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Sample records for e3 ligase complex

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

    PubMed Central

    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.

    2011-01-01

    SUMMARY 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. PMID:21504829

  2. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins

    PubMed Central

    Ju Shin, Yeong; Kyun Park, Seung; Jung Jung, Yoo; Na Kim, Ye; Sung Kim, Ki; Kyu Park, Ok; Kwon, Seung-Hae; Ho Jeon, Sung; Trinh, Le A.; Fraser, Scott E.; Kee, Yun; Joon Hwang, Byung

    2015-01-01

    Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein. PMID:26373678

  3. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

    PubMed Central

    Lechtenberg, Bernhard C.; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K.; Ware, Carl F.; Mace, Peter D.; Riedl, Stefan J.

    2015-01-01

    Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

  4. Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.

    PubMed

    Lechtenberg, Bernhard C; Rajput, Akhil; Sanishvili, Ruslan; Dobaczewska, Małgorzata K; Ware, Carl F; Mace, Peter D; Riedl, Stefan J

    2016-01-28

    Ubiquitination is a central process affecting all facets of cellular signalling and function. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases. The RBR family includes Parkin and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex). While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix-IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases. PMID:26789245

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

    PubMed Central

    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.

    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 factors by CRL4CRBN. Here we present the crystal structure of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes CRBN as a CRL4CRBN substrate receptor, which enantioselectively binds IMiDs. Through an unbiased screen we identify the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4CRBN. Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4CRBN when recruiting Ikaros/Aiolos for degradation. This dual activity implies that small molecules can principally modulate a ligase to up- or down-regulate the ubiquitination of proteins. PMID:25043012

  6. Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases

    PubMed Central

    Pruneda, Jonathan N.; Littlefield, Peter J.; Soss, Sarah E.; Nordquist, Kyle A.; Chazin, Walter J.; Brzovic, Peter S.; Klevit, Rachel E.

    2012-01-01

    Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 sidechain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates. PMID:22885007

  7. Novel Cul3 binding proteins function to remodel E3 ligase complexes

    PubMed Central

    2014-01-01

    Background Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. Results Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor. To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. Conclusion Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC

  8. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation

    PubMed Central

    Xiong, Hui; Wang, Danling; Chen, Linan; Choo, Yeun Su; Ma, Hong; Tang, Chengyuan; Xia, Kun; Jiang, Wei; Ronai, Ze’ev; Zhuang, Xiaoxi; Zhang, Zhuohua

    2009-01-01

    Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis. PMID:19229105

  9. The E3 ubiquitin ligase CHIP and the molecular chaperone Hsc70 form a dynamic, tethered complex.

    PubMed

    Smith, Matthew C; Scaglione, K Matthew; Assimon, Victoria A; Patury, Srikanth; Thompson, Andrea D; Dickey, Chad A; Southworth, Daniel R; Paulson, Henry L; Gestwicki, Jason E; Zuiderweg, Erik R P

    2013-08-13

    The E3 ubiquitin ligase CHIP (C-terminus of Hsc70 Interacting Protein, a 70 kDa homodimer) binds to the molecular chaperone Hsc70 (a 70 kDa monomer), and this complex is important in both the ubiquitination of Hsc70 and the turnover of Hsc70-bound clients. Here we used NMR spectroscopy, biolayer interferometry, and fluorescence polarization to characterize the Hsc70-CHIP interaction. We found that CHIP binds tightly to two molecules of Hsc70 forming a 210 kDa complex, with a Kd of approximately 60 nM, and that the IEEVD motif at the C-terminus of Hsc70 (residues 642-646) is both necessary and sufficient for binding. Moreover, the same motif is required for CHIP-mediated ubiquitination of Hsc70 in vitro, highlighting its functional importance. Relaxation-based NMR experiments on the Hsc70-CHIP complex determined that the two partners move independently in solution, similar to "beads on a string". These results suggest that a dynamic C-terminal region of Hsc70 provides for flexibility between CHIP and the chaperone, allowing the ligase to "search" a large space and engage in productive interactions with a wide range of clients. In support of this suggestion, we find that deleting residues 623-641 of the C-terminal region, while retaining the IEEVD motif, caused a significant decrease in the efficiency of Hsc70 ubiquitination by CHIP. PMID:23865999

  10. Polycomb-group complex 1 acts as an E3 ubiquitin ligase for Geminin to sustain hematopoietic stem cell activity

    PubMed Central

    Ohtsubo, Motoaki; Yasunaga, Shin'ichiro; Ohno, Yoshinori; Tsumura, Miyuki; Okada, Satoshi; Ishikawa, Nobutsune; Shirao, Kenichiro; Kikuchi, Akira; Nishitani, Hideo; Kobayashi, Masao; Takihara, Yoshihiro

    2008-01-01

    Polycomb-group (PcG) genes encode multimeric nuclear protein complexes, PcG complex 1 and 2. PcG complex 2 was proved to induce transcription repression and to further methylate histone H3 at lysine-27 (H3K27). Subsequently PcG complex 1 is recruited through recognition of methylated H3K27 and maintains the transcription silencing by mediating monoubiquitination of histone H2A at lysine-119. Genetic evidence demonstrated a crucial role for PcG complex 1 in stem cells, and Bmi1, a member of PcG complex 1, was shown to sustain adult stem cells through direct repression of the INK4a locus encoding cyclin-dependent kinase inhibitor, p16CKI, and p19ARF. The molecular functions of PcG complex 1, however, remain insufficiently understood. In our study, deficiency of Rae28, a member of PcG complex 1, was found to impair ubiquitin-proteasome-mediated degradation of Geminin, an inhibitor of DNA replication licensing factor Cdt1, and to increase protein stability. The resultant accumulation of Geminin, based on evidence from retroviral transduction experiments, presumably eliminated hematopoietic stem cell activity in Rae28-deficient mice. Rae28 mediates recruiting Scmh1, which provides PcG complex 1 an interaction domain for Geminin. Moreover, PcG complex 1 acts as the E3 ubiquitin ligase for Geminin, as we demonstrated in vivo as well as in vitro by using purified recombinant PcG complex 1 reconstituted in insect cells. Our findings suggest that PcG complex 1 supports the activity of hematopoietic stem cells, in which high-level Geminin expression induces quiescence securing genome stability, by enhancing cycling capability and hematopoietic activity through direct regulation of Geminin. PMID:18650381

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

    PubMed Central

    Deyter, Gary M.R.

    2014-01-01

    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-ACse4 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-ACse4 for degradation. The interaction between Psh1 and Spt16 is critical for both CENP-ACse4 ubiquitylation and its exclusion from euchromatin. We found that Psh1 cannot efficiently ubiquitylate CENP-ACse4 nucleosomes in vitro, suggesting that additional factors must facilitate CENP-ACse4 removal from chromatin in vivo. Consistent with this, a Psh1 mutant that cannot associate with FACT has a reduced interaction with CENP-ACse4 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-ACse4. PMID:25128498

  12. Crystal structures of two bacterial HECT-like E3 ligases in complex with a human E2 reveal atomic details of pathogen-host interactions

    SciTech Connect

    Lin, David Yin-wei; Diao, Jianbo; Chen, Jue

    2012-12-10

    In eukaryotes, ubiquitination is an important posttranslational process achieved through a cascade of ubiquitin-activating (E1), conjugating (E2), and ligase (E3) enzymes. Many pathogenic bacteria deliver virulence factors into the host cell that function as E3 ligases. How these bacterial 'Trojan horses' integrate into the eukaryotic ubiquitin system has remained a mystery. Here we report crystal structures of two bacterial E3s, Salmonella SopA and Escherichia coli NleL, both in complex with human E2 UbcH7. These structures represent two distinct conformational states of the bacterial E3s, supporting the necessary structural rearrangements associated with ubiquitin transfer. The E2-interacting surface of SopA and NleL has little similarity to those of eukaryotic E3s. However, both bacterial E3s bind to the canonical surface of E2 that normally interacts with eukaryotic E3s. Furthermore, we show that a glutamate residue on E3 is involved in catalyzing ubiquitin transfer from E3 to the substrate, but not from E2 to E3. Together, these results provide mechanistic insights into the ubiquitin pathway and a framework for understanding molecular mimicry in bacterial pathogenesis.

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

    PubMed

    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. PMID:25912140

  14. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new function for

  15. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new

  16. Structure of a glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface.

    PubMed

    Duda, David M; Olszewski, Jennifer L; Tron, Adriana E; Hammel, Michal; Lambert, Lester J; Waddell, M Brett; Mittag, Tanja; DeCaprio, James A; Schulman, Brenda A

    2012-08-10

    The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF(FBW7) complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition. PMID:22748924

  17. Structure of a Glomulin-RBX1-CUL1 complex: inhibition of a RING E3 ligase through masking of its E2-binding surface

    PubMed Central

    Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.; Hammel, Michal; Lambert, Lester J.; Waddell, M. Brett; Mittag, Tanja; DeCaprio, James A.; Schulman, Brenda A.

    2012-01-01

    Summary The ~300 human Cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1’s RING domain, regulates the RBX1-CUL1-containing SCFFBW7 complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN’s selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition. PMID:22748924

  18. Structure of a Glomulin-RBX1-CUL1 Complex: Inhibition of a RING E3 Ligase through Masking of Its E2-Binding Surface

    SciTech Connect

    Duda, David M.; Olszewski, Jennifer L.; Tron, Adriana E.; Hammel, Michal; Lambert, Lester J.; Waddell, M. Brett; Mittag, Tanja; DeCaprio, James A.; Schulman, Brenda A.

    2012-11-01

    The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF{sup FBW7} complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.

  19. The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes

    PubMed Central

    Ritterhoff, Tobias; Das, Hrishikesh; Hofhaus, Götz; Schröder, Rasmus R.; Flotho, Annette; Melchior, Frauke

    2016-01-01

    Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions. PMID:27160050

  20. Cullin E3 Ligases and Their Rewiring by Viral Factors

    PubMed Central

    Mahon, Cathal; Krogan, Nevan J.; Craik, Charles S.; Pick, Elah

    2014-01-01

    The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity. PMID:25314029

  1. Ubiquitin-conjugated degradation of golden 2-like transcription factor is mediated by CUL4-DDB1-based E3 ligase complex in tomato.

    PubMed

    Tang, Xiaofeng; Miao, Min; Niu, Xiangli; Zhang, Danfeng; Cao, Xulv; Jin, Xichen; Zhu, Yunye; Fan, Youhong; Wang, Hongtao; Liu, Ying; Sui, Yuan; Wang, Wenjie; Wang, Anquan; Xiao, Fangming; Giovannoni, Jim; Liu, Yongsheng

    2016-02-01

    CULLIN4-RING ubiquitin ligases (CRL4s) as well as their targets are fundamental regulators functioning in many key developmental and stress responses in eukaryotes. In tomato (Solanum lycopersicum), molecular cloning has revealed that the underlying genes of natural spontaneous mutations high pigment 1 (hp1), high pigment 2 (hp2) and uniform ripening (u) encode UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), DE-ETIOLATED 1 (DET1) and GOLDEN 2-LIKE (GLK2), respectively. However, the molecular basis of the opposite actions of tomato GLK2 vs CUL4-DDB1-DET1 complex on regulating plastid level and fruit quality remains unknown. Here, we provide molecular evidence showing that the tomato GLK2 protein is a substrate of the CUL4-DDB1-DET1 ubiquitin ligase complex for the proteasome degradation. SlGLK2 is degraded by the ubiquitin-proteasome system, which is mainly determined by two lysine residues (K11 and K253). SlGLK2 associates with the CUL4-DDB1-DET1 E3 complex in plant cells. Genetically impairing CUL4, DDB1 or DET1 results in a retardation of SlGLK2 degradation by the 26S proteasome. These findings are relevant to the potential of nutrient accumulation in tomato fruit by mediating the plastid level and contribute to a deeper understanding of an important regulatory loop, linking protein turnover to gene regulation. PMID:26352615

  2. Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin

    PubMed Central

    Lazarou, Michael; Jin, Seok Min; Kane, Lesley A.; Youle, Richard J.

    2012-01-01

    Summary 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 re-import of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation. PMID:22280891

  3. Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin.

    PubMed

    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. PMID:22280891

  4. Purification, crystallization and preliminary X-ray crystallographic studies of the complex between Smc5 and the SUMO E3 ligase Mms21

    SciTech Connect

    Duan, Xinyuan; Ye, Hong

    2010-03-04

    Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21-Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected to resolutions of 2.3 and 3.9 {angstrom} from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C2221, with unit-cell parameters a = 47.465, b = 97.574, c = 249.215 {angstrom} for the native crystals.

  5. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F.

    PubMed

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G Eric; Dobner, Thomas; Branton, Philip E; Blanchette, Paola

    2016-01-01

    The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  6. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  7. E3 ubiquitin ligases as novel targets for inflammatory diseases.

    PubMed

    Goru, Santosh Kumar; Pandey, Anuradha; Gaikwad, Anil Bhanudas

    2016-04-01

    Ubiquitination is one of the post translational modifications which decide the fate of various proteins in the cells, by either directing them towards proteasomal degradation or participation in several cell signalling pathways. Recently, the role of ubiquitination has been unravelled in pathogenesis and progression of various diseases, where inflammation is critical, like obesity, insulin resistance, atherosclerosis, angiotensin-II induced cardiac inflammation and asthma. E3 ligases are known to be instrumental in regulation of the inflammatory cascade. This review focuses on the role of different E3 ligases in the development of inflammatory diseases and thus may help us to target these E3 ligases in future drug discovery to prevent inflammation. PMID:26875639

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

    PubMed

    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. PMID:27312108

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

    PubMed Central

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

    2014-01-01

    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. PMID:24576094

  10. Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

    PubMed Central

    Landré, Vivien; Rotblat, Barak; Melino, Sonia; Bernassola, Francesca; Melino, Gerry

    2014-01-01

    The ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides. PMID:25237759

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

    PubMed Central

    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. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed ‘RING/HECT hybrid’ enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin’s cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein. PMID:23770887

  12. Composition, Roles, and Regulation of Cullin-Based Ubiquitin E3 Ligases

    PubMed Central

    Choi, Christina M.; Gray, William M.; Mooney, Sutton; Hellmann, Hanjo

    2014-01-01

    Due to their sessile nature, plants depend on flexible regulatory systems that allow them to adequately regulate developmental and physiological processes in context with environmental cues. The ubiquitin proteasome pathway, which targets a great number of proteins for degradation, is cellular tool that provides the necessary flexibility to accomplish this task. Ubiquitin E3 ligases provide the needed specificity to the pathway by selectively binding to particular substrates and facilitating their ubiquitylation. The largest group of E3 ligases known in plants is represented by CULLIN-REALLY INTERESTING NEW GENE (RING) E3 ligases (CRLs). In recent years, a great amount of knowledge has been generated to reveal the critical roles of these enzymes across all aspects of plant life. This review provides an overview of the different classes of CRLs in plants, their specific complex compositions, the variety of biological processes they control, and the regulatory steps that can affect their activities. PMID:25505853

  13. Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling.

    PubMed

    Swarup, Sharan; Verheyen, Esther M

    2011-06-14

    Drosophila Homeodomain-interacting protein kinase (Hipk) has been shown to regulate in vivo, the stability of Armadillo, the transcriptional effector of Wingless signaling. The Wingless pathway culminates in the stabilization of Armadillo that, in the absence of signaling, is sequentially phosphorylated, polyubiquitinated and degraded. Loss-of-function clones for hipk result in reduced stabilized Armadillo, whereas overexpression of hipk elevates Armadillo levels to promote Wingless-responsive target gene expression. Here, we show that overexpression of hipk can suppress the effects of negative regulators of Armadillo to prevent its degradation in the wing imaginal disc. Hipk acts to stabilize Armadillo by impeding the function of the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF)(Slimb), thereby inhibiting Armadillo ubiquitination and subsequent degradation. Vertebrate Hipk2 displays a similar ability to prevent β-catenin ubiquitination in a functionally conserved mechanism. We find that Hipk's ability to inhibit SCF(Slimb)-mediated ubiquitination is not restricted to Armadillo and extends to other substrates of SCF(Slimb), including the Hedgehog signaling effector Ci. Thus, similar to casein kinase 1 and glycogen synthase kinase 3, Hipk dually regulates both Wingless and Hedgehog signaling by controlling the stability of their respective signaling effectors, but it is the first kinase to our knowledge identified that promotes the stability of both Armadillo and Ci. PMID:21628596

  14. The CUL3–KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction

    PubMed Central

    Ohta, Akihito; Schumacher, Frances-Rose; Mehellou, Youcef; Johnson, Clare; Knebel, Axel; Macartney, Thomas J.; Wood, Nicola T.; Alessi, Dario R.; Kurz, Thimo

    2013-01-01

    The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating

  15. Entropy-driven mechanism of an E3 ligase.

    PubMed

    Truong, Khue; Su, Yang; Song, Jing; Chen, Yuan

    2011-06-28

    Ubiquitin-like modifications are macromolecular chemistry for which our understanding of the enzymatic mechanisms is lacking. Most E3 ligases in ubiquitin-like modifications do not directly participate in chemistry but are thought to confer allosteric effects; however, the nature of the allosteric effects has been elusive. Recent molecular dynamics simulations suggested that an E3 binding enhances the population of the conformational states of the E2·SUMO thioester that favor reactions. In this study, we conducted the first temperature-dependent enzyme kinetic analysis to investigate the role of an E3 on activation entropy and enthalpy. The small ubiquitin-like modifier (SUMO) E3, RanBP2, confers unusually large, favorable activation entropy to lower the activation energy of the reaction. Mutants of RanBP2, designed to alter the flexibilities of the E2·SUMO thioester, showed a direct correlation of their favorable entropic effects with their ability to restrict the conformational flexibility of the E2·SUMO thioester. While the more favorable activation entropy is consistent with the previously suggested role of E3 in conformational selection, the large positive entropy suggests a significant role of solvent in catalysis. Indeed, molecular dynamics simulations in explicit water revealed that the more stable E2·SUMO thioester upon E3 binding results in stabilization of a large number of bound water molecules. Liberating such structured water at the transition state can result in large favorable activation entropy but unfavorable activation enthalpy. The entropy-driven mechanism of the E3 is consistent with the lack of structural conservation among E3s despite their similar functions. This study also illustrates how proteins that bind both SUMO and E2 can function as E3s and how intrinsically unstructured proteins can enhance macromolecular chemistry in addition to their known advantages in protein--protein interactions. PMID:21568279

  16. Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.

    PubMed

    Yoo, Seung-Hee; Mohawk, Jennifer A; Siepka, Sandra M; Shan, Yongli; Huh, Seong Kwon; Hong, Hee-Kyung; Kornblum, Izabela; Kumar, Vivek; Koike, Nobuya; Xu, Ming; Nussbaum, Justin; Liu, Xinran; Chen, Zheng; Chen, Zhijian J; Green, Carla B; Takahashi, Joseph S

    2013-02-28

    Period determination in the mammalian circadian clock involves the turnover rate of the repressors CRY and PER. We show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss of function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals. PMID:23452855

  17. THE ROLE OF E3 LIGASES IN THE UBIQUITIN-DEPENDENT REGULATION OF SPERMATOGENESIS*

    PubMed Central

    Richburg, John H.; Myers, Jessica L.; Bratton, Shawn B.

    2014-01-01

    The ubiquitination of proteins is a post-translational modification that was first described as a means to target misfolded or unwanted proteins for degradation by the proteasome. It is now appreciated that the ubiquitination of proteins also serves as a mechanism to modify protein function and cellular functions such as protein trafficking, cell signaling, DNA repair, chromatin modifications, cell-cycle progression and cell death. The ubiquitination of proteins occurs through the hierarchal transfer of ubiquitin from an E1 ubiquitin-activating enzyme to an E2 ubiquitin-conjugating enzyme and finally to an E3 ubiquitin ligase that transfers the ubiquitin to its target protein. It is the final E3 ubiquitin ligase that confers the substrate specificity for ubiquitination and is the focus of this review. Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells undergo mitotic proliferation and expansion of the diploid spermatogonial population, differentiate into spermatocytes and progress through two meiotic divisions to produce haploid spermatids that proceed through a final morphogenesis to generate mature spermatozoa. The ubiquitination of proteins in the cells of the testis occurs in many of the processes required for the progression of mature spermatozoa. Since it is the E3 ubiquitin ligase that recognizes the target protein and provides the specificity and selectivity for ubiquitination, this review highlights known examples of E3 ligases in the testis and the differing roles that they play in maintaining functional spermatogenesis. PMID:24632385

  18. Identifying the ERAD ubiquitin E3 ligases for viral and cellular targeting of MHC class I.

    PubMed

    van den Boomen, D J H; Lehner, P J

    2015-12-01

    The human cytomegalovirus (HCMV) US2 and US11 gene products hijack mammalian ER-associated degradation (ERAD) to induce rapid degradation of major histocompatibility class I (MHC-I) molecules. The rate-limiting step in this pathway is thought to be the polyubiquitination of MHC-I by distinct host ERAD E3 ubiquitin ligases. TRC8 was identified as the ligase responsible for US2-mediated MHC-I degradation and shown to be required for the cleavage-dependent degradation of some tail-anchored proteins. In addition to MHC-I, plasma membrane profiling identified further immune receptors, which are also substrates for the US2/TRC8 complex. These include at least six α integrins, the coagulation factor thrombomodulin and the NK cell ligand CD112. US2's use of specific HCMV-encoded adaptors makes it an adaptable viral degradation hub. US11-mediated degradation is MHC-I-specific and genetic screens have identified TMEM129, an uncharacterised RING-C2 E3 ligase, as responsible for US11-mediated degradation. In a unique auto-regulatory loop, US11 readily responds to changes in cellular expression of MHC-I. Free US11 either rebinds more MHC-I or is itself degraded by the HRD1/SEL1L E3 ligase complex. While virally encoded US2 and US11 appropriate mammalian ERAD, the MHC-I complex also undergoes stringent cellular quality control and misfolded MHC-I is degraded by the HRD1/SEL1L complex. We discuss the identification and central role of E3 ubiquitin ligases in ER quality control and viral degradation of the MHC-I chain. PMID:26210183

  19. Identifying the ERAD ubiquitin E3 ligases for viral and cellular targeting of MHC class I

    PubMed Central

    van den Boomen, D.J.H.; Lehner, P.J.

    2015-01-01

    The human cytomegalovirus (HCMV) US2 and US11 gene products hijack mammalian ER-associated degradation (ERAD) to induce rapid degradation of major histocompatibility class I (MHC-I) molecules. The rate-limiting step in this pathway is thought to be the polyubiquitination of MHC-I by distinct host ERAD E3 ubiquitin ligases. TRC8 was identified as the ligase responsible for US2-mediated MHC-I degradation and shown to be required for the cleavage-dependent degradation of some tail-anchored proteins. In addition to MHC-I, plasma membrane profiling identified further immune receptors, which are also substrates for the US2/TRC8 complex. These include at least six α integrins, the coagulation factor thrombomodulin and the NK cell ligand CD112. US2’s use of specific HCMV-encoded adaptors makes it an adaptable viral degradation hub. US11-mediated degradation is MHC-I-specific and genetic screens have identified TMEM129, an uncharacterised RING-C2 E3 ligase, as responsible for US11-mediated degradation. In a unique auto-regulatory loop, US11 readily responds to changes in cellular expression of MHC-I. Free US11 either rebinds more MHC-I or is itself degraded by the HRD1/SEL1L E3 ligase complex. While virally encoded US2 and US11 appropriate mammalian ERAD, the MHC-I complex also undergoes stringent cellular quality control and misfolded MHC-I is degraded by the HRD1/SEL1L complex. We discuss the identification and central role of E3 ubiquitin ligases in ER quality control and viral degradation of the MHC-I chain. PMID:26210183

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

    PubMed

    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. PMID:27162139

  1. Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21

    PubMed Central

    Zhang, Cuiyan; Li, Xiaotong; Adelmant, Guillaume; Dobbins, Jessica; Geisen, Christoph; Oser, Matthew G.; Wucherpfenning, Kai W.; Marto, Jarrod A.; Kaelin, William G.

    2015-01-01

    EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCFFBXO21 polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells. PMID:26631746

  2. Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast*

    PubMed Central

    Tong, Zongtian; Kim, Min-Sik; Pandey, Akhilesh; Espenshade, Peter J.

    2014-01-01

    Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires endoplasmic reticulum (ER)-resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation. Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic activation of fungal sterol regulatory element-binding protein transcription factors. The Dsc E3 ligase contains five integral membrane subunits and structurally resembles ER-associated degradation E3 ligases. Saccharomyces cerevisiae codes for homologs of Dsc E3 ligase subunits, including the Dsc1 E3 ligase homolog Tul1 that functions in Golgi protein quality control. Interestingly, S. cerevisiae lacks sterol regulatory element-binding protein homologs, indicating that novel Tul1 E3 ligase substrates exist. Here, we show that the S. cerevisiae Tul1 E3 ligase consists of Tul1, Dsc2, Dsc3, and Ubx3 and define Tul1 complex architecture. Tul1 E3 ligase function required each subunit as judged by vacuolar sorting of the artificial substrate Pep12D. Genetic studies demonstrated that Tul1 E3 ligase was required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we performed quantitative diGly proteomics using stable isotope labeling by amino acids in cell culture to survey ubiquitylation in wild-type and tul1Δ cells. We identified 3116 non-redundant ubiquitylation sites, including 10 sites in candidate substrates. Quantitative proteomics found 4.5% of quantified proteins (53/1172) to be differentially expressed in tul1Δ cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, our data demonstrate that the Tul1 E3 ligase functions in

  3. Modulation of myocardin function by the ubiquitin E3 ligase UBR5.

    PubMed

    Hu, Guoqing; Wang, Xiaobo; Saunders, Darren N; Henderson, Michelle; Russell, Amanda J; Herring, B Paul; Zhou, Jiliang

    2010-04-16

    Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin

  4. Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

    PubMed

    Thomas, Jemima C; Matak-Vinkovic, Dijana; Van Molle, Inge; Ciulli, Alessio

    2013-08-01

    Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems. PMID:23837592

  5. Identification of an Unconventional E3 Binding Surface on the UbcH5 ∼ Ub Conjugate Recognized by a Pathogenic Bacterial E3 Ligase by a pathogenic bacterial E3 ligase.

