The Experts below are selected from a list of 1929 Experts worldwide ranked by ideXlab platform
Yi Sun - One of the best experts on this subject based on the ideXlab platform.
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selective inhibition of cullin 3 Neddylation through covalent targeting dcn1 protects mice from acetaminophen induced liver toxicity
Nature Communications, 2021Co-Authors: Haibin Zhou, Liu Liu, Denzil Bernard, Chao Yie Yang, Krishnapriya Chinnaswamy, Jeanne A Stuckey, Donna Mceachern, Hong Shen, Liangyou Rui, Yi SunAbstract:Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins. Neddylation of individual cullin proteins is essential for the activation of each CRL. We report herein the discovery of DI-1548 and DI-1859 as two potent, selective and covalent DCN1 inhibitors. These inhibitors selectively inhibit Neddylation of cullin 3 in cells at low nanomolar concentrations and are 2–3 orders of magnitude more potent than our previously reported reversible DCN1 inhibitor. Mass spectrometric analysis and co-crystal structures reveal that these compounds employ a unique mechanism of covalent bond formation with DCN1. DI-1859 induces a robust increase of NRF2 protein, a CRL3 substrate, in mouse liver and effectively protects mice from acetaminophen-induced liver damage. Taken together, this study demonstrates the therapeutic potential of selective inhibition of cullin Neddylation. Activation of cullin-RING ligases can be inhibited by targeting DCN1, but selective DCN1 inhibitors with in vivo activity are lacking. Here, the authors develop covalent DCN1 inhibitors that selectively and potently inhibit cullin-3 activation and downstream functions in cells and in mice.
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selective inhibition of cullin 3 Neddylation through covalent targeting dcn1 protects mice from acetaminophen induced liver toxicity
Nature Communications, 2021Co-Authors: Haibin Zhou, Liu Liu, Denzil Bernard, Chao Yie Yang, Krishnapriya Chinnaswamy, Jeanne A Stuckey, Donna Mceachern, Hong Shen, Liangyou Rui, Yi SunAbstract:Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins. Neddylation of individual cullin proteins is essential for the activation of each CRL. We report herein the discovery of DI-1548 and DI-1859 as two potent, selective and covalent DCN1 inhibitors. These inhibitors selectively inhibit Neddylation of cullin 3 in cells at low nanomolar concentrations and are 2-3 orders of magnitude more potent than our previously reported reversible DCN1 inhibitor. Mass spectrometric analysis and co-crystal structures reveal that these compounds employ a unique mechanism of covalent bond formation with DCN1. DI-1859 induces a robust increase of NRF2 protein, a CRL3 substrate, in mouse liver and effectively protects mice from acetaminophen-induced liver damage. Taken together, this study demonstrates the therapeutic potential of selective inhibition of cullin Neddylation.
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rnf111 facilitated Neddylation potentiates cgas mediated antiviral innate immune response
PLOS Pathogens, 2021Co-Authors: Lele Zhang, Yi Sun, Dong Qian, Mingxing Cheng, Ze Hong, Ye Cui, Quanyi Wang, Juanjuan Zhu, Wei Meng, Peng ZhangAbstract:The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) has emerged as a fundamental component fueling the anti-pathogen immunity. Because of its pivotal role in initiating innate immune response, the activity of cGAS must be tightly fine-tuned to maintain immune homeostasis in antiviral response. Here, we reported that Neddylation modification was indispensable for appropriate cGAS-STING signaling activation. Blocking Neddylation pathway using Neddylation inhibitor MLN4924 substantially impaired the induction of type I interferon and proinflammatory cytokines, which was selectively dependent on Nedd8 E2 enzyme Ube2m. We further found that deficiency of the Nedd8 E3 ligase Rnf111 greatly attenuated DNA-triggered cGAS activation while not affecting cGAMP induced activation of STING, demonstrating that Rnf111 was the Nedd8 E3 ligase of cGAS. By performing mass spectrometry, we identified Lys231 and Lys421 as essential Neddylation sites in human cGAS. Mechanistically, Rnf111 interacted with and polyneddylated cGAS, which in turn promoted its dimerization and enhanced the DNA-binding ability, leading to proper cGAS-STING pathway activation. In the same line, the Ube2m or Rnf111 deficiency mice exhibited severe defects in innate immune response and were susceptible to HSV-1 infection. Collectively, our study uncovered a vital role of the Ube2m-Rnf111 Neddylation axis in promoting the activity of the cGAS-STING pathway and highlighted the importance of Neddylation modification in antiviral defense.
