (United States)

    Yusof, Faridah; Mehde, Atheer Awad; Mehdi, Wesen Adel; Raus, Raha Ahmed; Ghazali, Hamid; Rahman, Azlina Abd

    2015-09-01

    Nephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage. Sixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria. The result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01). This study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis. Copyright © 2015 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  19. TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes.

    Science.gov (United States)

    Kelsall, Ian R; Duda, David M; Olszewski, Jennifer L; Hofmann, Kay; Knebel, Axel; Langevin, Frédéric; Wood, Nicola; Wightman, Melanie; Schulman, Brenda A; Alpi, Arno F

    2013-10-30

    RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7∼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.

  20. Inhibition of MARCH5 ubiquitin ligase abrogates MCL1-dependent resistance to BH3 mimetics via NOXA.

    Science.gov (United States)

    Subramanian, Aishwarya; Andronache, Adrian; Li, Yao-Cheng; Wade, Mark

    2016-03-29

    BH3 mimetic compounds induce tumor cell death through targeted inhibition of anti-apoptotic BCL2 proteins. Resistance to one such compound, ABT-737, is due to increased levels of anti-apoptotic MCL1. Using chemical and genetic approaches, we show that resistance to ABT-737 is abrogated by inhibition of the mitochondrial RING E3 ligase, MARCH5. Mechanistically, this is due to increased expression of pro-apoptotic BCL2 family member, NOXA, and is associated with MARCH5 regulation of MCL1 ubiquitylation and stability in a NOXA-dependent manner. MARCH5 expression contributed to an 8-gene signature that correlates with sensitivity to the preclinical BH3 mimetic, navitoclax. Furthermore, we observed a synthetic lethal interaction between MCL1 and MARCH5 in MCL1-dependent breast cancer cells. Our data uncover a novel level at which the BCL2 family is regulated; furthermore, they suggest targeting MARCH5-dependent signaling will be an effective strategy for treatment of BH3 mimetic-resistant tumors, even in the presence of high MCL1.

  1. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    Science.gov (United States)

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  2. Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

    Directory of Open Access Journals (Sweden)

    Barbara Mojsa

    2014-05-01

    Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

  3. The tomato DWD motif-containing protein DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase and plays a pivotal role in abiotic stress responses

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Min [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339 (United States); Zhu, Yunye [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Qiao, Maiju [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); Tang, Xiaofeng [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Zhao, Wei [School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China); Xiao, Fangming [Department of Plant, Soil and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339 (United States); Liu, Yongsheng, E-mail: liuyongsheng1122@hfut.edu.cn [Ministry of Education Key Laboratory for Bio-resource and Eco-environment, College of Life Science, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064 (China); School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009 (China)

    2014-08-08

    Highlights: • We identify DDI1 as a DAMAGED DNA BINDING PROTEIN1 (DDB1)-interacting protein. • DDI1 interacts with the CUL4–DDB1-based ubiquitin ligase in the nucleus. • DDI1 plays a positive role in regulating abiotic stress response in tomato. - Abstract: CULLIN4(CUL4)–DAMAGED DNA BINDING PROTEIN1 (DDB1)-based ubiquitin ligase plays significant roles in multiple physiological processes via ubiquitination-mediated degradation of relevant target proteins. The DDB1–CUL4-associated factor (DCAF) acts as substrate receptor in the CUL4–DDB1 ubiquitin ligase complex and determines substrate specificity. In this study, we identified a tomato (Solanum lycopersicum) DDB1-interacting (DDI1) protein as a DCAF protein involved in response to abiotic stresses, including UV radiation, high salinity and osmotic stress. Co-immunoprecipitation and bimolecular fluorescence complementation assay indicated that DDI1 associates with CUL4–DDB1 in the nucleus. Quantitative RT-PCR analysis indicated the DDI1 gene is induced by salt, mannitol and UV-C treatment. Moreover, transgenic tomato plants with overexpression or knockdown of the DDI1 gene exhibited enhanced or attenuated tolerance to salt/mannitol/UV-C, respectively. Thus, our data suggest that DDI1 functions as a substrate receptor of the CUL4–DDB1 ubiquitin ligase, positively regulating abiotic stress response in tomato.