    SciTech Connect

    Levin, Itay; Eakin, Catherine M.; Blanc, Marie-Pierre; Klevit, Rachel E.; Miller, Samuel I.; Brzovic, Peter S.

    2010-02-16

    Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ∼ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ∼ Ubn). Rapid generation of UbcH5 ∼ Ubn may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.

  6. UHRF2, another E3 ubiquitin ligase for p53

    SciTech Connect

    Bai, Lu; Wang, Xiaohui; Jin, Fangmin; Yang, Yan; Qian, Guanhua; Duan, Changzhu

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer UHRF2 associates with p53 in vivo and in vitro. Black-Right-Pointing-Pointer UHRF2 interacts with p53 through its SRA/YDG domain. Black-Right-Pointing-Pointer UHRF2 ubiquitinates p53 in vivo and in vitro. -- Abstract: UHRF2, ubiquitin-like with PHD and ring finger domains 2, is a nuclear E3 ubiquitin ligase, which is involved in cell cycle and epigenetic regulation. UHRF2 interacts with multiple cell cycle proteins, including cyclins (A2, B1, D1, and E1), CDK2, and pRb; moreover, UHRF2 could ubiquitinate cyclin D1 and cyclin E1. Also, UHRF2 has been shown to be implicated in epigenetic regulation by associating with DNMTs, G9a, HDAC1, H3K9me2/3 and hemi-methylated DNA. We found that UHRF2 associates with tumor suppressor protein p53, and p53 is ubiquitinated by UHRF2 in vivo and in vitro. Given that both UHRF2 and p53 are involved in cell cycle regulation, this study may suggest a novel signaling pathway on cell proliferation.

  7. UBXN2A regulates nicotinic receptor degradation by modulating the E3 ligase activity of CHIP.

    PubMed

    Teng, Yanfen; Rezvani, Khosrow; De Biasi, Mariella

    2015-10-15

    Neuronal nicotinic acetylcholine receptors (nAChRs) containing the α3 subunit are known for their prominent role in normal ganglionic transmission while their involvement in the mechanisms underlying nicotine addiction and smoking-related disease has been emerging only in recent years. The amount of information available on the maturation and trafficking of α3-containing nAChRs is limited. We previously showed that UBXN2A is a p97 adaptor protein that facilitates the maturation and trafficking of α3-containing nAChRs. Further investigation of the mechanisms of UBXN2A actions revealed that the protein interacts with CHIP (carboxyl terminus of Hsc70 interacting protein), whose ubiquitin E3 ligase activity regulates the degradation of several disease-related proteins. We show that CHIP displays E3 ligase activity toward the α3 nAChR subunit and contributes to its ubiquitination and subsequent degradation. UBXN2A interferes with CHIP-mediated ubiquitination of α3 and protects the nicotinic receptor subunit from endoplasmic reticulum associated degradation (ERAD). UBXN2A also cross-talks with VCP/p97 and HSC70/HSP70 proteins in a complex where α3 is likely to be targeted by CHIP. Overall,we identify CHIP as an E3 ligase for α3 and UBXN2A as a protein that may efficiently regulate the stability of CHIP's client substrates. PMID:26265139

  8. The Not4 RING E3 Ligase: A Relevant Player in Cotranslational Quality Control

    PubMed Central

    Collart, Martine A.

    2013-01-01

    The Not4 RING E3 ligase is a subunit of the evolutionarily conserved Ccr4-Not complex. Originally identified in yeast by mutations that increase transcription, it was subsequently defined as an ubiquitin ligase. Substrates for this ligase were characterized in yeast and in metazoans. Interestingly, some substrates for this ligase are targeted for polyubiquitination and degradation, while others instead are stable monoubiquitinated proteins. The former are mostly involved in transcription, while the latter are a ribosomal protein and a ribosome-associated chaperone. Consistently, Not4 and all other subunits of the Ccr4-Not complex are present in translating ribosomes. An important function for Not4 in cotranslational quality control has emerged. In the absence of Not4, the total level of polysomes is reduced. In addition, translationally arrested polypeptides, aggregated proteins, and polyubiquitinated proteins accumulate. Its role in quality control is likely to be related on one hand to its importance for the functional assembly of the proteasome and on the other hand to its association with the RNA degradation machines. Not4 is in an ideal position to signal to degradation mRNAs whose translation has been aborted, and this defines Not4 as a key player in the quality control of newly synthesized proteins. PMID:27335678

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

    NASA Astrophysics Data System (ADS)

    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.

  10. Ozz-E3 ubiquitin ligase targets sarcomeric embryonic myosin heavy chain during muscle development.

    PubMed

    Campos, Yvan; Qiu, Xiaohui; Zanoteli, Edmar; Moshiach, Simon; Vergani, Naja; Bongiovanni, Antonella; Harris, A John; d'Azzo, Alessandra

    2010-01-01

    Muscle contractile proteins are expressed as a series of developmental isoforms that are in constant dynamic remodeling during embryogenesis, but how obsolete molecules are recognized and removed is not known. Ozz is a developmentally regulated protein that functions as the adaptor component of a RING-type ubiquitin ligase complex specific to striated muscle. Ozz(-/-) mutants exhibit defects in myofibrillogenesis and myofiber differentiation. Here we show that Ozz targets the rod portion of embryonic myosin heavy chain and preferentially recognizes the sarcomeric rather than the soluble pool of myosin. We present evidence that Ozz binding to the embryonic myosin isoform within sarcomeric thick filaments marks it for ubiquitination and proteolytic degradation, allowing its replacement with neonatal or adult isoforms. This unique function positions Ozz within a system that facilitates sarcomeric myosin remodeling during muscle maturation and regeneration. Our findings identify Ozz-E3 as the ubiquitin ligase complex that interacts with and regulates myosin within its fully assembled cytoskeletal structure. PMID:20352047

  11. Characterization of the mammalian family of DCN-type NEDD8 E3 ligases

    PubMed Central

    Keuss, Matthew J.; Thomas, Yann; Mcarthur, Robin; Wood, Nicola T.; Knebel, Axel; Kurz, Thimo

    2016-01-01

    ABSTRACT Cullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression. PMID:26906416

  12. Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

    PubMed Central

    Spratt, Donald E.; Mercier, Pascal; Shaw, Gary S.

    2013-01-01

    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. PMID:24058416

  13. MDM2 Inhibits Axin-Induced p53 Activation Independently of its E3 Ligase Activity.

    PubMed

    He, Ying; Lian, Guili; Lin, Shuyong; Ye, Zhiyun; Li, Qinxi

    2013-01-01

    MDM2 plays a crucial role in negatively regulating the functions of tumor suppressor p53. Here we show that MDM2 can inhibit Axin-stimulated p53-dependent apoptosis by suppressing p53 phosphorylation at Ser 46 and apoptosis-related p53 transactivational activity. Interestingly, the ubiquitin E3 ligase activity of MDM2 is not required for this inhibitory effect. Mechanically, either wildtype MDM2 or its E3-dead mutant, disrupts the Axin-based HIPK2/p53 complex formation by blocking the binding of p53 and HIPK2 to Axin. MDM2Δp53, a deletion mutant that lacks p53 binding domain fails to exert the inhibitory effect, demonstrating that the interaction of MDM2 and p53, but not its E3 ligase activity toward p53 plays key role in suppressing Axin-stimulated p53 activation. Our results thus have revealed a novel aspect of the mechanism by which MDM2 regulates p53 activities. PMID:23826318

  14. Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly

    SciTech Connect

    Stanley,B.; Ehrlich, E.; Short, L.; Yu, Y.; Xiao, Z.; Yu, X.; Xiong, Y.

    2008-01-01

    Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

  15. Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation.

    PubMed

    Medvar, Barbara; Raghuram, Viswanathan; Pisitkun, Trairak; Sarkar, Abhijit; Knepper, Mark A

    2016-07-01

    Aquaporin-2 (AQP2) is regulated in part via vasopressin-mediated changes in protein half-life that are in turn dependent on AQP2 ubiquitination. Here we addressed the question, "What E3 ubiquitin ligase is most likely to be responsible for AQP2 ubiquitination?" using large-scale data integration based on Bayes' rule. The first step was to bioinformatically identify all E3 ligase genes coded by the human genome. The 377 E3 ubiquitin ligases identified in the human genome, consisting predominant of HECT, RING, and U-box proteins, have been used to create a publically accessible and downloadable online database (https://hpcwebapps.cit.nih.gov/ESBL/Database/E3-ligases/). We also curated a second database of E3 ligase accessory proteins that included BTB domain proteins, cullins, SOCS-box proteins, and F-box proteins. Using Bayes' theorem to integrate information from multiple large-scale proteomic and transcriptomic datasets, we ranked these 377 E3 ligases with respect to their probability of interaction with AQP2. Application of Bayes' rule identified the E3 ligases most likely to interact with AQP2 as (in order of probability): NEDD4 and NEDD4L (tied for first), AMFR, STUB1, ITCH, ZFPL1. Significantly, the two E3 ligases tied for top rank have also been studied extensively in the reductionist literature as regulatory proteins in renal tubule epithelia. The concordance of conclusions from reductionist and systems-level data provides strong motivation for further studies of the roles of NEDD4 and NEDD4L in the regulation of AQP2 protein turnover. PMID:27199454

  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.

    PubMed

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

    2015-02-01

    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 RNF8 stabilizes TPP1 to promote telomere end protection

    PubMed Central

    Rai, Rekha; Li, Ju-Mei; Zheng, Hong; Lok, Gabriel Tsz-Mei; Deng, Yu; Huen, Michael; Chen, Junjie; Jin, Jianping; Chang, Sandy

    2013-01-01

    TPP1, a component of the mammalian shelterin complex, plays essential roles in telomere maintenance. It forms a heterodimer with POT1 to repress ATR-dependent DNA damage signaling at telomeres, and recruits telomerase to chromosome ends. Here we show that the E3 ubiquitin ligase RNF8 localizes to and promotes the accumulation of DNA damage proteins 53BP1 and γ-H2AX to uncapped telomeres. TPP1 is unstable in the absence of RNF8, resulting in telomere shortening and chromosome fusions via the alternative non-homologous end joining (A-NHEJ)-mediated DNA repair pathway. The RNF8 ubiquitin ligase RING domain is essential for TPP1 stability and retention at telomeres. RNF8 physically interacts with TPP1 to generate Ubc13-dependent K63 polyubiquitin chains that stabilizes TPP1 at telomeres. The conserved TPP1 lysine residue 233 is essential for RNF8-mediated TPP1 ubiquitylation and localization to telomeres. Our results demonstrate that TPP1 is a novel substrate for RNF8, and suggest a previously unrecognized role for RNF8 in telomere end protection. We propose a model in which engagement of classical vs. A-NHEJ repair pathways at dysfunctional telomeres is controlled by the ubiquitin ligase functions of RNF8. PMID:22101936

  18. Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity.

    PubMed

    Koliopoulos, Marios G; Esposito, Diego; Christodoulou, Evangelos; Taylor, Ian A; Rittinger, Katrin

    2016-06-01

    TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N-terminal portion which comprises a canonical RING domain, one or two B-box domains and a coiled-coil region that mediates ligase dimerization. Self-association via the coiled-coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin-loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full-length protein. Our data reveal an unexpected diversity in the self-association mechanism of TRIMs that might be crucial for their biological function. PMID:27154206

  19. System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

    PubMed

    Zhang, Wei; Wu, Kuen-Phon; Sartori, Maria A; Kamadurai, Hari B; Ordureau, Alban; Jiang, Chong; Mercredi, Peter Y; Murchie, Ryan; Hu, Jicheng; Persaud, Avinash; Mukherjee, Manjeet; Li, Nan; Doye, Anne; Walker, John R; Sheng, Yi; Hao, Zhenyue; Li, Yanjun; Brown, Kevin R; Lemichez, Emmanuel; Chen, Junjie; Tong, Yufeng; Harper, J Wade; Moffat, Jason; Rotin, Daniela; Schulman, Brenda A; Sidhu, Sachdev S

    2016-04-01

    HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins. PMID:26949039

  20. Structural and functional insights into the E3 ligase, RNF126.

    PubMed

    Krysztofinska, Ewelina M; Martínez-Lumbreras, Santiago; Thapaliya, Arjun; Evans, Nicola J; High, Stephen; Isaacson, Rivka L

    2016-01-01

    RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm. PMID:27193484

  1. Structural and functional insights into the E3 ligase, RNF126

    PubMed Central

    Krysztofinska, Ewelina M.; Martínez-Lumbreras, Santiago; Thapaliya, Arjun; Evans, Nicola J.; High, Stephen; Isaacson, Rivka L.

    2016-01-01

    RNF126 is an E3 ubiquitin ligase that collaborates with the BAG6 sortase complex to ubiquitinate hydrophobic substrates in the cytoplasm that are destined for proteasomal recycling. Composed of a trimeric complex of BAG6, TRC35 and UBL4A the BAG6 sortase is also associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates. Here we solve the solution structure of the RNF126 zinc finger domain in complex with the BAG6 UBL domain. We also characterise an interaction between RNF126 and UBL4A and analyse the competition between SGTA and RNF126 for the N-terminal BAG6 binding site. This work sheds light on the sorting mechanism of the BAG6 complex and its accessory proteins which, together, decide the fate of stray hydrophobic proteins in the aqueous cytoplasm. PMID:27193484

  2. E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis.

    PubMed

    Du, Zhuo; He, Fei; Yu, Zidong; Bowerman, Bruce; Bao, Zhirong

    2015-02-15

    Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. Caenorhabditis elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two Cullin-RING ubiquitin ligase (CRL) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/β-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these

  3. E3 ubiquitin ligases promote progression of differentiation during C. elegans embryogenesis

    PubMed Central

    Du, Zhuo; He, Fei; Yu, Zidong; Bowerman, Bruce; Bao, Zhirong

    2014-01-01

    Regulated choice between cell fate maintenance and differentiation provides decision points in development to progress toward more restricted cell fates or to maintain the current one. C. elegans embryogenesis follows an invariant cell lineage where cell fate is generally more restricted upon each cell division. EMS is a progenitor cell in the four-cell embryo that gives rise to the endomesoderm. We recently found that when ubiquitin-mediated protein degradation is compromised, the anterior daughter of EMS, namely MS, reiterates the EMS fate. This observation demonstrates an essential function of ubiquitin-mediated protein degradation in driving the progression of EMS-to-MS differentiation. Here we report a genome-wide screen of the ubiquitin pathway and extensive lineage analyses. The results suggest a broad role of E3 ligases in driving differentiation progression. First, we identified three substrate-binding proteins for two CRL (Cullin-RING ubiquitin Ligase) E3 complexes that promote the progression from the EMS fate to MS, namely LIN-23/β-TrCP and FBXB-3 for the CRL1/SCF complex and ZYG-11/ZYG-11B for the CRL2 complex. Genetic analyses suggest these E3 ligases function through a multifunctional protein OMA-1 and the endomesoderm lineage specifier SKN-1 to drive differentiation. Second, we found that depletion of components of the CRL1/SCF complex induces fate reiteration in all major founder cell lineages. These data suggest that regulated choice between self-renewal and differentiation is widespread during C. elegans embryogenesis as in organisms with regulative development, and ubiquitin-mediated protein degradation drives the choice towards differentiation. Finally, bioinformatic analysis of time series gene expression data showed that expression of E3 genes is transiently enriched during time windows of developmental stage transitions. Transcription factors show similar enrichment, but not other classes of regulatory genes. Based on these findings we

  4. Identification of RFPL3 Protein as a Novel E3 Ubiquitin Ligase Modulating the Integration Activity of Human Immunodeficiency Virus, Type 1 Preintegration Complex Using a Microtiter Plate-based Assay*

    PubMed Central

    Tan, Beng Hui; Suzuki, Yasutsugu; Takahashi, Hirotaka; Ying, Pamela Ho Rui; Takahashi, Chikako; Han, Qi'En; Chin, Wei Xin; Chao, Sheng-Hao; Sawasaki, Tatsuya; Yamamoto, Naoki; Suzuki, Youichi

    2014-01-01

    Integration, one of the hallmarks of retrovirus replication, is mediated by a nucleoprotein complex called the preintegration complex (PIC), in which viral DNA is associated with many protein components that are required for completion of the early phase of infection. A striking feature of the PIC is its powerful integration activity in vitro. The PICs from a freshly isolated cytoplasmic extract of infected cells are able to insert viral DNA into exogenously added target DNA in vitro. Therefore, a PIC-based in vitro assay is a reliable system for assessing protein factors influencing retroviral integration. In this study, we applied a microtiter plate-based in vitro assay to a screening study using a protein library that was produced by the wheat germ cell-free protein synthesis system. Using a library of human E3 ubiquitin ligases, we identified RFPL3 as a potential stimulator of human immunodeficiency virus, type 1 (HIV-1) PIC integration activity in vitro. This enhancement of PIC activity by RFPL3 was likely to be attributed to its N-terminal RING domain. To further understand the functional role of RFPL3 in HIV infection, we created a human cell line overexpressing RFPL3. Immunoprecipitation analysis revealed that RFPL3 was associated with the human immunodeficiency virus, type 1 PICs in infected cells. More importantly, single-round HIV-1 infection was enhanced significantly by RFPL3 expression. Our proteomic approach displays an advantage in the identification of new cellular proteins affecting the integration activity of the PIC and, therefore, contributes to the understanding of functional interaction between retroviral integration complexes and host factors. PMID:25107902

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

    PubMed Central

    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. PMID:26431207

  6. The antiobesity factor WDTC1 suppresses adipogenesis via the CRL4WDTC1 E3 ligase.

    PubMed

    Groh, Beezly S; Yan, Feng; Smith, Matthew D; Yu, Yanbao; Chen, Xian; Xiong, Yue

    2016-05-01

    WDTC1/Adp encodes an evolutionarily conserved suppressor of lipid accumulation. While reduced WDTC1 expression is associated with obesity in mice and humans, its cellular function is unknown. Here, we demonstrate that WDTC1 is a component of a DDB1-CUL4-ROC1 (CRL4) E3 ligase. Using 3T3-L1 cell culture model of adipogenesis, we show that disrupting the interaction between WDTC1 and DDB1 leads to a loss of adipogenic suppression by WDTC1, increased triglyceride accumulation and adipogenic gene expression. We show that the CRL4(WDTC) (1) complex promotes histone H2AK119 monoubiquitylation, thus suggesting a role for this complex in transcriptional repression during adipogenesis. Our results identify a biochemical role for WDTC1 and extend the functional range of the CRL4 complex to the suppression of fat accumulation. PMID:27113764

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

    PubMed

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

    2012-09-01

    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 domain of rat 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 carboxy-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 stabilize the consequent tetrahedral transition-state intermediate. PMID:22842904

  8. TRIM32 is an E3 ubiquitin ligase for dysbindin.

    PubMed

    Locke, Matthew; Tinsley, Caroline L; Benson, Matthew A; Blake, Derek J

    2009-07-01

    Mutations in the gene encoding tripartite motif protein 32 (TRIM32) cause two seemingly diverse diseases: limb-girdle muscular dystrophy type 2H (LGMD2H) or sarcotubular myopathy (STM) and Bardet-Biedl syndrome type 11(BBS11). Although TRIM32 is involved in protein ubiquitination, its substrates and the molecular consequences of disease-causing mutations are poorly understood. In this paper, we show that TRIM32 is a widely expressed ubiquitin ligase that is localized to the Z-line in skeletal muscle. Using the yeast two-hybrid system, we found that TRIM32 binds and ubiquitinates dysbindin, a protein implicated in the genetic aetiology of schizophrenia, augmenting its degradation. Small-interfering RNA-mediated knock-down of TRIM32 in myoblasts resulted in elevated levels of dysbindin. Importantly, the LGMD2H/STM-associated TRIM32 mutations, D487N and R394H impair ubiquitin ligase activity towards dysbindin and were mislocalized in heterologous cells. These mutants were able to self-associate and also co-immunoprecipitated with wild-type TRIM32 in transfected cells. Furthermore, the D487N mutant could bind to both dysbindin and its E2 enzyme but was defective in monoubiquitination. In contrast, the BBS11 mutant P130S did not show any biochemical differences compared with the wild-type protein. Our data identify TRIM32 as a regulator of dysbindin and demonstrate that the LGMD2H/STM mutations may impair substrate ubiquitination. PMID:19349376

  9. Circulating E3 ligases are novel and sensitive biomarkers for diagnosis of acute myocardial infarction

    PubMed Central

    Han, Qiu-Yue; Wang, Hong-Xia; Liu, Xiao-Hong; Guo, Cai-Xia; Hua, Qi; Yu, Xiao-Hong; Li, Nan; Yang, Yan-Zong; Du, Jie

    2015-01-01

    Ubiquitin ligase (E3) is a decisive element of the ubiquitin-proteasome system (UPS), which is the main pathway for intracellular protein turnover. Recently, circulating E3 ligases have been increasingly considered as cancer biomarkers. In the present study, we aimed to determine if cardiac-specific E3 ligases in circulation can serve as novel predictors for early diagnosis of acute myocardial infarction (AMI). By screening and verifying their tissue expression patterns with microarray and real-time PCR analysis, six of 261 E3 ligases, including cardiac-specific Rnf207 and cardiac- and muscle-enriched Fbxo32/atrogin-1, Trim54/MuRF3, Trim63/MuRF1, Kbtbd10/KLHL41, Asb11 and Asb2 in mouse heart, were selected for the present study. In the AMI rats, the levels of five E3 ligases including Rnf207, Fbxo32, Trim54, Trim63 and Kbtbd10 in the plasma were significantly increased compared with control animals. Especially, the plasma levels of Rnf207 was markedly increased at 1 h, peaked at 3 h and decreased at 6–24 h after ligation. Further evaluation of E3 ligases in AMI patients confirmed that plasma Rnf207 level increased significantly compared with that in healthy people and patients without AMI, and showed a similar time course to that in AMI rats. Simultaneously, plasma level of cardiac troponin I (cTnI) was measured by ELISA assays. Finally, receiver operating characteristic (ROC) curve analysis indicated that Rnf207 showed a similar sensitivity and specificity to the classic biomarker troponin I for diagnosis of AMI. Increased cardiac-specific E3 ligase Rnf207 in plasma may be a novel and sensitive biomarkers for AMI in humans. PMID:25599194

  10. A Pathogen Type III Effector with a Novel E3 Ubiquitin Ligase Architecture

    PubMed Central

    Skarina, Tatiana; Xu, Xiaohui; Cui, Hong; Eschen-Lippold, Lennart; Egler, Monique; Srikumar, Tharan; Raught, Brian; Lee, Justin; Scheel, Dierk; Savchenko, Alexei; Bonas, Ulla

    2013-01-01

    Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity. PMID:23359647

  11. Human ITCH E3 ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease.

    PubMed

    Lohr, Naomi J; Molleston, Jean P; Strauss, Kevin A; Torres-Martinez, Wilfredo; Sherman, Eric A; Squires, Robert H; Rider, Nicholas L; Chikwava, Kudakwashe R; Cummings, Oscar W; Morton, D Holmes; Puffenberger, Erik G

    2010-03-12

    Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes. PMID:20170897

  12. Human ITCH E3 Ubiquitin Ligase Deficiency Causes Syndromic Multisystem Autoimmune Disease

    PubMed Central

    Lohr, Naomi J.; Molleston, Jean P.; Strauss, Kevin A.; Torres-Martinez, Wilfredo; Sherman, Eric A.; Squires, Robert H.; Rider, Nicholas L.; Chikwava, Kudakwashe R.; Cummings, Oscar W.; Morton, D. Holmes; Puffenberger, Erik G.

    2010-01-01

    Ubiquitin ligases play an important role in the regulation of the immune system. Absence of Itch E3 ubiquitin ligase in mice has been shown to cause severe autoimmune disease. Using autozygosity mapping in a large Amish kindred, we identified a linkage region on chromosome 20 and selected candidate genes for screening. We describe, in ten patients, identification of a mutation resulting in truncation of ITCH. These patients represent the first reported human phenotype associated with ITCH deficiency. These patients not only have multisystem autoimmune disease but also display morphologic and developmental abnormalities. This disorder underscores the importance of ITCH ubiquitin ligase in many cellular processes. PMID:20170897

  13. The E3 ubiquitin ligase WVIP2 highlights the versatility of protein ubiquitination

    PubMed Central

    Guerra, Davide; Cattivelli, Luigi; Mazzucotelli, Elisabetta

    2012-01-01

    Plant cells regulate many cellular processes controlling the half-life of critical proteins through ubiquitination. Previously, we characterized two interacting RING-type E3 ubiquitin ligases of Triticum durum, TdRF1 and WVIP2. We revealed their role in tolerance to dehydration, and existing knowledge about their partners also indicated their involvement in the regulation of some aspects of plant development. Here we located WVIP2 in the regulation of the ABA signaling, based on sequence similarities. Further we acquired general evidence about the versatility of ubiquitination in plant cells. A protein can be target of different E3 ligases for a perfect tuning of its abundance as well as the same E3 ligase can ubiquitinate different and unrelated proteins, thus representing a cross-connections between different signaling pathways for a global coordination of cellular processes. PMID:22899050

  14. GIDE is a mitochondrial E3 ubiquitin ligase that induces apoptosis and slows growth

    PubMed Central

    Zhang, Bicheng; Huang, Jun; Li, Hong-Liang; Liu, Ting; Wang, Yan-Yi; Waterman, Paul; Mao, Ai-Ping; Xu, Liang-Guo; Zhai, Zhonghe; Liu, Depei; Marrack, Philippa; Shu, Hong-Bing

    2011-01-01

    We report here the identification of GIDE, a mitochondrially located E3 ubiquitin ligase. GIDE contains a C-terminal Ring finger domain, which is mostly conserved with those of the IAP family members, and which is required for its E3 ligase activity. Overexpression of GIDE induces apoptosis via a pathway involving activation of caspases since the caspase inhibitors, XIAP and an inactive mutant of caspase-9 block GIDE-induced apoptosis. GIDE also activates JNK, and blockade of JNK activation inhibits GIDE-induced release of cytochrome c and Smac and apoptosis, suggesting that JNK activation precedes release of cytochrome c and Smac and is required for GIDE-induced apoptosis. These proapoptotic properties of GIDE require its E3 ligase activity. When somewhat over or underexpressed, GIDE slows or hastens cell growth respectively. These pro-apoptotic or growth rate effects of GIDE may account for its absence from tumor cells. PMID:18591963

  15. Discovery of Mdm2-MdmX E3 Ligase Inhibitors Using a Cell-Based Ubiquitination Assay

    PubMed Central

    Herman, Ariel G.; Hayano, Miki; Poyurovsky, Masha V.; Shimada, Kenichi; Skouta, Rachid; Prives, Carol; Stockwell, Brent R.