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targeting protein Neddylation to inactivate cullin ring ligases by gossypol a lucky hit or a new start
Drug Design Development and Therapy, 2021Co-Authors: Yi SunAbstract:Abstract Cullin-RING E3 ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for about 20% of the protein degradation by the ubiquitin-proteasome system (UPS). Given their vital roles in multiple cellular processes, and over-activation in many human cancers, CRLs are validated as promising targets for anti-cancer therapies. Activation of CRLs requires cullin Neddylation, a process catalysed by three Neddylation enzymes. Recently, our group established an AlphaScreen-based in vitro cullin Neddylation assay and employed it for high-throughput screening to search for small-molecule inhibitors targeting cullin Neddylation. During our pilot screen, gossypol, a natural product extracted from cottonseeds, was identified as one of the most potent Neddylation inhibitors of cullin-1 and cullin-5. We further demonstrated that gossypol blocks cullin Neddylation by binding to cullin-1/-5 to inactivate CRL1/5 ligase activity, leading to accumulation of MCL-1 and NOXA, the substrates of CRL1 and CRL5, respectively. The combination of gossypol and an MCL-1 inhibitor synergistically enhanced the anti-proliferative effect in multiple human cancer cell lines. Our study unveiled a rational combination of two previously known inhibitors of the Bcl-2 family for enhanced anti-cancer efficacy and identified a novel activity of gossypol as an inhibitor of CRL1 and CRL5 E3s, thus providing a new possibility in the development of novel CRL inhibitors for anti-cancer therapy.
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anticancer drug discovery by targeting cullin Neddylation
Acta Pharmaceutica Sinica B, 2020Co-Authors: Yihan Jiang, Yi SunAbstract:Protein Neddylation is a post-translational modification which transfers the ubiquitin-like protein NEDD8 to a lysine residue of the target substrate through a three-step enzymatic cascade. The best-known substrates of Neddylation are cullin family proteins, which are the core component of Cullin-RING E3 ubiquitin ligases (CRLs). Given that cullin Neddylation is required for CRL activity, and CRLs control the turn-over of a variety of key signal proteins and are often abnormally activated in cancers, targeting Neddylation becomes a promising approach for discovery of novel anti-cancer therapeutics. In the past decade, we have witnessed significant progress in the field of protein Neddylation from preclinical target validation, to drug screening, then to the clinical trials of Neddylation inhibitors. In this review, we first briefly introduced the nature of protein Neddylation and the regulation of Neddylation cascade, followed by a summary of all reported chemical inhibitors of Neddylation enzymes. We then discussed the structure-based targeting of protein-protein interaction in Neddylation cascade, and finally the available approaches for the discovery of new Neddylation inhibitors. This review will provide a focused, up-to-date and yet comprehensive overview on the discovery effort of Neddylation inhibitors.
Bhuvanesh Singh - One of the best experts on this subject based on the ideXlab platform.
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improvement of oral bioavailability of pyrazolo pyridone inhibitors of the interaction of dcn1 2 and ube2m
Journal of Medicinal Chemistry, 2021Co-Authors: Ho Shin Kim, Bhuvanesh Singh, Daniel C Scott, Jared T Hammill, Brenda A Schulman, Yizhe Chen, Amy L Rice, William Pistel, Kiplin R GuyAbstract:The cullin-RING ubiquitin ligases (CRLs) are ubiquitin E3 enzymes that play a key role in controlling proteasomal degradation and are activated by Neddylation. We previously reported inhibitors that target CRL activation by disrupting the interaction of defective in cullin Neddylation 1 (DCN1), a CRL Neddylation co-E3, and UBE2M, a Neddylation E2. Our first-generation inhibitors possessed poor oral bioavailability and fairly rapid clearance that hindered the study of acute inhibition of DCN-controlled CRL activity in vivo. Herein, we report studies to improve the pharmacokinetic performance of the pyrazolo-pyridone inhibitors. The current best inhibitor, 40, inhibits the interaction of DCN1 and UBE2M, blocks NEDD8 transfer in biochemical assays, thermally stabilizes cellular DCN1, and inhibits anchorage-independent growth in a DCN1 amplified squamous cell carcinoma cell line. Additionally, we demonstrate that a single oral 50 mg/kg dose sustains plasma exposures above the biochemical IC90 for 24 h in mice.
