The COP9 signalosome-mediated deneddylation is stimulated by caspases during apoptosis

In concert with the ubiquitin (Ub) proteasome system (UPS) the COP9 signalosome (CSN) controls the stability of cellular regulators. The CSN interacts with cullin-RING Ub ligases (CRLs) consisting of a specific cullin, a RING protein as Rbx1 and substrate recognition proteins. The Ub-like protein Nedd8 is covalently linked to cullins and removed by the CSN-mediated deneddylation. Cycles of neddylation and deneddylation regulate CRLs. Apoptotic stimuli cause caspase-dependent modifications of the UPS. However, little is known about the CSN during apoptosis. We demonstrate in vitro and in vivo that CSN6 is cleaved most effectively by caspase 3 at D₂₃ after 2–3 h of apoptosis induced by anti-Fas-Ab or etoposide. CSN6 processing occurs in CSN–CRL complexes and is followed by the cleavage of Rbx1, the direct interaction partner of CSN6. Caspase-dependent cutting of Rbx1 is accompanied by decrease of neddylated proteins in Jurkat T cells. Another functional consequence of CSN6 cleavage is the enhancement of CSN-mediated deneddylating activity causing deneddylation of cullin 1 in cells. The CSN-associated deubiquitinating as well as kinase activity remained unchanged in presence of active caspase 3. The cleavage of Rbx1 and increased deneddylation of cullins inactivate CRLs and presumably stabilize pro-apoptotic factors for final apoptotic steps. Bettina K. J. Hetfeld and Andreas Peth contributed equally.     

Consequences of COP9 signalosome and 26S proteasome interaction

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr-Pad1-N-terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag-CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull-down experiments revealed the formation of an intact Flag-CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull-downs also precipitated cullins indicating the existence of super-complexes consisting of the CSN, the 26S proteasome and cullin-based Ub ligases. Permanent expression of a chimerical subunit (Flag-CSN2-Rpn6) consisting of the N-terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag-CSN2 overexpression was de-novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c-Jun in B8 cells. The possible role of super-complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.     

Post-transcriptional fine-tuning of COP9 signalosome subunit biosynthesis is regulated by the c-Myc/Lin28B/let-7 pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 - CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.     

The COP9 Signalosome Mediates β-Catenin Degradation by Deneddylation and Blocks Adenomatous Polyposis coli Destruction via USP15

The Wnt/β-catenin signalling pathway has important roles in normal cellular proliferation, development and angiogenesis. Many malignant transformations, including sporadic colorectal tumours, are caused by constitutive activation of the Wnt route due to mutations in the tumour suppressor protein adenomatous polyposis coli (APC) or the β-catenin oncogene, ultimately resulting in reduced β-catenin degradation by the ubiquitin (Ub) proteasome system (UPS). The COP9 signalosome (CSN) regulates the UPS by controlling cullin-RING Ub ligases (CRLs). We show here that the CSN and the β-catenin destruction complex cooperate in targeting β-catenin for degradation by the UPS. Together with the CRL that ubiquitinates β-catenin, they form a supercomplex responsible for β-catenin degradation. Wnt3A, glycogen synthase kinase 3β inhibitors or mutation of CSN-mediated deneddylation induce the disassembly of the supercomplex and the accumulation of β-catenin. Likewise, downregulation of the CSN in HeLa cells leads to retarded degradation of β-catenin. Additionally, we found that the knockdown of the CSN causes accelerated proteolysis of APC, an essential component of the β-catenin destruction complex, which is degraded by the UPS as β-catenin. We show here that APC is stabilised by the Ub-specific protease 15 (USP15) associated with the CSN. This is demonstrated by over-expression of siRNA oligonucleotides against USP15 or by over-expression of an USP15 mutant, which is unable to degrade poly-Ub chains. Thus, the CSN controls the Wnt/β-catenin signalling by assisting the assembly of β-catenin-degrading supercomplexes by deneddylation and, simultaneously, by stabilising APC via CSN-associated USP15. The CSN regulates the balance between β-catenin and APC. Disturbance of this balance can cause cancer by driving cell transformation, tumour angiogenesis and metastasis. A model is provided that proposes a role of CSN-mediated deneddylation in the formation of the β-catenin-degrading supercomplex and the protection of complex-bound APC via CSN-associated USP15.     