    2011-01-01

    E3 ubiquitin ligases are of interest as drug targets for their ability to regulate protein stability and function. The oncogene Mdm2 is an attractive E3 ligase to target, as it is the key negative regulator of the tumor suppressor p53, which controls the transcription of genes involved in cell fate. Overexpression of Mdm2 facilitates tumorigenesis by inactivating p53, and through p53-independent oncogenic effects. We developed a high-throughput cellular Mdm2 auto-ubiquitination assay, which we used to discover a class of small molecule Mdm2 ligase activity inhibitors. These compounds inhibit Mdm2 and p53 ubiquitination in cells, reduce viability of cells with wild-type p53, and synergize with DNA-damaging agents to cause cell death. We determined that these compounds effectively inhibit the E3 ligase activity of the Mdm2-MdmX hetero-complex. This mechanism may be exploitable to create a new class of anti-tumor agents. PMID:22586610

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

    PubMed Central

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

    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. PMID:22200179

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

    PubMed Central

    Nielsen, Sofie V.; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus

    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 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 of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is

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

    PubMed Central

    2011-01-01

    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. PMID:21798009

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

    PubMed

    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. PMID:27197154

  20. Characterization and Promoter Analysis of a Cotton Ring-Type Ubiquitin Ligase (E3) Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus a...

  1. Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis.

    PubMed

    Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

    2014-12-01

    E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design. PMID:25301376

  2. E3Miner: a text mining tool for ubiquitin-protein ligases.

    PubMed

    Lee, Hodong; Yi, Gwan-Su; Park, Jong C

    2008-07-01

    Ubiquitination is a regulatory process critically involved in the degradation of >80% of cellular proteins, where such proteins are specifically recognized by a key enzyme, or a ubiquitin-protein ligase (E3). Because of this important role of E3s, a rapidly growing body of the published literature in biology and biomedical fields reports novel findings about various E3s and their molecular mechanisms. However, such findings are neither adequately retrieved by general text-mining tools nor systematically made available by such protein databases as UniProt alone. E3Miner is a web-based text mining tool that extracts and organizes comprehensive knowledge about E3s from the abstracts of journal articles and the relevant databases, supporting users to have a good grasp of E3s and their related information easily from the available text. The tool analyzes text sentences to identify protein names for E3s, to narrow down target substrates and other ubiquitin-transferring proteins in E3-specific ubiquitination pathways and to extract molecular features of E3s during ubiquitination. E3Miner also retrieves E3 data about protein functions, other E3-interacting partners and E3-related human diseases from the protein databases, in order to help facilitate further investigation. E3Miner is freely available through http://e3miner.biopathway.org. PMID:18483079

  3. The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress

    PubMed Central

    Melnik, Andre; Wilson-Zbinden, Caroline; Schellhaas, René; Kastner, Lisa; Piwko, Wojciech; Dees, Martina; Picotti, Paola; Maric, Marija; Labib, Karim; Luke, Brian; Peter, Matthias

    2016-01-01

    Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1’s replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks. PMID:26849847

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

    PubMed

    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. PMID:27215386

  5. Functional characterization of SAG/RBX2/ROC2/RNF7, an antioxidant protein and an E3 ubiquitin ligase

    PubMed Central

    Li, Hua

    2013-01-01

    SAG (Sensitive to Apoptosis Gene), also known as RBX2 (RING box protein 2), ROC2 (Regulator of Cullins 2), or RNF7 (RING Finger Protein 7), was originally cloned in our laboratory as a redox inducible antioxidant protein and later characterized as the second member of the RBX/ROC RING component of the SCF (SKP1-CUL-F-box Proteins) E3 ubiquitin ligase. When acting alone, SAG scavenges oxygen radicals by forming inter- and intra- molecular disulfide bonds, whereas by forming a complex with other components of the SCF E3 ligase, SAG promotes ubiquitination and degradation of a number of protein substrates, including c-JUN, DEPTOR, HIF-1α, IκBα, NF1, NOXA, p27, and procaspase-3, thus regulating various signaling pathways and biological processes. Specifically, SAG protects cells from apoptosis, confers radioresistance, and plays an essential and non-redundant role in mouse embryogenesis and vasculogenesis. Furthermore, stress-inducible SAG is overexpressed in a number of human cancers and SAG overexpression correlates with poor patient prognosis. Finally, SAG transgenic expression in epidermis causes an early stage inhibition, but later stage promotion, of skin tumorigenesis triggered by DMBA/TPA. Given its major role in promoting targeted degradation of tumor suppressive proteins, leading to apoptosis suppression and accelerated tumorigenesis, SAG E3 ligase appears to be an attractive anticancer target. PMID:23136067

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

    PubMed

    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. PMID:24388516

  7. Functional identification of MdSIZ1 as a SUMO E3 ligase in apple.

    PubMed

    Zhang, Rui-Fen; Guo, Ying; Li, Yuan-Yuan; Zhou, Li-Jie; Hao, Yu-Jin; You, Chun-Xiang

    2016-07-01

    SUMOylation, the conjugation of target proteins with SUMO (small ubiquitin-related modifier), is a type of post-translational modification in eukaryotes and involves the sequential action of activation (E1), conjugation (E2) and ligation (E3) enzymes. In Arabidopsis, the AtSIZ1 protein is a SUMO E3 ligase that promotes the conjugation of SUMO proteins to target substrates. Here, we isolated and identified a SUMO E3 ligase, MdSIZ1, in apple, which was similar to AtSIZ1. SUMOylation analysis showed that MdSIZ1 had SUMO E3 ligase activity in vitro and in vivo. SUMO conjugation was increased by high temperatures, low temperatures, and abscisic acid (ABA). The ectopic expression of MdSIZ1 in Arabidopsis siz1-2 mutant plants partially complemented the morphological mutant phenotype and enhanced the levels of SUMO conjugation. Taken together, these results suggest that MdSIZ1-mediated SUMO conjugation of target proteins is an important process that regulates the adaptation of apple plants to various environmental stresses. PMID:27152458

  8. Identification of TRIM22 as a RING finger E3 ubiquitin ligase

    SciTech Connect

    Duan Zhijian; Gao Bo; Xu Wei; Xiong Sidong

    2008-09-26

    TRIM22, a member of the TRIM family proteins which contain RING finger, B-box, and coiled-coil domains, has been reported as a transcriptional regulator and involved in various cellular processes. In this study, the E3 ubiquitin ligase activity, a novel property of TRIM22, was demonstrated. It was found that TRIM22 underwent self-ubiquitylation in vitro in combination with the E2 enzyme UbcH5B and the ubiquitylation was dependent on its RING finger domain. Further evidences showed that TRIM22 could also be self-ubiquitylated in vivo. Importantly, TRIM22 was conjugated with poly-ubiquitin chains and stabilized by the proteasome inhibitor in 293T cells, suggesting that TRIM22 targeted itself for proteasomal degradation through the poly-ubiquitylation. We also found that TRIM22 was located in the nucleus, indicating that TRIM22 might function as a nuclear E3 ubiquitin ligase.

  9. IAPs as E3 ligases of Rac1: shaping the move.

    PubMed

    Oberoi-Khanuja, Tripat Kaur; Rajalingam, Krishnaraj

    2012-01-01

    Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination-dependent inactivation of Rac1 signaling. PMID:22790203

  10. “Ubiquitylation: mechanism and functions“ Review series: RBR E3-ligases at work

    PubMed Central

    Smit, Judith J; Sixma, Titia K

    2014-01-01

    The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region. PMID:24469331

  11. Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase

    PubMed Central

    Cappadocia, Laurent; Pichler, Andrea; Lima, Christopher D.

    2015-01-01

    E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. Here, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. ZNF451 catalytic module contains tandem SUMO interaction motifs (SIMs) bridged by a Proline-Leucine-Arginine-Proline (PLRP) motif. The first SIM and PLRP motif engage thioester charged E2~SUMO while the next SIM binds a second molecule of SUMO bound to the backside of E2. We show that ZNF451 is SUMO2 specific and that SUMO-modification of ZNF451 may contribute to activity by providing a second molecule of SUMO that interacts with E2. Our results are consistent with ZNF451 functioning as a bona fide SUMO E3 ligase. PMID:26524494

  12. Arabidopsis HIGH PLOIDY2 Sumoylates and Stabilizes Flowering Locus C through Its E3 Ligase Activity

    PubMed Central

    Kwak, Jun S.; Son, Ga H.; Kim, Sung-Il; Song, Jong T.; Seo, Hak S.

    2016-01-01

    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. PMID:27148346

  13. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity

    PubMed Central

    Kane, Lesley A.; Lazarou, Michael; Fogel, Adam I.; Li, Yan; Yamano, Koji; Sarraf, Shireen A.; Banerjee, Soojay

    2014-01-01

    PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin’s ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity. PMID:24751536

  14. PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.

    PubMed

    Kane, Lesley A; Lazarou, Michael; Fogel, Adam I; Li, Yan; Yamano, Koji; Sarraf, Shireen A; Banerjee, Soojay; Youle, Richard J

    2014-04-28

    PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity. PMID:24751536

  15. Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

    PubMed Central

    Bulatov, Emil; Ciulli, Alessio

    2015-01-01

    In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

  16. Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

    PubMed Central

    Kumar, Atul; Aguirre, Jacob D; Condos, Tara EC; Martinez-Torres, R Julio; Chaugule, Viduth K; Toth, Rachel; Sundaramoorthy, Ramasubramanian; Mercier, Pascal; Knebel, Axel; Spratt, Donald E; Barber, Kathryn R; Shaw, Gary S; Walden, Helen

    2015-01-01

    The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2∼Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2∼Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin. PMID:26254304

  17. Release from myosin V via regulated recruitment of an E3 Ub ligase controls organelle localization

    PubMed Central

    Yau, Richard G.; Peng, Yutian; Valiathan, Rajeshwari R.; Birkeland, Shanda R.; Wilson, Thomas E.; Weisman, Lois S.

    2014-01-01

    Summary Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin-V, Myo2, binds the vacuole specific adaptor, Vac17, to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 which recruits the E3 ligase, Dma1, to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism which detaches myosin-V from select cargoes. PMID:24636257

  18. The Evolutionarily Conserved E3 Ubiquitin Ligase AtCHIP Contributes to Plant Immunity.

    PubMed

    Copeland, Charles; Ao, Kevin; Huang, Yan; Tong, Meixuizi; Li, Xin

    2016-01-01

    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, Caenorhabditis 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. PMID:27014328

  19. The Evolutionarily Conserved E3 Ubiquitin Ligase AtCHIP Contributes to Plant Immunity

    PubMed Central

    Copeland, Charles; Ao, Kevin; Huang, Yan; Tong, Meixuizi; Li, Xin

    2016-01-01

    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, Caenorhabditis 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. PMID:27014328

  20. Transcription factor RFX1 is ubiquitinated by E3 ligase STUB1 in systemic lupus erythematosus.

    PubMed

    Guo, Yu; Zhao, Ming; Lu, Qianjin

    2016-08-01

    Systemic lupus erythematosus (SLE) is a chronic autoimmune disease caused by complex interactions between genes and the environment. The expression level of transcription factor regulatory factor X 1 (RFX1) is reduced in T cells from SLE patients. RFX1 can regulate epigenetic modifications of CD70 and CD11a and plays an important role in the development of SLE. However, the mechanisms that mediate reduction of RFX1 in SLE are unclear. Here, we demonstrate that RFX1 protein expression can be tightly regulated by polyubiquitination-mediated proteosomal degradation via STIP1 homology and U-box containing protein 1 (STUB1). The E3 ligase STUB1 is upregulated in CD4(+)T cells of SLE patients compared to healthy subjects. Overexpression of STUB1 in CD4(+)T cells leads to upregulation of levels of CD70 and CD11a in T cells. The modulation of STUB1 activity may provide a novel therapeutic approach for SLE. PMID:27283392

  1. Structural Basis for Substrate Selectivity of the E3 Ligase COP1.

    PubMed

    Uljon, Sacha; Xu, Xiang; Durzynska, Izabela; Stein, Sarah; Adelmant, Guillaume; Marto, Jarrod A; Pear, Warren S; Blacklow, Stephen C

    2016-05-01

    COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation. PMID:27041596

  2. The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex

    PubMed Central

    Kozakova, Lucie; Vondrova, Lucie; Stejskal, Karel; Charalabous, Panagoula; Kolesar, Peter; Lehmann, Alan R; Uldrijan, Stjepan; Sanderson, Christopher M; Zdrahal, Zbynek; Palecek, Jan J

    2015-01-01

    The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5–6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex. PMID:25590999

  3. The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2

    PubMed Central

    Mokhonova, Ekaterina I.; Avliyakulov, Nuraly K.; Kramerova, Irina; Kudryashova, Elena; Haykinson, Michael J.; Spencer, Melissa J.

    2015-01-01

    Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation. PMID:25701873

  4. The E3 ubiquitin ligase TRIM32 regulates myoblast proliferation by controlling turnover of NDRG2.

    PubMed

    Mokhonova, Ekaterina I; Avliyakulov, Nuraly K; Kramerova, Irina; Kudryashova, Elena; Haykinson, Michael J; Spencer, Melissa J

    2015-05-15

    Limb girdle muscular dystrophy 2H is caused by mutations in the gene encoding the E3 ubiquitin ligase, TRIM32. Previously, we generated and characterized a Trim32 knockout mouse (T32KO) that displays both neurogenic and myopathic features. The myopathy in these mice is attributable to impaired muscle growth, associated with satellite cell senescence and premature sarcopenia. This satellite cell senescence is due to accumulation of the SUMO ligase PIASy, a substrate of TRIM32. The goal of this investigation was to identify additional substrates of TRIM32 using 2D fluorescence difference gel electrophoresis (2D-DIGE) in order to further explore its role in skeletal muscle. Because TRIM32 is an E3 ubiquitin ligase, we reasoned that TRIM32's substrates would accumulate in its absence. 2D-DIGE identified 19 proteins that accumulate in muscles from the T32KO mouse. We focused on two of these proteins, NDRG2 and TRIM72, due to their putative roles in myoblast proliferation and myogenesis. Follow-up analysis confirmed that both proteins were ubiquitinated by TRIM32 in vitro; however, only NDRG2 accumulated in skeletal muscle and myoblasts in the absence of TRIM32. NDRG2 overexpression in myoblasts led to reduced cell proliferation and delayed cell cycle withdrawal during differentiation. Thus, we identified NDRG2 as a novel target for TRIM32; these findings further corroborate the hypothesis that TRIM32 is involved in control of myogenic cells proliferation and differentiation. PMID:25701873

  5. The E3 ubiquitin ligase GREUL1 anteriorizes ectoderm during Xenopus development.

    PubMed

    Borchers, Annette G M; Hufton, Andrew L; Eldridge, Adam G; Jackson, Peter K; Harland, Richard M; Baker, Julie C

    2002-11-15

    We have identified a family of RING finger proteins that are orthologous to Drosophila Goliath (G1, Gol). One of the members, GREUL1 (Goliath Related E3 Ubiquitin Ligase 1), can convert Xenopus ectoderm into XAG-1- and Otx2-expressing cells in the absence of both neural tissue and muscle. This activity, combined with the finding that XGREUL1 is expressed within the cement gland, suggests a role for GREUL1 in the generation of anterior ectoderm. Although GREUL1 is not a direct inducer of neural tissue, it can activate the formation of ectopic neural cells within the epidermis of intact embryos. This suggests that GREUL1 can sensitize ectoderm to neuralizing signals. In this paper, we provide evidence that GREUL1 is an E3 ubiquitin ligase. Using a biochemical assay, we show that GREUL1 catalyzes the addition of polyubiquitin chains. These events are mediated by the RING domain since a mutation in two of the cysteines abolishes ligase activity. Mutation of these cysteines also compromises GREUL1's ability to induce cement gland. Thus, GREUL1's RING domain is necessary for both the ubiquitination of substrates and for the conversion of ectoderm to an anterior fate. PMID:12435366

  6. Model of the Ankyrin and SOCS Box Protein, ASB9, E3 Ligase Reveals a Mechanism for Dynamic Ubiquitin Transfer.

    PubMed

    Schiffer, Jamie M; Malmstrom, Robert D; Parnell, Jonathan; Ramirez-Sarmiento, Cesar; Reyes, Javiera; Amaro, Rommie E; Komives, Elizabeth A

    2016-08-01

    Cullin-RING E3 ligases (CRLs) are elongated and bowed protein complexes that transfer ubiquitin over 60 Å to proteins targeted for proteasome degradation. One such CRL contains the ankyrin repeat and SOCS box protein 9 (ASB9), which binds to and partially inhibits creatine kinase (CK). While current models for the ASB9-CK complex contain some known interface residues, the overall structure and precise interface of the ASB9-CK complex remains unknown. Through an integrative modeling approach, we report a third-generation model that reveals precisely the interface interactions and also fits the shape of the ASB9-CK complex as determined by small-angle X-ray scattering. We constructed an atomic model for the entire CK-targeting CRL to uncover dominant modes of motion that could permit ubiquitin transfer. Remarkably, only the correctly docked CK-containing E3 ligase and not incorrectly docked structures permitted close approach of ubiquitin to the CK substrate. PMID:27396830

  7. Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein aggregation

    PubMed Central

    2011-01-01

    Background Accumulation of aberrant proteins to form Lewy bodies (LBs) is a hallmark of Parkinson's disease (PD). Ubiquitination-mediated degradation of aberrant, misfolded proteins is critical for maintaining normal cell function. Emerging evidence suggests that oxidative/nitrosative stress compromises the precisely-regulated network of ubiquitination in PD, particularly affecting parkin E3 ligase activity, and contributes to the accumulation of toxic proteins and neuronal cell death. Results To gain insight into the mechanism whereby cell stress alters parkin-mediated ubiquitination and LB formation, we investigated the effect of oxidative stress. We found significant increases in oxidation (sulfonation) and subsequent aggregation of parkin in SH-SY5Y cells exposed to the mitochondrial complex I inhibitor 1-methyl-4-phenlypyridinium (MPP+), representing an in vitro cell-based PD model. Exposure of these cells to direct oxidation via pathological doses of H2O2 induced a vicious cycle of increased followed by decreased parkin E3 ligase activity, similar to that previously reported following S-nitrosylation of parkin. Pre-incubation with catalase attenuated H2O2 accumulation, parkin sulfonation, and parkin aggregation. Mass spectrometry (MS) analysis revealed that H2O2 reacted with specific cysteine residues of parkin, resulting in sulfination/sulfonation in regions of the protein similar to those affected by parkin mutations in hereditary forms of PD. Immunohistochemistry or gel electrophoresis revealed an increase in aggregated parkin in rats and primates exposed to mitochondrial complex I inhibitors, as well as in postmortem human brain from patients with PD with LBs. Conclusion These findings show that oxidative stress alters parkin E3 ligase activity, leading to dysfunction of the ubiquitin-proteasome system and potentially contributing to LB formation. PMID:21595948

  8. The Prp19 U-box Crystal Structure Suggests a Common Dimeric Architecture for a Class of Oligomeric E3 Ubiquitin Ligases †,‡

    PubMed Central

    Vander Kooi, Craig W.; Ohi, Melanie D.; Rosenberg, Joshua A.; Oldham, Michael L.; Newcomer, Marcia E.; Gould, Kathleen L.; Chazin, Walter J.

    2008-01-01

    Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homo-dimeric state of the Prp19 U-box was determined by x-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligmeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase mediated poly-ubiquitination and E4 poly-ubiquitin ligases. PMID:16388587

  9. Ring finger protein 146/Iduna is a Poly (ADP-ribose) polymer binding and PARsylation dependent E3 ubiquitin ligase

    PubMed Central

    Zhou, Zhi-dong; Chan, Christine Hui-shan; Xiao, Zhi-cheng

    2011-01-01

    Recent findings suggest that Ring finger protein 146 (RNF146), also called Iduna, have neuroprotective property due to its inhibition of Parthanatos via binding with Poly(ADP-ribose) (PAR). The Parthanatos is a PAR dependent cell death that has been implicated in many human diseases. RNF146/Iduna acts as a PARsylation-directed E3 ubquitin ligase to mediate tankyrase-dependent degradation of axin, thereby positively regulates Wnt signaling. RNF146/Iduna can also facilitate DNA repair and protect against cell death induced by DNA damaging agents or γ-irradiation. It can translocate to the nucleus after cellular injury and promote the ubiquitination and degradation of various nuclear proteins involved in DNA damage repair. The PARsylation-directed ubquitination mediated by RNF146/Iduna is analogous to the phosphorylation-directed ubquitination catalyzed by Skp1-Cul1-F-box (SCF) E3 ubiquitin complex. RNF146/Iduna has been found to be implicated in neurodegenerative disease and cancer development. Therefore modulation of the PAR-binding and PARsylation dependent E3 ligase activity of RNF146/Iduna could have therapeutic significance for diseases, in which PAR and PAR-binding proteins play key pathophysiologic roles. PMID:22274711

  10. Identification of HECT E3 ubiquitin ligase family genes involved in stem cell regulation and regeneration in planarians.

    PubMed

    Henderson, Jordana M; Nisperos, Sean V; Weeks, Joi; Ghulam, Mahjoobah; Marín, Ignacio; Zayas, Ricardo M

    2015-08-15

    E3 ubiquitin ligases constitute a large family of enzymes that modify specific proteins by covalently attaching ubiquitin polypeptides. This post-translational modification can serve to regulate protein function or longevity. In spite of their importance in cell physiology, the biological roles of most ubiquitin ligases remain poorly understood. Here, we analyzed the function of the HECT domain family of E3 ubiquitin ligases in stem cell biology and tissue regeneration in planarians. Using bioinformatic searches, we identified 17 HECT E3 genes that are expressed in the Schmidtea mediterranea genome. Whole-mount in situ hybridization experiments showed that HECT genes were expressed in diverse tissues and most were expressed in the stem cell population (neoblasts) or in their progeny. To investigate the function of all HECT E3 ligases, we inhibited their expression using RNA interference (RNAi) and determined that orthologs of huwe1, wwp1, and trip12 had roles in tissue regeneration. We show that huwe1 RNAi knockdown led to a significant expansion of the neoblast population and death by lysis. Further, our experiments showed that wwp1 was necessary for both neoblast and intestinal tissue homeostasis as well as uncovered an unexpected role of trip12 in posterior tissue specification. Taken together, our data provide insights into the roles of HECT E3 ligases in tissue regeneration and demonstrate that planarians will be a useful model to evaluate the functions of E3 ubiquitin ligases in stem cell regulation. PMID:25956527

  11. UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1

    PubMed Central

    Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

    2015-01-01

    The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV RESISTANCE LOCUS 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B. PMID:25817546

  12. UV-B induction of the E3 ligase ARIADNE12 depends on CONSTITUTIVELY PHOTOMORPHOGENIC 1.

    PubMed

    Xie, Lisi; Lang-Mladek, Christina; Richter, Julia; Nigam, Neha; Hauser, Marie-Theres

    2015-08-01

    The UV-B inducible ARIADNE12 (ARI12) gene of Arabidopsis thaliana is a member of the RING-between-RING (RBR) family of E3 ubiquitin ligases for which a novel ubiquitination mechanism was identified in mammalian homologs. This RING-HECT hybrid mechanism needs a conserved cysteine which is replaced by serine in ARI12 and might affect the E3 ubiquitin ligase activity. We have shown that under photomorphogenic UV-B, ARI12 is a downstream target of the classical ultraviolet B (UV-B) UV Resistance Locus 8 (UVR8) pathway. However, under high fluence rate of UV-B ARI12 was induced independently of UVR8 and the UV-A/blue light and red/far-red photoreceptors. A key component of several light signaling pathways is Constitutively Photomorphogenic 1 (COP1). Upon UV-B COP1 is trapped in the nucleus through interaction with UVR8 permitting the activation of genes that regulate the biosynthesis of UV-B protective metabolites and growth adaptations. To clarify the role of COP1 in the regulation of ARI12 mRNA expression and ARI12 protein stability, localization and interaction with COP1 was assessed with and without UV-B. We found that COP1 controls ARI12 in white light, low and high fluence rate of UV-B. Furthermore we show that ARI12 is indeed an E3 ubiquitin ligase which is mono-ubiquitinated, a prerequisite for the RING-HECT hybrid mechanism. Finally, genetic analyses with transgenes expressing a genomic pmARI12:ARI12-GFP construct confirm the epistatic interaction between COP1 and ARI12 in growth responses to high fluence rate UV-B. PMID:25817546

  13. E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

    PubMed

    Kumar, Yogesh; Kapoor, Isha; Khan, Kainat; Thacker, Gatha; Khan, Mohd Parvez; Shukla, Nidhi; Kanaujiya, Jitendra Kumar; Sanyal, Sabyasachi; Chattopadhyay, Naibedya; Trivedi, Arun Kumar

    2015-12-25

    Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before. PMID:26542806

  14. Recognition of p63 by the E3 ligase ITCH: Effect of an ectodermal dysplasia mutant.

    PubMed

    Bellomaria, A; Barbato, Gaetano; Melino, G; Paci, M; Melino, Sonia

    2010-09-15

    The E3 ubiquitin ligase Itch mediates the degradation of the p63 protein. Itch contains four WW domains which are pivotal for the substrate recognition process. Indeed, this domain is implicated in several signalling complexes crucially involved in human diseases including Muscular Dystrophy, Alzheimer's Disease and Huntington Disease. WW domains are highly compact protein-protein binding modules that interact with short proline-rich sequences. The four WW domains present in Itch belong to the Group I type, which binds polypeptides with a PY motif characterized by a PP xY consensus sequence, where x can be any residue. Accordingly, the Itch-p63 interaction results from a direct binding of Itch-WW2 domain with the PY motif of p63. Here, we report a structural analysis of the Itch-p63 interaction by fluorescence, CD and NMR spectroscopy. Indeed, we studied the in vitro interaction between Itch-WW2 domain and p63(534-551), an 18-mer peptide encompassing a fragment of the p63 protein including the PY motif. In addition, we evaluated the conformation and the interaction with Itch-WW2 of a site specific mutant of p63, I549T, that has been reported in both Hay-Wells syndrome and Rapp-Hodgkin syndrome. Based on our results, we propose an extended PP xY motif for the Itch recognition motif (P-P-P-Y-x(4)-[ST]-[ILV]), which includes these C-terminal residues to the PP xY motif. PMID:20855944

  15. The Membrane Associated RING-CH Proteins: A Family of E3 Ligases with Diverse Roles through the Cell

    PubMed Central

    Means, Robert E.

