CSN5i-3 induces degradation of selective CRL substrate receptors and accumulation of CRL substrates
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
DISEASE(S): Chronic Myeloid Leukemia
SUBMITTER: 
Brett Lomenick
LAB HEAD: Spiros D Garbis
PROVIDER: PXD027862 | Pride | 2022-09-05
REPOSITORIES: Pride
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Project description:The COP9 signalosome (CSN) is a conserved protein complex occurring in all eukaryotes. The mammalian CSN consists of eight subunits (CSN1 – CSN8) and possesses an intrinsic metalloprotease JAMM motif localised to CSN5. In addition, it is associated with kinases such as protein kinase CK2 and D and the ubiquitin (Ub) specific protease 15. It regulates cullin-RING Ub ligases (CRLs) that label proteins for proteolysis with poly-Ub chains in the Ub proteasome system (UPS). CRLs contain one of the scaffolding cullins 1 – 7, a RING domain protein e. g. Rbx1, and one of hundreds of substrate binding units such as F-box or BTB proteins that specifically target proteins for ubiquitination. The CSN forms functional supercomplexes with CRLs. It removes the Ub-like protein Nedd8 from cullins by its metalloprotease activity and thereby inactivates CRLs. On the other hand, it is essential for CRL function by protecting components and allowing reassembly of the complexes. As a regulator of the UPS the CSN is involved in cell cycle10, DNA repair and development. Overexpression of CSN subunits has been reported in tumour cells. Up to date nothing is known about CSN biogenesis and its regulation. Ten years ago we have observed that overexpression of selected CSN subunits caused a coordinated increase of all CSN subunits and a de novo biosynthesis of the entire complex. Here we show that overexpression of CSN1 mediates an increase of c-Myc which coordinates CSN subunit expression via microRNAs (miRNAs). miRNAs are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides that function as posttranscriptional gene regulators. We found that inhibitors of let-7 family miRNAs or upregulation of c-Myc, which represses let-7 miRNAs, resulted in a coordinated increase of CSN subunits and de novo CSN biogenesis. Keywords: Gene expression analysis of human RNA samples using topic-defined PIQOR™ Ubiquitin-PS Microarrays. Two different platforms were used (these are two different microarray batches which are identical in terms of the spotted cDNAs, but differ in the position of some cDNAs) Gene expression analysis of Hela cells overexpressing either CSN2 or CSN1 was performed to study CSN biogenesis and its regulation. Curcumin, Nr. 8 (Curcumin analogon) and Piceatannol were used for the treatment of Hela cells since they inhibit the CSN associated kinases. The aim was to investigate the effect on the signalosome. For each treatment 50 µM Curcumin, Nr. 8 and Piceatannol were used.
2011-10-26 | E-GEOD-19085 | biostudies-arrayexpress
Project description:The COP9 signalosome (CSN) is a conserved protein complex occurring in all eukaryotes. The mammalian CSN consists of eight subunits (CSN1 – CSN8) and possesses an intrinsic metalloprotease JAMM motif localised to CSN5. In addition, it is associated with kinases such as protein kinase CK2 and D and the ubiquitin (Ub) specific protease 15. It regulates cullin-RING Ub ligases (CRLs) that label proteins for proteolysis with poly-Ub chains in the Ub proteasome system (UPS). CRLs contain one of the scaffolding cullins 1 – 7, a RING domain protein e. g. Rbx1, and one of hundreds of substrate binding units such as F-box or BTB proteins that specifically target proteins for ubiquitination. The CSN forms functional supercomplexes with CRLs. It removes the Ub-like protein Nedd8 from cullins by its metalloprotease activity and thereby inactivates CRLs. On the other hand, it is essential for CRL function by protecting components and allowing reassembly of the complexes. As a regulator of the UPS the CSN is involved in cell cycle10, DNA repair and development. Overexpression of CSN subunits has been reported in tumour cells. Up to date nothing is known about CSN biogenesis and its regulation. Ten years ago we have observed that overexpression of selected CSN subunits caused a coordinated increase of all CSN subunits and a de novo biosynthesis of the entire complex. Here we show that overexpression of CSN1 mediates an increase of c-Myc which coordinates CSN subunit expression via microRNAs (miRNAs). miRNAs are evolutionarily conserved, endogenous, small, noncoding RNA molecules of about 22 nucleotides that function as posttranscriptional gene regulators. We found that inhibitors of let-7 family miRNAs or upregulation of c-Myc, which represses let-7 miRNAs, resulted in a coordinated increase of CSN subunits and de novo CSN biogenesis. Keywords: Gene expression analysis of human RNA samples using topic-defined PIQOR™ Ubiquitin-PS Microarrays. Two different platforms were used (these are two different microarray batches which are identical in terms of the spotted cDNAs, but differ in the position of some cDNAs)
2011-10-27 | GSE19085 | GEO
Project description:Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.
