30 results on '"MDMX"'
Search Results
2. Computational modeling of cyclic peptide inhibitor–MDM2/MDMX binding through global docking and Gaussian accelerated molecular dynamics simulations
- Author
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Tian-Lu Cheng and Yeng-Tseng Wang
- Subjects
chemistry.chemical_classification ,MDMX ,Gaussian ,Nuclear Proteins ,Cell Cycle Proteins ,Proto-Oncogene Proteins c-mdm2 ,Peptide ,General Medicine ,Interaction energy ,Molecular Dynamics Simulation ,Peptides, Cyclic ,Cyclic peptide ,Molecular dynamics ,symbols.namesake ,chemistry ,Structural Biology ,Docking (molecular) ,Proto-Oncogene Proteins ,Biophysics ,symbols ,Humans ,Tumor Suppressor Protein p53 ,Potential of mean force ,Molecular Biology ,Protein Binding - Abstract
MDM2 and MDMX are potential targets for p53-dependent cancer therapy. Peptides are key in cellular immunology and oncology, and cyclic peptides generally have higher half-life than their linear counterparts. However, prediction of cyclic peptide-protein binding is challenging with normal molecular simulation approaches because of high peptide flexibility. Here, we used global peptide docking, normal molecular dynamics, Gaussian accelerated molecular dynamics (GaMD), two-dimensional (2D) potential of mean force (PMF) profiles, and solvated interaction energy (SIE) techniques to investigate the interactions of MDM2/MDMX with three N-to-C-terminal cyclic peptide-based inhibitors. We determined the possible cyclic peptide-MDM2/MDMX complex structures via 2D PMF profiles and SIE calculations. Our findings increase the accuracy of peptide-protein structural prediction, which may facilitate cyclic peptide drug design. Advancements in the computational methods and computing power may further aid in addressing the challenges in cyclic peptide drug design. Communicated by Ramaswamy H. Sarma.
- Published
- 2020
3. Dual-target MDM2/MDMX inhibitor increases the sensitization of doxorubicin and inhibits migration and invasion abilities of triple-negative breast cancer cells through activation of TAB1/TAK1/p38 MAPK pathway
- Author
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Enxiao Li, Yu Shi, Yangwei Fan, Jiayu Jing, Yuan Hu, Mengya Li, Ke Ma, and Danfeng Dong
- Subjects
0301 basic medicine ,MAPK/ERK pathway ,Cancer Research ,MDMX ,Aftercare ,Cell Cycle Proteins ,Triple Negative Breast Neoplasms ,Kaplan-Meier Estimate ,Mice ,0302 clinical medicine ,Cell Movement ,Antineoplastic Combined Chemotherapy Protocols ,Breast ,RNA, Small Interfering ,Triple-negative breast cancer ,biology ,Chemistry ,Kinase ,Drug Synergism ,Proto-Oncogene Proteins c-mdm2 ,Middle Aged ,MAP Kinase Kinase Kinases ,Recombinant Proteins ,Gene Expression Regulation, Neoplastic ,Oncology ,Gene Knockdown Techniques ,030220 oncology & carcinogenesis ,Molecular Medicine ,Mdm2 ,Female ,Research Paper ,medicine.drug ,Adult ,Epithelial-Mesenchymal Transition ,MAP Kinase Signaling System ,Mice, Nude ,Young Adult ,03 medical and health sciences ,Cell Line, Tumor ,Proto-Oncogene Proteins ,medicine ,Animals ,Humans ,Neoplasm Invasiveness ,Doxorubicin ,Epithelial–mesenchymal transition ,Adaptor Proteins, Signal Transducing ,Aged ,Retrospective Studies ,Pharmacology ,Transplantation ,030104 developmental biology ,Drug Resistance, Neoplasm ,biology.protein ,Cancer research - Abstract
Triple-negative breast cancer (TNBC) has a poor prognosis mainly due to insensitivity or resistance to standard anthracycline- and taxane-based chemotherapy, urgently calling for new adjuvants to reverse drug resistance. Dual-target murine double minute 2 (MDM2) and murine double minute X (MDMX) inhibitor has been proved to play a critical part against cancer, particularly focusing on the tremendous potential to enhance the efficacy of doxorubicin (DOX), however little was reported in TNBC. In the present study, we investigated the synergistic antitumor effect of the MDM2/MDMX inhibitor with DOX using three TNBC cell lines, two in situ transplantation tumor models and 214 clinical samples. We observed that the MDM2/MDMX inhibitor combined with DOX could not only inhibit cell vitality and migration and invasion abilities, but also highly inhibit tumor growth in TNBC nude mice. Besides, co-treatment of MDM2/MDMX inhibitor and DOX suppressed epithelial to mesenchymal transition (EMT) through increasing the TAK1-binding protein 1 (TAB1), transforming growth factor β-activated kinase 1 (TAK1) and p38 mitogen-activated protein kinase (MAPK) expression. Small interfering RNA-mediated TAB1 knockdown induced the EMT, desensitized cells to DOX and enhanced the migration and invasion abilities. High MDM2/MDMX expression was positively associated with weak TAB1 expression in 214 TNBC tumor tissues confirmed by immumohistochemical staining and MDM2/MDMX/TAB1 expression was significantly related to TNBC patient survival. These findings indicate that dual-target MDM2/MDMX inhibitor could increase the sensitization of doxorubicin and inhibit migration and invasion abilities in TNBC cells through p38 MAPK pathway activation caused EMT suppression and hence could be useful in TNBC treatments in future.