    2014-01-01

    Since the discovery that conjugation of ubiquitin to proteins can drive proteolytic degradation, ubiquitination has been shown to perform a diverse range of functions in the cell. It plays an important role in endocytosis, signal transduction, trafficking of vesicles inside the cell, and even DNA repair. The process of ubiquitination-mediated control has turned out to be remarkably complex, involving a diverse array of proteins and many levels of control. This review focuses on a family of structurally related E3 ligases termed the membrane-associated RING-CH (MARCH) ubiquitin ligases, which were originally discovered as structural homologs to the virals E3s, K3, and K5 from Kaposi's sarcoma-associated herpesvirus (KSHV). These proteins contain a catalytic RING-CH finger and are typically membrane-bound, with some having up to 14 putative transmembrane domains. Despite several lines of evidence showing that the MARCH proteins play a complex and essential role in several cellular processes, this family remains understudied.

  16. Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

    PubMed

    Scott, Daniel C; Rhee, David Y; Duda, David M; Kelsall, Ian R; Olszewski, Jennifer L; Paulo, Joao A; de Jong, Annemieke; Ovaa, Huib; Alpi, Arno F; Harper, J Wade; Schulman, Brenda A

    2016-08-25

    Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates. Our data define ARIH1 as a component of the human CRL system, demonstrate that ARIH1 can efficiently and specifically mediate monoubiquitylation of several CRL substrates, and establish principles for how two distinctive E3s can reciprocally control each other for simultaneous and joint regulation of substrate ubiquitylation. These studies have broad implications for CRL-dependent proteostasis and mechanisms of E3-mediated UB ligation. PMID:27565346

  17. An E3 ubiquitin ligase, ERECT LEAF1, functions in brassinosteroid signaling of rice

    PubMed Central

    Sakamoto, Tomoaki; Kitano, Hidemi; Fujioka, Shozo

    2013-01-01

    A spontaneous rice mutant, erect leaf1 (elf1–1), produced a dwarf phenotype with erect leaves and short grains. Physiological analyses suggested that elf1–1 is brassinosteroid-insensitive, so we hypothesized that ELF1 encodes a positive regulator of brassinosteroid signaling. ELF1, identified by means of positional cloning, encodes a protein with both a U-box domain and ARMADILLO (ARM) repeats. U-box proteins have been shown to function as E3 ubiquitin ligases; in fact, ELF1 possessed E3 ubiquitin ligase activity in vitro. However, ELF1 itself does not appear to be polyubiquitinated. Mutant phenotypes of 2 more elf1 alleles indicate that the entire ARM repeats is indispensable for ELF1 activity. These results suggest that ELF1 ubiquitinates target proteins through an interaction mediated by ARM repeats. Similarities in the phenotypes of elf1 and d61 mutants (mutants of brassinosteroid receptor gene OsBRI1), and in the regulation of ELF1 and OsBRI1 expression, imply that ELF1 functions as a positive regulator of brassinosteroid signaling in rice. PMID:24299927

  18. F-box only protein 9 is an E3 ubiquitin ligase of PPARγ

    PubMed Central

    Lee, Kyeong Won; Kwak, Soo Heon; Koo, Young Do; Cho, Yun-Kyung; Lee, Hak Mo; Jung, Hye Seung; Cho, Young Min; Park, Young Joo; Chung, Sung Soo; Park, Kyong Soo

    2016-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of carbohydrate and lipid metabolism, adipocyte differentiation and inflammatory response. Post-translational modification of PPARγ and its degradation involve several pathways, including the ubiquitin–proteasome system. Here, we identified F-box only protein 9 (FBXO9) as an E3 ubiquitin ligase of PPARγ. We screened interacting partners of PPARγ using immunoprecipitation and mass spectrometric analysis and identified FBXO9 as an E3 ubiquitin ligase of PPARγ. FBXO9 directly interacted with PPARγ through the activation function-1 domain and ligand-binding domain. FBXO9 decreased the protein stability of PPARγ through induction of ubiquitination. We found that the F-box motif of FBXO9 was required for its ubiquitination function. The activity of PPARγ was significantly decreased by FBXO9 overexpression. Furthermore, FBXO9 overexpression in 3T3-L1 adipocytes resulted in decreased levels of endogenous PPARγ and suppression of adipogenesis. These results suggest that FBXO9 is an important enzyme that regulates the stability and activity of PPARγ through ubiquitination. PMID:27197753

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

    PubMed

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

    2016-09-12

    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 peroxisomal membrane proteins signals pexophagy; however, the E3 ligase responsible for mediating ubiquitination is not known. Here, we report that the peroxisomal E3 ubiquitin ligase peroxin 2 (PEX2) is the causative agent for mammalian pexophagy. Expression of PEX2 leads to gross ubiquitination of peroxisomes and degradation of peroxisomes in an NBR1-dependent autophagic process. We identify PEX5 and PMP70 as substrates of PEX2 that are ubiquitinated during amino acid starvation. We also find that PEX2 expression is up-regulated during both amino acid starvation and rapamycin treatment, suggesting that the mTORC1 pathway regulates pexophagy by regulating PEX2 expression levels. Finally, we validate our findings in vivo using an animal model. PMID:27597759

  20. Roles of E3 ubiquitin ligases in cell adhesion and migration.

    PubMed

    Huang, Cai

    2010-01-01

    Recent studies have demonstrated that a number of E3 ubiquitin ligases, including Cbl, Smurf1, Smurf2, HDM2, BCA2, SCF(beta-TRCP) and XRNF185, play important roles in cell adhesion and migration. Cbl negatively regulates cell adhesion via alpha integrin and Rap1 and inhibits actin polymerization by ubiquitinating mDab1 and WAVE2. Smurf1 regulates cell migration through ubiquitination of RhoA, talin head domain and hPEM2, while Smurf2 ubiquitinates Smurf1, TGFbeta type I receptor and RaplB to modulate cell migration and adhesion. HDM2 negatively regulates cell migration by targeting NFAT (a transcription factor) for ubiquitination and degradation, while SCF(beta-TRCP) ubiquitinates Snail (a transcriptional repressor of E-cadherin) to inhibit cell migration. TRIM32 promotes cell migration through ubiquitination of Abl interactor 2 (Abi2), a tumor suppressor. RNF5 and XRNF185 modulate cell migration by ubiquitinating paxillin. Thus, these E3 ubiquitin ligases regulate cell adhesion and (or) migration through ubiquitination of their specific substrates. PMID:20009572

  1. A conserved role for the ARC1 E3 ligase in Brassicaceae self-incompatibility.

    PubMed

    Indriolo, Emily; Goring, Daphne R

    2014-01-01

    Ubiquitination plays essential roles in the regulation of many processes in plants including pollen rejection in self-incompatible species. In the Brassicaceae (mustard family), self-incompatibility drives the rejection of self-pollen by preventing pollen hydration following pollen contact with the stigmatic surface. Self-pollen is recognized by a ligand-receptor pair: the pollen S-locus cysteine rich/S-locus protein 11 (SCR/SP11) ligand and the pistil S receptor kinase (SRK). Following self-pollen contact, the SCR/SP11 ligand on the pollen surface binds to SRK on the pistil surface, and the SRK-activated signaling pathway is initiated. This pathway includes the armadillo repeat containing 1 (ARC1) protein, a member of the plant U-box (PUB) family of E3 ubiquitin ligases. ARC1 is a functional E3 ligase and is required downstream of SRK for the self-incompatibility response. This mini review highlights our recent progress in establishing ARC1's conserved role in self-pollen rejection in Brassica and Arabidopsis species and discusses future research directions in this field. PMID:24847339

  2. The SUMO E3 ligase activity of Pc2 is coordinated through a SUMO interaction motif.

    PubMed

    Yang, Shen-hsi; Sharrocks, Andrew D

    2010-05-01

    Protein modification by SUMO conjugation has emerged to be an important regulatory event. Recently, the mechanisms through which SUMO elicits its effects on target proteins have been elucidated. One of these is the noncovalent association between SUMO and coregulatory proteins via SUMO interaction motifs (SIMs). We therefore searched for additional binding proteins to elucidate how SUMO acts as a signal to potentiate novel noncovalent interactions with SUMO-binding proteins. We identified an E3 ligase, Pc2, as a SUMO-binding protein with two functionally distinct SIMs. Here, we focus on the role of SIM2 and demonstrate that it is crucial for many of the documented Pc2 functions, which converge on determining its E3 ligase activity. One role of SUMO binding in this context is the subnuclear partitioning of the active form of Ubc9 (SUMO approximately Ubc9) by Pc2. The significance of the SIM2-dependent functions of Pc2 is demonstrated in the control of the precise expression of lineage-specific genes during embryonic stem cell differentiation. PMID:20176810

  3. A Tail of Two Sites: A Bipartite Mechanism for Recognition of Notch Ligands by Mind Bomb E3 Ligases

    PubMed Central

    McMillan, Brian J.; Schnute, Björn; Ohlenhard, Nadja; Zimmerman, Brandon; Miles, Laura; Beglova, Natalia; Klein, Thomas; Blacklow, Stephen C.

    2015-01-01

    Summary Mind bomb (Mib) proteins are large, multi-domain E3 ligases that promote ubiquitination of the cytoplasmic tails of Notch ligands. This ubiquitination step marks the ligand proteins for epsin-dependent endocytosis, which is critical for in vivo Notch receptor activation. We present here crystal structures of the substrate recognition domains of Mib1, both in isolation and in complex with peptides derived from Notch ligands. The structures, in combination with biochemical, cellular and in vivo assays, show that Mib1 contains two independent substrate recognition domains that engage two distinct epitopes from the cytoplasmic tail of the ligand Jagged1, one in the intracellular membrane proximal region and the other near the C-terminus. Together, these studies provide new insights into the mechanism of ubiquitin transfer by Mind bomb E3 ligases, illuminate a key event in ligand-induced activation of Notch receptors, and identify a potential new target for therapeutic modulation of Notch signal transduction in disease. PMID:25747658

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

    PubMed Central

    van den Boomen, Dick J. H.; Timms, Richard T.; Grice, Guinevere L.; Stagg, Helen R.; Skødt, Karsten; Dougan, Gordon; Nathan, James A.; Lehner, Paul J.

    2014-01-01

    The US11 gene product of human cytomegalovirus promotes viral immune evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US11 initiates dislocation of newly translocated MHC I from the ER to the cytosol for proteasome-mediated degradation. Despite the critical 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 of a novel mammalian ERAD complex. PMID:25030448

  5. Arabidopsis BPM Proteins Function as Substrate Adaptors to a CULLIN3-Based E3 Ligase to Affect Fatty Acid Metabolism in Plants[W

    PubMed Central

    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

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

    PubMed

    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. PMID:26766115

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

    PubMed Central

    Rodrigues, Elizabeth M.; Scudder, Samantha L.; Goo, Marisa S.

    2016-01-01

    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. SIGNIFICANCE STATEMENT 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. PMID:26843640

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

    PubMed

    Lin, Xiao-Li; Niu, De; Hu, Zi-Liang; Kim, Dae Heon; Jin, Yin Hua; Cai, Bin; Liu, Peng; Miura, Kenji; Yun, Dae-Jin; Kim, Woe-Yeon; Lin, Rongcheng; Jin, Jing Bo

    2016-04-01

    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. PMID:27128446

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

    PubMed Central

    Lin, Xiao-Li; Niu, De; Hu, Zi-Liang; Kim, Dae Heon; Jin, Yin Hua; Cai, Bin; Liu, Peng; Miura, Kenji; Yun, Dae-Jin; Kim, Woe-Yeon; Lin, Rongcheng

    2016-01-01

    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. PMID:27128446

  10. A mouse forward genetics screen identifies LISTERIN as an E3 ubiquitin ligase involved in neurodegeneration

    PubMed Central

    Chu, Jessie; Hong, Nancy A.; Masuda, Claudio A.; Jenkins, Brian V.; Nelms, Keats A.; Goodnow, Christopher C.; Glynne, Richard J.; Wu, Hua; Masliah, Eliezer; Joazeiro, Claudio A. P.; Kay, Steve A.

    2009-01-01

    A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders. PMID:19196968

  11. Merlin's tumor suppression linked to inhibition of the E3 ubiquitin ligase CRL4DCAF1

    PubMed Central

    Li, Wei

    2010-01-01

    The mechanism by which the FERM domain protein Merlin, encoded by the tumor suppressor NF2, restrains cell proliferation is poorly understood. Prior studies have suggested that Merlin exerts its antimitogenic effect by interacting with multiple signaling proteins located at or near the plasma membrane. We have recently observed that Merlin translocates into the nucleus and binds to and inhibits the E3 ubiquitin ligase CRL4DCAF1. Genetic evidence indicates that inactivation of Merlin induces oncogenic gene expression, hyperproliferation, and tumorigenicity by unleashing the activity of CRL4DCAF1. In addition to providing a potential explanation for the diverse effects that loss of Merlin exerts in multiple cell types, these findings suggest that compounds inhibiting CRL4DCAF1 may display therapeutic efficacy in Neurofibromatosis type 2 and other cancers driven by Merlin inactivation. PMID:21084862

  12. The Ubiquitin E3 Ligase NOSIP Modulates Protein Phosphatase 2A Activity in Craniofacial Development

    PubMed Central

    Hoffmeister, Meike; Prelle, Carola; Küchler, Philipp; Kovacevic, Igor; Moser, Markus; Müller-Esterl, Werner; Oess, Stefanie

    2014-01-01

    Holoprosencephaly is a common developmental disorder in humans characterised by incomplete brain hemisphere separation and midface anomalies. The etiology of holoprosencephaly is heterogeneous with environmental and genetic causes, but for a majority of holoprosencephaly cases the genes associated with the pathogenesis could not be identified so far. Here we report the generation of knockout mice for the ubiquitin E3 ligase NOSIP. The loss of NOSIP in mice causes holoprosencephaly and facial anomalies including cleft lip/palate, cyclopia and facial midline clefting. By a mass spectrometry based protein interaction screen we identified NOSIP as a novel interaction partner of protein phosphatase PP2A. NOSIP mediates the monoubiquitination of the PP2A catalytic subunit and the loss of NOSIP results in an increase in PP2A activity in craniofacial tissue in NOSIP knockout mice. We conclude, that NOSIP is a critical modulator of brain and craniofacial development in mice and a candidate gene for holoprosencephaly in humans. PMID:25546391

  13. New role of E3 ubiquitin ligase in the regulation of necroptosis.

    PubMed

    Seo, Jinho; Lee, Eun-Woo; Song, Jaewhan

    2016-05-01

    Necroptosis is a well-known form of caspase-independent cell death. Necroptosis can be triggered by various extrinsic stimuli, including death ligands in the presence of receptorinteracting protein kinase 3 (RIPK3), a key mediator of necroptosis induction. Our recent studies have revealed that C-terminus HSC-70 interacting protein (CHIP), an E3 ligase, can function as an inhibitor of necroptosis. CHIP-/- mouse embryonic fibroblast showed higher sensitivity to necrotic stimuli than wild-type mouse embryonic fibroblast cells. Deleterious effects of CHIP knockout MEFs were retrieved by RIPK3 depletion. We found that CHIP negatively regulated RIPK3 and RIPK1 by ubiquitylation- and lysosome- dependent degradation. In addition, CHIP-/- mice showed postnatal lethality with intestinal defects that could be rescued by crossing with RIPK3-/- mice. These results suggest that CHIP is a negative regulator of RIPK1 and RIPK3, thus inhibiting necroptosis. [BMB Reports 2016; 49(5): 247-248]. PMID:27099235

  14. Substrates of the ASB2α E3 ubiquitin ligase in dendritic cells

    PubMed Central

    Spinner, Camille A.; Uttenweiler-Joseph, Sandrine; Metais, Arnaud; Stella, Alexandre; Burlet-Schiltz, Odile; Moog-Lutz, Christel; Lamsoul, Isabelle; Lutz, Pierre G.

    2015-01-01

    Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2α, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2α substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies. PMID:26537633

  15. Trim17, a novel E3 ubiquitin-ligase, initiates neuronal apoptosis

    PubMed Central

    Lassot, Irina; Robbins, Ian; Kristiansen, Mark; Rahmeh, Rita; Jaudon, Fanny; Magiera, Maria M.; Mora, Stéphan; Vanhille, Laurent; Lipkin, Alexey; Pettmann, Brigitte; Ham, Jonathan; Desagher, Solange

    2010-01-01

    Accumulating data indicate that the ubiquitin-proteasome system controls apoptosis by regulating the level and the function of key regulatory proteins. In the present study, we identified Trim17, a member of the TRIM/RBCC protein family, as one of the critical E3 ubiquitin-ligases involved in the control of neuronal apoptosis upstream of mitochondria. We show that expression of Trim17 is increased both at the mRNA and protein level in several in vitro models of transcription-dependent neuronal apoptosis. Expression of Trim17 is controlled by the PI3K/Akt/GSK3 pathway in cerebellar granule neurons (CGN). Moreover, the Trim17 protein is expressed in vivo, in apoptotic neurons that naturally die during postnatal cerebellar development. Overexpression of active Trim17 in primary CGN was sufficient to induce the intrinsic pathway of apoptosis in survival conditions. This proapoptotic effect was abolished in Bax−/− neurons and depended on the E3 activity of Trim17 conferred by its RING domain. Furthermore, knock-down of endogenous Trim17 and overexpression of dominant-negative mutants of Trim17 blocked trophic factor withdrawal-induced apoptosis both in CGN and in sympathetic neurons. Collectively, our data are the first to assign a cellular function to Trim17 by showing that its E3 activity is both necessary and sufficient for the initiation of neuronal apoptosis. PMID:20559321

  16. A Novel Type of E3 Ligase for the Ufm1 Conjugation System*

    PubMed Central

    Tatsumi, Kanako; Sou, Yu-shin; Tada, Norihiro; Nakamura, Eri; Iemura, Shun-ichiro; Natsume, Tohru; Kang, Sung Hwan; Chung, Chin Ha; Kasahara, Masanori; Kominami, Eiki; Yamamoto, Masayuki; Tanaka, Keiji; Komatsu, Masaaki

    2010-01-01

    The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116·Ufm1 formation was greatly accelerated by Ufl1. The C20orf116·Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms. PMID:20018847

  17. Phosphorylation-dependent control of Pc2 SUMO E3 ligase activity by its substrate protein HIPK2.

    PubMed

    Roscic, Ana; Möller, Andreas; Calzado, Marco A; Renner, Florian; Wimmer, Verena C; Gresko, Ekaterina; Lüdi, Katharina Schmid; Schmitz, M Lienhard

    2006-10-01

    Sumoylation serves to control key cellular functions, but the regulation of SUMO E3 ligase activity is largely unknown. Here we show that the polycomb group protein Pc2 binds to and colocalizes with homeodomain interacting protein kinase 2 (HIPK2) and serves as a SUMO E3 ligase for this kinase. DNA damage-induced HIPK2 directly phosphorylates Pc2 at multiple sites, which in turn controls Pc2 sumoylation and intranuclear localization. Inducible phosphorylation of Pc2 at threonine 495 is required for its ability to increase HIPK2 sumoylation in response to DNA damage, thereby establishing an autoregulatory feedback loop between a SUMO substrate and its cognate E3 ligase. Sumoylation enhances the ability of HIPK2 to mediate transcriptional repression, thus providing a mechanistic link for DNA damage-induced transcriptional silencing. PMID:17018294

  18. Enzymatic Analysis of PTEN Ubiquitylation by WWP2 and NEDD4-1 E3 Ligases.

    PubMed

    Chen, Zan; Thomas, Stefani N; Bolduc, David M; Jiang, Xuejun; Zhang, Xiangbin; Wolberger, Cynthia; Cole, Philip A

    2016-07-01

    PTEN is a lipid phosphatase that converts phosphatidylinositol 3,4,5-phosphate (PIP3) to phosphatidylinositol 4,5-phosphate (PIP2) and plays a critical role in the regulation of tumor growth. PTEN is subject to regulation by a variety of post-translational modifications, including phosphorylation on a C-terminal cluster of four Ser/Thr residues (380, 382, 383, and 385) and ubiquitylation by various E3 ligases, including NEDD4-1 and WWP2. It has previously been shown that C-terminal phosphorylation of PTEN can increase its cellular half-life. Using in vitro ubiquitin transfer assays, we show that WWP2 is more active than NEDD4-1 in ubiquitylating unphosphorylated PTEN. The mapping of ubiquitylation sites in PTEN by mass spectrometry showed that both NEDD4-1 and WWP2 can target a broad range of Lys residues in PTEN, although NEDD4-1 versus WWP2 showed a stronger preference for ubiquitylating PTEN's C2 domain. Whereas tetraphosphorylation of PTEN did not significantly affect its ubiquitylation by NEDD4-1, it inhibited PTEN ubiquitylation by WWP2. Single-turnover and pull-down experiments suggested that tetraphosphorylation of PTEN appears to weaken its interaction with WWP2. These studies reveal how the PTEN E3 ligases WWP2 and NEDD4-1 exhibit distinctive properties in Lys selectivity and sensitivity to PTEN phosphorylation. Our findings also provide a molecular mechanism for the connection between PTEN Ser/Thr phosphorylation and PTEN's cellular stability. PMID:27295432

  19. SGR9, a RING type E3 ligase, modulates amyloplast dynamics important for gravity sensing.

    NASA Astrophysics Data System (ADS)

    Morita, Miyo T.; Nakamura, Moritaka; Tasaka, Masao

    Gravitropism is triggered when the directional change of gravity is sensed in the specific cells, called statocytes. In higher plants, statocytes contain sinking heavier amyloplasts which are particular plastids accumulating starch granules. The displacement of amyloplasts within the statocytes is thought to be the initial event of gravity perception. We have demonstrated that endodermal cells are most likely to be the statocytes in Arabidop-sis shoots. Live cell imaging of the endodermal cell of stem has shown that most amyloplasts are sediment to the direction of gravity but they are not static. Several amyloplasts move dynamically in an actin filament (F-actin) dependent manner. In the presence of actin poly-merization inhibitor, all amyloplasts become static and sediment to the direction of gravity. In addition, stems treated with the inhibitor can exhibit gravitropism. These results suggest that F-actin-dependent dynamic movement of amyloplasts is not essential for gravity sensing. sgr (shoot gravitropism) 9 mutant exhibits greatly reduced shoot gravitropism. In endodermal cells of sgr9, dynamic amyloplast movement was predominantly observed and amyloplasts did not sediment to the direction of gravity. Interestingly, inhibition of actin polymerization re-stored both gravitropism and amyloplast sedimentation in sgr9. The SGR9 encodes a novel RING finger protein, which is localized to amyloplasts in endodermal cells. SGR9 showed ubiq-uitin E3 ligase activity in vitro. Together with live cell imaging of amyloplasts and F-actin, our data suggest that SGR9 modulate interaction between amyloplasts and F-actin on amylo-plasts. SGR9 positively act on amyloplasts sedimentation, probably by releasing amyloplasts from F-actin. SGR9 that is localized to amyloplast, possibly degrades unknown substrates by its E3 ligase activity, and this might promote release of amyloplasts from F-actin.

