Structural analysis of MDM2 RING separates degradation from regulation of p53 transcription activity

MDM2–MDMX complexes bind the p53 tumor-suppressor protein, inhibiting p53's transcriptional activity and targeting p53 for proteasomal degradation. Inhibitors that disrupt binding between p53 and MDM2 efficiently activate a p53 response, but their use in the treatment of cancers that retain wild-type p53 may be limited by on-target toxicities due to p53 activation in normal tissue. Guided by a novel crystal structure of the MDM2–MDMX–E2(UbcH5B)–ubiquitin complex, we designed MDM2 mutants that prevent E2–ubiquitin binding without altering the RING-domain structure. These mutants lack MDM2's E3 activity but retain the ability to limit p53′s transcriptional activity and allow cell proliferation. Cells expressing these mutants respond more quickly to cellular stress than cells expressing wild-type MDM2, but basal p53 control is maintained. Targeting the MDM2 E3-ligase activity could therefore widen the therapeutic window of p53 activation in tumors

Regulation of Transcriptional Activators by DNA-Binding Domain Ubiquitination

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure–function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate

A Generic Platform for Cellular Screening Against Ubiquitin Ligases

Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence(1,2). Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies(3). To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma(4,5). We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth

Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

International audienceThe interplay between influenza A viruses (IAV) and the p53 pathway has been reported in several studies, highlighting the antiviral contribution of p53. Here, we investigated the impact of IAV on the E3-ubiquitin ligase Mdm2, a major regulator of p53, and observed that IAV targets Mdm2, notably via its non-structural protein (NS1), therefore altering Mdm2 stability, p53/Mdm2 interaction and regulatory loop during the time-course of infection. This study also highlights a new antiviral facet of Mdm2 possibly increasing the list of its many p53-independent functions. Altogether, our work contributes to better understand the mechanisms underlining the complex interactions between IAV and the p53 pathway, for which both NS1 and Mdm2 arise as key players

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

Studying kinetochore kinases

Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics of their localized activation and deactivation is therefore instrumental for understanding the spatiotemporal control of chromosome segregation

Effects of YM155 on survivin levels and viability in neuroblastoma cells with acquired drug resistance

Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug's mechanism of action

Genome-Wide Identification of Molecular Pathways and Biomarkers in Response to Arsenic Exposure in Zebrafish Liver

10.1371/journal.pone.0068737PLoS ONE87-POLN