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piperidinyl ureas chemically control defective in cullin Neddylation 1 dcn1 mediated cullin Neddylation
Journal of Medicinal Chemistry, 2018Co-Authors: Jared T Hammill, Bhuvanesh Singh, Daniel C Scott, Jaeki Min, Michele Connelly, Gloria Holbrook, Fangyi Zhu, Amy Matheny, Lei Yang, Brenda A SchulmanAbstract:We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin Neddylation 1 (DCN1)-dependent Neddylation of cullins. Here, we report preliminary structure–activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1–UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1–UBE2M protein–protein interaction in our TR-FRET binding assay and inhibit cullin Neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous...
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squamous cell carcinoma related oncogene sccro neddylates cul3 protein to selectively promote midbody localization and activity of cul3klhl21 protein complex during abscission
Journal of Biological Chemistry, 2017Co-Authors: Guochang Huang, Andrew J Kaufman, Katia Manova, Bhuvanesh SinghAbstract:Abstract SCCRO/DCUN1D1, a component of the Neddylation E3 complex, regulates the activity of the cullin-RING-ligase (CRL) type of ubiquitination E3s by promoting Neddylation of cullin family members. Studies have shown SCCRO regulates proliferation in vitro and in vivo. Here, we show that inactivation of SCCRO results in prolonged mitotic time due to delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in Neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates SCCRO's effects on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of Aurora B inhibitor at the midbody stage suggests Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting SCCRO selectively, rather than collectively, neddylates cullins in vivo. Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of Neddylation and CRL activity in vivo.
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squamous cell carcinoma related oncogene sccro neddylates cul3 protein to selectively promote midbody localization and activity of cul3klhl21 protein complex during abscission
Journal of Biological Chemistry, 2017Co-Authors: Guochang Huang, Andrew J Kaufman, Katia Manova, Bhuvanesh SinghAbstract:Squamous cell carcinoma–related oncogene (SCCRO)/DCUN1D1, a component of the Neddylation E3 complex, regulates the activity of the cullin–RING–ligase type of ubiquitination E3s by promoting Neddylation of cullin family members. Studies have shown that SCCRO regulates proliferation in vitro and in vivo. Here we show that inactivation of SCCRO results in prolonged mitotic time because of delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in Neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates the effects of SCCRO on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission, and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting that it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of an Aurora B inhibitor at the midbody stage suggests that Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting that SCCRO selectively, rather than collectively, neddylates cullins in vivo. Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of Neddylation and cullin–RING–ligase activity in vivo.
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squamous cell carcinoma related oncogene sccro family members regulate cell growth and proliferation through their cooperative and antagonistic effects on cullin Neddylation
Journal of Biological Chemistry, 2016Co-Authors: Joanne Sun, Guochang Huang, Andrew Kaufman, Jeffrey C Liu, Russell J H Ryan, Suresh Y Ramanathan, Tadmiri Venkatesh, Bhuvanesh SinghAbstract:SCCRO (squamous cell carcinoma-related oncogene; also known as DCUN1D1) is a highly conserved gene that functions as an E3 in Neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO(-/-) mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led us to assess whether compensation by SCCRO paralogues rescues lethality in SCCRO(-/-) mice. Using murine and Drosophila models, we assessed the in vivo activities of SCCRO and its paralogues in cullin Neddylation. We found that SCCRO family members have overlapping and antagonistic activity that regulates Neddylation and cell proliferation activities in vivo. In flies, both dSCCRO and dSCCRO3 promote Neddylation and cell proliferation, whereas dSCCRO4 negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones with a net decrease in Neddylation activity. We found that dSCCRO and, to a lesser extent, dSCCRO3 rescue the Neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promote, and human SCCRO3 inhibits, Neddylation and proliferation when expressed in flies. Our findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate Neddylation and cell proliferation.