- Published
- 2018
4. AMP-Activated Protein Kinase Induces p53 by Phosphorylating MDMX and Inhibiting Its Activity
- Author
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Zhijun Luo, Hua Lu, Shelya X. Zeng, X. Charlie Dong, Benoit Viollet, Jun-Ho Lee, Yi Wei Zhang, Geoffrey M. Wahl, Guifen He, and Yunyuan V. Wang
- Subjects
Male ,MDMX ,Molecular Sequence Data ,Cell Cycle Proteins ,Mice, Transgenic ,Serine ,Gene Knockout Techniques ,Mice ,AMP-activated protein kinase ,Stress, Physiological ,Proto-Oncogene Proteins ,Animals ,Humans ,Amino Acid Sequence ,Phosphorylation ,Protein kinase A ,Molecular Biology ,biology ,Adenylate Kinase ,HEK 293 cells ,Ubiquitination ,Nuclear Proteins ,AMPK ,Articles ,Cell Cycle Checkpoints ,Cell Biology ,HCT116 Cells ,Mice, Inbred C57BL ,HEK293 Cells ,14-3-3 Proteins ,Amino Acid Substitution ,Biochemistry ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,Protein Binding - Abstract
AMP-activated protein kinase (AMPK) has been shown to activate p53 in response to metabolic stress. However, the underlying mechanisms remain unclear. Here we show that metabolic stresses induce AMPK-mediated phosphorylation of human MDMX on Ser342 in vitro and in cells, leading to enhanced association between MDMX and 14-3-3. This markedly inhibits p53 ubiquitylation and significantly stabilizes and activates p53. By striking contrast, no phosphorylation of MDM2 by AMPK was noted. AMPK-mediated MDMX phosphorylation, MDMX-14-3-3 binding, and p53 activation were drastically reduced in mouse embryo fibroblasts harboring endogenous MDMX with S341A (mouse homologue of human serine 342), S367A, and S402A (mouse homologue of human serine 403) mutations. Moreover, deficiency of AMPK prevented MDMX-14-3-3 interaction and p53 activation. The activation of p53 through AMPK-mediated MDMX phosphorylation and inactivation was further confirmed by using cell and animal model systems with two AMPK activators, metformin and salicylate (the active form of aspirin). Together, the results unveil a mechanism by which metabolic stresses activate AMPK, which, in turn, phosphorylates and inactivates MDMX, resulting in p53 stabilization and activation.
- Published
- 2014
5. On the interaction mechanisms of a p53 peptide and nutlin with the MDM2 and MDMX proteins: A Brownian dynamics study
- Author
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Reidun Twarock, Chandra S. Verma, Thomas Leonard Joseph, David P. Lane, Karim M. ElSawy, and Leo S. D. Caves
- Subjects
MDMX ,Structural similarity ,Static Electricity ,Allosteric regulation ,Cell Cycle Proteins ,Plasma protein binding ,Molecular Dynamics Simulation ,Piperazines ,chemistry.chemical_compound ,Proto-Oncogene Proteins c-mdm2 ,Neoplasms ,Proto-Oncogene Proteins ,Humans ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Genetics ,Extra Views ,Binding Sites ,biology ,Cell Cycle ,Imidazoles ,Nuclear Proteins ,Cell Biology ,Nutlin ,chemistry ,biology.protein ,Biophysics ,Mdm2 ,Tumor Suppressor Protein p53 ,Sequence Alignment ,Protein Binding ,Developmental Biology - Abstract
The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53/nutlin interactions with MDM2 and MDMX and could potentially have a broader impact on anticancer drug optimization strategies.
