The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Jeffrey L Wrana - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin binding region of the smurf HECT Domain facilitates polyubiquitylation and binding of ubiquitylated substrates
Journal of Biological Chemistry, 2010Co-Authors: Abiodun A Ogunjimi, Douglas J Briant, Frank Sicheri, Silke Wiesner, Xaralabos Varelas, Julie D Formankay, Jeffrey L WranaAbstract:Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT Domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT Domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT Domain.
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autoinhibition of the HECT type ubiquitin ligase smurf2 through its c2 Domain
Cell, 2007Co-Authors: Silke Wiesner, Daniela Rotin, Abiodun A Ogunjimi, Frank Sicheri, Jeffrey L Wrana, Hongrui Wang, Julie D FormankayAbstract:Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-Domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT Domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-Domain E3s. Using NMR analysis the C2 Domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding Domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT Domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.
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regulation of cell polarity and protrusion formation by targeting rhoa for degradation
Science, 2003Co-Authors: Hongrui Wang, Abiodun A Ogunjimi, Jeffrey L Wrana, Yue Zhang, Barish Ozdamar, Evguenia Alexandrova, Gerald H ThomsenAbstract:The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such as cell shape changes, migration, and polarity. We found that Smurf1, a HECT Domain E3 ubiquitin ligase, regulated cell polarity and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein kinase C zeta (PKCζ), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia to prevent RhoA signaling during dynamic membrane movements.
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smad7 binds to smurf2 to form an e3 ubiquitin ligase that targets the tgfβ receptor for degradation
Molecular Cell, 2000Co-Authors: Peter A Kavsak, Richele K Rasmussen, Jeffrey L Wrana, Gerald H Thomsen, Carrie G Causing, Shirin Bonni, Haitao ZhuAbstract:Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT Domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.
Daniela Rotin - One of the best experts on this subject based on the ideXlab platform.
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comparison of substrate specificity of the ubiquitin ligases nedd4 and nedd4 2 using proteome arrays
Molecular Systems Biology, 2009Co-Authors: Avinash Persaud, Philipp Alberts, Eva Amsen, Xuejian Xiong, James D. Wasmuth, Zachary Saadon, Christopher Fladd, John Parkinson, Daniela RotinAbstract:Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 Domain, 2-4 WW Domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT Domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with approximately 8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.
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regulation of nedd4 2 self ubiquitination and stability by a py motif located within its HECT Domain
Biochemical Journal, 2008Co-Authors: Christine M Bruce, Voula Kanelis, Fatemeh Fouladkou, Anne Debonneville, Olivier Staub, Daniela RotinAbstract:Ubiquitin ligases play a pivotal role in substrate recognition and ubiquitin transfer, yet little is known about the regulation of their catalytic activity. Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated 4)-2 is an E3 ubiquitin ligase composed of a C2 Domain, four WW Domains (protein–protein interaction Domains containing two conserved tryptophan residues) that bind PY motifs (L/PPXY) and a ubiquitin ligase HECT (homologous with E6-associated protein C-terminus) Domain. In the present paper we show that the WW Domains of Nedd4-2 bind (weakly) to a PY motif (LPXY) located within its own HECT Domain and inhibit auto-ubiquitination. Pulse–chase experiments demonstrated that mutation of the HECT PY-motif decreases the stability of Nedd4-2, suggesting that it is involved in stabilization of this E3 ligase. Interestingly, the HECT PY-motif mutation does not affect ubiquitination or down-regulation of a known Nedd4-2 substrate, ENaC (epithelial sodium channel). ENaC ubiquitination, in turn, appears to promote Nedd4-2 self-ubiquitination. These results support a model in which the inter- or intra-molecular WW-Domain–HECT PY-motif interaction stabilizes Nedd4-2 by preventing self-ubiquitination. Substrate binding disrupts this interaction, allowing self-ubiquitination of Nedd4-2 and subsequent degradation, resulting in down-regulation of Nedd4-2 once it has ubiquitinated its target. These findings also point to a novel mechanism employed by a ubiquitin ligase to regulate itself differentially compared with substrate ubiquitination and stability.
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autoinhibition of the HECT type ubiquitin ligase smurf2 through its c2 Domain
Cell, 2007Co-Authors: Silke Wiesner, Daniela Rotin, Abiodun A Ogunjimi, Frank Sicheri, Jeffrey L Wrana, Hongrui Wang, Julie D FormankayAbstract:Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-Domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT Domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-Domain E3s. Using NMR analysis the C2 Domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding Domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT Domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.