2019-09-13 | PXD013001 | Pride
Project description:Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.
2019-09-13 | PXD013018 | Pride
Project description:The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that functions as a deneddylase to inactivate Cullin-RING E3 ligases for controlling protein ubiquitination. CSN possesses structural flexibility that is important for its activation upon binding to a diverse array of CRLs. The canonical and non-canonical CSN complexes consist of 8 (CSN1-8) and 9 (CSN1-9) subunits, respectively. Although CSN9 is not essential for CSN assembly and function, it appears to be important in non-catalytic regulation of CRLs by CSN. Here we employed a combinatory cross-linking mass spectrometry (XL-MS) approach to generate the largest PPI maps of human CSN complexes, which significantly enhanced the precision of integrative structural modeling. The resulting integrative structures allowed us not only to elucidate architectures of both complexes, but also to assess CSN structural dynamics that was not described in the crystal structure. In addition, we have determined CSN9 docking sites and its impact on the CSN structure. While CSN9 binding did not induce global conformational changes, it triggered subunit local reorientations that may be associated with CSN9 involvement in CSN-mediated steric regulation of CRLs.
2020-02-04 | PXD014673 | Pride
Project description:The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We report development of a synthetic antibody recognizing the active conformation of a NEDD8-linked cullin. We established a pipeline probing cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing CRLs, revealing the CRL complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, prime efficiency of targeted protein degradation, and are differentially rewired across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate development of degrader drugs.
2023-09-02 | PXD039649 | Pride
Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles
2009-03-24 | GSE14088 | GEO
Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles Ube2m and Ube2f are E2 enzymes that direct protein modification by NEDD8. Here we explore the specific functions of Ube2f and Ube2m by comparing gene expression profiles following knockdown of their function in NIH 3T3 cells. We used microarrays to detail the global programme of gene expression changes following knockdown of Ube2m and Ube2f in NIH 3T3 cells. NIH 3T3 cells were transduced with retroviral constructs containing shRNA directed against Ube2m or Ube2f. Three replicates of each condition were analyzed.
2009-03-23 | E-GEOD-14088 | biostudies-arrayexpress
Project description:The COP9 signalosome (CSN) is a highly conserved eukaryotic protein complex which regulates the Cullin-RING family of ubiquitin ligases and carries out a deneddylase activity that resides in subunit 5 (CSN5). The budding yeast CSN is biochemically active and deletion mutants of each of its subunits exhibit deficiency in deneddylation of cullins, although the biological context of this activity is still unknown in this organism. To further characterize CSN function in budding yeast, we present here a transcriptomic analysis of a S. cerevisiae strain deleted in CSN5/RRI1 gene (hereafter referred to as CSN5), coding for the only canonical subunit of the complex. We show that Csn5 is involved in the modulation of the genes controlling aminoacid and lipid metabolism, and especially ergosterol biosynthesis. These alterations in gene expression correlate with the lower ergosterol levels and increased intracellular zinc content which we observed in csn5 null mutant cells. Two biological replicates, csn5 deleted strain vs. isogenic wild-type strain W303
2013-10-23 | E-GEOD-51563 | biostudies-arrayexpress
Project description:NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation both in vitro and in cells. Furthermore, we identified SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in engineered SENP8-deficient cells. Importantly, loss of SENP8 function contributes to reduced CRL activity, accumulation of CRL substrates, and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels and CRL-dependent proteostasis, implicating this protease as a potential therapeutic target for inhibiting CRL activity.
2017-04-13 | MSV000080917 | MassIVE