COP9 Signalosome Interacts ATP-dependently with p97/Valosin-containing Protein (VCP) and Controls the Ubiquitination Status of Proteins Bound to p97/VCP

Ubiquitinated proteins can alternatively be delivered directly to the proteasome or via p97/VCP (valosin-containing protein). Whereas the proteasome degrades ubiquitinated proteins, the homohexameric ATPase p97/VCP seems to control the ubiquitination status of recruited substrates. The COP9 signalosome (CSN) is also involved in the ubiquitin/proteasome system (UPS) as exemplified by regulating the neddylation of ubiquitin E3 ligases. Here, we show that p97/VCP colocalizes and directly interacts with subunit 5 of the CSN (CSN5) in vivo and is associated with the entire CSN complex in an ATP-dependent manner. Furthermore, we provide evidence that the CSN and in particular the isopeptidase activity of its subunit CSN5 as well as the associated deubiquitinase USP15 are required for proper processing of polyubiquitinated substrates bound to p97/VCP. Moreover, we show that in addition to NEDD8, CSN5 binds to oligoubiquitin chains in vitro. Therefore, CSN and p97/VCP could form an ATP-dependent complex that resembles the 19 S proteasome regulatory particle and serves as a key mediator between ubiquitination and degradation pathways.     

Ubiquitin-dependent proteolysis of the microtubule end-binding protein 1, EB1, is controlled by the COP9 signalosome: possible consequences for microtubule filament stability

The COP9 signalosome (CSN) is a regulatory particle of the ubiquitin (Ub) proteasome system (UPS) consisting of eight subunits (CSN1-CSN8). We show that the CSN stabilizes the microtubule end-binding protein 1 (EB1) towards degradation by the UPS. EB1, the master regulator of microtubule plus ends, controls microtubule growth and dynamics. Therefore, regulation of EB1 stability by the CSN has consequences for microtubule function. EB1 binds the CSN via subunit CSN5. The C terminus of EB1 is sufficient for interaction with the CSN. Dimerization of EB1 is a prerequisite for complex association and subsequent CSN-mediated phosphorylation, as revealed by studies with the EB1I224A mutant, which is unable to dimerize. In cells, EB1 and CSN co-localize to the centrosome, as demonstrated by confocal fluorescence microscopy. EB1 is ubiquitinated and its proteolysis can be inhibited by MG132, demonstrating that it is a substrate of the UPS. Its degradation is accelerated by inhibition of CSN-associated kinases. HeLa cells permanently expressing siRNAs against CSN1 (siCSN1) or CSN3 (siCSN3) exhibit reduced levels of the CSN complex accompanied by lower steady-state concentrations of EB1. In siCSN1 cells, EB1 is less phosphorylated as compared with control cells, demonstrating that the protein is most likely protected towards the UPS by CSN-mediated phosphorylation. The CSN-dependent EB1 stabilization is not due to the CSN-associated deubiquitinating enzyme USP15. Treatment with nocodazole revealed a significantly increased sensitivity of siCSN1 and siCSN3 cells towards the microtubule depolymerizing drug accompanied by a collapse of microtubule filaments. A nocodazole-induced cell-cycle arrest was partially rescued by CSN1 or EB1. These data demonstrate that the CSN-dependent protection of EB1 is important for microtubule function.     

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.     