  20. AUTOUBIQUITINATION OF BCA2 RING E3 LIGASE REGULATES ITS OWN STABILITY AND AFFECTS CELL MIGRATION

    PubMed Central

    Amemiya, Yutaka; Azmi, Peter; Seth, Arun

    2009-01-01

    Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2 binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, majority of those were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitinA-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the N-terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared to wild-type BCA2. On the basis of these findings, we propose that BCA2 maybe an important factor regulating breast cancer cell migration/metastasis. We put-forward a novel model for BCA2 E3 ligase mediated cell regulation. PMID:18819927

  1. Solution structure of the E3 ligase HOIL-1 Ubl domain.

    PubMed

    Beasley, Steven A; Safadi, Susan S; Barber, Kathryn R; Shaw, Gary S

    2012-07-01

    The E3 ligases HOIL-1 and parkin are each comprised of an N-terminal ubiquitin-like (Ubl) domain followed by a zinc-binding region and C-terminal RING-In-between-RING-RING domains. These two proteins, involved in the ubiquitin-mediated degradation pathway, are the only two known E3 ligases to share this type of multidomain architecture. Further, the Ubl domain of both HOIL-1 and parkin has been shown to interact with the S5a subunit of the 26S proteasome. The solution structure of the HOIL-1 Ubl domain was solved using NMR spectroscopy to compare it with that of parkin to determine the structural elements responsible for S5a intermolecular interactions. The final ensemble of 20 structures had a β-grasp Ubl-fold with an overall backbone RMSD of 0.59 ± 0.10 Å in the structured regions between I55 and L131. HOIL-1 had a unique extension of both β1 and β2 sheets compared to parkin and other Ubl domains, a result of a four-residue insertion in this region. A similar 15-residue hydrophobic core in the HOIL-1 Ubl domain resulted in a comparable stability to the parkin Ubl, but significantly lower than that observed for ubiquitin. A comparison with parkin and other Ubl domains indicates that HOIL-1 likely uses a conserved hydrophobic patch (W58, V102, Y127, Y129) found on the β1 face, the β3-β4 loop and β5, as well as a C-terminal basic residue (R134) to recruit the S5a subunit as part of the ubiquitin-mediated proteolysis pathway. PMID:22517668

  2. Copy number variation of E3 ubiquitin ligase genes in peripheral blood leukocyte and colorectal cancer

    PubMed Central

    Bi, Haoran; Tian, Tian; Zhu, Lin; Zhou, Haibo; Hu, Hanqing; Liu, Yanhong; Li, Xia; Hu, Fulan; Zhao, Yashuang; Wang, Guiyu

    2016-01-01

    Given that E3 ubiquitin ligases (E3) regulate specific protein degradation in many cancer-related biological processes. E3 copy number variation (CNV) may affect the development and prognosis of colorectal cancer (CRC). Therefore, we detected CNVs of five E3 genes in 518 CRC patients and 518 age, gender and residence matched controls in China, and estimated the association between E3 gene CNVs and CRC risk and prognosis. We also estimated their interactions with environmental factors and CRC risk. We find a significant association between the CNVs of MDM2 and CRC risk (amp v.s. wt: odds ratio = 14.37, 95% confidence interval: 1.27, 163.74, P = 0.032), while SKP2 CNVs may significantly decrease CRC risk (del v.s. wt: odds ratio = 0.32, 95% confidence interval: 0.10, 1.00, P = 0.050). However, we find no significant association between the CNVs of other genes and CRC risk. The only significant gene-environment interaction effects are between SKP2 CNVs and consumption of fish and/or fruit (P = 0.014 and P = 0.035) and between FBXW7 CNVs and pork intake (P = 0.040). Finally, we find marginally significant association between β-TRCP CNVs and CRC prognosis (amp v.s. wt, hazard ratio = 0.42, 95% confidence interval: 0.19, 0.97, P = 0.050). PMID:27417709

  3. Copy number variation of E3 ubiquitin ligase genes in peripheral blood leukocyte and colorectal cancer.

    PubMed

    Bi, Haoran; Tian, Tian; Zhu, Lin; Zhou, Haibo; Hu, Hanqing; Liu, Yanhong; Li, Xia; Hu, Fulan; Zhao, Yashuang; Wang, Guiyu

    2016-01-01

    Given that E3 ubiquitin ligases (E3) regulate specific protein degradation in many cancer-related biological processes. E3 copy number variation (CNV) may affect the development and prognosis of colorectal cancer (CRC). Therefore, we detected CNVs of five E3 genes in 518 CRC patients and 518 age, gender and residence matched controls in China, and estimated the association between E3 gene CNVs and CRC risk and prognosis. We also estimated their interactions with environmental factors and CRC risk. We find a significant association between the CNVs of MDM2 and CRC risk (amp v.s. wt: odds ratio = 14.37, 95% confidence interval: 1.27, 163.74, P = 0.032), while SKP2 CNVs may significantly decrease CRC risk (del v.s. wt: odds ratio = 0.32, 95% confidence interval: 0.10, 1.00, P = 0.050). However, we find no significant association between the CNVs of other genes and CRC risk. The only significant gene-environment interaction effects are between SKP2 CNVs and consumption of fish and/or fruit (P = 0.014 and P = 0.035) and between FBXW7 CNVs and pork intake (P = 0.040). Finally, we find marginally significant association between β-TRCP CNVs and CRC prognosis (amp v.s. wt, hazard ratio = 0.42, 95% confidence interval: 0.19, 0.97, P = 0.050). PMID:27417709

  4. An E3 ligase possessing an iron-responsive hemerythrin domain is a regulator of iron homeostasis.

    PubMed

    Salahudeen, Ameen A; Thompson, Joel W; Ruiz, Julio C; Ma, He-Wen; Kinch, Lisa N; Li, Qiming; Grishin, Nick V; Bruick, Richard K

    2009-10-30

    Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5's differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis. PMID:19762597

  5. The Pallbearer E3 Ligase Promotes Actin Remodeling via RAC in Efferocytosis by Degrading the Ribosomal Protein S6

    PubMed Central

    Xiao, Hui; Wang, Hui; Silva, Elizabeth; Thompson, James; Guillou, Aurélien; Yates, John R.; Buchon, Nicolas; Franc, Nathalie C.

    2014-01-01

    Clearance of apoptotic cells (efferocytosis) is achieved through phagocytosis by professional or amateur phagocytes. It is critical for tissue homeostasis and remodeling in all animals. Failure in this process can contribute to the development of inflammatory autoimmune or neurodegenerative diseases. We previously found that the PALL-SCF E3-Ubiquitin ligase complex promotes apoptotic cell clearance, yet it remained unclear as to how it did so. Here, we show that the F-Box protein PALL interacts with phosphorylated Ribosomal protein S6 (RpS6) to promote its ubiquitylation and proteasomal degradation. This leads to RAC2 GTPase up-regulation and activation and F-actin remodeling that promotes efferocytosis. We further show that the specific role of PALL in efferocytosis is driven by its apoptotic cell-induced nuclear export. Finding a role for RpS6 in negatively regulating efferocytosis provides the opportunity to develop new strategies to regulate this process. PMID:25533207

  6. The Pallbearer E3 ligase promotes actin remodeling via RAC in efferocytosis by degrading the ribosomal protein S6.

    PubMed

    Xiao, Hui; Wang, Hui; Silva, Elizabeth A; Thompson, James; Guillou, Aurélien; Yates, John R; Buchon, Nicolas; Franc, Nathalie C

    2015-01-12

    Clearance of apoptotic cells (efferocytosis) is achieved through phagocytosis by professional or amateur phagocytes. It is critical for tissue homeostasis and remodeling in all animals. Failure in this process can contribute to the development of inflammatory autoimmune or neurodegenerative diseases. We found previously that the PALL-SCF E3-ubiquitin ligase complex promotes apoptotic cell clearance, but it remained unclear how it did so. Here we show that the F-box protein PALL interacts with phosphorylated ribosomal protein S6 (RpS6) to promote its ubiquitylation and proteasomal degradation. This leads to RAC2 GTPase upregulation and activation and F-actin remodeling that promotes efferocytosis. We further show that the specific role of PALL in efferocytosis is driven by its apoptotic cell-induced nuclear export. Finding a role for RpS6 in the negative regulation of efferocytosis provides the opportunity to develop new strategies to regulate this process. PMID:25533207

  7. Cross-functional E3 ligases Parkin and C-terminus Hsp70-interacting protein in neurodegenerative disorders.

    PubMed

    Kumar, Pravir; Pradhan, Kaveri; Karunya, R; Ambasta, Rashmi K; Querfurth, Henry W

    2012-02-01

    The study of neurodegenerative disorders has had a major impact on our understanding of more fundamental mechanisms underlying neurobiology. Breakthroughs in the genetics of Alzheimer's (AD) and Parkinson's diseases (PD) has resulted in new knowledge in the areas of axonal transport, energy metabolism, protein trafficking/clearance and synaptic physiology. The major neurodegenerative diseases have in common a regional or network pathology associated with abnormal protein accumulation(s) and various degrees of motor or cognitive decline. In AD, β-amyloids are deposited in extracellular diffuse and compacted plaques as well as intracellularly. There is a major contribution to the disease by the co-existence of an intraneuronal tauopathy. Additionally, PD-like Lewy Bodies (LBs) bearing aggregated α-synuclein is present in 40-60% of all AD cases, especially involving amygdala. Amyloid deposits can be degraded or cleared by several mechanisms, including immune-mediated and transcytosis across the blood-brain barrier. Another avenue for disposal involves the lysosome pathway via autophagy. Enzymatic pathways include insulin degradative enzyme and neprilysin. Finally, the co-operative actions of C-terminus Hsp70 interacting protein (CHIP) and Parkin, components of a multiprotein E3 ubiquitin ligase complex, may be a portal to proteasome-mediated degradation. Mutations in the Parkin gene are the most common genetic link to autosomal recessive Parkinson's disease. Parkin catalyzes the post-translational modification of proteins with polyubiquitin, targeting them to the 26S proteasome. Parkin reduces intracellular Aβ(1-42) peptide levels, counteracts its effects on cell death, and reverses its effect to inhibit the proteasome. Additionally, Parkin has intrinsic cytoprotective activity to promote proteasome function and defend against oxidative stress to mitochondria. Parkin and CHIP are also active in amyloid clearance and cytoprotection in vivo. Parkin has cross

  8. The E3 Ligase APC/C-Cdh1 Is Required for Associative Fear Memory and Long-Term Potentiation in the Amygdala of Adult Mice

    ERIC Educational Resources Information Center

    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…

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

    PubMed Central

    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. The RING E3 Ligase KEEP ON GOING Modulates JASMONATE ZIM-DOMAIN12 Stability1[OPEN

    PubMed Central

    Pauwels, Laurens; Ritter, Andrés; Goossens, Jonas; Durand, Astrid Nagels; Liu, Hongxia; Gu, Yangnan; Geerinck, Jan; Boter, Marta; Vanden Bossche, Robin; De Clercq, Rebecca; Van Leene, Jelle; Gevaert, Kris; De Jaeger, Geert; Solano, Roberto; Stone, Sophia; Innes, Roger W.; Callis, Judy; Goossens, Alain

    2015-01-01

    Jasmonate (JA) signaling in plants is mediated by the JASMONATE ZIM-DOMAIN (JAZ) proteins that repress the activity of several transcription factors regulating JA-inducible gene expression. The hormone JA-isoleucine triggers the interaction of JAZ repressor proteins with the F-box protein CORONATINE INSENSITIVE1 (COI1), part of an S-phase kinase-associated protein1/Cullin1/F-box protein COI1 (SCFCOI1) E3 ubiquitin ligase complex, and their degradation by the 26S proteasome. In Arabidopsis (Arabidopsis thaliana), the JAZ family consists of 13 members. The level of redundancy or specificity among these members is currently not well understood. Here, we characterized JAZ12, encoded by a highly expressed JAZ gene. JAZ12 interacted with the transcription factors MYC2, MYC3, and MYC4 in vivo and repressed MYC2 activity. Using tandem affinity purification, we found JAZ12 to interact with SCFCOI1 components, matching with observed in vivo ubiquitination and with rapid degradation after treatment with JA. In contrast to the other JAZ proteins, JAZ12 also interacted directly with the E3 RING ligase KEEP ON GOING (KEG), a known repressor of the ABSCISIC ACID INSENSITIVE5 transcription factor in abscisic acid signaling. To study the functional role of this interaction, we circumvented the lethality of keg loss-of-function mutants by silencing KEG using an artificial microRNA approach. Abscisic acid treatment promoted JAZ12 degradation, and KEG knockdown led to a decrease in JAZ12 protein levels. Correspondingly, KEG overexpression was capable of partially inhibiting COI1-mediated JAZ12 degradation. Our results provide additional evidence for KEG as an important factor in plant hormone signaling and a positive regulator of JAZ12 stability. PMID:26320228

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

    PubMed

    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 SCF(SEL-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

  12. The E3 Ubiquitin Ligase MID1 Catalyzes Ubiquitination and Cleavage of Fu

    PubMed Central

    Schweiger, Susann; Dorn, Stephanie; Fuchs, Melanie; Köhler, Andrea; Matthes, Frank; Müller, Eva-Christina; Wanker, Erich; Schneider, Rainer; Krauß, Sybille

    2014-01-01

    SHH (Sonic Hedgehog)-GLI signaling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signaling. A protein complex containing the ubiquitin ligase MID1 and protein phosphatase 2A regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signaling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyzes the ubiquitination and proteasomal cleavage of the GLI3 regulator Fu. Our data suggest that Fu ubiquitination and cleavage is one of the key elements connecting the MID1-PP2A protein complex with GLI3 activity control. PMID:25278022

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

    PubMed

    Hannah, Jeffrey; Zhou, Pengbo

    2015-11-15

    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

  14. Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is diffe...

  15. Heterologous expression of rice SUMO E3 ligase (OsSIZ1) enhances drought and heat tolerance in transgenic cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis gene AtSIZ1 encodes a SUMO E3 ligase that plays important roles in plant response to abiotic stresses such as drought, heat, cold, salt, and nutrient starvation. Loss of function in AtSIZ1 leads to increased sensitivity to drought, heat, and salt stresses, whereas overexpression of t...

  16. Atypical ubiquitination by E3 ligase WWP1 inhibits the proteasome-mediated degradation of mutant huntingtin.

    PubMed

    Lin, Li; Jin, Zhenzhen; Tan, Huiping; Xu, Qiaoqiao; Peng, Ting; Li, He

    2016-07-15

    Huntington's disease (HD) is caused by the expansion of CAG trinucleotide repeats in exon 1 of HD gene encoding huntingtin (Htt), which is characterized by aggregation and formation of mutant Htt containing expanded polyglutamine (polyQ) repeats. Dysfunction of the ubiquitin-proteasome system (UPS) plays a critical role in the pathogenesis of HD. As the linkage mediator between ubiquitin and specific target proteins, E3 ubiquitin ligases have been suggested to be involved in mHtt degradation and HD pathology. However, the potential involvement of the E3 ligase WWP1 in HD has not been explored. The present study determined whether WWP1 is involved in the development of HD in both in vivo and in vitro models. The results showed that in contrast to several other E3 ligases, expression of WWP1 is enhanced in mice and N2a cells expressing mutant Htt (160Q) and co-localized with mHtt protein aggregates. In addition, expression of WWP1 positively regulates mutan Htt levels, aggregate formation, and cell toxicity. Further analysis revealed that WWP1 ubiquitinated mHtt at an atypical position of Lys-63, which may have inhibited degradation of mutant Htt through the ubiquitin-proteasome pathway. In conclusion, these results suggested that the E3 ligase WWP1 is involved in the pathogenesis of HD; therefore, it may be a novel target for therapeutic intervention. PMID:27107943

  17. The E3 ligase CHIP mediates ubiquitination and degradation of mixed-lineage kinase 3.

    PubMed

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

    2014-08-01

    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 Hsp70 overexpression promoted endogenous MLK3 ubiquitination and induced a decline in MLK3 protein levels in cells with Hsp90 inhibition. Furthermore, CHIP overexpression caused a proteasome-dependent reduction in exogenous MLK3 protein. Geldanamycin (GA), heat shock, and osmotic shock treatments also reduced the level of MLK3 protein via a CHIP-dependent mechanism. In addition, CHIP depletion in ovarian cancer SKOV3 cells increased cell invasion, and the enhancement of invasiveness was abrogated by small interfering RNA (siRNA)-mediated knockdown of MLK3. Thus, CHIP modulates MLK3 protein levels in response to GA and stress stimuli, and CHIP-dependent regulation of MLK3 is required for suppression of SKOV3 ovarian cancer cell invasion. PMID:24912674

  18. Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1

    PubMed Central

    Park, Bong Soo; Song, Jong Tae; Seo, Hak Soo

    2011-01-01

    Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis. PMID:21772271

  19. MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification

    PubMed Central

    Kwon, Duk-Hwa; Eom, Gwang Hyeon; Ko, Jeong Hyeon; Shin, Sera; Joung, Hosouk; Choe, Nakwon; Nam, Yoon Seok; Min, Hyun-Ki; Kook, Taewon; Yoon, Somy; Kang, Wanseok; Kim, Yong Sook; Kim, Hyung Seok; Choi, Hyuck; Koh, Jeong-Tae; Kim, Nacksung; Ahn, Youngkeun; Cho, Hyun-Jai; Lee, In-Kyu; Park, Dong Ho; Suk, Kyoungho; Seo, Sang Beom; Wissing, Erin R.; Mendrysa, Susan M.; Nam, Kwang-Il; Kook, Hyun

    2016-01-01

    Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC. PMID:26832969

  20. The ubiquitin E3 ligase TRAF6 exacerbates pathological cardiac hypertrophy via TAK1-dependent signalling

    PubMed Central

    Ji, Yan-Xiao; Zhang, Peng; Zhang, Xiao-Jing; Zhao, Yi-Chao; Deng, Ke-Qiong; Jiang, Xi; Wang, Pi-Xiao; Huang, Zan; Li, Hongliang

    2016-01-01

    Tumour necrosis factor receptor-associated factor 6 (TRAF6) is a ubiquitin E3 ligase that regulates important biological processes. However, the role of TRAF6 in cardiac hypertrophy remains unknown. Here, we show that TRAF6 levels are increased in human and murine hypertrophied hearts, which is regulated by reactive oxygen species (ROS) production. Cardiac-specific Traf6 overexpression exacerbates cardiac hypertrophy in response to pressure overload or angiotensin II (Ang II) challenge, whereas Traf6 deficiency causes an alleviated hypertrophic phenotype in mice. Mechanistically, we show that ROS, generated during hypertrophic progression, triggers TRAF6 auto-ubiquitination that facilitates recruitment of TAB2 and its binding to transforming growth factor beta-activated kinase 1 (TAK1), which, in turn, enables the direct TRAF6–TAK1 interaction and promotes TAK1 ubiquitination. The binding of TRAF6 to TAK1 and the induction of TAK1 ubiquitination and activation are indispensable for TRAF6-regulated cardiac remodelling. Taken together, we define TRAF6 as an essential molecular switch leading to cardiac hypertrophy in a TAK1-dependent manner. PMID:27249171

  1. Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

    PubMed Central

    Xu, Congfeng; Feng, Kuan; Zhao, Xiaonan; Huang, Shiqian; Cheng, Yiji; Qian, Liu; Wang, Yanan; Sun, Hongxing; Jin, Min; Chuang, Tsung-Hsien; Zhang, Yanyun

    2015-01-01

    Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases. PMID:25484083

  2. E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis.

    PubMed

    Liu, Ju; Zhang, C; Wang, X L; Ly, P; Belyi, V; Xu-Monette, Z Y; Young, K H; Hu, W; Feng, Z

    2014-11-01

    Tumor suppressor p53 has a key role in maintaining genomic stability and preventing tumorigenesis through its regulation of cellular stress responses, including apoptosis, cell cycle arrest and senescence. To ensure its proper levels and functions in cells, p53 is tightly regulated mainly through post-translational modifications, such as ubiquitination. Here, we identified E3 ubiquitin ligase TRIM32 as a novel p53 target gene and negative regulator to regulate p53-mediated stress responses. In response to stress, such as DNA damage, p53 binds to the p53 responsive element in the promoter of the TRIM32 gene and transcriptionally induces the expression of TRIM32 in cells. In turn, TRIM32 interacts with p53 and promotes p53 degradation through ubiquitination. Thus, TRIM32 negatively regulates p53-mediated apoptosis, cell cycle arrest and senescence in response to stress. TRIM32 is frequently overexpressed in different types of human tumors. TRIM32 overexpression promotes cell oncogenic transformation and tumorigenesis in mice in a largely p53-dependent manner. Taken together, our results demonstrated that as a novel p53 target and a novel negative regulator for p53, TRIM32 has an important role in regulation of p53 and p53-mediated cellular stress responses. Furthermore, our results also revealed that impairing p53 function is a novel mechanism for TRIM32 in tumorigenesis. PMID:25146927

  3. The E3 Ubiquitin Ligase COP1 Regulates Thermosensory Flowering by Triggering GI Degradation in Arabidopsis

    PubMed Central

    Jang, Kiyoung; Gil Lee, Hong; Jung, Su-Jin; Paek, Nam-Chon; Joon Seo, Pil

    2015-01-01

    Floral transition is influenced by environmental factors such as light and temperature. Plants are capable of integrating photoperiod and ambient temperature signaling into their developmental program. Despite extensive investigations on individual genetic pathways, little is known about the molecular components that integrate both pathways. Here, we demonstrate that the RING finger–containing E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) acts as an integrator of photoperiod and ambient temperature signaling. In addition to the role in photoperiodic destabilization of CONSTANS (CO), COP1 also regulates temperature sensitivity by controlling the degradation of GIGANTEA (GI). COP1-impaired mutants showed reduced sensitivity to low ambient temperature. Notably, COP1 is more stabilized at low temperature and accelerates GI turnover in a 26S proteasome-dependent manner. The direct association of GI with the promoter of FLOWERING LOCUS T (FT) was reduced because of its ambient temperature-dependent protein stability control, and thus COP1-triggered GI turnover delays flowering at low temperatures via a CO-independent pathway. Taken together, our findings indicate that environmental conditions regulate the stability of COP1, and conditional specificity of its target selection stimulates proper developmental responses and ensures reproductive success. PMID:26159740

  4. RBCK1, an E3 Ubiquitin Ligase, Interacts with and Ubiquinates the Human Pregnane X Receptor

    PubMed Central

    Rana, Ritu; Coulter, Sherry; Kinyamu, Harriet

    2013-01-01

    The pregnane X receptor (PXR, NR1I2) plays a pivotal role in the disposition and detoxification of numerous foreign and endogenous chemicals by increasing transcription of numerous target genes, including phase I and II drug-metabolizing enzymes and transporters. In the present study, yeast two-hybrid screening identified an E3 ubiquitin ligase, RBCK1 (Ring-B-box-coiled-coil protein interacting with protein kinase C-1), as a human pregnane X receptor (hPXR)–interacting protein. Coimmunoprecipitation studies confirmed the interaction between RBCK1 and hPXR when both were ectopically expressed in AD-293 cells. Domain mapping studies showed that the interaction between RBCK1 and hPXR involves all RBCK1 domains. We further demonstrate that RBCK1 ubiquitinates hPXR, and this may target hPXR for degradation by the ubiquitin-proteasome pathway. Simultaneous ectopic overexpression of RBCK1 and PXR decreased PXR levels in AD-293 cells, and this decrease was inhibited by the proteasomal inhibitor MG-132 (carbobenzoxy-Leu-Leu-leucinal). Furthermore, overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 cells. Of importance, ectopic overexpression and silencing of endogenous RBCK1 in primary human hepatocytes resulted in a decrease and increase, respectively, in endogenous PXR protein levels and in the induction of PXR target genes by rifampicin. These results suggest that RBCK1 is important for the ubiquitination of PXR and may play a role in its proteasomal degradation. PMID:23160820

  5. HAX1 regulates E3 ubiquitin ligase activity of cIAPs by promoting their dimerization.

    PubMed

    Choi, Jin Sun; Park, Byoung Chul; Chi, Seung Wook; Bae, Kwang-Hee; Kim, Sunhong; Cho, Sayeon; Son, Woo-Chan; Myung, Pyung Keun; Kim, Jeong-Hoon; Park, Sung Goo

    2014-10-30

    HS-1-associated protein X-1 (HAX1) is a multi-functional protein which was first identified as a Hematopoietic cell specific Lyn Substrate 1 (HS1)-binding protein. Although the roles of HAX1 in apoptosis have been unraveled and HAX1 has been proposed to be involved in several diseases, additional roles of HAX1 are still being identified. Here, we demonstrated that HAX1 directly interacted with cellular Inhibitor of Apoptosis Proteins (cIAPs), ubiquitin E3 ligases which regulate the abundance of cellular proteins, via ubiquitin-dependent proteasomal degradation. We showed that HAX1 promotes auto-ubiquitination and degradation of cIAPs by facilitating the intermolecular homodimerization of RING finger domain. Moreover, HAX1 regulates the non-canonical Nuclear Factor-κB (NF-κB) signaling pathway by modulating the stability of NF-κB-Inducing Kinase (NIK), which is one of the substrates of cIAPs. Taken together, these results unveil a novel role of HAX1 in the non-canonical NF-κB pathway, and provide an important clue that HAX1 is a potential therapeutic target for the treatment of cancer. PMID:25275296

  6. Arabidopsis nitrate reductase activity is stimulated by the E3 SUMO ligase AtSIZ1.

    PubMed

    Park, Bong Soo; Song, Jong Tae; Seo, Hak Soo

    2011-01-01

    Small ubiquitin-related modifier (SUMO) is a small polypeptide that modulates protein activity and regulates hormone signalling, abiotic and biotic responses in plants. Here we show that AtSIZ regulates nitrogen assimilation in Arabidopsis through its E3 SUMO ligase function. Dwarf plants of siz1-2 flower early, show abnormal seed development and have high salicylic acid content and enhanced resistance to bacterial pathogens. These mutant phenotypes are reverted to wild-type phenotypes by exogenous ammonium but not by nitrate, phosphate or potassium. Decreased nitrate reductase activity in siz1-2 plants resulted in low nitrogen concentrations, low nitric oxide production and high nitrate content in comparison with wild-type plants. The nitrate reductases, NIA1 and NIA2, are sumoylated by AtSIZ1, which dramatically increases their activity. Both sumoylated and non-sumoylated NIA1 and NIA2 can form dimers. Our results indicate that AtSIZ1 positively controls nitrogen assimilation by promoting sumoylation of NRs in Arabidopsis. PMID:21772271

  7. MDM2 E3 ligase-mediated ubiquitination and degradation of HDAC1 in vascular calcification.

    PubMed

    Kwon, Duk-Hwa; Eom, Gwang Hyeon; Ko, Jeong Hyeon; Shin, Sera; Joung, Hosouk; Choe, Nakwon; Nam, Yoon Seok; Min, Hyun-Ki; Kook, Taewon; Yoon, Somy; Kang, Wanseok; Kim, Yong Sook; Kim, Hyung Seok; Choi, Hyuck; Koh, Jeong-Tae; Kim, Nacksung; Ahn, Youngkeun; Cho, Hyun-Jai; Lee, In-Kyu; Park, Dong Ho; Suk, Kyoungho; Seo, Sang Beom; Wissing, Erin R; Mendrysa, Susan M; Nam, Kwang-Il; Kook, Hyun

    2016-01-01

    Vascular calcification (VC) is often associated with cardiovascular and metabolic diseases. However, the molecular mechanisms linking VC to these diseases have yet to be elucidated. Here we report that MDM2-induced ubiquitination of histone deacetylase 1 (HDAC1) mediates VC. Loss of HDAC1 activity via either chemical inhibitor or genetic ablation enhances VC. HDAC1 protein, but not mRNA, is reduced in cell and animal calcification models and in human calcified coronary artery. Under calcification-inducing conditions, proteasomal degradation of HDAC1 precedes VC and it is mediated by MDM2 E3 ubiquitin ligase that initiates HDAC1 K74 ubiquitination. Overexpression of MDM2 enhances VC, whereas loss of MDM2 blunts it. Decoy peptide spanning HDAC1 K74 and RG 7112, an MDM2 inhibitor, prevent VC in vivo and in vitro. These results uncover a previously unappreciated ubiquitination pathway and suggest MDM2-mediated HDAC1 ubiquitination as a new therapeutic target in VC. PMID:26832969

  8. HERC2 is an E3 ligase that targets BRCA1 for degradation.

    PubMed

    Wu, Wenwen; Sato, Ko; Koike, Ayaka; Nishikawa, Hiroyuki; Koizumi, Hirotaka; Venkitaraman, Ashok R; Ohta, Tomohiko

    2010-08-01

    The breast cancer suppressor BRCA1 forms a stable heterodimeric E3 ubiquitin ligase with BARD1. Each protein controls the abundance and stability of the other, and loss of the interaction leads to BRCA1 degradation. Here, we show that HERC2, a protein recently implicated in DNA damage repair, targets BARD1-uncoupled BRCA1 for degradation. HERC2 shuttles between the nucleus and the cytoplasm. Its COOH-terminal HECT-containing domain interacts with an NH(2)-terminal degron domain in BRCA1. HERC2 ubiquitinates BRCA1; this reaction depends on Cys(4762) of HERC2, the catalytic ubiquitin binding site, and the degron of BRCA1. The HERC2-BRCA1 interaction is maximal during the S phase of the cell cycle and rapidly diminishes as cells enter G(2)-M, inversely correlated with the steady-state level of BRCA1. Significantly, HERC2 depletion antagonizes the effects of BARD1 depletion by restoring BRCA1 expression and G(2)-M checkpoint activity. Conversely, BARD1 protects BRCA1 from HERC2-mediated ubiquitination. Collectively, our findings identify a function for HERC2 in regulating BRCA1 stability in opposition to BARD1. The HERC2 expression in breast epithelial cells and breast carcinomas suggests that this mechanism may play a role in breast carcinogenesis. PMID:20631078

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

    PubMed

    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

    2014-10-01

    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

  10. Ubiquitination and Degradation of the Hominoid-Specific Oncoprotein TBC1D3 Is Mediated by CUL7 E3 Ligase

    PubMed Central

    Kong, Chen; Samovski, Dmitri; Srikanth, Priya; Wainszelbaum, Marisa J.; Charron, Audra J.; Liu, Jialiu; Lange, Jeffrey J.; Chen, Pin-I; Pan, Zhen-Qiang; Su, Xiong; Stahl, Philip D.