Guochang Huang - One of the best experts on this subject based on the ideXlab platform.
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squamous cell carcinoma related oncogene sccro neddylates cul3 protein to selectively promote midbody localization and activity of cul3klhl21 protein complex during abscission
Journal of Biological Chemistry, 2017Co-Authors: Guochang Huang, Andrew J Kaufman, Katia Manova, Bhuvanesh SinghAbstract:Abstract SCCRO/DCUN1D1, a component of the Neddylation E3 complex, regulates the activity of the cullin-RING-ligase (CRL) type of ubiquitination E3s by promoting Neddylation of cullin family members. Studies have shown SCCRO regulates proliferation in vitro and in vivo. Here, we show that inactivation of SCCRO results in prolonged mitotic time due to delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in Neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates SCCRO's effects on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of Aurora B inhibitor at the midbody stage suggests Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting SCCRO selectively, rather than collectively, neddylates cullins in vivo. Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of Neddylation and CRL activity in vivo.
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squamous cell carcinoma related oncogene sccro neddylates cul3 protein to selectively promote midbody localization and activity of cul3klhl21 protein complex during abscission
Journal of Biological Chemistry, 2017Co-Authors: Guochang Huang, Andrew J Kaufman, Katia Manova, Bhuvanesh SinghAbstract:Squamous cell carcinoma–related oncogene (SCCRO)/DCUN1D1, a component of the Neddylation E3 complex, regulates the activity of the cullin–RING–ligase type of ubiquitination E3s by promoting Neddylation of cullin family members. Studies have shown that SCCRO regulates proliferation in vitro and in vivo. Here we show that inactivation of SCCRO results in prolonged mitotic time because of delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in Neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates the effects of SCCRO on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission, and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting that it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of an Aurora B inhibitor at the midbody stage suggests that Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting that SCCRO selectively, rather than collectively, neddylates cullins in vivo. Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of Neddylation and cullin–RING–ligase activity in vivo.
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squamous cell carcinoma related oncogene sccro family members regulate cell growth and proliferation through their cooperative and antagonistic effects on cullin Neddylation
Journal of Biological Chemistry, 2016Co-Authors: Joanne Sun, Guochang Huang, Andrew Kaufman, Jeffrey C Liu, Russell J H Ryan, Suresh Y Ramanathan, Tadmiri Venkatesh, Bhuvanesh SinghAbstract:SCCRO (squamous cell carcinoma-related oncogene; also known as DCUN1D1) is a highly conserved gene that functions as an E3 in Neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO(-/-) mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led us to assess whether compensation by SCCRO paralogues rescues lethality in SCCRO(-/-) mice. Using murine and Drosophila models, we assessed the in vivo activities of SCCRO and its paralogues in cullin Neddylation. We found that SCCRO family members have overlapping and antagonistic activity that regulates Neddylation and cell proliferation activities in vivo. In flies, both dSCCRO and dSCCRO3 promote Neddylation and cell proliferation, whereas dSCCRO4 negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones with a net decrease in Neddylation activity. We found that dSCCRO and, to a lesser extent, dSCCRO3 rescue the Neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promote, and human SCCRO3 inhibits, Neddylation and proliferation when expressed in flies. Our findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate Neddylation and cell proliferation.
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SCCRO (DCUN1D1) Promotes Nuclear Translocation and Assembly of the Neddylation E3 Complex
Journal of Biological Chemistry, 2011Co-Authors: Guochang Huang, Andrew Kaufman, Y. Ramanathan, Bhuvanesh SinghAbstract:SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin Neddylation 1 domain containing 1/defective in cullin Neddylation) serves as an accessory E3 in Neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin Neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased Neddylation and proliferation in SCCRO−/− mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, Neddylation, and defective proliferation in SCCRO−/− mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and Neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed Neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote Neddylation. These findings suggest that SCCRO has an essential role in Neddylation in vivo involving nuclear localization of Neddylation components and recruitment and proper positioning of Ubc12.