  4. Ubiquitin

    DEFF Research Database (Denmark)

    Vinther-Jensen, T.; Simonsen, A. H.; Budtz-Jorgensen, E.;

    2015-01-01

    , as well as aid in identifying an optimal time point for initiating a potential therapeutic intervention. METHODS: This explorative proteomics study evaluated cerebrospinal fluid from 94 Huntington's disease gene-expansion carriers (39 premanifest and 55 manifest) and 27 Huntington's disease gene......-expansion negative individuals using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry. Differences in peak intensity from SELDI-TOF spectra were evaluated. RESULTS: Levels of 10 peaks were statistically significantly different between manifest gene-expansion carriers......-expansion carriers. CONCLUSIONS AND RELEVANCE: Multiple studies have shown that the ubiquitin-proteasome system is involved in Huntington's disease pathogenesis and understanding of this involvement may have therapeutic potential in humans. This is the first study on cerebrospinal fluid to confirm the involvement...

  5. BPM-CUL3 E3 ligase modulates thermotolerance by facilitating negative regulatory domain-mediated degradation of DREB2A in Arabidopsis.

    Science.gov (United States)

    Morimoto, Kyoko; Ohama, Naohiko; Kidokoro, Satoshi; Mizoi, Junya; Takahashi, Fuminori; Todaka, Daisuke; Mogami, Junro; Sato, Hikaru; Qin, Feng; Kim, June-Sik; Fukao, Yoichiro; Fujiwara, Masayuki; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2017-09-18

    DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN 2A (DREB2A) acts as a key transcription factor in both drought and heat stress tolerance in Arabidopsis and induces the expression of many drought- and heat stress-inducible genes. Although DREB2A expression itself is induced by stress, the posttranslational regulation of DREB2A, including protein stabilization, is required for its transcriptional activity. The deletion of a 30-aa central region of DREB2A known as the negative regulatory domain (NRD) transforms DREB2A into a stable and constitutively active form referred to as DREB2A CA. However, the molecular basis of this stabilization and activation has remained unknown for a decade. Here we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the Cullin3 (CUL3)-based E3 ligase, as DREB2A-interacting proteins. We observed that DREB2A and BPMs interact in the nuclei, and that the NRD of DREB2A is sufficient for its interaction with BPMs. BPM-knockdown plants exhibited increased DREB2A accumulation and induction of DREB2A target genes under heat and drought stress conditions. Genetic analysis indicated that the depletion of BPM expression conferred enhanced thermotolerance via DREB2A stabilization. Thus, the BPM-CUL3 E3 ligase is likely the long-sought factor responsible for NRD-dependent DREB2A degradation. Through the negative regulation of DREB2A stability, BPMs modulate the heat stress response and prevent an adverse effect of excess DREB2A on plant growth. Furthermore, we found the BPM recognition motif in various transcription factors, implying a general contribution of BPM-mediated proteolysis to divergent cellular responses via an accelerated turnover of transcription factors.

  6. Phosphorylation of Arabidopsis ubiquitin ligase ATL31 is critical for plant carbon/nitrogen nutrient balance response and controls the stability of 14-3-3 proteins.

    Science.gov (United States)

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-05-30

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr(209), Ser(247), Ser(270), and Ser(303) as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr(209) and Ser(247) on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr(209) peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.

  7. Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p

    Directory of Open Access Journals (Sweden)

    Abderazzaq Kareem

    2002-08-01

    Full Text Available Abstract Background SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes. Results We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity. Conclusion Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.

  8. The polyglutamine-expanded androgen receptor responsible for spinal and bulbar muscular atrophy inhibits the APC/C(Cdh1) ubiquitin ligase complex.

    Science.gov (United States)

    Bott, Laura C; Salomons, Florian A; Maric, Dragan; Liu, Yuhong; Merry, Diane; Fischbeck, Kenneth H; Dantuma, Nico P

    2016-06-17

    Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/C(Cdh1) complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/C(Cdh1) by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/C(Cdh1) and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA.

  9. The polyglutamine-expanded androgen receptor responsible for spinal and bulbar muscular atrophy inhibits the APC/CCdh1 ubiquitin ligase complex

    Science.gov (United States)

    Bott, Laura C.; Salomons, Florian A.; Maric, Dragan; Liu, Yuhong; Merry, Diane; Fischbeck, Kenneth H.; Dantuma, Nico P.