    2012-01-01

    Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8. PMID:23029530

  11. IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase, Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis[C][W][OPEN

    PubMed Central

    Shin, Lung-Jiun; Lo, Jing-Chi; Chen, Guan-Hong; Callis, Judy; Fu, Hongyong; Yeh, Kuo-Chen

    2013-01-01

    Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, IRON-REGULATED TRANSPORTER1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 DEGRADATION FACTOR1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity. PMID:23995086

  12. Molecular characterization of Oryza sativa arsenic-induced RING E3 ligase 1 (OsAIR1): Expression patterns, localization, functional interaction, and heterogeneous overexpression.

    PubMed

    Hwang, Sun-Goo; Park, Hyeon Mi; Han, A-Reum; Jang, Cheol Seong

    2016-02-01

    High levels of arsenic (As) in plants are a serious threat to human health, and arsenic accumulation affects plant metabolism and ultimately photosynthesis, growth, and development. We attempted to isolate As-responsive Really Interesting New Gene (RING) E3 ubiquitin ligase genes from rice, and we have designated one such gene Oryza sativa arsenic-induced RING E3 ligase 1 (OsAIR1). OsAIR1 expression was induced under abiotic stress conditions, including drought, salt, heat, and As exposure. Results from an in vitro ubiquitination assay showed that OsAIR1 possesses E3 ligase activity. Within the cell, the expression of this gene was found to be localized to the vacuole. In a network-based analysis, we found significantly enriched gene ontology (GO) functions, which included ribonucleoprotein complexes such as ribosomes, suggesting that the function of OsAIR1 are related to translation. Differences in the proportion of seedlings with expanded cotyledons and root lengths, and the lack of differences in germination rates between OsAIR1-overexpressing lines and control plants under AsV stress, suggest that OsAIR1 may positively regulate post-germination plant growth under stress conditions. PMID:26788958

  13. PARC and CUL7 form atypical cullin RING ligase complexes.

    PubMed

    Skaar, Jeffrey R; Florens, Laurence; Tsutsumi, Takeya; Arai, Takehiro; Tron, Adriana; Swanson, Selene K; Washburn, Michael P; DeCaprio, James A

    2007-03-01

    CUL7 and the p53-associated, PARkin-like cytoplasmic protein (PARC) were previously reported to form homodimers and heterodimers, the first demonstration of cullin dimerization. Although a CUL7-based SKP1/CUL1/F-box (SCF)-like complex has been observed, little is known about the existence of a PARC-based SCF-like complex and how PARC interacts with CUL7-based complexes. To further characterize PARC-containing complexes, we examined the ability of PARC to form an SCF-like complex. PARC binds RBX1 and is covalently modified by NEDD8, defining PARC as a true cullin. However, PARC fails to bind SKP1 or F-box proteins, including the CUL7-associated FBXW8. To examine the assembly of PARC- and CUL7-containing complexes, tandem affinity purification followed by multidimensional protein identification technology were used. Multidimensional protein identification technology analysis revealed that the CUL7 interaction with FBXW8 was mutually exclusive of CUL7 binding to PARC or p53. Notably, although heterodimers of CUL7 and PARC bind p53, p53 is not required for the dimerization of CUL7 and PARC. The observed physical separation of FBXW8 and PARC is supported functionally by the generation of Parc-/-, Fbxw8-/- mice, which do not show exacerbation of the Fbxw8-/- phenotype. Finally, all of the PARC and CUL7 subcomplexes examined exhibit E3 ubiquitin ligase activity in vitro. Together, these findings indicate that the intricate assembly of PARC- and CUL7-containing complexes is highly regulated, and multiple subcomplexes may exhibit ubiquitin ligase activity. PMID:17332328

  14. RMND5 from Xenopus laevis Is an E3 Ubiquitin-Ligase and Functions in Early Embryonic Forebrain Development

    PubMed Central

    Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K.; Menssen, Ruth; Wolf, Dieter H.; Hollemann, Thomas

    2015-01-01

    In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development. PMID:25793641

  15. Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection

    PubMed Central

    Srivastava, Smita; Swanson, Selene K.; Manel, Nicolas; Florens, Laurence; Washburn, Michael P.; Skowronski, Jacek

    2008-01-01

    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. PMID:18464893

  16. E3-ubiquitin ligase Nedd4 determines the fate of AID-associated RNA polymerase II in B cells.

    PubMed

    Sun, Jianbo; Keim, Celia D; Wang, Jiguang; Kazadi, David; Oliver, Paula M; Rabadan, Raul; Basu, Uttiya

    2013-08-15

    Programmed mutagenesis of the immunoglobulin locus of B lymphocytes during class switch recombination (CSR) and somatic hypermutation requires RNA polymerase II (polII) transcription complex-dependent targeting of the DNA mutator activation-induced cytidine deaminase (AID). AID deaminates cytidine residues on substrate sequences in the immunoglobulin (Ig) locus via a transcription-dependent mechanism, and this activity is stimulated by the RNA polII stalling cofactor Spt5 and the 11-subunit cellular noncoding RNA 3'-5' exonucleolytic processing complex RNA exosome. The mechanism by which the RNA exosome recognizes immunoglobulin locus RNA substrates to stimulate AID DNA deamination activity on its in vivo substrate sequences is an important question. Here we report that E3-ubiquitin ligase Nedd4 destabilizes AID-associated RNA polII by a ubiquitination event, leading to generation of 3' end free RNA exosome RNA substrates at the Ig locus and other AID target sequences genome-wide. We found that lack of Nedd4 activity in B cells leads to accumulation of RNA exosome substrates at AID target genes and defective CSR. Taken together, our study links noncoding RNA processing following RNA polII pausing with regulation of the mutator AID protein. Our study also identifies Nedd4 as a regulator of noncoding RNAs that are generated by stalled RNA polII genome-wide. PMID:23964096

  17. Redox regulation of E3 ubiquitin ligases and their role in skeletal muscle atrophy.

    PubMed

    Olaso-Gonzalez, Gloria; Ferrando, Beatriz; Derbre, Frederic; Salvador-Pascual, Andrea; Cabo, Helena; Pareja-Galeano, Helios; Sabater-Pastor, Frederic; Gomez-Cabrera, Mari Carmen; Vina, Jose

    2014-10-01

    Muscle atrophy is linked to reactive oxygen species (ROS) production during hindlimb-unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of our study was to determine the mechanism by which ROS cause muscle atrophy and its possible prevention by allopurinol, a well-known inhibitor of XO widely used in clinical practice, and indomethacin, a nonsteroidal anti-inflammatory drug. We studied the activation of p38 MAP Kinase and NF-?B pathways, and the expression of two E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFb) and Muscle RING Finger-1 (MuRF-1). Male Wistar rats (3 mold) conditioned by 14 days of hindlimb unloading (n=18), with or without the treatment, were compared with freely ambulating controls (n=18). After the experimental intervention, soleus muscles were removed, weighted and analyzed to determine oxidative stress and inflammatory parameters. We found that hindlimb unloading induced a significant increase in XO activity in plasma (39%, p=0.001) and in the protein expression of CuZnSOD and Catalase in skeletal muscle. Inhibitionof XO partially prevented protein carbonylation, both in plasma and in soleus muscle, in the unloaded animals. The most relevant new fact reported is that allopurinol prevents soleus muscle atrophy by ~20% after hindlimb unloading. Combining allopurinol and indomethacin we found a further prevention in the atrophy process. This is mediated by the inhibition of the p38 MAPK-MAFbx and NF-?B -MuRF-1 pathways. Our data point out the potential benefit of allopurinol and indomethacin administration for bedridden, astronauts, sarcopenic and cachexic patients. PMID:26461377

  18. The E3 ubiquitin ligase CHIP mediates ubiquitination and proteasomal degradation of PRMT5.

    PubMed

    Zhang, Huan-Tian; Zeng, Ling-Fei; He, Qing-Yu; Tao, W Andy; Zha, Zhen-Gang; Hu, Chang-Deng

    2016-02-01

    Protein arginine methyltransferase 5 (PRMT5) is an important member of the protein arginine methyltransferase family that regulates many cellular processes through epigenetic control of target gene expression. Because of its overexpression in a number of human cancers and its essential role in cell proliferation, transformation, and cell cycle progression, PRMT5 has been recently proposed to function as an oncoprotein in cancer cells. However, how its expression is regulated in cancer cells remains largely unknown. We have previously demonstrated that the transcription of PRMT5 can be negatively regulated by the PKC/c-Fos signaling pathway through modulating the transcription factor NF-Y in prostate cancer cells. In the present study, we demonstrated that PRMT5 undergoes polyubiquitination, possibly through multiple lysine residues. We also identified carboxyl terminus of heat shock cognate 70-interacting protein (CHIP), an important chaperone-dependent E3 ubiquitin ligase that couples protein folding/refolding to protein degradation, as an interacting protein of PRMT5 via mass spectrometry. Their interaction was further verified by co-immuoprecipitation, GST pull-down, and bimolecular fluorescence complementation (BiFC) assay. In addition, we provided evidence that the CHIP/chaperone system is essential for the negative regulation of PRMT5 expression via K48-linked ubiquitin-dependent proteasomal degradation. Given that down-regulation of CHIP and overexpression of PRMT5 have been observed in several human cancers, our finding suggests that down-regulation of CHIP may be one of the mechanisms underlying PRMT5 overexpression in these cancers. PMID:26658161

  19. The Pellino E3 Ubiquitin Ligases Recognize Specific Phosphothreonine Motifs and Have Distinct Substrate Specificities

    PubMed Central

    2015-01-01

    The four mammalian Pellinos (Pellinos 1, 2, 3a, and 3b) are E3 ubiquitin ligases that are emerging as critical mediators for a variety of immune signaling pathways, including those activated by Toll-like receptors, the T-cell receptor, and NOD2. It is becoming increasingly clear that each Pellino has a distinct role in facilitating immune receptor signaling. However, the underlying mechanisms by which these highly homologous proteins act selectively in these signaling pathways are not clear. In this study, we investigate whether Pellino substrate recognition contributes to the divergent functions of Pellinos. Substrate recognition of each Pellino is mediated by its noncanonical forkhead-associated (FHA) domain, a well-characterized phosphothreonine-binding module. Pellino FHA domains share very high sequence identity, so a molecular basis for differences in substrate recognition is not immediately apparent. To explore Pellino substrate specificity, we first identify a high-affinity Pellino2 FHA domain-binding motif in the Pellino substrate, interleukin-1 receptor-associated kinase 1 (IRAK1). Analysis of binding of the different Pellinos to a panel of phosphothreonine-containing peptides derived from the IRAK1-binding motif reveals that each Pellino has a distinct phosphothreonine peptide binding preference. We observe a similar binding specificity in the interaction of Pellinos with a number of known Pellino substrates. These results argue that the nonredundant roles that Pellinos play in immune signaling are in part due to their divergent substrate specificities. This new insight into Pellino substrate recognition could be exploited for pharmacological advantage in treating inflammatory diseases that have been linked to the aberrant regulation of Pellinos. PMID:25027698

  20. p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch

    PubMed Central

    Melino, Sonia; Bellomaria, Alessia; Nepravishta, Ridvan; Paci, Maurizio; Melino, Gerry

    2014-01-01

    Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition. PMID:25485500

  1. p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch.

    PubMed

    Melino, Sonia; Bellomaria, Alessia; Nepravishta, Ridvan; Paci, Maurizio; Melino, Gerry

    2014-01-01

    Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition. PMID:25485500

  2. Transcription Factor hDREF Is a Novel SUMO E3 Ligase of Mi2α.

    PubMed

    Yamashita, Daisuke; Moriuchi, Takanobu; Osumi, Takashi; Hirose, Fumiko

    2016-05-27

    The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation. PMID:27068747

  3. TRIM28 Is an E3 Ligase for ARF-Mediated NPM1/B23 SUMOylation That Represses Centrosome Amplification

    PubMed Central

    Neo, Shu Hui; Itahana, Yoko; Alagu, Jennifer; Kitagawa, Mayumi; Guo, Alvin Kunyao; Lee, Sang Hyun; Tang, Kai

    2015-01-01

    The tumor suppressor ARF enhances the SUMOylation of target proteins; however, the physiological function of ARF-mediated SUMOylation has been unclear due to the lack of a known, associated E3 SUMO ligase. Here we uncover TRIM28/KAP1 as a novel ARF-binding protein and SUMO E3 ligase for NPM1/B23. ARF and TRIM28 cooperate to SUMOylate NPM1, a nucleolar protein that regulates centrosome duplication and genomic stability. ARF-mediated SUMOylation of NPM1 was attenuated by TRIM28 depletion and enhanced by TRIM28 overexpression. Coexpression of ARF and TRIM28 promoted NPM1 centrosomal localization by enhancing its SUMOylation and suppressed centrosome amplification; these functions required the E3 ligase activity of TRIM28. Conversely, depletion of ARF or TRIM28 increased centrosome amplification. ARF also counteracted oncogenic Ras-induced centrosome amplification. Centrosome amplification is often induced by oncogenic insults, leading to genomic instability. However, the mechanisms employed by tumor suppressors to protect the genome are poorly understood. Our findings suggest a novel role for ARF in maintaining genome integrity by facilitating TRIM28-mediated SUMOylation of NPM1, thus preventing centrosome amplification. PMID:26055329

  4. Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage.

    PubMed

    Voiniciuc, Catalin; Dean, Gillian H; Griffiths, Jonathan S; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L; Estelle, Mark; Haughn, George W

    2013-03-01

    Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

  5. FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification in Arabidopsis Seed Mucilage[W

    PubMed Central

    Voiniciuc, Cătălin; Dean, Gillian H.; Griffiths, Jonathan S.; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L.; Estelle, Mark; Haughn, George W.

    2013-01-01

    Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca2+ or completely rescued using alkaline Ca2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1–yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

  6. Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin

    PubMed Central

    Fiesel, Fabienne C.; Caulfield, Thomas R.; Moussaud-Lamodière, Elisabeth L.; Ogaki, Kotaro; Dourado, Daniel F.A.R.; Flores, Samuel C.; Ross, Owen A.; Springer, Wolfdieter

    2015-01-01

    Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility and protein aggregation. However pathogenic effects can also result from misregulation of Parkin auto-inhibition and of its enzymatic functions. In addition, interference of binding to co-enzymes, substrates and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin. PMID:25939424

  7. Lipopolysaccharide Primes the NALP3 Inflammasome by Inhibiting Its Ubiquitination and Degradation Mediated by the SCFFBXL2 E3 Ligase*

    PubMed Central

    Han, SeungHye; Lear, Travis B.; Jerome, Jacob A.; Rajbhandari, Shristi; Snavely, Courtney A.; Gulick, Dexter L.; Gibson, Kevin F.; Zou, Chunbin; Chen, Bill B.; Mallampalli, Rama K.

    2015-01-01

    The inflammasome is a multiprotein complex that augments the proinflammatory response by increasing the generation and cellular release of key cytokines. Specifically, the NALP3 inflammasome requires two-step signaling, priming and activation, to be functional to release the proinflammatory cytokines IL-1β and IL-18. The priming process, through unknown mechanisms, increases the protein levels of NALP3 and pro-IL-1β in cells. Here we show that LPS increases the NALP3 protein lifespan without significantly altering steady-state mRNA in human cells. LPS exposure reduces the ubiquitin-mediated proteasomal processing of NALP3 by inducing levels of an E3 ligase component, FBXO3, which targets FBXL2. The latter is an endogenous mediator of NALP3 degradation. FBXL2 recognizes Trp-73 within NALP3 for interaction and targets Lys-689 within NALP3 for ubiquitin ligation and degradation. A unique small molecule inhibitor of FBXO3 restores FBXL2 levels, resulting in decreased NALP3 protein levels in cells and, thereby, reducing the release of IL-1β and IL-18 in human inflammatory cells after NALP3 activation. Our findings uncover NALP3 as a molecular target for FBXL2 and suggest that therapeutic targeting of the inflammasome may serve as a platform for preclinical intervention. PMID:26037928

  8. Interaction Proteomics Identify NEURL4 and the HECT E3 Ligase HERC2 as Novel Modulators of Centrosome Architecture*

    PubMed Central

    Al-Hakim, Abdallah K.; Bashkurov, Mikhail; Gingras, Anne-Claude; Durocher, Daniel; Pelletier, Laurence

    2012-01-01

    Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture. PMID:22261722

  9. EM Structure of human APC/CCDH1-EMI1 reveals multimodal mechanism of E3 ligase shutdown

    PubMed Central

    Frye, Jeremiah J.; Brown, Nicholas G.; Petzold, Georg; Watson, Edmond R.; Grace, Christy R. R.; Nourse, Amanda; Jarvis, Marc A.; Kriwacki, Richard W.; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A.

    2013-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) is a ~1.5 MDa multiprotein E3 ligase enzyme that regulates cell division by promoting timely ubiquitin-mediated proteolysis of key cell cycle regulatory proteins. Inhibition of human APC/CCDH1 during interphase by Early Mitotic Inhibitor 1 (EMI1) is essential for accurate coordination of DNA synthesis and mitosis. Here, we report a hybrid structural approach involving NMR, electron microscopy, and enzymology, which reveal that EMI1’s 143-residue C-terminal domain inhibits multiple APC/CCDH1 functions. The intrinsically disordered D-box, Linker, and Tail elements, together with a structured zinc-binding domain, bind distinct regions of APC/CCDH1 to synergistically both block the substrate-binding site and inhibit ubiquitin chain elongation. The functional importance of intrinsic structural disorder is explained by enabling a small inhibitory domain to bind multiple sites to shut down multiple functions of a “molecular machine” nearly 100 times its size. PMID:23708605

  10. Red Light-Mediated Degradation of CONSTANS by the E3 Ubiquitin Ligase HOS1 Regulates Photoperiodic Flowering in Arabidopsis

    PubMed Central

    Lazaro, Ana; Mouriz, Alfonso; Piñeiro, Manuel; Jarillo, José A.

    2015-01-01

    The regulation of CONSTANS (CO) gene expression is crucial to accurately measure changes in daylength, which influences flowering time in Arabidopsis thaliana. CO expression is under both transcriptional and posttranslational control mechanisms. We previously showed that the E3 ubiquitin ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) physically interacts with CO in Arabidopsis. This interaction is required to precisely modulate the timing of CO accumulation and, consequently, to maintain low levels of FLOWERING LOCUS T expression during the first part of the day. The data presented here demonstrate that HOS1 is involved in the red light-mediated degradation of CO that takes place in the early stages of the daylight period. Our results show that phytochrome B (phyB) is able to regulate flowering time, acting in the phloem companion cells, as previously described for CO and HOS1. Moreover, we reveal that phyB physically interacts with HOS1 and CO, indicating that the three proteins may be present in a complex in planta that is required to coordinate a correct photoperiodic response in Arabidopsis. PMID:26373454

  11. Interaction proteomics identify NEURL4 and the HECT E3 ligase HERC2 as novel modulators of centrosome architecture.

    PubMed

    Al-Hakim, Abdallah K; Bashkurov, Mikhail; Gingras, Anne-Claude; Durocher, Daniel; Pelletier, Laurence

    2012-06-01

    Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as pericentriolar material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of pericentriolar material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of pericentriolar material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture. PMID:22261722

  12. Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins

    PubMed Central

    Takahashi, Hirotaka; Uematsu, Atsushi; Yamanaka, Satoshi; Imamura, Mei; Nakajima, Tatsuro; Doi, Kousuke; Yasuoka, Saki; Takahashi, Chikako; Takeda, Hiroyuki; Sawasaki, Tatsuya

    2016-01-01

    Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3). Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1) targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3—which there have been no report to bind p53—were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein. PMID:27249653

  13. A SUMO-targeted ubiquitin ligase is involved in the degradation of the nuclear pool of the SUMO E3 ligase Siz1

    PubMed Central

    Westerbeck, Jason W.; Pasupala, Nagesh; Guillotte, Mark; Szymanski, Eva; Matson, Brooke C.; Esteban, Cecilia; Kerscher, Oliver

    2014-01-01

    The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure–function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5∆ cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase. PMID:24196836

  14. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis.

    PubMed

    Zhang, M; Zhu, X; Zhang, Y; Cai, Y; Chen, J; Sivaprakasam, S; Gurav, A; Pi, W; Makala, L; Wu, J; Pace, B; Tuan-Lo, D; Ganapathy, V; Singh, N; Li, H

    2015-12-01

    The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis. PMID:25952549

  15. h-Goliath, paralog of GRAIL, is a new E3 ligase protein, expressed in human leukocytes.

    PubMed

    Guais, Adeline; Siegrist, Sylvie; Solhonne, Brigitte; Jouault, Hélène; Guellaën, Georges; Bulle, Frédérique

    2006-06-01

    In Drosophila, the RING finger protein d-Goliath was originally identified as a transcription factor involved in the embryo mesoderm formation [Bouchard, M.L., Cote, S., 1993. The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein. Gene 125, 205-209]. In mouse, the m-Goliath mRNA level was shown to be increased in growth factor withdrawal-induced apoptosis of myeloid cells [Baker, S.J., Reddy, E.P., 2000. Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1. Gene 248, 33-40]. Due to its putative function of transcription factor in apoptosis, we cloned the human cDNA for h-Goliath and characterized the expression of the protein in blood and bone marrow cells. The human protein of 419 aa (44 kDa) contains a protease-associated domain, a transmembrane domain and a RING-H2 motif. This structure classifies h-Goliath as a new member of a human family of ubiquitin ligases with GRAIL (gene related to anergy in lymphocytes) as founder. This E3 ligase controls the development of T cell clonal anergy by ubiquitination [Anandasabapathy, N., Ford, G.S., Bloom, D., Holness, C., Paragas, V., Seroogy, C., Skrenta, H., Hollenhorst, M., Fathman, C.G., Soares, L., 2003. GRAIL: an E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells. Immunity 18, 535-547]. In vitro ubiquitination studies support the E3 ubiquitin ligase activity of h-Goliath. In human, the protein is expressed under 3 isoforms, a major one at 28 kDa and two others at 46 and 55 kDa. These proteins come from a common precursor (44 kDa) as we observed using in vitro transcription-translation. Using immunohistochemistry on blood or bone marrow smears, of healthy or leukemia samples, we found that the protein expression was restricted to the cytoplasm of progenitors and fully differentiated leukocyte populations. We did not observe any modification of h-Goliath expression or localization in leukemia. In these cells

  16. Structure of the Siz/PIAS SUMO E3 Ligase Siz1 and Determinants Required for SUMO Modification of PCNA

    SciTech Connect

    Yunus, Ali A.; Lima, Christopher D.

    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.