Interaction Between Syntaxin 8 and HECTd3, a HECT Domain Ligase

Ubiquitination of proteins and their degradation within the proteasome has emerged as the major proteolytic mechanism used by mammalian cells to regulate cytosolic and nuclear protein levels. Substrate ubiquitylation is mediated by ubiquitin (Ub) ligases, also called E3 Ub ligases. HECT-E3 Ub ligases are characterized by the presence of a C-terminal HECT domain that contains the active site for Ub transfer onto substrates. Among the many E3 Ub ligases, the family homologous to E6-Ap C-terminus (HECT) E3 Ub ligases, which includes the yeast protein Rsp5p and the mammalian homolog NEDD4, AIP4/Itch, and Smurf, has been shown to ubiquitylate membrane proteins and, in some instances, to induce their degradation. In this report, we have identified Syntaxin 8 as a binding protein to a novel HECT domain protein, HECT domain containing 3 (HECTd3), by yeast two-hybrid screen. Besides HECT domain, HECTd3 contains an anaphase-promoting complex, subunit 10 (APC10) domain. Our co-immunoprecipitation experiments show that Syntaxin 8 directly interacts with HECTd3 and that the overexpression of HECTd3 promotes the ubiquitination of Syntaxin 8. Immunofluorescence results show that Syntaxin 8 and HECTd3 have similar subcellular localization.     

TRIB2 modulates proteasome function to reduce ubiquitin stability and protect liver cancer cells against oxidative stress

The regulation of homeostasis in the Ubiquitin (Ub) proteasome system (UPS) is likely to be important for the development of liver cancer. Tribbles homolog 2 (TRIB2) is known to affect Ub E3 ligases (E3s) in liver cancer. However, whether TRIB2 regulates the UPS in other ways and the relevant mechanisms are still unknown. Here, we reveal that TRIB2 decreased Ub levels largely by stimulating proteasome degradation of Ub. In the proteasome, proteasome 20S subunit beta 5 (PSMB5) was critical for the function of TRIB2, although it did not directly interact with TRIB2. However, poly (rC) binding protein 2 (PCBP2), which was identified by mass spectrometry, directly interacted with both TRIB2 and PSMB5. PCBP2 was a prerequisite for the TRIB2 induction of PSMB5 activity and decreased Ub levels. A significant correlation between TRIB2 and PCBP2 was revealed in liver cancer specimens. Interestingly, TRIB2 suppressed the K48-ubiquitination of PCBP2 to increase its level. Therefore, a model showing that TRIB2 cooperates and stimulates PCBP2 to reduce Ub levels was established. Additionally, the reduction in Ub levels induced by TRIB2 and PCBP2 was dependent on K48-ubiquitination. PCBP2 was one of the possible downstream factors of TRIB2 and their interaction relied on the DQLVPD element of TRIB2 and the KH3 domain of PCBP2. This interaction was necessary to maintain the viability of the liver cancer cells and promote tumor growth. Mechanistically, glutathione peroxidase 4 functioned as one of the terminal effectors of TRIB2 and PCBP2 to protect liver cancer cells from oxidative damage. Taken together, the data indicate that, in addition to affecting E3s, TRIB2 plays a critical role in regulating UPS by modulating PSMB5 activity in proteasome to reduce Ub flux, and that targeting TRIB2 might be helpful in liver cancer treatments by enhancing the oxidative damage induced by therapeutic agents.Subject terms: Oncogenes, Cancer     

F-box proteins: the key to protein degradation

Summary The eukaryotic protein degradation pathway involves the ubiquitin (Ub) modification of substrates targeted for degradation by the 26S proteasome. The addition of Ub, a process called ubiquitination, is mediated by enzymes including the E3 Ub ligases which transfer the Ub to targeted substrates. A major type of E3 Ub ligases, the SCF (Skp–Cullin–F-box) complex, is composed of four major components: Skp1, Cul1/Cdc53, Roc1/Rbx1/Hrt1, and an F-box protein. The F-box component of the SCF machineries is responsible for recognizing different substrates for ubiquitination. Interaction with components of the SCF complex is mediated through the F-box motif of the F-box protein while it associates with phosphorylated substrates through its second protein–protein interaction motif such as Trp–Asp (WD) repeats or leucine-rich repeats (LRRs). By targeting diverse substrates, F-box proteins exert controls over stability of proteins and regulate the mechanisms for a wide-range of cellular processes. Here we discuss the importance of F-box proteins by providing a general overview and examples of how F-box proteins function in various cellular settings such as tissue development, cell proliferation, and cell death, in the modeling organism Drosophila.     

