The Essential Functions of NEDD8 Are Mediated via Distinct Surface Regions, and Not by Polyneddylation in Schizosaccharomyces pombe

The ubiquitin-like protein NEDD8 is highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. NEDD8 conjugation to cullin proteins is a prerequisite for cullin based E3 ubiquitin ligase activity, and essential for S. pombe viability. Here, we have performed alanine scanning mutagenesis of all conserved surface residues and show that the majority of essential residues were located around the hydrophobic patch and the C-terminus. However, we further identified essential residues not previously reported to be involved in ubiquitin ligase regulation that importantly do not prevent Ned8p conjugation. We also find that mutation of all conserved lysine residues in Ned8p, did not affect yeast viability, suggesting that mono-neddylation is sufficient for yeast viability under most conditions

Targeting the NEDP1 enzyme to ameliorate ALS phenotypes through stress granule disassembly

The elimination of aberrant inclusions is regarded as a therapeutic approach in neurodegeneration. In amyotro-phic lateral sclerosis (ALS), mutations in proteins found within cytoplasmic condensates called stress granules (SGs) are linked to the formation of pathological SGs, aberrant protein inclusions, and neuronal toxicity. We found that inhibition of NEDP1, the enzyme that processes/deconjugates the ubiquitin-like molecule NEDD8, promotes the disassembly of physiological and pathological SGs. Reduction in poly(ADP-ribose) polymerase1 activity through hyper-NEDDylation is a key mechanism for the observed phenotype. These effects are related to improved cell survival in human cells, and in C. elegans, nedp1 deletion ameliorates ALS phenotypes related to animal motility. Our studies reveal NEDP1 as potential therapeutic target for ALS, correlated to the disassembly of pathological SGs

The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity

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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

Interplay between SUMOylation and NEDDylation regulates RPL11 localization and function

The ribosomal protein L11 (RPL11) integrates different types of stress into a p53-mediated response. Here, we analyzed the impact of the ubiquitin-like protein SUMO on the RPL11-mouse double-minute 2 homolog-p53 signaling. We show that small ubiquitin-related modifier (SUMO)1 and SUMO2 covalently modify RPL11. We find that SUMO negatively modulates the conjugation of the ubiquitin-like protein neural precursor cell-expressed developmentally downregulated 8 (NEDD8) to RPL11 and promotes the translocation of the RP outside of the nucleoli. Moreover, the SUMO-conjugating enzyme, Ubc9, is required for RPL11-mediated activation of p53. SUMOylation of RPL11 is triggered by ribosomal stress, as well as by alternate reading frame protein upregulation. Collectively, our data identify SUMO protein conjugation to RPL11 as a new regulator of the p53-mediated cellular response to different types of stress and reveal a previously unknown SUMO-NEDD8 interplay.-El Motiam, A., Vidal, S., de la Cruz-Herrera, C. F., Da Silva-Alvarez, S., Baz-Martinez, M., Seoane, R., Vidal, A., Rodriguez, M. S., Xirodimas, D. P., Carvalho, A. S., Beck, H. C., Matthiesen, R., Collado, M., Rivas, C. Interplay between SUMOylation and NEDDylation regulates RPL11 localization and function

MAGE-A cancer/testis antigens inhibit MDM2 ubiquitylation function and promote increased levels of MDM4

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically

Interplay between SUMOylation and NEDDylation regulates RPL11 localization and function

The ribosomal protein L11 (RPL11) integrates different types of stress into a p53‐mediated response. Here, we analyzed the impact of the ubiquitin‐like protein SUMO on the RPL11‐mouse double‐minute 2 homolog‐p53 signaling. We show that small ubiquitin‐related modifier (SUMO)1 and SUMO2 covalently modify RPL11. We find that SUMO negatively modulates the conjugation of the ubiquitin‐like protein neural precursor cell‐expressed developmentally downregulated 8 (NEDD8) to RPL11 and promotes the translocation of the RP outside of the nucleoli. Moreover, the SUMO‐conjugating enzyme, Ubc9, is required for RPL11‐mediated activation of p53. SUMOylation of RPL11 is triggered by ribosomal stress, as well as by alternate reading frame protein upregulation. Collectively, our data identify SUMO protein conjugation to RPL11 as a new regulator of the p53‐mediated cellular response to different types of stress and reveal a previously unknown SUMO‐NEDD8 interplay

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