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

    PubMed

    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. PMID:27050259

  18. Fbs2 is a new member of the E3 ubiquitin ligase family that recognizes sugar chains.

    PubMed

    Yoshida, Yukiko; Tokunaga, Fuminori; Chiba, Tomoki; Iwai, Kazuhiro; Tanaka, Keiji; Tai, Tadashi

    2003-10-31

    F-box proteins are substrate recognition components of Skp1-Cullin1-F-box protein-Roc1 (SCF) E3 ubiquitin-protein ligases. We reported previously that Fbs1 (F-box protein that recognizes sugar chains; equivalent to Fbx2 or NFB42) binds specifically to proteins attached with high mannose oligosaccharides and subsequently contributes to elimination of N-glycoproteins in cytosol (Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and Tai, T. (2002) Nature 418, 438-442). Here we report the identification of another F-box protein that recognizes N-glycan, Fbs2 (called Fbx6b or FBG2 previously). Although the expression of Fbs1 was restricted to the adult brain and testis, the Fbs2 transcript was widely expressed. The Fbs2 protein forms an SCFFbs2 ubiquitinligase complex that targets sugar chains in N-glycoproteins for ubiquitylation. Only glycoproteins bound to concanavalin A lectin and not to wheat germ agglutinin or Ricinus communis agglutinin interacted with Fbs2 in various tissues and cell lines. Pull-down analysis using various oligosaccharides revealed that Man3-9GlcNAc2 glycans were required for efficient Fbs2 binding, whereas modifications of mannose residues by other sugars or deletion of inner GlcNAc reduced Fbs2 binding. Fbs2 interacted with N-glycans of T-cell receptor alpha-subunit (TCRalpha), a typical substrate of the endoplasmic reticulum-associated degradation (ERAD) pathway, and the forced expression of mutant Fbs2DeltaF, which lacks the F-box domain essential for forming the SCF complex, and decrease of endogenous Fbs2 by small interfering RNA led to inhibition of TCRalpha degradation in cells. Thus, Fbs2 is a novel member of F-box protein family that recognizes N-glycans and plays a role in ERAD. PMID:12939278

  19. Inactivation of SAG E3 Ubiquitin Ligase Blocks Embryonic Stem Cell Differentiation and Sensitizes Leukemia Cells to Retinoid Acid

    PubMed Central

    Yang, Ruiguo; Xi, Ning; Sun, Yi

    2011-01-01

    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. PMID:22110742

  20. CHIP: a quality-control E3 ligase collaborating with molecular chaperones.

    PubMed

    Murata, Shigeo; Chiba, Tomoki; Tanaka, Keiji

    2003-05-01

    It is notable that both the chaperone and ubiquitin-proteasome systems are required for removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. Carboxyl-terminus of Hsc70 interacting protein (CHIP), originally identified as a co-chaperone of Hsc70, has both a tetratricopeptide repeat (TPR) motif and a U-box domain. The TPR motif associates with Hsc70 and Hsp90, while the U-box domain executes a ubiquitin ligase activity. Thus, CHIP is an ideal molecule acting as a protein quality-control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. Accumulating evidence from in vitro studies indicates that this is apparently the case. Here, we present and discuss several unresolved but critical issues related to the molecular mechanism and in vivo roles of CHIP. PMID:12672450

  1. Determinants of Small Ubiquitin-like Modifier 1 (SUMO1) Protein Specificity, E3 Ligase, and SUMO-RanGAP1 Binding Activities of Nucleoporin RanBP2

    SciTech Connect

    Gareau, Jaclyn R.; Reverter, David; Lima, Christopher D.

    2012-02-16

    The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.

  2. Disinhibition of the HECT E3 ubiquitin ligase WWP2 by polymerized Dishevelled

    PubMed Central

    Mund, Thomas; Graeb, Michael; Mieszczanek, Juliusz; Gammons, Melissa; Pelham, Hugh R. B.; Bienz, Mariann

    2015-01-01

    Dishevelled is a pivot in Wnt signal transduction, controlling both β-catenin-dependent transcription to specify proliferative cell fates, and cell polarity and other non-nuclear events in post-mitotic cells. In response to Wnt signals, or when present at high levels, Dishevelled forms signalosomes by dynamic polymerization. Its levels are controlled by ubiquitylation, mediated by various ubiquitin ligases, including NEDD4 family members that bind to a conserved PPxY motif in Dishevelled (mammalian Dvl1–3). Here, we show that Dvl2 binds to the ubiquitin ligase WWP2 and unlocks its ligase activity from autoinhibition. This disinhibition of WWP2 depends on several features of Dvl2 including its PPxY motif and to a lesser extent its DEP domain, but crucially on the ability of Dvl2 to polymerize, indicating that WWP2 is activated in Wnt signalosomes. We show that Notch intracellular domains are substrates for Dvl-activated WWP2 and their transcriptional activity is consequently reduced, providing a molecular mechanism for cross-talk between Wnt and Notch signalling. These regulatory interactions are conserved in Drosophila whose WWP2 orthologue, Suppressor-of-deltex, downregulates Notch signalling upon activation by Dishevelled in developing wing tissue. Attentuation of Notch signalling by Dishevelled signalosomes could be important during the transition of cells from the proliferative to the post-mitotic state. PMID:26701932

  3. Deficiency of the E3 ubiquitin ligase TRIM32 in mice leads to a myopathy with a neurogenic component.

    PubMed

    Kudryashova, Elena; Wu, Jun; Havton, Leif A; Spencer, Melissa J

    2009-04-01

    Limb-girdle muscular dystrophy type 2H (LGMD2H) and sarcotubular myopathy are hereditary skeletal muscle disorders caused by mutations in TRIM32. We previously identified TRIM32 as an E3 ubiquitin ligase that binds to myosin and ubiquitinates actin. To date four TRIM32 mutations have been linked to LGMD2H, all of which occur in the C-terminal NHL domains. Unexpectedly, a fifth mutation in the B-box of TRIM32 causes a completely different, multisystemic disorder, Bardet-Biedl syndrome type 11. It is not understood how allelic mutations in TRIM32 can create such diverse phenotypic outcomes. To generate a tool for elucidating the complex in vivo functions of TRIM32, we created the first murine Trim32 knock-out model (T32KO). Histological analysis of T32KO skeletal muscles revealed mild myopathic changes. Electron microscopy showed areas with Z-line streaming and a dilated sarcotubular system with vacuoles -- the latter being a prominent feature of sarcotubular myopathy. Therefore, our model replicates phenotypes of LGMD2H and sarcotubular myopathy. The level of Trim32 expression in normal mouse brain exceeds that observed in skeletal muscle by more than 100 times, as we demonstrated by real-time PCR. Intriguingly, analysis of T32KO neural tissue revealed a decreased concentration of neurofilaments and a reduction in myelinated motoraxon diameters. The axonal changes suggest a shift toward a slower motor unit type. Not surprisingly, T32KO soleus muscle expressed an elevated type I slow myosin isotype with a concomitant reduction in the type II fast myosin. These data suggest that muscular dystrophy due to TRIM32 mutations involves both neurogenic and myogenic characteristics. PMID:19155210

  4. A MARCH6 and IDOL E3 Ubiquitin Ligase Circuit Uncouples Cholesterol Synthesis from Lipoprotein Uptake in Hepatocytes

    PubMed Central

    Loregger, Anke; Cook, Emma Claire Laura; Nelson, Jessica Kristin; Moeton, Martina; Sharpe, Laura Jane; Engberg, Susanna; Karimova, Madina; Lambert, Gilles; Brown, Andrew John

    2015-01-01

    Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake. PMID:26527619

  5. Impaired Neurite Contact Guidance in Ubiquitin Ligase E3a (Ube3a)-Deficient Hippocampal Neurons on Nanostructured Substrates.

    PubMed

    Tonazzini, I; Meucci, S; Van Woerden, G M; Elgersma, Y; Cecchini, M

    2016-04-01

    Recent discoveries indicate that during neuronal development the signaling processes that regulate extracellular sensing (e.g., adhesion, cytoskeletal dynamics) are important targets for ubiquitination-dependent regulation, in particular through E3 ubiquitin ligases. Among these, Ubiquitin E3a ligase (UBE3A) has a key role in brain functioning, but its function and how its deficiency results in the neurodevelopmental disorder Angelman syndrome is still unclear. Here, the role of UBE3A is investigated in neurite contact guidance during neuronal development, in vitro. The microtopography sensing of wild-type and Ube3a-deficient hippocampal neurons is studied by exploiting gratings with different topographical characteristics, with the aim to compare their capabilities to read and follow physical directional stimuli. It is shown that neuronal contact guidance is defective in Ube3a-deficient neurons, and this behavior is linked to an impaired activation of the focal adhesion signaling pathway. Taken together, the results suggest that the neuronal contact sensing machinery might be affected in Angelman syndrome. PMID:26845073

  6. E3 ubiquitin ligase RFWD2 controls lung branching through protein-level regulation of ETV transcription factors.

    PubMed

    Zhang, Yan; Yokoyama, Shigetoshi; Herriges, John C; Zhang, Zhen; Young, Randee E; Verheyden, Jamie M; Sun, Xin

    2016-07-01

    The mammalian lung is an elaborate branching organ, and it forms following a highly stereotypical morphogenesis program. It is well established that precise control at the transcript level is a key genetic underpinning of lung branching. In comparison, little is known about how regulation at the protein level may play a role. Ring finger and WD domain 2 (RFWD2, also termed COP1) is an E3 ubiquitin ligase that modifies specific target proteins, priming their degradation via the ubiquitin proteasome system. RFWD2 is known to function in the adult in pathogenic processes such as tumorigenesis. Here, we show that prenatal inactivation of Rfwd2 gene in the lung epithelium led to a striking halt in branching morphogenesis shortly after secondary branch formation. This defect is accompanied by distalization of the lung epithelium while growth and cellular differentiation still occurred. In the mutant lung, two E26 transformation-specific (ETS) transcription factors essential for normal lung branching, ETS translocation variant 4 (ETV4) and ETV5, were up-regulated at the protein level, but not at the transcript level. Introduction of Etv loss-of-function alleles into the Rfwd2 mutant background attenuated the branching phenotype, suggesting that RFWD2 functions, at least in part, through degrading ETV proteins. Because a number of E3 ligases are known to target factors important for lung development, our findings provide a preview of protein-level regulatory network essential for lung branching morphogenesis. PMID:27335464

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

    PubMed

    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 regulator of ABA signalling, and stimulate its ubiquitination and degradation. Genetic analysis shows that MYB96 is epistatic to MIEL1 in the control of ABA sensitivity in seeds. While MIEL1 acts primarily via MYB96 in seed germination, MIEL1 regulates protein turnover of both MYB96 and MYB30 in vegetative tissues. We find that ABA regulates the expression of MYB30-responsive genes during pathogen infection and this regulation is partly dependent on MIEL1. These results suggest that MIEL1 may facilitate crosstalk between ABA and biotic stress signalling. PMID:27615387

  8. COP1 Controls Abiotic Stress Responses by Modulating AtSIZ1 Function through Its E3 Ubiquitin Ligase Activity

    PubMed Central

    Kim, Joo Y.; Jang, In-Cheol; Seo, Hak S.

    2016-01-01

    Ubiquitination and sumoylation are essential post-translational modifications that regulate growth and development processes in plants, including control of hormone signaling mechanisms and responses to stress. This study showed that COP1 (Constitutive photomorphogenic 1) regulated the activity of Arabidopsis E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 through its E3 ubiquitin ligase activity. Yeast two hybrid analysis demonstrated that COP1 and AtSIZ1 directly interacted with one another, and subcellular localization assays indicated that COP1 and AtSIZ1 co-localized in nuclear bodies. Analysis of ubiquitination showed that AtSIZ1 was polyubiquitinated by COP1. The AtSIZ1 level was higher in cop1-4 mutants than in wild-type seedlings under light or dark conditions, and overexpression of a dominant-negative (DN)-COP1 mutant led to a substantial increase in AtSIZ1 accumulation. In addition, under drought, cold, and high salt conditions, SUMO-conjugate levels were elevated in DN-COP1-overexpressing plants and cop1-4 mutant plants compared to wild-type plants. Taken together, our results indicate that COP1 controls responses to abiotic stress by modulation of AtSIZ1 levels and activity. PMID:27536318

  9. Arabidopsis COP1 SUPPRESSOR 2 Represses COP1 E3 Ubiquitin Ligase Activity through Their Coiled-Coil Domains Association

    PubMed Central

    Jiang, Yan; Ling, Junjie; Hettiarachchi, Chamari; Tellgren-Roth, Christian; Wei, Ning; Deng, Xing Wang

    2015-01-01

    CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) functions as an E3 ubiquitin ligase and mediates a variety of developmental processes in Arabidopsis by targeting a number of key regulators for ubiquitination and degradation. Here, we identify a novel COP1 interacting protein, COP1 SUPPRESSOR 2 (CSU2). Loss of function mutations in CSU2 suppress the constitutive photomorphogenic phenotype of cop1-6 in darkness. CSU2 directly interacts with COP1 via their coiled-coil domains and is recruited by COP1 into nuclear speckles in living plant cells. Furthermore, CSU2 inhibits COP1 E3 ubiquitin ligase activity in vitro, and represses COP1 mediated turnover of HY5 in cell-free extracts. We propose that in csu2 cop1-6 mutants, the lack of CSU2’s repression of COP1 allows the low level of COP1 to exhibit higher activity that is sufficient to prevent accumulation of HY5 in the dark, thus restoring the etiolated phenotype. In addition, CSU2 is required for primary root development under normal light growth condition. PMID:26714275

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

    PubMed Central

    Kamanova, Jana; Sun, Hui; Lara-Tejero, Maria; Galán, Jorge E.

    2016-01-01

    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. PMID:27058235

  11. COP1 Controls Abiotic Stress Responses by Modulating AtSIZ1 Function through Its E3 Ubiquitin Ligase Activity.

    PubMed

    Kim, Joo Y; Jang, In-Cheol; Seo, Hak S

    2016-01-01

    Ubiquitination and sumoylation are essential post-translational modifications that regulate growth and development processes in plants, including control of hormone signaling mechanisms and responses to stress. This study showed that COP1 (Constitutive photomorphogenic 1) regulated the activity of Arabidopsis E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 through its E3 ubiquitin ligase activity. Yeast two hybrid analysis demonstrated that COP1 and AtSIZ1 directly interacted with one another, and subcellular localization assays indicated that COP1 and AtSIZ1 co-localized in nuclear bodies. Analysis of ubiquitination showed that AtSIZ1 was polyubiquitinated by COP1. The AtSIZ1 level was higher in cop1-4 mutants than in wild-type seedlings under light or dark conditions, and overexpression of a dominant-negative (DN)-COP1 mutant led to a substantial increase in AtSIZ1 accumulation. In addition, under drought, cold, and high salt conditions, SUMO-conjugate levels were elevated in DN-COP1-overexpressing plants and cop1-4 mutant plants compared to wild-type plants. Taken together, our results indicate that COP1 controls responses to abiotic stress by modulation of AtSIZ1 levels and activity. PMID:27536318

  12. Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP).

    PubMed

    Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D; Blackburn, Elizabeth A; Ball, Kathryn L

    2015-11-01

    CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

  13. Stability of HIB-Cul3 E3 ligase adaptor HIB Is Regulated by Self-degradation and Availability of Its Substrates

    PubMed Central

    Zhou, Zizhang; Xu, Congyu; Chen, Ping; Liu, Chen; Pang, Shu; Yao, Xia; Zhang, Qing

    2015-01-01

    The HIB-Cul3 complex E3 ligase regulates physiological homeostasis through regulating its substrate stability and its activity can be modulated by changing HIB abundance. However, regulation of HIB remains elusive. Here we provide evidence that HIB is degraded through the proteasome by Cul3-mediated polyubiquitination in K48 manner in Drosophila. Strikingly, HIB is targeted for degradation by itself. We further identify that three degrons (52LKSS56T, 76LDEE80S and 117MESQ121R) and K185 and K198 of HIB are essential for its auto-degradation. Finally, we demonstrate that HIB-Cul3 substrates, Ci and Puc, can effectively protect HIB from HIB-Cul3-mediated degradation. Taken together, our study indicates that there is an exquisite equilibrium between the adaptor and targets to achieve the tight control of the HIB, which is essential for maintaining suitable Hh and JNK signaling. And the mechanism of adaptor self-degradation and reciprocal control of the abundance between adaptor and its substrates is also applied to BTB-Cul3 E3 ligase adaptor dKeap1, dDiablo and dKLHL18. PMID:26263855

  14. Interaction of APC/C-E3 Ligase with Swi6/HP1 and Clr4/Suv39 in Heterochromatin Assembly in Fission Yeast*S⃞♦

    PubMed Central

    Dubey, Rudra Narayan; Nakwal, Nandni; Bisht, Kamlesh Kumar; Saini, Ashok; Haldar, Swati; Singh, Jagmohan

    2009-01-01

    Heterochromatin assembly in fission yeast is initiated by binding of Swi6/HP1 to the Lys-9-dimethylated H3 followed by spreading via cooperative recruitment of Swi6/HP1. Recruitment of Cohesin by Swi6/HP1 further stabilizes the heterochromatin structure and integrity. Subsequently, polyubiquitylation of Cut2 by anaphase-promoting complex-cyclosome (APC/C)-ubiquitin-protein isopeptide ligase (E3 ligase) followed by degradation of Cut2 releases Cut1, which cleaves the Rad21 subunit of Cohesin, facilitating sister chromatid separation during mitosis. Here, we demonstrate a surprising role of APC/C in assembly of heterochromatin and silencing at mating type, centromere, and ribosomal DNA loci. Coincidentally with the loss of silencing, recruitment of Swi6, H3-Lys-9-Me2, and Clr4 at dg-dh repeats at cen1 and the K region of mat locus is abrogated in mutants cut4, cut9, and nuc2. Surprisingly, both Cut4 and Cut9 are also highly enriched at these regions in wild type and depleted in swi6Δ mutant. Cut4 and Cut9 interact directly with Swi6/HP1 and Clr4, whereas the mutant Cut4 does not, suggesting that a direct physical interaction of APC subunits Cut4 and Cut9 with Swi6 and Clr4 is instrumental in heterochromatin assembly. The silencing defect in APC mutants is causally related to ubiquitylation activity of APC-E3 ligase. Like swi6 mutant, APC mutants are also defective in Cohesin recruitment and exhibit defects like lagging chromosomes, chromosome loss, and aberrant recombination in the mat region. In addition, APC mutants exhibit a bidirectional expression of dh repeats, suggesting a role in the RNA interference pathway. Thus, APC and heterochromatin proteins Swi6 and Clr4 play a mutually cooperative role in heterochromatin assembly, thereby ensuring chromosomal integrity, inheritance, and segregation during mitosis and meiosis. PMID:19117951

  15. HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction.

    PubMed

    Tran, Hoanh; Bustos, Daisy; Yeh, Ronald; Rubinfeld, Bonnee; Lam, Cynthia; Shriver, Stephanie; Zilberleyb, Inna; Lee, Michelle W; Phu, Lilian; Sarkar, Anjali A; Zohn, Irene E; Wertz, Ingrid E; Kirkpatrick, Donald S; Polakis, Paul

    2013-02-01

    The adenomatous polyposis coli (APC) protein functions as a negative regulator of the Wnt signaling pathway. In this capacity, APC forms a "destruction complex" with Axin, CK1α, and GSK3β to foster phosphorylation of the Wnt effector β-catenin earmarking it for Lys-48-linked polyubiquitylation and proteasomal degradation. APC is conjugated with Lys-63-linked ubiquitin chains when it is bound to Axin, but it is unclear whether this modification promotes the APC-Axin interaction or confers upon APC an alternative function in the destruction complex. Here we identify HectD1 as a candidate E3 ubiquitin ligase that modifies APC with Lys-63 polyubiquitin. Knockdown of HectD1 diminished APC ubiquitylation, disrupted the APC-Axin interaction, and augmented Wnt3a-induced β-catenin stabilization and signaling. These results indicate that HectD1 promotes the APC-Axin interaction to negatively regulate Wnt signaling. PMID:23277359

  16. Recognition mechanism of p63 by the E3 ligase Itch: novel strategy in the study and inhibition of this interaction.

    PubMed

    Bellomaria, Alessia; Barbato, Gaetano; Melino, Gerry; Paci, Maurizio; Melino, Sonia

    2012-10-01

    The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer's or Huntington's diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly (13)C-(15)N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534-551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase. PMID:22935697

  17. The Arabidopsis F-box E3 ligase RIFP1 plays a negative role in abscisic acid signalling by facilitating ABA receptor RCAR3 degradation.

    PubMed

    Li, Ying; Zhang, Liang; Li, Dekuan; Liu, Zhibin; Wang, Jianmei; Li, Xufeng; Yang, Yi

    2016-03-01

    The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability. PMID:26386272

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

    PubMed

    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. PMID:26232501

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

    PubMed Central

    Chesarino, Nicholas M.; McMichael, Temet M.; Yount, Jacob S.

    2015-01-01

    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. PMID:26263374

  20. The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

    PubMed Central

    Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

    2015-01-01

    The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

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

    PubMed Central

    Zhou, Weihua; Wei, Wenyi; Sun, Yi

    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. PMID:23528706

  2. The E3 ubiquitin ligase Itch controls the protein stability of p63

    PubMed Central

    Rossi, Mario; Aqeilan, Rami I.; Neale, Michael; Candi, Eleonora; Salomoni, Paolo; Knight, Richard A.; Croce, Carlo M.; Melino, Gerry

    2006-01-01

    p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile α motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steady-state level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions. PMID:16908849

  3. Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP.

    PubMed

    Soss, Sarah E; Rose, Kristie L; Hill, Salisha; Jouan, Sophie; Chazin, Walter J

    2015-01-01

    The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate. PMID:26010904

  4. RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties.

    PubMed

    Horn, Elizabeth J; Albor, Amador; Liu, Yuangang; El-Hizawi, Sally; Vanderbeek, Gretchen E; Babcock, Melissa; Bowden, G Tim; Hennings, Henry; Lozano, Guillermina; Weinberg, Wendy C; Kulesz-Martin, Molly

    2004-02-01

    Tripartite motif protein 32, Trim32, mRNA and protein expression was elevated in independently transformed and tumorigenic keratinocytes of a mouse epidermal carcinogenesis model, in ultraviolet B (UVB)-induced squamous cell carcinomas (SCC), and in approximately 20-25% of chemically induced mouse papillomas and human head and neck SCCs. This suggests that elevated Trim32 expression occurs frequently in experimental epidermal carcinogenesis and is relevant to human cancer. Transduced Trim32 increased colony number in an epidermal in vitro transformation assay and epidermal thickening in vivo when skin-grafted to athymic nu/nu mice. These effects were not associated with proliferation and were not sufficient for tumorigenesis, even with 12-O-tetradecanoylphorbol-13-acetate treatment or defects in the tumor suppressor p53. However, transduced Trim32 inhibited the synergistic effect of tumor necrosis factor alpha (TNFalpha) on UVB-induced apoptosis of keratinocytes in vitro and the apoptotic response of keratinocyte grafts exposed to UVB-light in vivo. Consistent with its RING domain, Trim32 exhibited characteristics of E3-ubiquitin ligases, including being ubiquitylated itself and interacting with ubiquitylated proteins, with increases in these properties following treatment of cultured keratinocytes with TNFalpha/UVB. Interestingly, missense point mutation of human TRIM32 has been reported in Limb-Girdle Muscular Dystrophy Type 2H, an autosomal recessive disease. We propose a model in which Trim32 activities as an E3-ubiquitin ligase favor initiated cell survival in carcinogenesis by blocking UVB-induced TNFalpha apoptotic signaling. PMID:14578165

  5. A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis.

    PubMed

    Kabelitz, Tina; Brzezinka, Krzysztof; Friedrich, Thomas; Górka, Michał; Graf, Alexander; Kappel, Christian; Bäurle, Isabel

    2016-05-01

    Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity. PMID:26979329

  6. RNF4, a SUMO-targeted ubiquitin E3 ligase, promotes DNA double-strand break repair.

    PubMed

    Galanty, Yaron; Belotserkovskaya, Rimma; Coates, Julia; Jackson, Stephen P

    2012-06-01

    Protein ubiquitylation and sumoylation play key roles in regulating cellular responses to DNA double-strand breaks (DSBs). Here, we show that human RNF4, a small ubiquitin-like modifier (SUMO)-targeted ubiquitin E3 ligase, is recruited to DSBs in a manner requiring its SUMO interaction motifs, the SUMO E3 ligases PIAS1 and PIAS4, and various DSB-responsive proteins. Furthermore, we reveal that RNF4 depletion impairs ubiquitin adduct formation at DSB sites and causes persistent histone H2AX phosphorylation (γH2AX) associated with defective DSB repair, hypersensitivity toward DSB-inducing agents, and delayed recovery from radiation-induced cell cycle arrest. We establish that RNF4 regulates turnover of the DSB-responsive factors MDC1 and replication protein A (RPA) at DNA damage sites and that RNF4-depleted cells fail to effectively replace RPA by the homologous recombination factors BRCA2 and RAD51 on resected DNA. Consistent with previous data showing that RNF4 targets proteins to the proteasome, we show that the proteasome component PSMD4 is recruited to DNA damage sites in a manner requiring its ubiquitin-interacting domains, RNF4 and RNF8. Finally, we establish that PSMD4 binds MDC1 and RPA1 in a DNA damage-induced, RNF4-dependent manner and that PSMD4 depletion cause MDC1 and γH2AX persistence in irradiated cells. RNF4 thus operates as a DSB response factor at the crossroads between the SUMO and ubiquitin systems. PMID:22661229

  7. Structural model of ubiquitin transfer onto an artificial RING finger as an E3 ligase

    NASA Astrophysics Data System (ADS)

    Miyamoto, Kazuhide

    2014-10-01

    The artificial WSTF PHD_EL5 RING finger was designed via ``α-helical region substitution'', and its structural model for the attachment of activated ubiquitin has been demonstrated. Chemical modifications of Cys residues, the circular dichroism spectra, and substrate-independent ubiquitination assays illustrated that the WSTF PHD_EL5 RING finger has E3 activity, and it is ubiquitinated via Lys14. Homology modeling calculations revealed that the WSTF PHD_EL5 RING finger possesses a classical RING fold for specific E2-E3 binding. The docking poses of the WSTF PHD_EL5 RING finger with the UbcH5b-ubiquitin conjugate provided insight into its functional E2 interaction and development of ubiquitination at the atomic level. The structural model of the artificial WSTF PHD_EL5 RING finger proposed by the present work is useful and may help to extend the strategy of α-helical region substitution.

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

    PubMed Central

    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

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

    PubMed

    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

  10. Skp1, a component of E3 ubiquitin ligase, is necessary for growth, sporulation, development and pathogenicity in rice blast fungus (Magnaporthe oryzae).

    PubMed

    Prakash, Chandra; Manjrekar, Johannes; Chattoo, Bharat B

    2016-08-01

    Ubiqitination is an important process in eukaryotic cells involving E3 ubiquitin ligase, which co-ordinates with cell cycle proteins and controls various cell functions. Skp1 (S-phase kinase-associated protein 1) is a core component of the SCF (Skp1-Cullin 1-F-box) E3 ubiquitin ligase complex necessary for protein degradation by the 26S proteasomal pathway. The rice blast fungus Magnaporthe oryzae has a single MoSKP1(MGG_04978) required for viability. Skp1 has multiple functions; however, its roles in growth, sporulation and appressorial development are not understood. MoSKP1 complements Skp1 function in the fission yeast temperature-sensitive mutant skp1 A7, restoring the normal length of yeast cells at restrictive temperature. The MoSkp1 protein in M. oryzae is present in spores and germ tubes, and is abundantly expressed in appressoria. Various RNA interference (RNAi) and antisense transformants of MoSKP1 in B157 show reduced sporulation, defective spore morphology, lesser septation and diffuse nuclei. Further, they show elongated germ tubes and are unable to form appressoria. Transformants arrested in G1/S stage during initial spore germination show a similar phenotype to wild-type spores treated with hydroxyurea (HU). Reduced MoSkp1 transcript and protein levels in knockdown transformants result in atypical germ tube development. MoSkp1 interacts with the putative F-box protein (MGG_06351) revealing the ability to form protein complexes. Our investigation of the role of MoSKP1 suggests that a decrease in MoSkp1 manifests in decreased total protein ubiquitination and, consequently, defective cell cycle and appressorial development. Thus, MoSKP1 plays important roles in growth, sporulation, appressorial development and pathogenicity of M. oryzae. PMID:26575697

  11. The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

    PubMed

    Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

    2007-07-01

    Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy. PMID:17592138

  12. Lack of Cul4b, an E3 Ubiquitin Ligase Component, Leads to Embryonic Lethality and Abnormal Placental Development

    PubMed Central

    Yuan, Jupeng; Qian, Yanyan; Sun, Wenjie; Zou, Yongxin; Guo, Chenhong; Chen, Bingxi; Shao, Changshun; Gong, Yaoqin

    2012-01-01

    Cullin-RING ligases (CRLs) complexes participate in the regulation of diverse cellular processes, including cell cycle progression, transcription, signal transduction and development. Serving as the scaffold protein, cullins are crucial for the assembly of ligase complexes, which recognize and target various substrates for proteosomal degradation. Mutations in human CUL4B, one of the eight members in cullin family, are one of the major causes of X-linked mental retardation. We here report the generation and characterization of Cul4b knockout mice, in which exons 3 to 5 were deleted. In contrast to the survival to adulthood of human hemizygous males with CUL4B null mutation, Cul4b null mouse embryos show severe developmental arrest and usually die before embryonic day 9.5 (E9.5). Accumulation of cyclin E, a CRL (CUL4B) substrate, was observed in Cul4b null embryos. Cul4b heterozygotes were recovered at a reduced ratio and exhibited a severe developmental delay. The placentas in Cul4b heterozygotes were disorganized and were impaired in vascularization, which may contribute to the developmental delay. As in human CUL4B heterozygotes, Cul4b null cells were selected against in Cul4b heterozygotes, leading to various degrees of skewed X-inactivation in different tissues. Together, our results showed that CUL4B is indispensable for embryonic development in the mouse. PMID:22606329

  13. The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass.

    PubMed

    Rom, Oren; Reznick, Abraham Z

    2016-09-01

    The ubiquitin-proteasome system (UPS) is the main regulatory mechanism of protein degradation in skeletal muscle. The ubiquitin-ligase enzymes (E3s) have a central role in determining the selectivity and specificity of the UPS. Since their identification in 2001, the muscle specific E3s, muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx), have been shown to be implicated in the regulation of skeletal muscle atrophy in various pathological and physiological conditions. This review aims to explore the involvement of MuRF-1 and MAFbx in catabolism of skeletal muscle during various pathologies, such as cancer cachexia, sarcopenia of aging, chronic kidney disease (CKD), diabetes, and chronic obstructive pulmonary disease (COPD). In addition, the effects of various lifestyle and modifiable factors (e.g. nutrition, exercise, cigarette smoking, and alcohol) on MuRF-1 and MAFbx regulation will be discussed. Finally, evidence of potential strategies to protect against skeletal muscle wasting through inhibition of MuRF-1 and MAFbx expression will be explored. PMID:26738803

  14. The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth.

    PubMed

    Cao, Juxiang; Dai, Xiangpeng; Wan, Lixin; Wang, Hongshen; Zhang, Jinfang; Goff, Philip S; Sviderskaya, Elena V; Xuan, Zhenyu; Xu, Zhixiang; Xu, Xiaowei; Hinds, Philip; Flaherty, Keith T; Faller, Douglas V; Goding, Colin R; Wang, Yongjun; Wei, Wenyi; Cui, Rutao

    2015-09-01

    The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma. PMID:26329581

  15. Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase*

    PubMed Central

    Matsumura, Yoshihiro; Sakai, Juro; Skach, William R.

    2013-01-01

    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. PMID:23990462

  16. Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

    PubMed Central

    Hartmann, Thomas; Xu, Xinsong; Kronast, Mira; Muehlich, Susanne; Meyer, Kathleen; Zimmermann, Wolfgang; Hurwitz, Jerard; Pan, Zhen-Qiang; Engelhardt, Stefan; Sarikas, Antonio

    2014-01-01

    Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT–CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LTΔ69–83), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways. PMID:24550499

  17. Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

    PubMed Central

    Moussaud-Lamodière, Elisabeth L.; Dourado, Daniel F. A. R.; Flores, Samuel C.; Springer, Wolfdieter

    2014-01-01

    Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through

  18. SUMOylation of Wor1 by a novel SUMO E3 ligase controls cell fate in Candida albicans.

    PubMed

    Yan, Minghui; Nie, Xinyi; Wang, Huafeng; Gao, Ning; Liu, Haoping; Chen, Jiangye

    2015-10-01

    Candida albicans is the most common human fungal pathogen, yet is a normal commensal resident of the human gut. CO(2) levels in the gut are much higher than in air, and it is known that elevated CO(2) concentration promotes C. albicans cells to undergo a phenotypic switch from white to opaque phase. Wor1, the master regulator of opaque cell formation, is required for both the white to opaque transition and opaque maintenance. To elucidate the regulatory mechanism of Wor1, we set out to identify Wor1-interacting proteins using a yeast two-hybrid screen. A SUMO E3 ligase named Wos1 (Wor1 SUMO-ligase 1) was identified to interact with Wor1 and regulate Wor1 SUMOylation. WOS1 expression is upregulated in response to high CO(2), and the induction by CO(2) is dependent on the transcription factor Flo8. Under high CO(2) conditions, Wos1 is required for the white to opaque switch and acts downstream of Flo8. At atmospheric CO(2) levels, overexpression of Wos1 enhances Wor1 SUMOylation and promotes the white to opaque switch. Wor1 is found to be SUMOylated at lysine 385, and loss of this mark by point mutation leads to a defect in CO(2) -mediated opaque cell induction. Together, our genetic and biological data show that Wos1-mediated Wor1 SUMOylation contributes to the regulation of CO(2) -induced white to opaque switching as well as heritable maintenance of the opaque cell type. PMID:26112173

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

    PubMed

    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. PMID:24525351

  20. Transcription factor single-minded 2 (SIM2) is ubiquitinated by the RING-IBR-RING-type E3 ubiquitin ligases.

    PubMed

    Okui, Michiyo; Yamaki, Akiko; Takayanagi, Atsushi; Kudoh, Jun; Shimizu, Nobuyoshi; Shimizu, Yoshiko

    2005-09-10

    Human single-minded 2 (SIM2) is a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors and is associated with the etiology of Down syndrome phenotype. Here, we examined a possibility of the post-translational modification of SIM2 protein by transfecting various expression constructs followed by the analysis with immunoprecipitation and Western blotting. In fact, transient expression of SIM2 cDNA in HEK293 cells revealed poly-ubiquitination of SIM2 protein. In the stable transfectants, a proteasome inhibitor MG132 protected the poly-ubiquitinated SIM2 protein from degradation. Furthermore, in the cells co-transfected with SIM2 and each of four different E3 ubiquitin ligases, SIM2 was immunoprecipitated with the RING-IBR-RING-type E3 ubiquitin ligases, Parkin and HHARI, but it was not immunoprecipitated with other E3 ligases, such as one RING-type Siah-1 and the PHD type AIRE. A series of deletion constructs revealed that Parkin actually binds to SIM2 with the IBR (294-377)-RING2 (378-465) domains and that the sites for poly-ubiquitination of SIM2 reside within the PAS1-PAS2 region (aa 141-289). We postulated that transcription factor SIM2 and E3 ubiquitin ligase Parkin may interact each other to play an important physiological role in the brain development which is controlled by ubiquitination. PMID:15963499

  1. Pto Kinase Binds Two Domains of AvrPtoB and Its Proximity to the Effector E3 Ligase Determines if It Evades Degradation and Activates Plant Immunity

    PubMed Central

    Mathieu, Johannes; Schwizer, Simon; Martin, Gregory B.

    2014-01-01

    The tomato—Pseudomonas syringae pv. tomato (Pst)—pathosystem is one of the best understood models for plant-pathogen interactions. Certain wild relatives of tomato express two closely related members of the same kinase family, Pto and Fen, which recognize the Pst virulence protein AvrPtoB and activate effector-triggered immunity (ETI). AvrPtoB, however, contains an E3 ubiquitin ligase domain in its carboxyl terminus which causes degradation of Fen and undermines its ability to activate ETI. In contrast, Pto evades AvrPtoB-mediated degradation and triggers ETI in response to the effector. It has been reported recently that Pto has higher kinase activity than Fen and that this difference allows Pto to inactivate the E3 ligase through phosphorylation of threonine-450 (T450) in AvrPtoB. Here we show that, in contrast to Fen which can only interact with a single domain proximal to the E3 ligase of AvrPtoB, Pto binds two distinct domains of the effector, the same site as Fen and another N-terminal domain. In the absence of E3 ligase activity Pto binds to either domain of AvrPtoB to activate ETI. However, the presence of an active E3 ligase domain causes ubiquitination of Pto that interacts with the domain proximal to the E3 ligase, identical to ubiquitination of Fen. Only when Pto binds its unique distal domain can it resist AvrPtoB-mediated degradation and activate ETI. We show that phosphorylation of T450 is not required for Pto-mediated resistance in vivo and that a kinase-inactive version of Pto is still capable of activating ETI in response to AvrPtoB. Our results demonstrate that the ability of Pto to interact with a second site distal to the E3 ligase domain in AvrPtoB, and not a higher kinase activity or T450 phosphorylation, allows Pto to evade ubiquitination and to confer immunity to Pst. PMID:25058029

  2. A novel ubiquitin-protein ligase E3 functions as a modulator of immune response against lipopolysaccharide in Pacific oyster, Crassostrea gigas.

    PubMed

    Cheng, Qi; Wang, Hao; Jiang, Shuai; Wang, Lingling; Xin, Lusheng; Liu, Conghui; Jia, Zhihao; Song, Linsheng; Zhu, Beiwei

    2016-07-01

    Ubiquitination is an important post-translational protein modification and plays a crucial role in various processes such as cell cycle, signal transduction, and transcriptional regulation. In the present study, a novel ubiquitin (Ub)-protein ligase E3 (designed as CgE3Rv1) was identified from Crassostrea gigas, and its ubiquitination regulation in the immune response against lipopolysaccharide (LPS) stimulation was investigated. The open reading frame of CgE3Rv1 gene was of 1455 bp encoding a polypeptide of 484 amino acids with the predicted molecular mass of 54.89 kDa. There were two transmembrane regions and a RING-variant (RINGv) domain identified in CgE3Rv1. CgE3Rv1 shared similar C4HC3 zinc-finger-like motif with those RINGv domain Ub-protein ligases E3s identified from vertebrates and invertebrates, and it was closely clustered with the membrane-associated RING-CH2 (MARCH2) Ub-protein ligases E3s in the phylogenetic tree. The mRNA transcript of CgE3Rv1 was highest expressed in gonads and hemolymph (p < 0.05), and its mRNA expression level in hemocytes was significantly increased at 6 h (p < 0.01) after the stimulation of LPS, while the up-regulated mRNA expression was significantly decreased (p < 0.01) after acetylcholine stimulation. No significant changes of CgE3Rv1 expression were observed after peptidoglycan or mannan stimulation. Immunohistochemistry and in situ hybridization assays revealed that CgE3Rv1 protein and mRNA were dominantly distributed in the gonad. In the hemocytes, CgE3Rv1 was mainly located around the nucleus, and slightly distributed in the cytoplasm and on the cell membrane. Recombinant CgE3Rv1 RINGv domain protein (rCgE3Rv1-RINGv) was confirmed to activate the Ub reaction system in vitro with the aid of Ub-activating enzyme E1 and Ub-conjugating enzyme E2. These results demonstrated that CgE3Rv1 was an Ub-protein ligase E3, which was involved in the immune response against LPS and the interaction with cell surface signal

  3. The E3 SUMO ligase Nse2 regulates sumoylation and nuclear-to-cytoplasmic translocation of skNAC-Smyd1 in myogenesis.

    PubMed

    Berkholz, Janine; Michalick, Laura; Munz, Barbara

    2014-09-01

    Skeletal and heart muscle-specific variant of the α subunit of nascent polypeptide associated complex (skNAC; encoded by NACA) is exclusively found in striated muscle cells. Its function, however, is largely unknown. Previous reports have demonstrated that skNAC binds to m-Bop/Smyd1, a multi-functional protein that regulates myogenesis both through the control of transcription and the modulation of sarcomerogenesis, and that both proteins undergo nuclear-to-cytoplasmic translocation at the later stages of myogenic differentiation. Here, we show that skNAC binds to the E3 SUMO ligase mammalian Mms21/Nse2 and that knockdown of Nse2 expression inhibits specific aspects of myogenic differentiation, accompanied by a partial blockade of the nuclear-to-cytoplasmic translocation of the skNAC-Smyd1 complex, retention of the complex in promyelocytic leukemia (PML)-like nuclear bodies and disturbed sarcomerogenesis. In addition, we show that the skNAC interaction partner Smyd1 contains a putative sumoylation motif and is sumoylated in muscle cells, with depletion of Mms21/Nse2 leading to reduced concentrations of sumoylated Smyd1. Taken together, our data suggest that the function, specifically the balance between the nuclear and cytosolic roles, of the skNAC-Smyd1 complex might be regulated by sumoylation. PMID:25002400

  4. The ubiquitin E3 ligase ITCH enhances breast tumor progression by inhibiting the Hippo tumor suppressor pathway

    PubMed Central

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

    2014-01-01

    The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis. Recently, we reported that the ubiquitin E3 ligase ITCH negatively regulates LATS1, thereby increasing YAP activity, which leads to increased cell proliferation and decreased apoptosis. Here, we investigated the role of ITCH in breast tumorigenesis. In particular, we show that ITCH enhances epithelial-to-mesenchymal transition (EMT) through boosting YAP oncogenic function. By contrast, a point mutation in the catalytic domain or WW1 domain of ITCH abolished its EMT-mediated effects. Furthermore, while overexpression of ITCH expression in breast cells is associated with increased incidence of mammary tumor formation and progression, its knockdown inhibited breast cancer cell tumorigenicity and metastasis. Importantly, YAP knockdown was able to attenuate ITCH pro-tumorigenic functions. Lastly, we found that ITCH expression is significantly upregulated in invasive and metastatic breast cancer cases and is associated with worse survival. Together, our results reveal that ITCH pro-tumorigenic functions in breast cancer are mediated, at least in part, through inactivation of the Hippo tumor suppressor pathway. PMID:25350971

  5. TRIM56 Is a Virus- and Interferon-Inducible E3 Ubiquitin Ligase That Restricts Pestivirus Infection ▿ †

    PubMed Central

    Wang, Jie; Liu, Baoming; Wang, Nan; Lee, Young-Min; Liu, Chunming; Li, Kui

    2011-01-01

    The tripartite motif (TRIM) protein family comprises more than 60 members that have diverse functions in various biological processes. Although a small number of TRIM proteins have been shown to regulate innate immunity, much remains to be learned about the functions of the majority of the TRIM proteins. Here we identify TRIM56 as a cellular protein associated with the N-terminal protease (Npro) of bovine viral diarrhea virus (BVDV), a pestiviral interferon antagonist which degrades interferon regulatory factor 3 (IRF3) through the proteasome. We found that TRIM56 was constitutively expressed in most tissues, and its abundance was further upregulated moderately by interferon or virus. The manipulation of TRIM56 abundance did not affect the protein turnover of Npro and IRF3. Rather, ectopic expression of TRIM56 substantially impaired, while knockdown of TRIM56 expression greatly enhanced, BVDV replication in cell culture. The antiviral activity of TRIM56 depended on its E3 ubiquitin ligase activity as well as the integrity of its C-terminal region but was not attributed to a general augmentation of the interferon antiviral response. Overexpression of TRIM56 did not inhibit the replication of vesicular stomatitis virus or hepatitis C virus, a virus closely related to BVDV. Together, our data demonstrate that TRIM56 is a novel antiviral host factor that restricts pestivirus infection. PMID:21289118

  6. E3 ubiquitin ligase E6AP negatively regulates adipogenesis by downregulating proadipogenic factor C/EBPalpha.

    PubMed

    Pal, Pooja; Lochab, Savita; Kanaujiya, Jitendra Kumar; Kapoor, Isha; Sanyal, Sabyasachi; Behre, Gerhard; Trivedi, Arun Kumar

    2013-01-01

    CCAAT/Enhancer Binding Protein Alpha (C/EBPα) is a key transcription factor involved in the adipocyte differentiation. Here for the first time we demonstrate that E6AP, an E3 ubiquitin ligase inhibits adipocyte differentiation in 3T3-L1 cells as revealed by reduced lipid staining with oil red. Knock down of E6AP in mouse 3T3L1 preadipocytes is sufficient to convert them to adipocytes independent of external hormonal induction. C/EBPα protein level is drastically increased in E6AP deficient 3T3L1 preadipocytes while inverse is observed when wild type E6AP is over expressed. We show that transient transfection of wild type E6AP downregulates C/EBPα protein expression in a dose dependent manner while catalytically inactive E6AP-C843A rather stabilizes it. In addition, wild type E6AP inhibits expression of proadipogenic genes while E6AP-C843A enhances them. More importantly, overexpression of E6AP-C843A in mesenchymal progenitor cells promotes accumulation of lipid droplets while there is drastically reduced lipid droplet formation when E6AP is over expressed. Taken together, our finding suggests that E6AP may negatively control adipogenesis by inhibiting C/EBPα expression by targeting it to ubiquitin-proteasome pathway for degradation. PMID:23762344

  7. Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signalling§

    PubMed Central

    Kruse, Michael; Hartmann, Thomas; Lempart, Justine; Mühlich, Susanne; Pfeiffer, Andreas F. H.; Field, Loren J.; Charron, Maureen J.; Pan, Zhen-Qiang; Engelhardt, Stefan; Sarikas, Antonio

    2014-01-01

    Dysfunctional regulation of signalling pathways downstream of the insulin receptor plays a pivotal role in the pathogenesis of insulin resistance and type 2 diabetes. In this study we report both in vitro and in vivo experimental evidence for a role of Cullin-RING E3 ubiquitin ligase 7 (CRL7) in the regulation of insulin signalling and glucose homeostasis. We show that Cul7−/− mouse embryonic fibroblasts displayed enhanced AKT and Erk MAP kinase phosphorylation upon insulin stimulation. Depletion of CUL7 by RNA interference in C2C12 myotubes led to increased activation of insulin signalling pathways and cellular glucose uptake, as well as a reduced capacity of these cells to execute insulin-induced degradation of insulin receptor substrate 1 (IRS1). In vivo, heterozygosity of either Cul7 or Fbxw8, both key components of CRL7, resulted in elevated PI3 kinase / AKT activation in skeletal muscle tissue upon insulin stimulation when compared to wild-type controls. Finally, Cul7+/− or Fbxw8+/− mice exhibited enhanced insulin sensitivity and plasma glucose clearance. Collectively, our findings point to a yet unrecognized role of CRL7 in insulin-mediated control of glucose homeostasis by restraining PI3 kinase / AKT activities in skeletal muscle cells. PMID:24219910

  8. The E3 ligase synoviolin controls body weight and mitochondrial biogenesis through negative regulation of PGC-1β

    PubMed Central

    Fujita, Hidetoshi; Yagishita, Naoko; Aratani, Satoko; Saito-Fujita, Tomoko; Morota, Saori; Yamano, Yoshihisa; Hansson, Magnus J; Inazu, Masato; Kokuba, Hiroko; Sudo, Katsuko; Sato, Eiichi; Kawahara, Ko-ichi; Nakajima, Fukami; Hasegawa, Daisuke; Higuchi, Itsuro; Sato, Tomoo; Araya, Natsumi; Usui, Chie; Nishioka, Kenya; Nakatani, Yu; Maruyama, Ikuro; Usui, Masahiko; Hara, Naomi; Uchino, Hiroyuki; Elmer, Eskil; Nishioka, Kusuki; Nakajima, Toshihiro

    2015-01-01

    Obesity is a major global public health problem, and understanding its pathogenesis is critical for identifying a cure. In this study, a gene knockout strategy was used in post-neonatal mice to delete synoviolin (Syvn)1/Hrd1/Der3, an ER-resident E3 ubiquitin ligase with known roles in homeostasis maintenance. Syvn1 deficiency resulted in weight loss and lower accumulation of white adipose tissue in otherwise wild-type animals as well as in genetically obese (ob/ob and db/db) and adipose tissue-specific knockout mice as compared to control animals. SYVN1 interacted with and ubiquitinated the thermogenic coactivator peroxisome proliferator-activated receptor coactivator (PGC)-1β, and Syvn1 mutants showed upregulation of PGC-1β target genes and increase in mitochondrion number, respiration, and basal energy expenditure in adipose tissue relative to control animals. Moreover, the selective SYVN1 inhibitor LS-102 abolished the negative regulation of PGC-1β by SYVN1 and prevented weight gain in mice. Thus, SYVN1 is a novel post-translational regulator of PGC-1β and a potential therapeutic target in obesity treatment. PMID:25698262

  9. Complementary genetic screens identify the E3 ubiquitin ligase CBLC, as a modifier of PARP inhibitor sensitivity

    PubMed Central

    Brough, Rachel; Hodny, Zdenek; Ashworth, Alan; Bartek, Jiri; Lord, Christopher J.

    2015-01-01

    Based on a series of basic, preclinical and clinical studies, the Poly (ADP-ribose) Polymerase 1 (PARP1) inhibitor, olaparib, has recently been approved for use in ovarian cancer patients with BRCA1 or BRCA2 mutations. By identifying novel predictive biomarkers of tumour cell sensitivity to olaparib, it is possible that the utility of PARP inhibitors could be extended beyond this patient subgroup. Many of the known genetic determinants of PARP inhibitor response have key roles in DNA damage response (DDR) pathways. Although protein ubiquitylation is known to play an important role in regulating the DDR, the exact mechanisms by which this occurs are not fully understood. Using two parallel RNA interference-based screening approaches, we identified the E3 ubiquitin ligase, CBLC, as a candidate biomarker of response to olaparib. We validated this observation by demonstrating that silencing of CBLC causes increased sensitivity to olaparib in breast cancer cell line models and that defective homologous recombination (HR) DNA repair is the likely cause. This data provides an example of how defects in the ubiquitin machinery have the potential to influence the response of tumour cells to PARP inhibitors. PMID:25883215

  10. Ubiquitylation of Rad51d Mediated by E3 Ligase Rnf138 Promotes the Homologous Recombination Repair Pathway

    PubMed Central

    Han, Deqiang; Liang, Junbo; Lu, Yalan; Xu, Longchang; Miao, Shiying; Lu, Lin-Yu; Song, Wei; Wang, Linfang

    2016-01-01

    Ubiquitylation has an important role as a signal transducer that regulates protein function, subcellular localization, or stability during the DNA damage response. In this study, we show that Ring domain E3 ubiquitin ligases RNF138 is recruited to DNA damage site quickly. And the recruitment is mediated through its Zinc finger domains. We further confirm that RNF138 is phosphorylated by ATM at Ser124. However, the phosphorylation was dispensable for recruitment to the DNA damage site. Our findings also indicate that RAD51 assembly at DSB sites following irradiation is dramatically affected in RNF138-deficient cells. Hence, RNF138 is likely involved in regulating homologous recombination repair pathway. Consistently, efficiency of homologous recombination decreased observably in RNF138-depleted cells. In addition, RNF138-deficient cell is hypersensitive to DNA damage insults, such as IR and MMS. And the comet assay confirmed that RNF138 directly participated in DNA damage repair. Moreover, we find that RAD51D directly interacted with RNF138. And the recruitment of RAD51D to DNA damage site is delayed and unstable in RNF138-depleted cells. Taken together, these results suggest that RNF138 promotes the homologous recombination repair pathway. PMID:27195665