The Experts below are selected from a list of 9201 Experts worldwide ranked by ideXlab platform
Brenda A Schulman - One of the best experts on this subject based on the ideXlab platform.
-
NEDD8 nucleates a multivalent cullin ring ube2d ubiquitin ligation assembly
Nature, 2020Co-Authors: Kheewoong Baek, David T Krist, Rajan J Prabu, Spencer Hill, Maren Felizitas Klugel, Lisamarie Neumaier, Susanne Von Gronau, Gary Kleiger, Brenda A SchulmanAbstract:Eukaryotic cell biology depends on cullin-RING E3 ligase (CRL)-catalysed protein ubiquitylation1, which is tightly controlled by the modification of cullin with the ubiquitin-like protein NEDD82-6. However, how CRLs catalyse ubiquitylation, and the basis of NEDD8 activation, remain unknown. Here we report the cryo-electron microscopy structure of a chemically trapped complex that represents the ubiquitylation intermediate, in which the neddylated CRL1β-TRCP promotes the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme UBE2D to its recruited substrate, phosphorylated IκBα. NEDD8 acts as a nexus that binds disparate cullin elements and the RING-activated ubiquitin-linked UBE2D. Local structural remodelling of NEDD8 and large-scale movements of CRL domains converge to juxtapose the substrate and the ubiquitylation active site. These findings explain how a distinctive ubiquitin-like protein alters the functions of its targets, and show how numerous NEDD8-dependent interprotein interactions and conformational changes synergistically configure a catalytic CRL architecture that is both robust, to enable rapid ubiquitylation of the substrate, and fragile, to enable the subsequent functions of cullin-RING proteins.
-
structure of a ring e3 trapped in action reveals ligation mechanism for the ubiquitin like protein NEDD8
Cell, 2014Co-Authors: Daniel C Scott, Wade J Harper, Vladislav O Sviderskiy, Julie K Monda, John R Lydeard, Brenda A SchulmanAbstract:Most E3 ligases use a RING domain to activate a thioester-linked E2∼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2∼UBL-target intermediate representing RBX1-UBC12∼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2∼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2∼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.
-
structural insights into NEDD8 activation of cullin ring ligases conformational control of conjugation
Cell, 2008Co-Authors: David M Duda, Harold W Hunt, Daniel C Scott, Laura A Borg, Michal Hammel, Brenda A SchulmanAbstract:Summary Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8∼Cul5 ctd -Rbx1 and SAXS analysis of NEDD8∼Cul1 ctd -Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.
-
basis for a ubiquitin like protein thioester switch toggling e1 e2 affinity
Nature, 2007Co-Authors: Brenda A Schulman, Danny T Huang, Harold W Hunt, Min Zhuang, Melanie D Ohi, James M HoltonAbstract:Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1–E2–E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1~UBL thioester intermediate, and generating a thioester-linked E2~UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1–UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1–E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2~NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.
Dimitris P Xirodimas - One of the best experts on this subject based on the ideXlab platform.
-
the NEDD8 cycle controlled by nedp1 upon dna damage is a regulatory module of the hsp70 atpase activity
bioRxiv, 2019Co-Authors: Aymeric P Bailly, Chantal M. Maghames, Aurelien Perrin, Helene Trauchessec, Marina Serranomacia, Orsolya Leidecker, Maria L Martinezchantar, Anton Gartner, Dimitris P XirodimasAbstract:Summary Ubiquitin and ubiquitin-like chains are finely balanced by the action of conjugating and de-conjugating enzymes. Alterations in this balance trigger signalling events required for the response to stress conditions and are often observed in pathologies. How such changes are detected is not well-understood. We show that upon DNA damage the induction of the de-NEDDylating enzyme NEDP1 restricts the formation of poly-NEDD8 chains, mainly through lysines K11/K48. This promotes APAF1 oligomerisation and apoptosis induction, a step that requires the HSP70 ATPase activity. We found that HSP70 binds to NEDD8 and in vitro, mono-NEDD8 stimulates the ATPase activity of HSP70, counteracted upon poly-NEDDylation. This effect is independent of NEDD8 conjugation onto substrates. The studies identify the HSP70 chaperone as sensor of changes in the NEDD8 cycle, providing mechanistic insights for a cytoplasmic role of NEDD8 in the DNA damage induced apoptosis. They also indicate that the balance between mono- versus poly-NEDDylation is a regulatory module of HSP70 function. The above findings may be important in tumorigenesis, as we find that NEDP1 levels are downregulated in Hepatocellular Carcinoma with concomitant accumulation of NEDD8 conjugates.
-
NEDDylation promotes nuclear protein aggregation and protects the Ubiquitin Proteasome System upon proteotoxic stress.
Nature Communications, 2018Co-Authors: Chantal M. Maghames, Sofia Lobato-gil, Aurelien Perrin, Helene Trauchessec, Manuel S. Rodriguez, Serge Urbach, Philippe Marin, Dimitris P XirodimasAbstract:Spatial management of stress-induced protein aggregation is an integral part of the proteostasis network. Protein modification by the ubiquitin-like molecule NEDD8 increases upon proteotoxic stress and it is characterised by the formation of hybrid NEDD8/ubiquitin conjugates. However, the biological significance of this response is unclear. Combination of quantitative proteomics with biological analysis shows that, during proteotoxic stress, NEDDylation promotes nuclear protein aggregation, including ribosomal proteins as a major group. This correlates with protection of the nuclear Ubiquitin Proteasome System from stress-induced dysfunction. Correspondingly, we show that NEDD8 compromises ubiquitination and prevents targeting and processing of substrates by the proteasome. Moreover, we identify HUWE1 as a key E3-ligase that is specifically required for NEDDylation during proteotoxic stress. The study reveals a specific role for NEDD8 in nuclear protein aggregation upon stress and is consistent with the concept that transient aggregate formation is part of a defence mechanism against proteotoxicity.
-
regulation of cancer related pathways by protein neddylation and strategies for the use of NEDD8 inhibitors in the clinic
Endocrine-related Cancer, 2015Co-Authors: Naima Abidi, Dimitris P XirodimasAbstract:Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) controls a vast if not every biological process in the cell. It is not surprising that deregulation in ubiquitin and UBL signalling has been implicated in the pathogenesis of many diseases and that these pathways are considered as major targets for therapeutic intervention. In this review, we summarise recent advances in our understanding of the role of the UBL neural precursor cell expressed developmentally downregulated-8 (NEDD8) in cancer-related processes and potential strategies for the use of NEDD8 inhibitors as chemotherapeutics.
-
the ubiquitin e1 enzyme ube1 mediates NEDD8 activation under diverse stress conditions
Cell Cycle, 2012Co-Authors: Orsolya Leidecker, Ivan Matic, Bidesh Mahata, Emmanuelle Pion, Dimitris P XirodimasAbstract:Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. Here, we present the unexpected finding that under stress conditions, the ubiquitin E1 enzyme Ube1 mediates conjugation of the ubiquitin-like molecule NEDD8. Inhibition of the 26S proteasome, heat shock and oxidative stress cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1-activating enzyme, but rather on Ube1. A common event in the tested stress conditions is the depletion of “free” ubiquitin. A decrease in “free” ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. Further analysis on the NEDD8 proteome shows that the modified NEDDylated proteins are simultaneously ubiquitinated. Mass spectrometry on the complex proteome under str...
-
regulation of nucleolar signalling to p53 through neddylation of l11
EMBO Reports, 2009Co-Authors: Anders Sundqvist, Geng Liu, Antonis Mirsaliotis, Dimitris P XirodimasAbstract:Several studies have shown that ribosomal proteins (RPs) are important mediators of p53 activation in response to nucleolar disruption; however, the pathways that control this signalling function of RPs are currently unknown. We have recently shown that RPs are targets for the ubiquitin-like molecule NEDD8, and that NEDDylation protects RPs from destabilization. Here, we identify NEDD8 as a crucial regulator of L11 RP signalling to p53. A decrease in L11 NEDDylation during nucleolar stress causes relocalization of L11 from the nucleolus to the nucleoplasm. This not only provides the signal for p53 activation, but also makes L11 susceptible to degradation. Mouse double minute 2 (MDM2) -mediated NEDDylation protects L11 from degradation and this is required for p53 stabilization during nucleolar stress. By controlling the correct localization and stability of L11, NEDD8 acts as a crucial, new regulator of nucleolar signalling to p53.
Mark J Banfield - One of the best experts on this subject based on the ideXlab platform.
-
the molecular basis of ubiquitin like protein NEDD8 deamidation by the bacterial effector protein cif
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: A Crow, Frederic Taieb, Richard K. Hughes, Eric Oswald, Mark J BanfieldAbstract:The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The “occluding loop” in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.
-
the molecular basis of ubiquitin like protein NEDD8 deamidation by the bacterial effector protein cif
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: A Crow, Frederic Taieb, Richard K. Hughes, Eric Oswald, Mark J BanfieldAbstract:The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The “occluding loop” in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.
Chengting Chien - One of the best experts on this subject based on the ideXlab platform.
-
neddylation and deneddylation regulate cul1 and cul3 protein accumulation
Nature Cell Biology, 2005Co-Authors: Junetai Wu, Chengting Chien, Yenchen HuAbstract:Cullin family proteins organize ubiquitin ligase (E3) complexes to target numerous cellular proteins for proteasomal degradation. Neddylation, the process that conjugates the ubiquitin-like polypeptide NEDD8 to the conserved lysines of cullins, is essential for in vivo cullin-organized E3 activities1,2. Deneddylation, which removes the NEDD8 moiety, requires the isopeptidase activity of the COP9 signalosome (CSN)3,4. Here we show that in cells deficient for CSN activity, cullin1 (Cul1) and cullin3 (Cul3) proteins are unstable, and that to preserve their normal cellular levels, CSN isopeptidase activity is required. We further show that neddylated Cul1 and Cul3 are unstable — as suggested by the evidence that NEDD8 promotes the instability of both cullins — and that the unneddylatable forms of cullins are stable. The protein stability of NEDD8 is also subject to CSN regulation and this regulation depends on its cullin-conjugating ability, suggesting that NEDD8-conjugated cullins are degraded en bloc. We propose that while NEDD8 promotes cullin activation through neddylation, neddylation also renders cullins unstable. Thus, CSN deneddylation recycles the unstable, neddylated cullins into stable, unneddylated ones, and promotes cullin-organized E3 activity in vivo.
-
cul1 and cul3 mediate distinct protein degradation mechanisms to control ci stability in drosophila eye development
Journal of Genetics and Molecular Biology, 2002Co-Authors: Chanyen Ou, Yingjiun Chen, Chengting ChienAbstract:The ubiquitin-like protein, NEDD8, covalently modifies members of the Cullin family. Cullins are the major components of a series of ubiquitin ligases that control the degradation of a broad range of proteins. We found that NEDD8 modifies Cul 1 in Drosophila. In Drosophila NEDD8 and Cul 1 mutants, protein levels of the signal transduction effectors, Cubitus nterruptus (Ci) and Armadillo (Arm), and the cell cycle regulator, Cyclin E (CycE), are highly accumulated, suggesting that the Cul I-based SCF complex requires NEDD8 modification for the degradation processes of Ci, Arm, and CycE in vivo. We further show that two distinct degradation mechanisms odulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the NEDD8-modified Cull-based SCFslimb complex. In the posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul 3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires NEDD8 modification, is activated by hedgehog (Hh) signaling and PKA-independent.
-
distinct protein degradation mechanisms mediated by cul1 and cul3 controlling ci stability in drosophila eye development
Genes & Development, 2002Co-Authors: Chanyen Ou, Yingjiun Chen, Chengting ChienAbstract:The ubiquitin-like protein, NEDD8, covalently modifies members of the Cullin family. Cullins are the major components of a series of ubiquitin ligases that control the degradation of a broad range of proteins. We found that NEDD8 modifies Cul1 in Drosophila .I nDrosophila NEDD8 and Cul1 mutants, protein levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo (Arm), and the cell cycle regulator, Cyclin E (CycE), are highly accumulated, suggesting that the Cul1-based SCF complex requires NEDD8 modification for the degradation processes of Ci, Arm, and CycE in vivo. We further show that two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the NEDD8-modified Cul1-based SCF Slimb complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires NEDD8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent.
Wuhan Xiao - One of the best experts on this subject based on the ideXlab platform.
-
Zebrafish NEDD8 facilitates ovarian development and the maintenance of female secondary sexual characteristics via suppression of androgen receptor activity.
Development, 2020Co-Authors: Xing Liu, Gang Ouyang, Zhu Chen, Wuhan XiaoAbstract:NEDD8 is a ubiquitin-like protein that covalently conjugates to target proteins through neddylation. In addition to cullin-RING ligases, neddylation also modifies non-cullin proteins to regulate protein activity, stability and localization. However, the roles of NEDD8 remain largely unknown in vivo Here, we found that loss of NEDD8 in female zebrafish led to defects in oogenesis, disrupted oocyte maturation and stimulated growth of the breeding tubercles (BTs) on the pectoral fins. The BTs are normally present in males, not females. However, the loss of one copy of ar can partially rescue the phenotypes displayed by NEDD8-null female zebrafish. Further assays indicated that NEDD8 conjugates to Ar and Ar is neddylated at lysine 475 and lysine 862. Moreover, NEDD8 conjugation efficiently suppressed Ar transcriptional activity. Lysine 862 (K862) of Ar is the key site modified by neddylation to modulate Ar transcriptional activity. Thus, our results not only demonstrated that NEDD8 modulates ovarian maturation and the maintenance of female secondary sexual characteristics of female zebrafish in vivo, but also indicated that androgen signaling is strictly regulated by NEDD8.
-
neddylation facilitates the antiviral response in zebrafish
Frontiers in Immunology, 2019Co-Authors: Xing Liu, Gang Ouyang, Jinhua Tang, Wuhan XiaoAbstract:Neddylation is a type of post-translational protein modifications, in which neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is covalently conjugated to the lysine residues of target substrates. The best characterized principal substrates of neddylation are the cullin-RING ligases (CRLs). In addition, neddylation also modifies non-cullin proteins to affect gene regulation, cell survival, organ development, and stress response. However, the role of neddylation in antiviral innate immunity remain largely unknown. Here, we found that when neddylation was blocked by the NEDD8 activating enzyme E1 (NAE) inhibitor, MLN4924, the cellular and organismal antiviral response was suppressed. Moreover, the disruption of NEDD8 increased the sensitivity of zebrafish to SVCV infection. Further assays indicated that blocking or silencing neddylation significantly downregulated key antiviral genes after poly (I:C) stimulation or SVCV infection, but dramatically increased SVCV replication. Neddylation of Irf3 and Irf7 was readily detected, but not of Mda5, Mavs, and Tbk1. Thus, our results not only demonstrated that neddylation facilitated the antiviral response in vitro and in vivo, but also revealed a novel role of NEDD8 in antiviral innate immunity.
-
zebrafish NEDD8 facilitates ovarian development and the maintenance of female secondary sexual characteristics via suppression of androgen receptor activity
Social Science Research Network, 2019Co-Authors: Xing Liu, Gang Ouyang, Zhu Chen, Dawei Zhang, Jing Wang, Wuhan XiaoAbstract:NEDD8 is an ubiquitin-like protein that covalently conjugates to target proteins through neddylation. In addition to cullin-RING ligases, neddylation also modifies non-cullin proteins to regulate protein activity, stability, and localization. However, the roles of NEDD8 remain largely unknown in vivo. Here, we found that loss of NEDD8 in female zebrafish led to defects in ovarian development, disrupted oocyte maturation and stimulated growth of the breeding tubercles (BTs) on the pectoral fins. However, the loss of one copy of ar can partially rescued the phenotypes displayed by NEDD8-null female zebrafish. Further assays indicated that Ar conjugated to NEDD8 and Ar is neddylated at lysine 76 and lysine 558. Moreover, NEDD8 conjugation efficiently suppressed Ar transcriptional activity. Thus, our results not only demonstrated that NEDD8 modulates ovarian development and the formation of male secondary sexual characteristics in female zebrafish in vivo, but also indicated that androgen signaling is strictly regulated by NEDD8.
