NEDD8 Ultimate Buster Research Articles

Low cytoplasmic NUB1 protein exerts hypoxic cell death with poorer prognosis in oestrogen receptor negative breast cancer patients

Current prognostic biomarkers fall short in stratifying Oestrogen receptor (ER)-negative breast cancer patients regarding tumour progression risk at diagnosis. The role of AIPL1 in activating its tumour suppressor client protein, NEDD8 Ultimate Buster-1 (NUB1) remains unknown in cancer. Our study demonstrated how downregulated AIPL1 results in the deactivated NUB1 protein under hypoxic conditions. We examined the AIPL1-NUB1 pathwayin vitro using cell lines i.e. MCF-7, MDA-MB-231, RCC4 etc. NUB1 expression was assessed using Oncomine, and cBioPortal was performed to assess NUB1′s prognostic significance in human cancers. In the John Radcliffe Hospital cohort (n = 122), immunohistochemistry analysis revealed downregulated AIPL1 (Log2 fold change=-0.28; p < 0.001) and upregulated NUB1 transcripts (Log2 fold change=0.59; p < 0.001) compared to adjacent normal tissues. In severe chronic hypoxia, multimerised AIPL1 localisedin the cytoplasm while NUB1 protein migrated to the nucleus, where the absence of NUB1 nuclear localisation led to cell cycle arrest. Biopsies showed that patients with lower cytoplasmic NUB1 expression (n = 57) had poorer overall survival compared to those with higher cytoplasmic expression (n = 57), HR=1.78; 95 % CI=1.01–3.35, p = 0.048. Low NUB1 protein levels in both normoxic and hypoxic conditions were associated with cell cycle arrest and upregulation ofp21 and p27 in breast cancer cell lines, correlating significantly withpoorer survival outcomes in all breast cancer and ER-negative breast cancer patients.

MLN4924 Inhibits Defective Ribosomal Product Antigen Presentation Independently of Direct NEDDylation of Protein Antigens.

Successful direct MHC class I Ag presentation is dependent on the protein degradation machinery of the cell to generate antigenic peptides that can be loaded onto MHC class I molecules for surveillance by CD8+ T cells of the immune system. Most often this process involves the ubiquitin (Ub)-proteasome system; however, other Ub-like proteins have also been implicated in protein degradation and direct Ag presentation. In this article, we examine the role of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) in direct Ag presentation in mouse cells. NEDD8 is the Ub-like protein with highest similarity to Ub, and fusion of NEDD8 to the N terminus of a target protein can lead to the degradation of target proteins. We find that appending NEDD8 to the N terminus of the model Ag OVA resulted in degradation by both the proteasome and the autophagy protein degradation pathways, but only proteasomal degradation, involving the proteasomal subunit NEDD8 ultimate buster 1, resulted in peptide presentation. When directly compared with Ub, NEDD8 fusion was less efficient at generating peptides. However, inactivation of the NEDD8-conugation machinery by treating cells with MLN4924 inhibited the presentation of peptides from the defective ribosomal product-derived form of a model Ag. These results demonstrate that NEDD8 activity in the cell is important for direct Ag presentation, but not by directly targeting proteins for degradation.

NUB1 and FAT10 Proteins as Potential Novel Biomarkers in Cancer: A Translational Perspective.

Cancer increases the global disease burden substantially, but it remains a challenge to manage it. The search for novel biomarkers is essential for risk assessment, diagnosis, prognosis, prediction of treatment response, and cancer monitoring. This paper examined NEDD8 ultimate buster-1 (NUB1) and F-adjacent transcript 10 (FAT10) proteins as novel biomarkers in cancer. This literature review is based on the search of the electronic database, PubMed. NUB1 is an interferon-inducible protein that mediates apoptotic and anti-proliferative actions in cancer, while FAT10 is a ubiquitin-like modifier that promotes cancer. The upregulated expression of both NUB1 and FAT10 has been observed in various cancers. NUB1 protein binds to FAT10 non-covalently to promote FAT10 degradation. An overexpressed FAT10 stimulates nuclear factor-kappa β, activates the inflammatory pathways, and induces the proliferation of cancer. The FAT10 protein interacts with the mitotic arrest deficient 2 protein, causing chromosomal instability and breast tumourigenesis. FAT10 binds to the proliferating cell nuclear antigen protein and inhibits the DNA damage repair response. In addition, FAT10 involves epithelial–mesenchymal transition, invasion, apoptosis, and multiplication in hepatocellular carcinoma. Our knowledge about them is still limited. There is a need to further develop NUB1 and FAT10 as novel biomarkers.