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sccro dcun1d1 is an essential component of the e3 complex for Neddylation
Journal of Biological Chemistry, 2008Co-Authors: Alexander Y Kim, Guochang Huang, Andrew Kaufman, Claire C Bommelje, Benjamin Lee, Yoshihiro Yonekawa, Lydia Choi, Luc G T Morris, Russel J H RyanAbstract:Covalent modification of cullins by the ubiquitin-like protein NEDD8 (Neddylation) regulates protein ubiquitination by promoting the assembly of cullin-RING ligase E3 complexes. Like ubiquitination, Neddylation results from an enzymatic cascade involving the sequential activity of a dedicated E1 (APPBP1/Uba3), E2 (Ubc12), and an ill-defined E3. We show that SCCRO (also known as DCUN1D1) binds to the components of the Neddylation pathway (Cullin-ROC1, Ubc12, and CAND1) and augments but is not required for cullin Neddylation in reactions using purified recombinant proteins. We also show that SCCRO recruits Ubc12∼NEDD8 to the CAND1-Cul1-ROC1 complex but that this is not sufficient to dissociate or overcome the inhibitory effects of CAND1 on cullin Neddylation in purified protein assays. In contrast to findings in cellular systems where no binding is seen, we show that SCCRO and CAND1 can bind to the neddylated Cul1-ROC1 complex in assays using purified recombinant proteins. Although neddylated (not unneddylated) Cul1-ROC1 is released from CAND1 upon incubation with testis lysate from SCCRO+/+ mice, the addition of recombinant SCCRO is required to achieve the same results in lysate from SCCRO–/– mice. Combined, these results suggest that SCCRO is an important component of the Neddylation E3 complex that functions to recruit charged E2 and is involved in the release of inhibitory effects of CAND1 on cullin-RING ligase E3 complex assembly and activity.
Matthias Peter - One of the best experts on this subject based on the ideXlab platform.
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The TFIIH subunit Tfb3 regulates cullin Neddylation.
Molecular Cell, 2011Co-Authors: Gwénaël Rabut, Gaëlle Le Dez, Rati Verma, Taras Makhnevych, Axel Knebel, Thimo Kurz, Charles Boone, Raymond Deshaies, Matthias PeterAbstract:Cullin proteins are scaffolds for the assembly of multisubunit ubiquitin ligases, which ubiquitylate a large number of proteins involved in widely varying cellular functions. Multiple mechanisms cooperate to regulate cullin activity, including Neddylation of their C-terminal domain. Interestingly, we found that the yeast Cul4-type cullin Rtt101 is not only neddylated but also ubiquitylated, and both modifications promote Rtt101 function in vivo. Surprisingly, proper modification of Rtt101 neither correlated with catalytic activity of the RING domain of Hrt1 nor required the Nedd8 ligase Dcn1. Instead, ubiquitylation of Rtt101 was dependent on the ubiquitin-conjugating enzyme Ubc4, while efficient Neddylation involves the RING domain protein Tfb3, a subunit of the transcription factor TFIIH. Tfb3 also controls Cul3 Neddylation and activity in vivo, and physically interacts with Ubc4 and the Nedd8-conjugating enzyme Ubc12 and the Hrt1/Rtt101 complex. Together, these results suggest that the conserved RING domain protein Tfb3 controls activation of a subset of cullins.
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cullin Neddylation and substrate adaptors counteract scf inhibition by the cand1 like protein lag2 in saccharomyces cerevisiae
The EMBO Journal, 2009Co-Authors: Edyta Siergiejuk, Thimo Kurz, Kay Hofmann, Daniel C Scott, Brenda A Schulman, Matthias PeterAbstract:Cullin-based E3 ubiquitin ligases are activated through covalent modification of the cullin subunit by the ubiquitin-like protein Nedd8. Cullin Neddylation dissociates the ligase assembly inhibitor Cand1, and promotes E2 recruitment and ubiquitin transfer by inducing a conformational change. Here, we have identified and characterized Lag2 as a likely Saccharomyces cerevisiae orthologue of mammalian Cand1. Similar to Cand1, Lag2 directly interacts with non-neddylated yeast cullin Cdc53 and prevents its Neddylation in vivo and in vitro. Binding occurs through a conserved C-terminal β-hairpin structure that inserts into the Skp1-binding pocket on the cullin, and an N-terminal motif that covers the Neddylation lysine. Interestingly, Lag2 is itself neddylated in vivo on a lysine adjacent to this N-terminal-binding site. Overexpression of Lag2 inhibits Cdc53 activity in strains defective for Skp1 or Neddylation functions, implying that these activities are important to counteract Lag2 in vivo. Our results favour a model in which binding of substrate-specific adaptors triggers release of Cand1/Lag2, whereas subsequent Neddylation of the cullin facilitates the removal and prevents re-association of Lag2/Cand1.