泛素/26S蛋白酶体途径与显花植物自交不亲和反应

植物的生长和发育离不开短命调控蛋白的有选择性降解, 其中一种重要的降解方式就是泛素/26S蛋白酶体途径。在这个途径中, 泛素(ubiquitin)和26S蛋白酶体起着至关重要的作用, 需要被降解的蛋白会通过E1-E2-E3酶接合反应由Ub进行标记, 随后标记蛋白会被26S蛋白酶体识别并降解。自交不亲和反应也正是通过此途径实现的, ARC1(arm repeat containing 1)和SCFs (skp1-cul1-F-box-proteins)作为E3s分别在孢子体自交不亲和和配子体自交不亲和反应中起作用。本文综述了就泛素/26S蛋白酶体途径的组成及其在自交不亲和反应中的作用。     

泛素/26S蛋白酶体途径与显花植物自交不亲和反应

植物的生长和发育离不开短命调控蛋白的有选择性降解,其中一种重要的降解方式就是泛素,26S蛋白酶体途径。在这个途径中,泛素（ubiquitin）和26S蛋白酶体起着至关重要的作用,需要被降解的蛋白会通过E1-E2-E3酶接合反应由Ub进行标记,随后标记蛋白会被26s蛋白酶体识别并降解。自交不亲和反应也正是通过此途径实现的,ARC1（arm repeat containing 1）和SCFs（skp1-cul1-F-box-proteins）作为E3s分别在孢子体自交不亲和和配子体自交不亲和反应中起作用。本文综述了就泛素/26S蛋白酶体途径的组成及其在自交不亲和反应中的作用。     

COP9 signalosome function in the DDR

The COP9 signalosome (CSN) is a platform for protein communication in eukaryotic cells. It has an intrinsic metalloprotease that removes the ubiquitin (Ub)-like protein Nedd8 from cullins. CSN-mediated deneddylation regulates culling-RING Ub ligases (CRLs) and controls ubiquitination of proteins involved in DNA damage response (DDR). CSN forms complexes with CRLs containing cullin 4 (CRL4s) which act on chromatin playing crucial roles in DNA repair, checkpoint control and chromatin remodeling. Furthermore, via associated kinases the CSN controls the stability of DDR effectors such as p53 and p27 and thereby the DDR outcome. DDR is a protection against cancer and deregulation of CSN function causes cancer making it an attractive pharmacological target. Here we review current knowledge on CSN function in DDR.     

The RAD23 Family Provides an Essential Connection between the 26S Proteasome and Ubiquitylated Proteins in Arabidopsis

The ubiquitin (Ub)/26S proteasome system (UPS) directs the turnover of numerous regulatory proteins, thereby exerting control over many aspects of plant growth, development, and survival. The UPS is directed in part by a group of Ub-like/Ub-associated (UBL/UBA) proteins that help shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Here, we describe the collection of UBL/UBA proteins in Arabidopsis thaliana, including four isoforms that comprise the RADIATION SENSITIVE23 (RAD23) family. The nuclear-enriched RAD23 proteins bind Ub conjugates, especially those linked internally through Lys-48, via their UBA domains, and associate with the 26S proteasome Ub receptor RPN10 via their N-terminal UBL domains. Whereas homozygous mutants individually affecting the four RAD23 genes are without phenotypic consequences (rad23a, rad23c, and rad23d) or induce mild phyllotaxy and sterility defects (rad23b), higher-order mutant combinations generate severely dwarfed plants, with the quadruple mutant displaying reproductive lethality. Both the synergistic effects of a rad23b-1 rpn10-1 combination and the response of rad23b plants to mitomycin C suggest that RAD23b regulates cell division. Taken together, RAD23 proteins appear to play an essential role in the cell cycle, morphology, and fertility of plants through their delivery of UPS substrates to the 26S proteasome.     