Targeted protein degradation via NEDD8 conjugation does not enhance direct MHC class I antigen presentation.

Abstract Knowledge of different pathways contributing to peptide generation for direct MHC class I antigen presentation is important to the field of immunotherapy. Here we investigated the role of the ubiquitin-like protein NEDD8 in antigen presentation. We show that proteins tagged N-terminally with NEDD8 undergo rapid degradation via the proteasomal, and autophagy-lysosomal pathways. To determine if protein NEDDylation increased peptide presentation, we fused NEDD8 to the N-terminus of a cytosolic form of ovalbumin (OVA) and measured presentation of the SIINFEKL peptide via the murine MHC class I molecule K b , and compared the results to ovalbumin N-terminally modified with ubiquitin. While NEDD8-OVA was processed for degradation via the proteasomal and autophagosomal pathway Ub-OVA was only degraded via the proteasome. Peptide presentation from NEDD8-OVA was dependent on NEDD8-ultimate buster 1 (NUB1), while autophagy did not contribute peptides antigen presentation. Overall, we found that N-terminal Ub conjugation was far more effective at generating antigenic peptides than N-terminal NEDD8 conjugation. However, upon inhibition of NEDDYylation by treatment with MLN4924, we did detect a decrease in peptide presentation from a model antigen used specifically to track Defective Ribosomal Product (DRiP) presentation. These data demonstrate that while NEDD8 can cause the rapid degradation of substrates it is not as efficient as Ubiquitin for generating antigenic peptides. Our data provides a basis for understanding the role of NEDD8 in protein degradation and antigen presentation, especially in conditions of NEDD8 dysregulation, such as in some cancers and protein folding pathologies.

Phosphorylated NUB1 distinguishes α-synuclein in Lewy bodies from that in glial cytoplasmic inclusions in multiple system atrophy.

Posttranslational modifications by phosphorylation, ubiquitination, neddylation and other pathways have emerged as major regulators of cellular functions. NEDD8 ultimate buster 1, NUB1, is an adaptor protein, which negatively regulates the levels of the ubiquitin-like protein NEDD8 as well as neddylated proteins through proteasomal degradation. We previously reported that NUB1 is highly involved in the pathogenesis of synucleinopathy including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). In general, since phosphorylation is strongly related to the alteration of protein propensity, we examined if the fundamental function of NUB1 can be modulated by its phosphorylation. We created a series of phosphomimic mutants of NUB1. Among them, we found that phosphorylation of NUB1 at S46 (P-NUB46) efficiently degrades aggregates using a cell-based assay. Immunohistochemical studies have shown that specific antibodies against P-NUB46 reacted with Lewy bodies in PD and DLB but not with glial cytoplasmic inclusions in MSA. Moreover, P-NUB46 levels were significantly higher in the brains of patients with DLB than in control brains, and P-NUB46 was extracted in an insoluble fraction of DLB. These findings suggest that the phosphorylation of NUB1 is modulated during the pathological process of Lewy body disease.

PO-479 NUB1 as a prognostic marker in breast cancer: a retrospective, integrated genomic, transcriptomic, and protein analysis

IntroductionAryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) protein interacts with and restricts NEDD8 ultimate buster-1 (NUB1) protein from entering nucleus. The interferon-induced NUB1 protein causes degradation of neddylated proteins through the ubiquitin proteasome system. The AIPL1 transcripts were down regulated in various cancers compared to adjacent normal tissues. The mechanistic roles of AIPL1 and NUB1 protein in cancer cell cycle regulation remain unexplored.Material and methodsProtein expression was examined by immunoblotting and immunochemistry. Cells were cultured in the recommended conditions. The NUB1 monoclonal antibody and the DAKO Envision Plus system were used to evaluate FFPE. Survival curves was analysed by kaplan meier methods and compared by the log rank test. All statistical tests were two sided.Results and discussionsThe meta-analysis of cancer dataset showed the transcripts expression of AIPL1 were down regulated in lung, pancreatic and breast cancer in relative to the adjacent normal tissues. The upregulated NUB1 transcripts were observed in the breast cancer. METABRIC cohort highlighted that patients with low NUB1 transcripts had a poorer survival in the ER-negative subgroup of breast cancer patients [hazard ratio (HR)=0.66, 95% confidence interval (CI)=0.5–0.87, p=0.003] and triple negative subgroup (HR=0.67, 95% CI=0.47–0.96, p=0.028). NUB1 knockdown inhibits in vitro cell growth in MDA-MB-231. AIPL1 protein forms multimers in cancer cells. NUB1 protein moved into the nucleus in hypoxia (0.1% O248 hours). p21 and p27 proteins accumulated in NUB1-knockdown MDA-MB-231 cells. The prolonged G0/G1 cell cycle arrests resulted in cell death through the neddylation-dependent CUL1-p27-p21 and CUL2-VHL axis. We also demonstrated that HIF1α protein could be neddylated upon reoxygenation. In a retrospective study of Oxford breast cancer cohort, the lower cytoplasmic expression (n=57) prognosed worse overall survival than higher cytoplasmic expression (n=57) (HR=1.779, 95% CI=1.006–3.346, p=0.048).ConclusionNUB1 protein depletion cause G0/G1 cell cycle arrest in breast cancer. Low cytoplasmic NUB1 protein prognosed worse survival of breast cancer patients. NUB1 depletion causes cell cycle arrest via the deactivated ubiquitin E3 ligase pathway.

THE ROLE OF NUB1 IN THE CLEARANCE OF TAU AGGREGATES IN ALZHEIMER'S DISEASE

BACKGROUND: Intraneuronal aggregations of abnormal hyperphosphorylated tau are a hallmark of Alzheimer’s disease. Previous data revealed that NEDD8 ultimate buster 1 (NUB1) reduces the aggregation and phosphorylation of tau in an in vitro model. NUB1 is a ubiquitin-like (UBL)/ ubiquitin-associated (UBA) protein that targets the down regulation of ubiquitin-like modifiers (NEDD8, FAT10), and aggregation-prone proteins leading to neurodegenerative disease (synphilin-1, huntingtin). The aim of the study is to understand how NUB1 affects tau aggregation and phosphorylation. METHODS: A GFP-tau inducible neuroblastoma cell line was generated and treated with proteasome inhibitor to induce the formation of aggresomes. GFP-tau cells were transduced with NUB1 lentiviral vectors both in the absence and presence of proteasome inhibitor, followed by western blotting, filter trap assays, immunoprecipitation and immunofluorescence. RESULTS: Inhibition of the proteasome induced the formation of GFP-tau aggresomes that were positive for phospho-tau (p-tau) markers (AT8, pS396). Filter trap assays revealed that NUB1 significantly decreased the ratio of p-tau to tau in the total cellular fraction (both soluble and insoluble), as well as in only the detergent insoluble fraction, both with and without proteasome inhibition. However, the ratio of tau and p-tau in the insoluble fraction to that in the total fraction revealed that the NUB1-mediated decrease in the levels of detergent insoluble tau and p-tau exceeded that of the overall decline in total levels, suggesting NUB1 can target aggregation-prone tau and p-tau. This effect of NUB1 was more pronounced for p-tau following proteasome inhibition. Blocking the proteasome induced the upregulation of p62 and increased the ratio of LC3B-II to LC3B-I. NUB1 induced further upregulation of autophagy following proteasome inhibition as seen by an increase in the LC3B-II/LC3B-I ratio and changes in LC3B positive puncta in cells. Finally, GFP-tau aggresomes were positive for p62 and NUB1 overexpression promoted the interaction between GFP-tau and p62. CONCLUSIONS: These data suggest that NUB1 may play a role in the clearance of aggregation-prone tau and p-tau following inhibition of the proteasome via autophagy.

Regulation of NUB1 Activity through Non-Proteolytic Mdm2-Mediated Ubiquitination.