    2016-01-01

    Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/CCdh1 complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/CCdh1 by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/CCdh1 and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA. PMID:27312068

  10. Escape, or Vanish: Control the Fate of p53 through MDM2-Mediated Ubiquitination.

    Science.gov (United States)

    Wei, Jinlian; Yang, Yingrui; Lu, Mengchen; Xu, Lili; Liu, Fang; Yuan, Zhenwei; Bao, Qichao; Jiang, Zhengyu; Xu, Xiaoli; Guo, Xiaoke; Zhang, Xiaojin; You, Qidong; Sun, Haopeng

    2015-01-01

    p53 protein is a prominent tumor suppressor to induce cell cycle arrest, apoptosis and senescence, which attracts significant interest to cancer treatment. Therefore, it would be particularly important to restore the wild-type p53 that retains latent functions in the approximately 50% of tumors. MDM2 (murine double minute 2), the principal cellular antagonist of p53, has long been believed to suppress p53 activity through two main mechanisms: promoting degradation via its E3 ligase activity and masking p53 transcriptional activation by direct binding. Targeting MDM2 E3 ligase activity is becoming a potential antitumor strategy resulting from MDM2's decisive role in controlling the fate of p53: p53 is going to degradation when entrapped into MDM2-mediated ubiquitination, where p53 can escape by abrogating MDM2 E3 ligase activity using regulators. The intensive focus on regulating MDM2 ubiquitin E3 ligase activity has led to the rapid progress of its inhibitors, which may be possible to help p53 escape from degradation and restore its function to control tumor growth. This review summarizes the current inhibitors of MDM2 E3 ligase in cancer therapy based on the understanding the regulation of MDM2 E3 ubiquitin ligase activity, including post-translational modification, interactions between MDM2 and its cofactors, and regulation of MDM2 stability.

  11. Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

    DEFF Research Database (Denmark)

    Suryadinata, Randy; Holien, Jessica K; Yang, George

    2013-01-01

    The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however......, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines....... Evaluation of the relative importance of different residues positioned -2, -1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the -1 and -2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter...

  12. Ubiquitin ligase RNF123 mediates degradation of heterochromatin protein 1α and β in lamin A/C knock-down cells.

    Directory of Open Access Journals (Sweden)

    Pankaj Chaturvedi

    Full Text Available BACKGROUND: The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1. However, the pathways of proteasomal degradation have not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells. CONCLUSIONS/SIGNIFICANCE: Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of

  13. E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

    OpenAIRE

    2008-01-01

    The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC ...

  14. 1α,25(OH)2D3 downregulates gene expression levels of muscle ubiquitin ligases MAFbx and MuRF1 in human myotubes.

    Science.gov (United States)

    Hayakawa, Naohiko; Fukumura, Junko; Yasuno, Hideyuki; Fujimoto-Ouchi, Kaori; Kitamura, Hidemitsu

    2015-01-01

    Clinical trials involving in patients with osteoporosis have reported that activated vitamin D3 (1α,25(OH)2D3, calcitriol) can prevent falling by acting on the skeletal muscles. However, pharmacological mechanisms of 1α,25(OH)2D3 with respect to skeletal muscle hypertrophy or atrophy are still poorly understood. Therefore, we examined changes in the expression of several related genes in human myotubes to test whether 1α,25(OH)2D3 influences hypertrophy and atrophy of skeletal muscle. Myotubes treated with 1α,25(OH)2D3 increased interleukin-6 (IL-6) expression and inhibited expression of tumor necrosis factor alpha (TNF-α), whereas the expression of insulin-like growth factor-1 (IGF-1) that is involved in muscle hypertrophy was not affected. However, 1α,25(OH)2D3 treatment significantly inhibited the expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), ubiquitin ligases involved in muscle atrophy. The analysis of pathways using microarray data suggested that 1α,25(OH)2D3 upregulates AKT-1 by inhibiting the expression of protein phosphatase 2 (PP2A), a phosphatase acting on AKT-1, in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, thereby inhibiting the expression of ubiquitin ligases. Thus, this study showed that 1α,25(OH)2D3 might have an inhibitory effect on the expression of MAFbx and MuRF1 in skeletal muscle and a suppressive effect on muscle degradation in patients with osteoporosis.