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the human dcn1 like protein dcnl3 promotes cul3 Neddylation at membranes
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Nathalie Meyerschaller, Thimo Kurz, Kay Hofmann, Yangchieh Chou, Frank Sicheri, Izabela Sumara, Dale D O Martin, Nadja Katheder, Luc G Berthiaume, Matthias PeterAbstract:Cullin (Cul)-based E3 ubiquitin ligases are activated through the attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective in Cul Neddylation 1 protein) functions as a scaffold-like Nedd8 E3-ligase by interacting with its Cul substrates and the Nedd8 E2 Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each containing a C-terminal potentiating Neddylation domain but distinct amino-terminal extensions. Although the UBA-containing DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, DCNL3 also interacts with human Culs and is able to complement the Neddylation defect of yeast dcn1Δ cells. DCNL3 down-regulation by RNAi decreases Cul Neddylation, and overexpression of a Cul3 mutant deficient in DCNL3 binding interferes with Cul3 function in vivo. Interestingly, DCNL3 accumulates at the plasma membrane through a conserved, lipid-modified motif at the N terminus. Membrane-bound DCNL3 is able to recruit Cul3 to membranes and is functionally important for Cul3 Neddylation in vivo. We conclude that DCNL proteins function as nonredundant Cul Nedd8-E3 ligases. Moreover, the diversification of the N termini in mammalian Dcn1 homologues may contribute to substrate specificity by regulating their subcellular localization.
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function and regulation of protein Neddylation
EMBO Reports, 2008Co-Authors: Gwénaël Rabut, Matthias PeterAbstract:Neddylation is the post-translational protein modification that is most closely related to ubiquitination. However, ubiquitination is known to regulate a myriad of processes in eukaryotic cells, whereas only a limited number of Neddylation substrates have been described to date. Here, we review the principles of protein Neddylation and highlight the mechanisms that ensure the specificity of Neddylation over ubiquitination. As numerous Neddylation substrates probably remain to be discovered, we propose some criteria that could be used as guidelines for the characterization of neddylated proteins.
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dcn1 functions as a scaffold type e3 ligase for cullin Neddylation
Molecular Cell, 2008Co-Authors: Thimo Kurz, Matthias Peter, Andrew Willems, Yangchieh Chou, Nathalie Meyerschaller, Marielyn Hecht, Mike Tyers, Frank SicheriAbstract:Cullin-based E3 ubiquitin ligases are activated through modification of the cullin subunit with the ubiquitin-like protein Nedd8. Dcn1 regulates cullin Neddylation and thus ubiquitin ligase activity. Here we describe the 1.9 A X-ray crystal structure of yeast Dcn1 encompassing an N-terminal ubiquitin-binding (UBA) domain and a C-terminal domain of unique architecture, which we termed PONY domain. A conserved surface on Dcn1 is required for direct binding to cullins and for Neddylation. The reciprocal binding site for Dcn1 on Cdc53 is located ∼18 A from the site of Neddylation. Dcn1 does not require cysteine residues for catalytic function, and directly interacts with the Nedd8 E2 Ubc12 on a surface that overlaps with the E1-binding site. We show that Dcn1 is necessary and sufficient for cullin Neddylation in a purified recombinant system. Taken together, these data demonstrate that Dcn1 is a scaffold-like E3 ligase for cullin Neddylation.
Andrew Kaufman - One of the best experts on this subject based on the ideXlab platform.