NUB1 (Nedd8 ultimate buster 1) is an adaptor protein which negatively regulates the ubiquitin-like protein Nedd8 as well as neddylated proteins levels through proteasomal degradation. However, molecular mechanisms underlying this function are not completely understood. Here, we report that the oncogenic E3 ubiquitin ligase Mdm2 is a new NUB1 interacting protein which induces its ubiquitination. Interestingly, we found that Mdm2-mediated ubiquitination of NUB1 is not a proteolytic signal. Instead of promoting the conjugation of polyubiquitin chains and the subsequent proteasomal degradation of NUB1, Mdm2 rather induces its di-ubiquitination on lysine 159. Importantly, mutation of lysine 159 into arginine inhibits NUB1 activity by impairing its negative regulation of Nedd8 and of neddylated proteins. We conclude that Mdm2 acts as a positive regulator of NUB1 function, by modulating NUB1 ubiquitination on lysine 159.

Inhibition of NEDD8 and FAT10 ligase activities through the degrading enzyme NEDD8 ultimate buster 1: A potential anticancer approach.

The capabilities of tumour cells to survive through deregulated cell cycles and evade apoptosis are hallmarks of cancer. The ubiquitin-like proteins (UBL) proteasome system is important in regulating cell cycles via signaling proteins. Deregulation of the proteasomal system can lead to uncontrolled cell proliferation. The Skp, Cullin, F-box containing complex (SCF complex) is the predominant E3 ubiquitin ligase, and has diverse substrates. The ubiquitin ligase activity of the SCF complexes requires the conjugation of neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to cullin proteins. A tumour suppressor and degrading enzyme named NEDD8 ultimate buster 1 (NUB1) is able to recruit HLA-F-adjacent transcript 10 (FAT10)- and NEDD8-conjugated proteins for proteasomal degradation. Ubiquitination is associated with neddylation and FAT10ylation. Although validating the targets of UBLs, including ubiquitin, NEDD8 and FAT10, is challenging, understanding the biological significance of such substrates is an exciting research prospect. This present review discusses the interplay of these UBLs, as well as highlighting their inhibition through NUB1. Knowledge of the mechanisms by which NUB1 is able to downregulate the ubiquitin cascade via NEDD8 conjugation and the FAT10 pathway is essential. This will provide insights into potential cancer therapy that could be used to selectively suppress cancer growth.

Structural and mechanistic insights into the arginine/lysine-rich peptide motifs that interact with P97/VCP

P97 protein, also referred to as valosin-containing protein (VCP), is an AAA-ATPase (ATPase associated with a variety of cellular activities) that mediates vital cellular activities with the cooperation of many cofactors. A group of cofactors interact with the N-terminal domain of P97 (P97N) through their Arg/Lys-rich peptide motifs. We investigated the interactions between P97 and these motifs, including VCP-binding motif (VBM) and VCP-interacting motif (VIM). The solution structures of the VBM motif from HRD1 and the VIM motif from SVIP are both comprised mainly of a single α-helix. The VIM motifs generally have stronger P97N-binding affinities than the VBMs, and SVIP (VIM) can compete with HRD1–VBM for the interaction, providing a possibility that VIM-containing proteins (such as SVIP) act as competitors against VBM-containing proteins (such as HRD1) for interacting with P97. Based on biochemical features of the VBM motifs, we also identified NUB1L (NEDD8 ultimate buster-1 long) as a novel VBM-containing protein, which is involved in proteasomal degradation of NEDD8 through the P97 pathway.

Structural studies on AIPL1 and its functional interactions with NUB1 to identify key interacting residues in LCA4

Leber congenital amaurosis (LCA) is an autosomal recessive disorder that causes visual impairment in children due to fifteen different gene mutations. Of these, mutations in Aryl-Hydrocarbon Receptor Interacting Protein-like 1 (AIPL1) cause the most severe form of LCA (LCA4) leading to the degeneration of photoreceptor cells. NEDD8 Ultimate Buster 1 (NUB1), a protein that regulates cell cycle progression, interacts with AIPL1 to prevent the over expression of NUB1. In the case of over expression, cell cycle progression is disrupted and may lead to LCA. The studies on interactions between these two proteins will aid in identifying potential modulators for this condition. Since no three-dimensional structure is currently available for these two proteins, in this study we predicted the structures of these two proteins by molecular modelling methods. Moreover, we also modelled the three proven significant mutant forms of AIPL1 spanning the tetratricopeptide domain. Finally, both the modelled wild and mutant structures of AIPL1 (A197P, C239R and G262S) were computationally docked to NUB1, so as to map the potential molecular interactions. This is the first study on modelling the structure-function relationship of AIPL1-NUB1 interactions which shall aid in discovery of novel therapeutic agents.