Hepatic Deficiency of COP9 Signalosome Subunit 8 Induces Ubiquitin-Proteasome System Impairment and Bim-Mediated Apoptosis in Murine Livers

The COP9 signalosome (CSN), an evolutionally highly conserved protein complex composed of 8 unique subunits (CSN1 through CSN8) in higher eukaryotes, is purported to modulate protein degradation mediated by the ubiquitin-proteasome system (UPS) but this has not been demonstrated in a critical mitotic parenchymal organ of vertebrates. Hepatocyte-specific knockout of the Cops8 gene (HS-Csn8KO) was shown to cause massive hepatocyte apoptosis and liver malfunction but the underlying mechanism remains unclear. Here, we report that Csn8/CSN exerts profound impacts on hepatic UPS function and is critical to the stability of the pro-apoptotic protein Bim. Significant decreases in CIS (cytokine-inducible Src homology 2 domain-containing protein), a Bim receptor of a cullin2-based ubiquitin ligase, were found to co-exist with a marked increase of Bim proteins. Csn8 deficiency also significantly decreased 19S proteasome subunit Rpt5 and markedly increased high molecular weight neddylated and ubiquitinated proteins. The use of a surrogate UPS substrate further reveals severe impairment of UPS-mediated proteolysis in HS-Csn8KO livers. Inclusion body-like materials were accumulated in Csn8 deficient hepatocytes. In addition to Bim, massive hepatocyte apoptosis in HS-Csn8KO livers is also associated with elevated expression of other members of the Bcl2 family, including pro-apoptotic Bax as well as anti-apoptotic Bcl2 and Bcl-XL. Increased interaction between Bcl2 and Bim, but not between Bcl2 and Bax, was detected. Hence, it is concluded that hepatic CSN8 deficiency impairs the UPS in the liver and the resultant Bim upregulation likely plays an important role in triggering hepatocyte apoptosis via sequestering Bcl2 away from Bax.     

The cyclophilin-like domain of Ran-binding protein-2 modulates selectively the activity of the ubiquitin-proteasome system and protein biogenesis

The ubiquitin-proteasome system (UPS) plays a critical role in protein degradation. The 19S regulatory particle (RP) of the 26S proteasome mediates the recognition, deubiquitylation, unfolding, and channeling of ubiquitylated substrates to the 20S proteasome. Several subunits of the 19S RP interact with a growing number of factors. The cyclophilin-like domain (CLD) of Ran-binding protein-2 (RanBP2/Nup358) associates specifically with at least one subunit, S1, of the base subcomplex of the 19S RP, but the functional implications of this interaction on the UPS activity are elusive. This study shows the CLD of RanBP2 promotes selectively the accumulation of a subset of reporter substrates of the UPS, such as the ubiquitin (Ub)-fusion yellow fluorescent protein (YFP) degradation substrate, Ub(G76V)-YFP, and the N-end rule substrate, Ub-R-YFP. Conversely, the degradation of endoplasmic reticulum and misfolded proteins, and of those linked to UPS-independent degradation, is not affected by CLD. The selective effect of CLD on the UPS in vivo is independent of, and synergistic with, proteasome inhibitors, and CLD does not affect the intrinsic proteolytic activity of the 20S proteasome. The inhibitory activity of CLD on the UPS resides in a purported SUMO binding motif. We also found two RanBP2 substrates, RanGTPase-activating protein and retinitis pigmentosa GTPase regulator interacting protein-1alpha1, whose steady-state levels are selectively modulated by CLD. Hence, the CLD of RanBP2 acts as a novel auxiliary modulator of the UPS activity; it may contribute to the molecular and subcellular compartmentation of the turnover of properly folded proteins and modulation of the expressivity of several neurological diseases.     

Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipitation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to DeltaCSN3(111-403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun-Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation.