The proteasome lid triggers COP9 signalosome activity during the transition of Saccharomyces cerevisiae cells into quiescence.

The class of Cullin–RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8– Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1–CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53–Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes

A Tobacco Homolog of DCN1 is Involved in Cellular Reprogramming and in Developmental Transitions

Plant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein destruction regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8, and associates with cullin. When knocked down by RNAi, tobacco pollen formation stopped and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1, however, accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is specifically required for transitions during pollen development and embryogenesis, and for pollen tube tip growth

The COP9 SIGNALOSOME is required for postembryonic meristem maintenance in Arabidopsis thaliana

Cullin-RING E3 ligases (CRLs) regulate different aspects of plant development, and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 (NEural precursor cell expressed Developmentally Down-regulated 8) (neddylation) and deactivated by NEDD8 removal (deneddylation). The CONSTITUTIVELY PHOTOMORPHOGENIC9 (COP9) signalosome (CSN) acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis (albeit at a slower pace than wild type) and are able to germinate (albeit at a reduced rate), they progressively loose meristem activity upon germination, until they become unable to sustain growth. We also show that the majority of cullin proteins is progressively neddylated during the late stages of seed maturation and becomes deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis

Obscurin and KCTD6 regulate cullin-dependent small ankyrin-1 (sAnk1.5) protein turnover.

Protein turnover through cullin-3 is tightly regulated by posttranslational modifications, the COP9 signalosome, and BTB/POZ-domain proteins that link cullin-3 to specific substrates for ubiquitylation. In this paper, we report how potassium channel tetramerization domain containing 6 (KCTD6) represents a novel substrate adaptor for cullin-3, effectively regulating protein levels of the muscle small ankyrin-1 isoform 5 (sAnk1.5). Binding of sAnk1.5 to KCTD6, and its subsequent turnover is regulated through posttranslational modification by nedd8, ubiquitin, and acetylation of C-terminal lysine residues. The presence of the sAnk1.5 binding partner obscurin, and mutation of lysine residues increased sAnk1.5 protein levels, as did knockdown of KCTD6 in cardiomyocytes. Obscurin knockout muscle displayed reduced sAnk1.5 levels and mislocalization of the sAnk1.5/KCTD6 complex. Scaffolding functions of obscurin may therefore prevent activation of the cullin-mediated protein degradation machinery and ubiquitylation of sAnk1.5 through sequestration of sAnk1.5/KCTD6 at the sarcomeric M-band, away from the Z-disk-associated cullin-3. The interaction of KCTD6 with ankyrin-1 may have implications beyond muscle for hereditary spherocytosis, as KCTD6 is also present in erythrocytes, and erythrocyte ankyrin isoforms contain its mapped minimal binding site

SCF E3 Ligase Substrates Switch from CAN-D to Can-ubiquitylate

Liu et al. (2018) report a mathematical model predicting how the cellular repertoire of SCF E3 ligases is assembled by “adaptive exchange on demand,” with the limited pool of CUL1 scanning the vast sea of F-box proteins for those with substrates demanding ubiquitylation

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

BACKGROUND We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases

Proteasome Lid Bridges Mitochondrial Stress with Cdc53/Cullin1 NEDDylation Status

Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26 S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis

Neddylation dysfunction in Alzheimer's disease

Ubiquitin-dependent proteolysis is a major mechanism that downregulates misfolded proteins or those that have finished a programmed task. In the last two decades, neddylation has emerged as a major regulatory pathway for ubiquitination. Central to the neddylation pathway is the amyloid precursor protein (APP)-binding protein APP-BP1, which together with Uba3, plays an analogous role to the ubiquitin-activating enzyme E1 in nedd8 activation. Activated nedd8 covalently modifies and activates a major class of ubiquitin ligases called Cullin-RING ligases (CRLs). New evidence suggests that neddylation also modifies Type-1 transmembrane receptors such as APP. Here we review the functions of neddylation and summarize evidence suggesting that dysfunction of neddylation is involved in Alzheimer's disease.National Institutes of Health (U.S.) (NIH/NIA RO1 AG034980