NUB1 modulation of GSK3β reduces tau aggregation

Abnormal phosphorylation of the microtubule-associated protein tau in neurodegenerative disorders, including Alzheimer's disease (AD) and frontotemporal lobar degeneration, is associated with disrupted axonal transport and synaptic dysfunction ultimately manifesting as histopathological lesions of protein aggregates. Glycogen synthase kinase 3β (GSK3β) may be critical for the pathological hyperphosphorylation of tau. Here, we examined the role of the proteasome-associated protein Nedd8 ultimate buster 1 (NUB1) in the neuropathogenic phosphorylation and aggregation of tau. We reveal that NUB1 interacted with both tau and GSK3β to disrupt their interaction, and abolished recruitment of GSK3β to tau inclusions. Moreover, NUB1 reduced GSK3β-mediated phosphorylation of tau and aggregation of tau in intracellular inclusions. Strikingly, NUB1 induced GSK3β degradation. Deletion of the NUB1 ubiquitin-like (UBL) domain did not impair the interaction with tau and GSK3β, and the ability to suppress the phosphorylation and aggregation of tau was not affected. However, the UBL motif was necessary for GSK3β degradation. Deletion of the NUB1 ubiquitin-associated (UBA) domain abrogated the ability of NUB1 to interact with and degrade GSK3β. Moreover, the UBA domain was required to suppress the aggregation of tau. Silencing of NUB1 in cells stabilized endogenous GSK3β and exacerbated tau phosphorylation. Thus, we propose that NUB1, by regulating GSK3β levels, modulates tau phosphorylation and aggregation, and is a key player in neurodegeneration associated with tau pathology. Moreover, NUB1 regulation of GSK3β could modulate numerous signalling pathways in which GSK3β is a centrally important effector.

FAT10 and NUB1L bind to the VWA domain of Rpn10 and Rpn1 to enable proteasome-mediated proteolysis

FAT10 is the only ubiquitin-like modifier that can target proteins for degradation by the proteasome in a ubiquitin-independent manner. The degradation of FAT10-linked proteins by the proteasome is strongly accelerated by the ubiquitin-like-ubiquitin-associated protein NEDD8 ultimate buster-1 long (NUB1L). Here we show how FAT10 and NUB1L dock with the 26S proteasome to initiate proteolysis. We identify the 26S proteasome subunit hRpn10/S5a as the receptor for FAT10, whereas NUB1L can bind to both Rpn10 and Rpn1/S2. Unexpectedly, FAT10 and NUB1L both interact with hRpn10 via the VWA domain. FAT10 degradation in yeast shows that human Rpn10 can functionally reconstitute Rpn10-deficient yeast and that the VWA domain of hRpn10 suffices to enable FAT10 degradation. Depletion of hRpn10 causes an accumulation of FAT10-conjugates also in human cells. In conclusion, we identify the VWA domain of hRpn10 as a receptor for ubiquitin-like proteins within the 26S proteasome and elucidate how FAT10 mediates efficient proteolysis by the proteasome.

Incipient intranuclear inclusion body disease in a 78-year-old woman

We report an incipient case of intranuclear inclusion body disease (INIBD) in a 78-year-old woman. No apparent neurological symptoms were noticed during the clinical course. Post mortem examination revealed widespread occurrence of eosinophilic intranuclear inclusions in neuronal and glial cells of the central and peripheral nervous systems, as well as in parenchymal cells of the visceral organs. The inclusions were observed more frequently in glial cells than in neuronal cells. Ultrastructurally, the inclusions consisted of granular and filamentous material. Immunohistochemically, the inclusions were positive for ubiquitin, ubiquitin-related proteins (NEDD8 ultimate buster 1, small ubiquitin modifier-1, small ubiquitin modifier-2 and p62), promyelocytic leukemia protein and abnormally expanded polyglutamine. Consistent with previous studies, the vast majority of inclusion-bearing glial cells were astrocytes. Furthermore, p25α-positive oligodendrocytes rarely contained intranuclear inclusions. These findings suggest that INIBD may occur in non-demented elderly individuals and that oligodendrocyte is also involved in the disease process of INIBD.