- Published
- 2013
6. Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding
- Author
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Ernst Schönbrunn, Jiandong Chen, Shaofang Wu, Lihong Chen, and Andreas Becker
- Subjects
MDMX ,DNA damage ,Molecular Sequence Data ,Plasma protein binding ,Biology ,Cell Line ,Mice ,Protein structure ,Cell Line, Tumor ,Animals ,Humans ,Amino Acid Sequence ,Protein Interaction Maps ,Phosphorylation ,Binding site ,Molecular Biology ,Binding Sites ,Casein Kinase Ialpha ,Proto-Oncogene Proteins c-mdm2 ,Articles ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,Biochemistry ,Casein kinase 1 ,Tumor Suppressor Protein p53 ,DNA Damage ,Protein Binding ,P53 binding - Abstract
MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1α (CK1α) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1α stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1α binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1α complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1α is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1α-MDMX interaction and reducing MDMX-p53 binding affinity.
- Published
- 2012
7. MdmX Is Required for p53 Interaction with and Full Induction of the Mdm2 Promoter after Cellular Stress
- Author
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James L. Manley, Masha V. Poyurovsky, Lynn Biderman, Carol Prives, and Yael Assia
- Subjects
Transcriptional Activation ,MDMX ,DNA damage ,Cell Line ,Transactivation ,Proto-Oncogene Proteins c-mdm2 ,Cell Line, Tumor ,Neoplasms ,Phosphoprotein Phosphatases ,Animals ,Humans ,RNA, Messenger ,RNA, Small Interfering ,Promoter Regions, Genetic ,Molecular Biology ,Regulation of gene expression ,biology ,Promoter ,Articles ,Cell Biology ,Cell biology ,Gene Expression Regulation, Neoplastic ,Protein Phosphatase 2C ,biology.protein ,Cancer research ,Mdm2 ,Tumor Suppressor Protein p53 ,DNA Damage ,P53 binding - Abstract
The activity of the tumor suppressor p53 is tightly controlled by its main negative regulator, Mdm2, which inhibits p53's transcriptional activity and targets it for degradation via the proteasome pathway. The closely related Mdm2 homolog, MdmX, is also considered to be a general inhibitor of transactivation by p53, through binding to the p53 activation domain. We show here that, unexpectedly, upon DNA damage and ribosomal stress, MdmX plays a positive role in p53-mediated activation of the Mdm2 gene, but not of numerous other p53 target genes including p21. Downregulation of MdmX results in lower levels of mature and nascent Mdm2 transcripts following cellular stress. This correlates with a longer p53 half-life following DNA damage. In vitro, Mdm2 inhibits the binding of p53 to DNA to a much greater extent than does MdmX, although MdmX does not stimulate p53 interaction with Mdm2 promoter DNA. Strikingly, however, MdmX is required for optimal p53 binding to the Mdm2 promoter in vivo. Thus, we have described a new mechanism by which MdmX can suppress p53, which is through transcriptional activation of p53's principal negative regulator, Mdm2.
- Published
- 2012
8. p53 regulation: Teamwork between RING domains of Mdm2 and MdmX
- Author
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Xinjiang Wang
- Subjects
Aging ,MDMX ,Ubiquitin-Protein Ligases ,Proteolysis ,Cell Cycle Proteins ,Gene mutation ,Models, Biological ,law.invention ,Mice ,Protein structure ,Ubiquitin ,law ,Proto-Oncogene Proteins ,medicine ,Animals ,Humans ,Molecular Biology ,biology ,medicine.diagnostic_test ,TOR Serine-Threonine Kinases ,Ubiquitination ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,Ubiquitin ligase ,Gene Expression Regulation ,Biochemistry ,Multiprotein Complexes ,biology.protein ,Mdm2 ,Suppressor ,Tumor Suppressor Protein p53 ,Developmental Biology - Abstract
p53 is a major tumor suppressor frequently inactivated through direct gene mutation and alternative mechanisms including overexpression of Mdm2 and MdmX. Both Mdm2 and MdmX are essential for negative regulation of p53 in vivo in a mutually dependent manner. The RING domain dependent E3 ligase activity of Mdm2 has been shown to be essential for negative regulation of p53. The prevailing model has dubbed MdmX as an inhibitor of p53 transcriptional activity through direct binding of its N-terminal domain to p53. However, recent findings established an essential role of the RING domain of MdmX in p53 degradation in vitro and in vivo. Biochemically, Mdm2 on its own is a monoubiquitinatuion E3 ligase, however, MdmX can convert Mdm2 into a polyubiquitination E3 ligase necessary for p53 proteasomal degradation in cells, through their RING-RING interactions. While Mdm2 is the catalytic components of Mdm2/MdmX E3 complex, MdmX is both the activating component and a substrate of the holoenzyme. Knock-in of RING mutant MdmX in mice causes p53-dependent embryonic lethality in a similar manner as knockout of MdmX whole gene. The new advance of the field assigned an essential role of the RING domain of MdmX in negative regulation of p53 in vivo, just like Mdm2 RING domain, through p53 degradation.