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regulation of the epithelial na channel by nedd4 and ubiquitination
Kidney International, 2000Co-Authors: Olivier Staub, Voula Kanelis, Hugues Abriel, Pamela J Plant, Toru Ishikawa, Reza Saleki, Jeandaniel Horisberger, Laurent Schild, Daniela RotinAbstract:The epithelial Na+ channel (ENaC) is comprised of three subunits, alpha, beta and gamma, and plays an essential role in Na+ and fluid absorption in the kidney, colon and lung. We had identified proline-rich sequences at the C termini of alpha beta gamma ENaC, which include the sequence PPxY, the PY motif. This sequence in beta or gamma ENaC is deleted or mutated in Liddle's syndrome, a hereditary form of arterial hypertension. Our previous work demonstrated that these PY motifs bind to the WW Domains of Nedd4, a ubiquitin protein ligase containing a C2 Domain, three or four WW Domains and a ubiquitin protein ligase HECT Domain. Accordingly, we have recently demonstrated that Nedd4 regulates ENaC function by controlling the number of channels at the cell surface, that this regulation is impaired in ENaC bearing Liddle's syndrome mutations, and that ENaC stability and function are regulated by ubiquitination. The C2 Domain is responsible for localizing Nedd4 to the plasma membrane in a Ca(2+)-dependent manner, and in polarized epithelial MDCK cells this localization is primarily apical. In accordance, electrophysiological characterization of ENaC expressed in MDCK cells revealed inhibition of channel activity by elevated intracellular Ca2+ levels. Thus, in response to Ca2+, Nedd4 may be mobilized to the apical membrane via its C2 Domain, where it binds ENaC via Nedd4-WW:ENaC-PY motifs' interactions, leading to ubiquitination of the channel by the Nedd4-HECT Domain and subsequent channel endocytosis and lysosomal degradation. This process may be at least partially impaired in Liddle's syndrome due to reduced Nedd4 binding, leading to increased retention of ENaC at the cell surface.
Frank Sicheri - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin binding region of the smurf HECT Domain facilitates polyubiquitylation and binding of ubiquitylated substrates
Journal of Biological Chemistry, 2010Co-Authors: Abiodun A Ogunjimi, Douglas J Briant, Frank Sicheri, Silke Wiesner, Xaralabos Varelas, Julie D Formankay, Jeffrey L WranaAbstract:Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT Domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT Domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT Domain.
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autoinhibition of the HECT type ubiquitin ligase smurf2 through its c2 Domain
Cell, 2007Co-Authors: Silke Wiesner, Daniela Rotin, Abiodun A Ogunjimi, Frank Sicheri, Jeffrey L Wrana, Hongrui Wang, Julie D FormankayAbstract:Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-Domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT Domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-Domain E3s. Using NMR analysis the C2 Domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding Domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT Domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.
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regulation of smurf2 ubiquitin ligase activity by anchoring the e2 to the HECT Domain
Molecular Cell, 2005Co-Authors: Abiodun A Ogunjimi, Douglas J Briant, Nadia Pecebarbara, Christine Le Roy, Gianni M Di Guglielmo, Peter A Kavsak, Richele K Rasmussen, Bruce T Seet, Frank SicheriAbstract:The conjugation of ubiquitin to proteins involves a cascade of activating (E1), conjugating (E2), and ubiquitin-ligating (E3) type enzymes that commonly signal protein destruction. In TGFβ signaling the inhibitory protein Smad7 recruits Smurf2, an E3 of the C2-WW-HECT Domain class, to the TGFβ receptor complex to facilitate receptor degradation. Here, we demonstrate that the amino-terminal Domain (NTD) of Smad7 stimulates Smurf activity by recruiting the E2, UbcH7, to the HECT Domain. A 2.1 A resolution X-ray crystal structure of the Smurf2 HECT Domain reveals that it has a suboptimal E2 binding pocket that could be optimized by mutagenesis to generate a HECT Domain that functions independently of Smad7 and potently inhibits TGFβ signaling. Thus, E2 enzyme recognition by an E3 HECT enzyme is not constitutively competent and provides a point of control for regulating the ubiquitin ligase activity through the action of auxiliary proteins.
Silke Wiesner - One of the best experts on this subject based on the ideXlab platform.
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beta sheet augmentation is a conserved mechanism of priming HECT e3 ligases for ubiquitin ligation
Journal of Molecular Biology, 2018Co-Authors: Magnus Jackl, Silke Wiesner, Elena Maspero, Simona Polo, Carsten Stollmaier, Timo Strohaker, Karolina HyzAbstract:Abstract Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT Domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT Domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe ~ Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the β-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub.
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structural and functional framework for the autoinhibition of nedd4 family ubiquitin ligases
Structure, 2014Co-Authors: Sara Mari, Natalia Ruetalo, Elena Maspero, Mira C Stoffregen, Sebastiano Pasqualato, Simona Polo, Silke WiesnerAbstract:Summary Nedd4-family ubiquitin ligases are key regulators of cell surface receptor signaling. Their dysregulation is associated with several human diseases, including cancer. Under normal conditions, the activity of various Nedd4 E3s is controlled through an autoinhibitory interaction of the N-terminal C2 Domain with the C-terminal catalytic HECT Domain. Here, we report the structural and functional framework for this intramolecular interaction. Our nuclear magnetic resonance (NMR) data and biochemical analyses on Smurf2 and Nedd4 show that the C2 Domain has the potential to regulate E3 activity by maintaining the HECT Domain in a low-activity state where its ability for transthiolation and noncovalent Ub binding are impaired.
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the ubiquitin binding region of the smurf HECT Domain facilitates polyubiquitylation and binding of ubiquitylated substrates
Journal of Biological Chemistry, 2010Co-Authors: Abiodun A Ogunjimi, Douglas J Briant, Frank Sicheri, Silke Wiesner, Xaralabos Varelas, Julie D Formankay, Jeffrey L WranaAbstract:Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT Domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT Domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT Domain.
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autoinhibition of the HECT type ubiquitin ligase smurf2 through its c2 Domain
Cell, 2007Co-Authors: Silke Wiesner, Daniela Rotin, Abiodun A Ogunjimi, Frank Sicheri, Jeffrey L Wrana, Hongrui Wang, Julie D FormankayAbstract:Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-Domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT Domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-Domain E3s. Using NMR analysis the C2 Domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding Domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT Domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.
Jon M Huibregtse - One of the best experts on this subject based on the ideXlab platform.
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structure and function of a HECT Domain ubiquitin binding site
EMBO Reports, 2011Co-Authors: Hyung Cheol Kim, Alanna M Steffen, Michael L Oldham, Jue Chen, Jon M HuibregtseAbstract:The Rsp5 ubiquitin ligase contains a non-covalent binding site for ubiquitin within the amino-terminal lobe (N-lobe) of the HECT Domain, and the X-ray crystal structure of the HECT–ubiquitin complex has been determined. Hydrophobic patch residues of ubiquitin (L8, I44, V70) were crucial for interaction with Rsp5, and amino-acid alterations at the Rsp5-binding interface resulted in defects in polyubiquitination. Our results support a model in which the N-lobe-binding site acts to localize and orient the distal end of the ubiquitin chain to promote conjugation of the next ubiquitin molecule.
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expression and assay of HECT Domain ligases
Methods in Enzymology, 2005Co-Authors: Sylvie Beaudenon, Anahita Dastur, Jon M HuibregtseAbstract:Abstract HECT Domain ubiquitin ligases (HECT E3s), typified by human E6AP and yeast Rsp5p, are unique among the several classes of known ubiquitin ligases in that they participate directly in the chemistry of substrate ubiquitination reactions. This chapter discusses strategies for the expression of active HECT E3s and the assays that are available for analyzing E2 interaction, ubiquitin‐thioester formation, and substrate ubiquitination.
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structure of an e6ap ubch7 complex insights into ubiquitination by the e2 e3 enzyme cascade
Science, 1999Co-Authors: Lan Huang, Sylvie Beaudenon, Jon M Huibregtse, Elspeth Kinnucan, Guangli Wang, Peter M Howley, Nikola P PavletichAbstract:The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic HECT Domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP HECT Domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.
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rsp5 ubiquitin protein ligase mediates dna damage induced degradation of the large subunit of rna polymerase ii in saccharomyces cerevisiae
Molecular and Cellular Biology, 1999Co-Authors: Sylvie Beaudenon, Guangli Wang, Maria R Huacani, Donald P Mcdonnell, Jon M HuibregtseAbstract:Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the HECT Domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW Domain-containing HECT (WW-HECT) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-HECT E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.