369 NUB1, AN INTERFERON-INDUCIBLE PROTEIN, MEDIATES ANTI-PROLIFERATIVE ACTIONS AND APOPTOSIS IN RENAL CELL CARCINOMA CELLS THROUGH CELL CYCLE REGULATION

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2010369 NUB1, AN INTERFERON-INDUCIBLE PROTEIN, MEDIATES ANTI-PROLIFERATIVE ACTIONS AND APOPTOSIS IN RENAL CELL CARCINOMA CELLS THROUGH CELL CYCLE REGULATION Tomoaki Tanaka, Tomoko Hosono, Katsuyuki Kuratsukuri, and Tatsuya Nakatani Tomoaki TanakaTomoaki Tanaka More articles by this author , Tomoko HosonoTomoko Hosono More articles by this author , Katsuyuki KuratsukuriKatsuyuki Kuratsukuri More articles by this author , and Tatsuya NakataniTatsuya Nakatani More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.436AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES NEDD8 is a ubiquitin-like protein that regulates fundamental biological events via target protein conjugation. NEDD8 ultimate buster 1 (NUB1) is an interferon-inducible protein, which down-regulates NEDD8 expression and its conjugation system. Although overexpression of NUB1 induces a growth-inhibitory effect in cells, the mechanisms underlying the anti-mitogenic actions of NUB1 in cancer cells remain uncertain. We investigated the anticancer effects of NUB1 in human renal cell carcinoma (RCC) cells. METHODS Nine human RCC cell lines including ACHN, Caki-1, A-498, 786-0, 10RGB, OS-RC2, 4TUHR, OCUU1, and OCUU3 were used for this study. Cell-growth inhibition by exposure to interferon alpha (IFNα) was determined using the water-soluble tetrazolium salt (WST-1) assay. The expression levels of NUB1 mRNA and protein in those cell lines were measured by real-time RT-PCR and western blot analysis, respectively. A498 cells were transfected with pcDNA3/FLAG-NUB1 or pcDNA3 (control) and an apoptosis assay was performed by flow cytometry (FCM). To examine the process associated with the apoptotic effect of NUB1, cell-cycle analysis using FCM and western blot analysis for cell-cycle regulators was performed in A498 cells transfected with pcDNA3/FLAG-NUB1 or empty control vector. The effect of NUB1 knock-down on cell-growth in RCC cells was examined using the RNA interference technique. RESULTS First, both NUB1 messenger RNA and protein were significantly induced by IFNα in 7 out of 9 selected RCC cell lines, and the NUB1 expressions induced by IFNα correlated positively with the cell growth inhibition rate. Second, overexpression of NUB1 remarkably induced S-phase transition during the cell cycle and apoptosis in IFNα-resistant A498 cells, in which NUB1 is not induced by IFNα. Third, the expression levels of two cell-cycle regulator proteins, cyclin E and p27, were increased under the aforementioned conditions. Forth, knock-down of NUB1 clearly enhanced A498 cell-growth as compared with control cells. CONCLUSIONS Our results suggest that NUB1-mediated apoptosis might result from cell cycle arrest in S-phase via regulation of cyclin E and p27. Thus, NUB1 may be a key factor involved in IFNα-induced apoptosis in human RCC cells, even IFNα-resistant RCC cells. Osaka, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e146 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoaki Tanaka More articles by this author Tomoko Hosono More articles by this author Katsuyuki Kuratsukuri More articles by this author Tatsuya Nakatani More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

NUB1, an interferon-inducible protein, mediates anti-proliferative actions and apoptosis in renal cell carcinoma cells through cell-cycle regulation

Background:NEDD8 ultimate buster 1 (NUB1) is an interferon (IFN)-inducible protein that downregulates NEDD8 expression and its conjugation system. Although overexpression of NUB1 induces a growth-inhibitory effect in cells, the mechanisms underlying the anti-mitogenic actions of NUB1 in cancer cells remain uncertain. We investigated the anti-cancer effects of NUB1 in human renal cell carcinoma (RCC) cells.Methods:Nine human RCC cells were used for this study. The proliferation of RCC cells exposed to IFN-α was measured by water-soluble tetrazolium salt assay. The expression level of NUB1 in cells was measured by quantitative reverse transcriptase PCR or western blot analysis. Apoptosis and cell-cycle analysis were performed by flow cytometry. Silencing of NUB1 was performed using a small interfering RNA.Results:Both NUB1 messenger RNA and protein were significantly induced by IFN-α in seven out of nine selected RCC cell lines, and the NUB1 expressions induced by IFN-α correlated positively with cell growth inhibition. Overexpression of NUB1 remarkably induced S-phase transition during cell cycle and apoptosis in IFN-α-resistant A498 cells, in which NUB1 is not induced by IFN-α. The expression levels of two cell-cycle regulator proteins, cyclin E and p27, were increased under the aforementioned conditions. The knockdown of NUB1 enhanced cell proliferation of IFN-α-resistant A498 cells and suppressed IFN-α-induced growth inhibition in IFN-α-sensitive 4TUHR cells.Conclusion:NUB1 may be a key factor involved not only in cell growth inhibition by IFN-α in RCC cells but also in the anti-cancer effect against IFN-α-resistant RCC cells.

Degradation of FAT10 by the 26S proteasome is independent of ubiquitylation but relies on NUB1L

The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome, a process accelerated by the UBL-UBA domain protein NEDD8 ultimate buster 1-long. Here, we show that FAT10-mediated degradation occurs independently of poly-ubiquitylation as purified 26S proteasome readily degraded FAT10-dihydrofolate reductase (DHFR) but not ubiquitin-DHFR in vitro. Interestingly, the 26S proteasome could only degrade FAT10-DHFR when NUB1L was present. Knock-down of NUB1L attenuated the degradation of FAT10-DHFR in intact cells suggesting that NUB1L determines the degradation rate of FAT10-linked proteins. In conclusion, our data establish FAT10 as a ubiquitin-independent but NUB1L-dependent targeting signal for proteasomal degradation.

The UBA Domains of NUB1L Are Required for Binding but Not for Accelerated Degradation of the Ubiquitin-like Modifier FAT10

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.

The Leber Congenital Amaurosis Protein AIPL1 Modulates the Nuclear Translocation of NUB1 and Suppresses Inclusion Formation by NUB1 Fragments

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) cause the blinding disease Leber congenital amaurosis (LCA). The similarity of AIPL1 to AIP has led to suggestions that AIPL1 could function in a similar manner to AIP in facilitating protein translocation and as a component of chaperone complexes. AIPL1 interacts with the cell cycle regulator NEDD8 ultimate buster protein 1 (NUB1). As AIPL1 is predominantly cytoplasmic and NUB1 is predominantly nuclear, we tested the hypothesis that AIPL1 could modulate the nuclear translocation of NUB1. Co-transfection of AIPL1 with GFP-NUB1 resulted in a shift of GFP-NUB1 subcellular distribution toward the cytoplasm. Interestingly, AIPL1 was able to act in a chaperone-like fashion to efficiently suppress inclusion formation by NUB1 fragments. Co-transfection of AIPL1 with GFP-NUB1-N and GFP-NUB1-C resulted in an AIPL1-dependent suppression of GFP-NUB1-N perinuclear inclusions and GFP-NUB1-C intranuclear inclusions leading to the redistribution of these fragments in the cytoplasm. This chaperone-like function of AIPL1 was specific for NUB1, since AIPL1 was unable to suppress the inclusion formation by unrelated aggregation-prone proteins and AIP had no effect on NUB1 localization or inclusion formation. We examined the effect of a range of pathogenic and engineered mutations on the ability of AIPL1 to modulate NUB1 localization or inclusion formation. With the exception of W278X, which formed non-functional SDS-insoluble inclusions, all of the pathogenic mutations studied were soluble and could modulate NUB1 with varying efficiency compared with the wild-type protein. The effect of AIPL1 on NUB1 required the C-terminal region of AIPL1, as engineered C-terminal truncation mutations had no effect on NUB1. These data show that AIPL1 can modulate protein translocation and act in a chaperone-like manner and suggest that AIPL1 is an important modulator of NUB1 cellular function.

Role of AIP and its homologue the blindness-associated protein AIPL1 in regulating client protein nuclear translocation.

Mutations in the AIPL1 (aryl hydrocarbon receptor interacting protein-like 1) cause the blinding disease Leber's congenital amaurosis. AIPL1 is a homologue of the AIP. AIP functions as part of a chaperone heterocomplex to facilitate signalling by the AhR and plays an important role in regulating the nuclear translocation of the receptor. We review the evidence for the role of AIP in protein translocation and compare the potential functions of AIPL1 in the translocation of its interacting partner the NEDD8 ultimate buster protein 1.