  15. The role of Smad Ubiquitin Regulatory Factor-1: SMURF1 in the differentiation 3T3-L1

    OpenAIRE

    Muñiz Diego, María del Carmen

    2013-01-01

    The Smad Ubiquitin Regulatory Factor-1: SMURF1 is an E3 ligase. This E3 ligase has been linked to several important biological pathways, including the bone morphogenetic protein pathway, the non-canonical Wnt pathway and the mitogen-activated protein kinase pathway. Multiple functions of Smurf1 have been discovered in cell growth and morphogenesis, cell migration, cell polarity and autophagy. Previous studies, in 2003, have demonstrated that overexpression of Smurf1 induces proteasomal degrad...

  16. Core-binding factor β increases the affinity between human Cullin 5 and HIV-1 Vif within an E3 ligase complex.

    Science.gov (United States)

    Salter, Jason D; Lippa, Geoffrey M; Belashov, Ivan A; Wedekind, Joseph E

    2012-11-06

    HIV-1 Vif masquerades as a receptor for a cellular E3 ligase harboring Elongin B, Elongin C, and Cullin 5 (EloB/C/Cul5) proteins that facilitate degradation of the antiretroviral factor APOBEC3G (A3G). This Vif-mediated activity requires human core-binding factor β (CBFβ) in contrast to cellular substrate receptors. We observed calorimetrically that Cul5 binds tighter to full-length Vif((1-192))/EloB/C/CBFβ (K(d) = 5 ± 2 nM) than to Vif((95-192))/EloB/C (K(d) = 327 ± 40 nM), which cannot bind CBFβ. A comparison of heat capacity changes supports a model in which CBFβ prestabilizes Vif((1-192)) relative to Vif((95-192)), consistent with a stronger interaction of Cul5 with Vif's C-terminal Zn(2+)-binding motif. An additional interface between Cul5 and an N-terminal region of Vif appears to be plausible, which has therapeutic design implications.

  17. Mechanism of ubiquitylation by dimeric RING ligase RNF4

    Science.gov (United States)

    Plechanovová, Anna; Jaffray, Ellis G.; McMahon, Stephen A.; Johnson, Kenneth A.; Navrátilová, Iva; Naismith, James H.; Hay, Ronald T.

    2012-01-01

    Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model the “Ile44 hydrophobic patch” of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis. PMID:21857666

  18. Genetic interactions between the ESS1 prolyl-isomerase and the RSP5 ubiquitin ligase reveal opposing effects on RNA polymerase II function.

    Science.gov (United States)

    Wu, X; Chang, A; Sudol, M; Hanes, S D

    2001-12-01

    Transcription of protein-coding genes by RNA polymerase II (pol II) is a highly coordinated process that requires the stepwise association of distinct protein complexes with the C-terminal domain (CTD) of Rpbl, the largest subunit of RNA pol II. Interaction of these complexes with the CTD might be subject to regulation by proteins such as Ess1 and Rsp5. Ess1, a prolyl-isomerase, binds the CTD and is thought to play a positive role in pol II transcription by generating conformational isomers of the CTD. Rsp5, a ubiquitin ligase, binds the CTD and is thought to play a negative role in transcription by mediating Rpbl ubiquitination and degradation. In this paper, we demonstrate that ESS1 and RSP5 interact genetically and that these interactions occur via RPBI. We show that over-expression of RSP5 enhances the growth defect of ess1ts cells and this effect is reversed by introducing extra copies of RPB1. Over-expression of RSP5 also mimics the sensitivity of ess1ts mutant cells to the toxicity of plasmids carrying dominant-negative CTD mutations, whereas mutations in RSP5 suppress this effect. Using a modified two-hybrid assay, we also demonstrate that Essl and Rsp5 compete directly for binding to the CTD. The results suggest a model in which Essl and Rsp5 act opposingly on pol II function to control the level of pol II available for transcription.

  19. The multiple levels of regulation by p53 ubiquitination

    OpenAIRE

    Lee, JT; Gu, W

    2010-01-01

    p53 is a central integrator of a plethora of signals and outputs these signals in the form of tumor suppression. It is well accepted that ubiquitination plays a major part in p53 regulation. Nonetheless, the molecular mechanisms by which p53 activity is controlled by ubiquitination are complex. Mdm2, a RING oncoprotein, was once thought to be the sole E3 ubiquitin ligase for p53, however recent studies have shown that p53 is stabilized but still degraded in the cells of Mdm2-null mice. Althou...

  20. The archipelago ubiquitin ligase subunit acts in target tissue to restrict tracheal terminal cell branching and hypoxic-induced gene expression.

    Directory of Open Access Journals (Sweden)

    Nathan T Mortimer

    Full Text Available The Drosophila melanogaster gene archipelago (ago encodes the F-box/WD-repeat protein substrate specificity factor for an SCF (Skp/Cullin/F-box-type polyubiquitin ligase that inhibits tumor-like growth by targeting proteins for degradation by the proteasome. The Ago protein is expressed widely in the fly embryo and larva and promotes degradation of pro-proliferative proteins in mitotically active cells. However the requirement for Ago in post-mitotic developmental processes remains largely unexplored. Here we show that Ago is an antagonist of the physiologic response to low oxygen (hypoxia. Reducing Ago activity in larval muscle cells elicits enhanced branching of nearby tracheal terminal cells in normoxia. This tracheogenic phenotype shows a genetic dependence on sima, which encodes the HIF-1α subunit of the hypoxia-inducible transcription factor dHIF and its target the FGF ligand branchless (bnl, and is enhanced by depletion of the Drosophila Von Hippel Lindau (dVHL factor, which is a subunit of an oxygen-dependent ubiquitin ligase that degrades Sima/HIF-1α protein in metazoan cells. Genetic reduction of ago results in constitutive expression of some hypoxia-inducible genes in normoxia, increases the sensitivity of others to mild hypoxic stimulus, and enhances the ability of adult flies to recover from hypoxic stupor. As a molecular correlate to these genetic data, we find that Ago physically associates with Sima and restricts Sima levels in vivo. Collectively, these findings identify Ago as a required element of a circuit that suppresses the tracheogenic activity of larval muscle cells by antagonizing the Sima-mediated transcriptional response to hypoxia.

  1. CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

    Directory of Open Access Journals (Sweden)

    Yu-Chen Lin

    2017-05-01

    Full Text Available Influenza A virus (IAV RNA segments are individually packaged with viral nucleoprotein (NP and RNA polymerases to form a viral ribonucleoprotein (vRNP complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4, which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication.

  2. Overexpression of the Rice SUMO E3 Ligase Gene OsSIZ1 in Cotton Enhances Drought and Heat Tolerance, and Substantially Improves Fiber Yields in the Field under Reduced Irrigation and Rainfed Conditions

    Science.gov (United States)

    Mishra, Neelam; Sun, Li; Zhu, Xunlu; Smith, Jennifer; Prakash Srivastava, Anurag; Yang, Xiaojie; Pehlivan, Necla; Esmaeili, Nardana; Luo, Hong; Shen, Guoxin; Jones, Don; Auld, Dick; Burke, John

    2017-01-01

    The Arabidopsis SUMO E3 ligase gene AtSIZ1 plays important roles in plant response to abiotic stresses as loss of function in AtSIZ1 leads to increased sensitivity to drought, heat and salt stresses. Overexpression of the AtSIZ1 rice homolog, OsSIZ1, leads to increased heat and drought tolerance in bentgrass, suggesting that the function of the E3 ligase SIZ1 is highly conserved in plants and it plays a critical role in abiotic stress responses. To test the possibility that the SUMO E3 ligase could be used to engineer drought- and heat-tolerant crops, the rice gene OsSIZ1 was overexpressed in cotton. We report here that overexpression of OsSIZ1 in cotton results in higher net photosynthesis and better growth than wild-type cotton under drought and thermal stresses in growth chamber and greenhouse conditions. Additionally, this tolerance to abiotic stresses was correlated with higher fiber yield in both controlled-environment and field trials carried out under reduced irrigation and rainfed conditions. These results suggest that OsSIZ1 is a viable candidate gene to improve crop yields under water-limited and rainfed agricultural production systems. PMID:28340002

  3. Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65

    Science.gov (United States)

    Kazlauskaite, Agne; Kondapalli, Chandana; Gourlay, Robert; Campbell, David G.; Ritorto, Maria Stella; Hofmann, Kay; Alessi, Dario R.; Knebel, Axel; Trost, Matthias; Muqit, Miratul M. K.

    2014-01-01

    We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65

  4. Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival*

    Science.gov (United States)

    Peng, Lirong; Yuan, Zhigang; Li, Yixuan; Ling, Hongbo; Izumi, Victoria; Fang, Bin; Fukasawa, Kenji; Koomen, John; Chen, Jiandong; Seto, Edward

    2015-01-01

    Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR. PMID:25670865

  5. Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Jiazhang; Sheedlo, Michael J.; Yu, Kaiwen; Tan, Yunhao; Nakayasu, Ernesto S.; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2016-04-06

    Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. The E1/E2-independent ubiquitination catalyzed by these enzymes requires NAD but not ATP and Mg2+. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. These results establish that ubiquitination can be catalyzed by a single enzyme.

  6. Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage

    DEFF Research Database (Denmark)

    Thorslund, Tina; Ripplinger, Anita; Hoffmann, Saskia

    2015-01-01

    DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also...

  7. Upregulation of SIRT1 by 17β-estradiol depends on ubiquitin-proteasome degradation of PPAR-γ mediated by NEDD4-1

    OpenAIRE

    Han, Limin; Pan WANG; Zhao, Ganye; Wang, Hui; Wang, Meng; Chen, Jun; Tong, Tanjun

    2013-01-01

    17β-estradiol (E2) treatment of cells results in an upregulation of SIRT1 and a down-regulation of PPARγ. The decrease in PPARγ expression is mediated by increased degradation of PPARγ. Here we report that PPARγ is ubiquitinated by HECT E3 ubiquitin ligase NEDD4-1 and degraded, along with PPARγ, in response to E2 stimulation. The PPARγ interacts with ubiquitin ligase NEDD4-1 through a conserved PPXY-WW binding motif. The WW3 domain in NEDD4-1 is critical for binding to PPARΓ. NEDD4-1 overexpr...

  8. Dynamics of ubiquitin-mediated signalling: insights from mathematical modelling and experimental studies.

    Science.gov (United States)

    Nguyen, Lan K

    2016-05-01

    Post-translational modification of cellular proteins by ubiquitin is a pivotal regulatory event that controls not only protein degradation, but also a variety of non-proteolytic functions. Ubiquitination is involved in a broad array of physiological processes, and its dysregulation has been associated with many human diseases, including neuronal disorders and cancers. Ubiquitin-mediated signalling has thus come to the forefront of biomedical research. It is increasingly apparent that ubiquitination is a highly complex and dynamic process, evidenced by a myriad of ways of ubiquitin chain formation, tightly regulatory mechanisms involving E3 ligases and deubiquitinating enzymes and extensive crosstalk with other post-translational modifications. To unravel the complexity of ubiquitination and understand the dynamic properties of ubiquitin-mediated signalling are challenging, but critical topics in ubiquitin research, which will undoubtedly benefit our effort in developing strategies that could target ubiquitin signalling for therapeutics. Computational modelling and model-based approaches are emerging as promising tools that help tackle the complexity and provide useful frameworks for quantitative and dynamical analysis of ubiquitin signalling. In this article, I will discuss recent advances in our understanding of the dynamic behaviour of ubiquitination from both theoretical and experimental studies, and aspects of ubiquitin signalling that may have major dynamical consequences. It is expected the discussed issues will be of relevant interest to both the ubiquitin and systems biology fields.

  9. HERC 1 Ubiquitin Ligase Mutation Affects Neocortical, CA3 Hippocampal and Spinal Cord Projection Neurons: An Ultrastructural Study.

    Science.gov (United States)

    Ruiz, Rocío; Pérez-Villegas, Eva María; Bachiller, Sara; Rosa, José Luis; Armengol, José Angel

    2016-01-01

    The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tambaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic loss of cerebellar Purkinje cells. Interestingly, signs of autophagy are absent in both interneurons and neuroglia cells. Affected neurons have in common that they are projection neurons which receive strong and varied synaptic inputs, and possess the highest degree of neuronal activity. Therefore, because the integrity of the ubiquitin-proteasome system is essential for protein degradation and hence, for normal protein turnover, it could be hypothesized that the deleterious effects of the misrouting of these pathways would depend directly on the neuronal activity.

  10. HERC 1 ubiquitin ligase mutation affects neocortical, CA3 hippocampal and spinal cord projection neurons. An ultrastructural study

    Directory of Open Access Journals (Sweden)

    Rocío eRuiz

    2016-04-01

    Full Text Available The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tambaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic l