New insights into the roles of CUL1 in mouse placenta development.

CULLIN1 (CUL1) protein, as a scaffold protein in Skp1-CUL1-F box (SCF) E3 ligases complex, was reported involved in different cellular functions to regulate the early embryonic development. In our previous study, we have demonstrated that CUL1 promote trophoblast cell invasion at the maternal-fetal interface in human and the CUL1 protein significantly decreased in preeclampsia (PE) placenta, but how CUL1 involved in placentation is still obscure. Due to the embryo lethal in CUL1 knockout mice, the lentivirus mediated placenta-specific CUL1 knockdown mice model was constructed to uncover the potential role of CUL1 in placentation. In this study, CUL1 was first detected in mouse placenta. CUL1 mainly expressed in trophoblast giant cell at E9.5, and spongiotrophoblast at E11.5 and E13.5 by using immunohistochemistry and int situ hybridization. In lentivirus mediated placenta specific mouse model, the number of implanted embryos was reduced in CUL1 shRNA group at E13.5 and E18.5 compared to control group. Based on the morphological analysis of histologic staining, we observed that spongiotrophoblast layer is expanded, fetal angiogenesis in labyrinth was obstructed and fetus blood cells were accumulated in vessels. These results indicated that decreased expression of CUL1 affect placentation of mice, which give new insights into the cause of gestational diseases, but the exactly mechanism still needs further study.

Cullin 3 overexpression inhibits lung cancer metastasis and is associated with survival of lung adenocarcinoma.

Cullin 3 (CUL3), a molecular scaffold of Cullin-RING ubiquitin ligase, plays an important role in regulating biological processes through modulating the ubiquitylation and degradation of various protein substrates. Dysfunction of CUL3 is implicated in the development of several human diseases. However, the clinical significance and prognostic value of CUL3 in lung cancer have not been investigated. This study investigated the CUL3-modulating potential of non-small cell lung cancer cell lines, H1299, H358, H2170 and H520, by using immunoblotting, MTT, migration, invasion, colony formation and in vivo tumorigenicity assays. The prognostic significance of CUL3 was measured by public KM plotter database (http://kmplot.com/analysis/index.php?p=service&cancer=breast) and tissue immunohistochemistry analysis. The public online database analysis revealed that elevated mRNA expression of CUL3 was associated with better prognosis for non-small cell lung cancer and lung adenocarcinoma. In vitro experiments showed that ectopic overexpression of CUL3 significantly inhibited lung adenocarcinoma cell proliferation and migration, and the tumor-suppressive effect of CUL3 was dependent on the Nrf2/RhoA axis. In vivo mice model demonstrated that overexpression of CUL3 lead to a significant reduction of lung adenocarcinoma growth and metastasis. Importantly, tissue immunohistochemistry analysis showed that about 47% of non-small cell lung cancer tissues were expressed of CUL3 at high levels. Overexpression of CUL3 predicted favorable overall survival in non-small cell lung cancer patients, especially in lung adenocarcinoma, but not in lung squamous cell carcinoma patients. CUL3 could serve as a prognostic biomarker for lung adenocarcinoma. Loss of CUL3 might be driving tumorigenesis by activating the Nrf2/RhoA pathway.

High Expressions of CUL4A and TP53 in Colorectal Cancer Predict Poor Survival.

BACKGROUND/AIMS: Cullin 4A (CUL4A) is vital in cell survival, development, growth and cell cycle, it plays an important role in chaperone-mediated ubiquitination and interacts with TP53 in carcinogenesis. However, the clinicopathologic significance of CUL4A expression in colorectal cancer is unknown; in particular, the prognostic value of CUL4A combined with TP53 expression has not been explored. METHODS: We analyzed the expression of CUL4A in both public database (Oncomine) and 180 cases of colorectal cancer and paired normal tissues by real-time polymerase chain reaction and western blotting. Colony formation, wound healing, migration and invasion assays and tumorigenesis in nude mice were used to explore the function of CUL4A in CRC proliferation and metastasis in vitro and in vivo. Markers of epithelial to mesenchymal transition (EMT) were evaluated by western blotting. Immunohistochemistry (IHC) was used to analyse the relationship between CUL4A expression and E-cadherin expression. RESULTS: CUL4A and TP53 protein expression was significantly higher in cancerous tissues compared to normal tissues. Significant correlation between CUL4A and TP53 expression was observed. CUL4A expression was an independent prognostic factor for overall survival (OS) and disease-free survival (DFS). Interestingly, patients with tumors that had both CUL4A overexpression and mutant TP53 protein accumulation relapsed and died within a significantly short period after surgery (P < 0.001). Multivariate analysis showed that patients with both CUL4A+ and TP53+ positive tumors had extremely poor OS and DFS. Knockdown of CUL4A by a short interfering RNA (siRNA) significantly suppressed the progression of EMT, proliferation, migration, and invasion of colon cancer cells in vitro and tumor growth in vivo. ZEB1 silencing blocked CUL4A-driven these processes. CONCLUSION: CUL4A expression correlated positively with the prognosis of colorectal cancer. Mechanistically, ZEB1 was confirmed to mediate the function of CUL4A in regulating the EMT. The assessment of both CUL4A and mutant TP53 expression will be helpful in predicting colon cancer prognosis.

Overexpression of Rabl3 and Cullin7 is associated with pathogenesis and poor prognosis in hepatocellular carcinoma.

The expression of Rabl3 and Cullin7 is relevant to the carcinogenesis of certain cancers. However, the relationship of this expression with hepatocellular carcinoma remains unclear. To study the protein expression of Rabl3 and Cullin7 and to evaluate their role in hepatocarcinogenesis, in 162 cases of hepatocellular carcinoma, we used immunohistochemistry to investigate the expression of Rabl3 and Cullin7 in both the cancer tissues and the normal hepatic tissues around the hepatocellular carcinoma. The results demonstrated that the rates of positive Rabl3 and Cullin7 expression were 80.2% and 69.1%, respectively, in hepatocellular carcinoma tissues. However, the rates of positive Rabl3 and Cullin7 expression were 31.5% and 29.0%, respectively, in adjacent normal hepatic tissues. Rabl3 and Cullin7 were expressed at significantly higher rates in hepatocellular carcinoma compared with adjacent normal hepatic tissues (P<.01). The rates of positive Rabl3 and Cullin7 expression were higher in the hepatocellular carcinoma tissues of patients with lymph node metastasis, tumor thrombi in the portal vein and an advanced clinical stage (P<.05). A positive correlation between the expression of Rabl3 and the expression of Cullin7 (r=0.27, P<.001) was also observed in our hepatocellular carcinoma cohort. Moreover, patients with positive expression for both Rabl3 and Cullin7 had a remarkably shorter survival time compared with patients with negative expression for both proteins (P<.05). Therefore, the expression of the Rabl3 and Cullin7 proteins may play an important role in the pathogenesis and progression of hepatocellular carcinoma and could be used as a prognostic indicator in patients with hepatocellular carcinoma.

Integrative analysis of multi-omics data reveals distinct impacts of DDB1-CUL4 associated factors in human lung adenocarcinomas.

Many DDB1-CUL4 associated factors (DCAFs) have been identified and serve as substrate receptors. Although the oncogenic role of CUL4A has been well established, specific DCAFs involved in cancer development remain largely unknown. Here we infer the potential impact of 19 well-defined DCAFs in human lung adenocarcinomas (LuADCs) using integrative omics analyses, and discover that mRNA levels of DTL, DCAF4, 12 and 13 are consistently elevated whereas VBRBP is reduced in LuADCs compared to normal lung tissues. The transcriptional levels of DCAFs are significantly correlated with their gene copy number variations. SKIP2, DTL, DCAF6, 7, 8, 13 and 17 are frequently gained whereas VPRBP, PHIP, DCAF10, 12 and 15 are frequently lost. We find that only transcriptional level of DTL is robustly, significantly and negatively correlated with overall survival across independent datasets. Moreover, DTL-correlated genes are enriched in cell cycle and DNA repair pathways. We also identified that the levels of 25 proteins were significantly associated with DTL overexpression in LuADCs, which include significant decreases in protein level of the tumor supressor genes such as PDCD4, NKX2-1 and PRKAA1. Our results suggest that different CUL4-DCAF axis plays the distinct roles in LuADC development with possible relevance for therapeutic target development.

Dysregulation of the miR-194-CUL4B negative feedback loop drives tumorigenesis in non-small-cell lung carcinoma.

Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, is overexpressed in many types of cancers and represses many tumor suppressors through epigenetic mechanisms. However, the mechanisms by which CUL4B is upregulated remain to be elucidated. Here, we show that CUL4B is upregulated in non-small-cell lung carcinoma (NSCLC) tissues and is critically required for cell proliferation and migration in vitro and for xenograft tumor formation in vivo. We found that microRNA-194 (miR-194) and CUL4B protein were inversely correlated in cancer specimens and demonstrated that miR-194 could downregulate CUL4B by directly targeting its 3'-UTR. We also showed that CUL4B could be negatively regulated by p53 in a miR-194-dependent manner. miR-194 was further shown to attenuate the malignant phenotype of lung cancer cells by downregulating CUL4B. Interestingly, CRL4B also epigenetically represses miR-194 by catalyzing monoubiquitination at H2AK119 and by coordinating with PRC2 to promote trimethylation at H3K27 at the gene clusters encoding miR-194. RBX1, another component in CRL4B complex, is also targeted by miR-194 in NSCLC cells. Our results thus establish a double-negative feedback loop between miR-194 and CRL4B, dysregulation of which contributes to tumorigenesis. The function of miR-194 as a negative regulator of CUL4B has therapeutic implications in lung cancer.

Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.

Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.

Cullin1 is up-regulated and associated with poor patients' survival in hepatocellular carcinoma.

Cullin1 (Cul1) is a scaffold protein of the ubiquitin E3 ligase Skp1/Cullin1/Rbx1/F-box protein complex, which ubiquitinates a broad range of proteins involved in cell-cycle progression, signal transduction, and transcription. To investigate the role of Cul1 in the development of hepatocellular carcinoma (HCC), we evaluated the Cul1 expression by immunohistochemistry using a tissue microarray (TMA) containing 90 cases HCC tissues and paired adjacent non-cancerous tissues. We analyzed the correlation between Cul1 expression and clinicopathologic variables and patients survival using two independent HCC cohorts TMA. Our data showed that Cul1 expression was apparently increased in HCC tissues compared with paired adjacent non-tumor tissues. We also demonstrated that Cul1 staining was significantly correlated with tumor size, histology grade and TNM stage. Furthermore, we showed a strong correlation between high Cul1 expression and worse 5-year overall and disease-specific survival rates in HCC patients. Finally, univariate and multivariate Cox proportional hazards regression analysis investigated that high Cul1 expression was a strong independent prognostic indicator of HCC. Our data indicated that Cul1 may be an important prognosis marker for human HCC.

Cullin-1 promotes cell proliferation via cell cycle regulation and is a novel in prostate cancer.

BACKGROUND: There is no reliable marker available for early detection, diagnostic confirmation, or disease prognosis available of prostate cancer (PCa). We aimed to evaluate the function of Cullin-1 and unravel its underlying molecular mechanism to develop novel treatment options equivalent to PCa. METHOD: We used immunohistochemistry to analyze the correlation between Cullin-1 expression and clinicopathologic variables and patient survival. The Cullin-1 level was tested in PCa cells. The role of regulation of Cullin-1 in PCa was applied in vitro and vivo. In addition, we further investigated the signaling pathway of Cullin-1 in prostate cancer cell proliferation. RESULT: We first discovered that Cullin-1 expression was upregulated in human PCa tissues and inversely related with PCa differentiation. We then found that high expression of Cullin-1 protein suggested a poor prognosis in PCa patients. Also, Cullin-1 promotes PCa cell proliferation in vitro and tumor growth in vivo. We then found that the mechanism of Cullin-1 regulation on cell-cycle progression is due to increased expression of p21 and p27, and decreased expression of cyclin D1 and cyclin E after Cullin-1 knockdown. CONCLUSION: Cullin-1 exerts multiple biological effects in the PCa cell line. Through promoting proliferation and by countering cisplatin-induced apoptosis, Cullin-1 has been deeply implicated in the pathogenesis and development of PCa.

Novel Cul3 binding proteins function to remodel E3 ligase complexes.

BACKGROUND: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein. RESULTS: Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor.To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex. CONCLUSION: Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

High expression of Cullin1 indicates poor prognosis for NSCLC patients.

BACKGROUND: Cullin1 is a scaffold protein of the ubiquitin E3 ligase Skp1/Cullin1/Rbx1/F-box protein complex which ubiquitinates a broad range of proteins participating in biochemical events like cell-cycle progression, signal transduction, and transcription. Cullin1 is involved in the progression of several cancers, such as melanoma, breast cancer, and gastric cancer. METHODS: To investigate the role of Cullin1 in the development of non-small-cell lung cancer (NSCLC), we examined the expression of Cullin1 in 8-paired fresh NSCLC tissues. We then constructed immunohistochemistry (IHC) on 114 paraffin-embedded slices and evaluated the correlation between Cullin1 expression and clinicopathologic variables, as well as patients' overall survival. RESULTS: We found that Cullin1 was highly expressed in NSCLC tissues and significantly associated with NSCLC's histological differentiation (P=0.002), clinical stage (P=0.010) and Ki-67 (P=0.021). Furthermore, we showed a strong correlation between high Cullin1 expression and worse overall survival rates in NSCLC patients (P<0.001). Cox regression analysis revealed that Cullin1 expression was an independent prognostic factor to predict 5-year patient outcome in NSCLC cancer (P=0.033). CONCLUSION: These data suggested that Cullin1 might promote the progression of NSCLC and be a biotarget for NSCLC's therapy.

Cullin 4B is a novel prognostic marker that correlates with colon cancer progression and pathogenesis.

Cullin 4B (CUL4B), a scaffold protein of the Cullin4B-RING E3 ligase complex, functions in proteolysis. The present study aims to investigate its expression pattern and evaluate whether CUL4B expression was associated with histopathological and prognosis in the patients with colon cancer. Real-time PCR and western blot were used to identify CUL4B expression in tumor tissue and the paired adjacent normal mucosa from patients with colon cancer. Immunohistochemistry on a tissue microarray containing 203 cases of colon cancer was performed to analyze the association between CUL4B expression and clinicopathological features. Results indicated that CUL4B mRNA and protein levels in tumor tissues were both higher than that in normal mucosae (P < 0.001). Immunohistochemical study displayed that high CUL4B expression was significantly associated with the depth of tumor invasion, lymph node metastasis, distant metastasis, histological differentiation, vascular invasion, and advanced tumor stage. Patients with CUL4B-positive tumors had a higher recurrence rate and poorer survival than patients with CUL4B-negative tumors. In multivariate analyses, CUL4B expression was an independent factor for determining colon cancer prognosis after surgery. In conclusion, CUL4B might promote the progression of colon cancer and can be served as a novel independent prognostic marker for the prediction of recurrence in colon cancer.

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations.

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.

Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes--evidence for an autoregulatory mechanism.

RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of Cul3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with Cul3. The interaction requires the N-terminal region of Cul3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of Cul3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.

Regulation and role of Arabidopsis CUL4-DDB1A-DDB2 in maintaining genome integrity upon UV stress.

Plants use the energy in sunlight for photosynthesis, but as a consequence are exposed to the toxic effect of UV radiation especially on DNA. The UV-induced lesions on DNA affect both transcription and replication and can also have mutagenic consequences. Here we investigated the regulation and the function of the recently described CUL4-DDB1-DDB2 E3 ligase in the maintenance of genome integrity upon UV-stress using the model plant Arabidopsis. Physiological, biochemical, and genetic evidences indicate that this protein complex is involved in global genome repair (GGR) of UV-induced DNA lesions. Moreover, we provide evidences for crosstalks between GGR, the plant-specific photo reactivation pathway and the RAD1-RAD10 endonucleases upon UV exposure. Finally, we report that DDB2 degradation upon UV stress depends not only on CUL4, but also on the checkpoint protein kinase Ataxia telangiectasia and Rad3-related (ATR). Interestingly, we found that DDB1A shuttles from the cytoplasm to the nucleus in an ATR-dependent manner, highlighting an upstream level of control and a novel mechanism of regulation of this E3 ligase.

Altered pattern of Cul-1 protein expression and neddylation in human lung tumours: relationships with CAND1 and cyclin E protein levels.

The Cul-1 protein is the scaffold element of SCF complexes that are involved in the proteasomal degradation of numerous proteins regulating cell cycle progression. Owing to this central role in cell growth control, aberrant expression of the components of SCF is thought to play a role during tumourigenesis. Nothing is known about Cul-1 expression in human tumours. In this study, we have analysed its status in a series of 128 human lung carcinomas, comprising 50 non-small cell lung cancers (NSCLCs; 29 squamous cell carcinomas and 21 adenocarcinomas) and 78 neuroendocrine (NE) lung tumours (24 typical and atypical carcinoids, 19 large cell NE carcinomas and 35 small cell lung carcinomas), using immunohistochemistry. We report for the first time an altered pattern of Cul-1 expression in human tumours; indeed, we show that Cul-1 expression is up-regulated in 40% (51/128) of all lung tumours as compared to normal lung tissues, including 34% (17/50), 75% (18/24) and 30% (16/54) of NSCLCs, carcinoids and high grade neuroendocrine lung carcinomas, respectively. Furthermore, we demonstrate that high levels of Cul-1 protein are associated with a low KI67 proliferative index (p = 0.005) and with a decrease in the cyclin E oncoprotein (p = 0.0003), one of the major targets of SCF complexes. These data suggest that up-regulation of Cul-1 could protect cells from hyperproliferative signals through cyclin E down-regulation. Cul-1 is modified by neddylation, a post-translational modification that grafts ubiquitin-like Nedd8/Rub1 residues and controls Cul-1 activity. We also provide evidence that neddylated forms of Cul-1 are specifically expressed in high-grade NE lung tumours and are associated with down-regulation of the Cul-1 inhibitor CAND1 (p = 0.03) and a high level of cyclin E (p = 0.0002). These data support the notion that alterations in the Cul-1 neddylation/deneddylation pathway could contribute to the development of these highly aggressive lung tumours.

Glucosamine and its N-acetyl-phenylalanine derivative prevent TNF-alpha-induced transcriptional activation in human chondrocytes.

OBJECTIVES: Glucosamine (GlcN) is used in the treatment of osteoarthritis as symptomatic slow-acting drug, but its mode of action is not completely known. We analyzed the influence of GlcN and its N-acetyl-phenylalanine derivative (NAPA) on mRNA transcription level of TNF-alpha-stimulated genes in cell culture. METHODS: Human immortalized chondrocyte cell line lbpva55 was stimulated with TNF-alpha and treated with GlcN and NAPA. mRNA transcription level of several genes, identified by complementary DNA microarray (cDNA microarray), was validated by Quantitative Real-Time Polymerase Chain Reaction (Q-RT-PCR). RESULTS: Several genes, whose mRNA level was increased by TNF-alpha treatment and significantly reduced by GlcN and NAPA in lbpva55 cells, were identified. These include cytokine receptors TNF-R1 and TNF-R2, their associated factor TRAF-6, signaling intermediates IGFB-6 and Rnd1, as well as cell cycle regulating proteins CUL-2 and G1S protein 1. Down- regulation of mRNA expression level of some of these genes is in accordance with inactivation of NF-kB transcription factor. Moreover, we found down-regulation of c-jun mRNA level, a component of AP-1 transcription factor. CONCLUSIONS: Our study suggests that GlcN and NAPA interfere with activation of NF-kB and AP-1 transcription factors, which are responsible for the expression of genes involved in diverse biological processes, such as cell growth and death, inflammatory and stress responses, accounting for the beneficial effects of GlcN in osteoarthritis.

The DDB1-CUL4ADDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites.

Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4A(DDB2). The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4A(DDB2) complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4A(DDB2) ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.