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squamous cell carcinoma related oncogene sccro family members regulate cell growth and proliferation through their cooperative and antagonistic effects on cullin Neddylation
Journal of Biological Chemistry, 2016Co-Authors: Joanne Sun, Guochang Huang, Andrew Kaufman, Jeffrey C Liu, Russell J H Ryan, Suresh Y Ramanathan, Tadmiri Venkatesh, Bhuvanesh SinghAbstract:SCCRO (squamous cell carcinoma-related oncogene; also known as DCUN1D1) is a highly conserved gene that functions as an E3 in Neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO(-/-) mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led us to assess whether compensation by SCCRO paralogues rescues lethality in SCCRO(-/-) mice. Using murine and Drosophila models, we assessed the in vivo activities of SCCRO and its paralogues in cullin Neddylation. We found that SCCRO family members have overlapping and antagonistic activity that regulates Neddylation and cell proliferation activities in vivo. In flies, both dSCCRO and dSCCRO3 promote Neddylation and cell proliferation, whereas dSCCRO4 negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones with a net decrease in Neddylation activity. We found that dSCCRO and, to a lesser extent, dSCCRO3 rescue the Neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promote, and human SCCRO3 inhibits, Neddylation and proliferation when expressed in flies. Our findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate Neddylation and cell proliferation.
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SCCRO (DCUN1D1) Promotes Nuclear Translocation and Assembly of the Neddylation E3 Complex
Journal of Biological Chemistry, 2011Co-Authors: Guochang Huang, Andrew Kaufman, Y. Ramanathan, Bhuvanesh SinghAbstract:SCCRO/DCUN1D1/DCN1 (squamous cell carcinoma-related oncogene/defective in cullin Neddylation 1 domain containing 1/defective in cullin Neddylation) serves as an accessory E3 in Neddylation by binding to cullin and Ubc12 to allow efficient transfer of Nedd8. In this work we show that SCCRO has broader, pleiotropic effects that are essential for cullin Neddylation in vivo. Reduced primary nuclear localization of Cul1 accompanying decreased Neddylation and proliferation in SCCRO−/− mouse embryonic fibroblasts led us to investigate whether compartmentalization plays a regulatory role. Decreased nuclear localization, Neddylation, and defective proliferation in SCCRO−/− mouse embryonic fibroblasts were rescued by transgenic expression of SCCRO. Expression of reciprocal SCCRO and Cul1-binding mutants confirmed the requirement for SCCRO in nuclear translocation and Neddylation of cullins in vivo. Nuclear translocation of Cul1 by tagging with a nuclear localization sequence allowed Neddylation independent of SCCRO, but at a lower level. We found that in the nucleus, SCCRO enhances recruitment of Ubc12 to Cul1 to promote Neddylation. These findings suggest that SCCRO has an essential role in Neddylation in vivo involving nuclear localization of Neddylation components and recruitment and proper positioning of Ubc12.
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sccro dcun1d1 is an essential component of the e3 complex for Neddylation
Journal of Biological Chemistry, 2008Co-Authors: Alexander Y Kim, Guochang Huang, Andrew Kaufman, Claire C Bommelje, Benjamin Lee, Yoshihiro Yonekawa, Lydia Choi, Luc G T Morris, Russel J H RyanAbstract:Covalent modification of cullins by the ubiquitin-like protein NEDD8 (Neddylation) regulates protein ubiquitination by promoting the assembly of cullin-RING ligase E3 complexes. Like ubiquitination, Neddylation results from an enzymatic cascade involving the sequential activity of a dedicated E1 (APPBP1/Uba3), E2 (Ubc12), and an ill-defined E3. We show that SCCRO (also known as DCUN1D1) binds to the components of the Neddylation pathway (Cullin-ROC1, Ubc12, and CAND1) and augments but is not required for cullin Neddylation in reactions using purified recombinant proteins. We also show that SCCRO recruits Ubc12∼NEDD8 to the CAND1-Cul1-ROC1 complex but that this is not sufficient to dissociate or overcome the inhibitory effects of CAND1 on cullin Neddylation in purified protein assays. In contrast to findings in cellular systems where no binding is seen, we show that SCCRO and CAND1 can bind to the neddylated Cul1-ROC1 complex in assays using purified recombinant proteins. Although neddylated (not unneddylated) Cul1-ROC1 is released from CAND1 upon incubation with testis lysate from SCCRO+/+ mice, the addition of recombinant SCCRO is required to achieve the same results in lysate from SCCRO–/– mice. Combined, these results suggest that SCCRO is an important component of the Neddylation E3 complex that functions to recruit charged E2 and is involved in the release of inhibitory effects of CAND1 on cullin-RING ligase E3 complex assembly and activity.