- Published
- 2011
9. Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73
- Author
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Benedykt Wladyka, Sylwia Kedracka-Krok, Stefan Jankowski, Katarzyna Pustelny, Michal Zdzalik, Adam Dubin, Aleksandra Pecak, Jan Potempa, Krzysztof Huben, and Grzegorz Dubin
- Subjects
MDMX ,tumor suppressor protein p53 ,protein-protein interactions ,Cell Cycle Proteins ,proto-oncogene proteins ,Plasma protein binding ,Biology ,Protein–protein interaction ,Transactivation ,Protein structure ,Proto-Oncogene Proteins ,Humans ,Tumor Protein p73 ,neoplasms ,Molecular Biology ,Transcription factor ,Genetics ,Tumor Suppressor Proteins ,p53/p63/p73 family of transcription factors ,Tryptophan ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,DNA-Binding Proteins ,Mdm2/Mdmx regulators ,Trans-Activators ,biology.protein ,Thermodynamics ,Mdm2 ,Tumor Suppressor Protein p53 ,Protein Binding ,Transcription Factors ,Developmental Biology - Abstract
The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer. Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation. Two p53 homologues, p63 and p73, have been described in humans. Unlike p53, these proteins regulate developmental processes rather than genome stability. Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx. Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined. Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.
- Published
- 2010
10. p53 inactivation by MDM2 and MDMX negative feedback loops in testicular germ cell tumors
- Author
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Baozong Li, Zhenyu Li, Qian Cheng, and Jiandong Chen
- Subjects
Male ,Chromatin Immunoprecipitation ,MDMX ,Blotting, Western ,Cell ,Cell Cycle Proteins ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,Article ,Malignant transformation ,chemistry.chemical_compound ,Testicular Neoplasms ,Cell Line, Tumor ,Proto-Oncogene Proteins ,medicine ,Humans ,Nuclear protein ,Molecular Biology ,Mutation ,Gene knockdown ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Nutlin ,Neoplasms, Germ Cell and Embryonal ,medicine.anatomical_structure ,chemistry ,Cancer research ,biology.protein ,Mdm2 ,RNA Interference ,Tumor Suppressor Protein p53 ,Developmental Biology - Abstract
Testicular germ cell tumors (TGCT) are unique in their excellent response to DNA-damaging chemotherapy. Mutation of p53 is rare in both untreated and relapsed TGCTs, suggesting that p53 fails to respond effectively against malignant transformation in germ cells. Previous studies implicated the presence of a poorly defined TGCT-specific mechanism of p53 inactivation. Here we show that disruption of p53-mdm2 binding using the MDM2-specific inhibitor Nutlin activates p53 in TGCT cells and is sufficient to induce strong apoptosis. Knockdown of MDMX cooperates with Nutlin to activate p53. Surprisingly, we found that p53 activation induced a two-fold increase in MDMX mRNA and protein expression in TGCT cells. A p53-responsive promoter is identified in MDMX intron 1 that contains a functional p53-binding site, suggesting that MDMX also functions as a negative feedback regulator of p53 in a cell line-dependent fashion. These findings suggest that MDM2 and MDMX are responsible for the functional inactivation of p53 in TGCT. Furthermore, TGCT cells are unique in having a strong apoptosis response to p53. Direct activation of p53 by targeting MDM2 and MDMX may provide a backup approach for the treatment of TGCTs resistant to DNA-damaging drugs.
- Published
- 2010
11. Structures of low molecular weight inhibitors bound to MDMX and MDM2 reveal new approaches for p53-MDMX/MDM2 antagonist drug discovery
- Author
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Grzegorz M. Popowicz, Tad A. Holak, Siglinde Wolf, Kan Wang, Anna Czarna, Alexander Dömling, and Wei Wang
- Subjects
Models, Molecular ,Indoles ,MDMX ,Antineoplastic Agents ,Ligands ,Drug Discovery ,Humans ,neoplasms ,Molecular Biology ,chemistry.chemical_classification ,DNA ligase ,biology ,P53 pathway ,Drug discovery ,Antagonist ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Small molecule ,Anticancer drug ,Molecular Weight ,Kinetics ,enzymes and coenzymes (carbohydrates) ,chemistry ,biology.protein ,Cancer research ,Biophysics ,Mdm2 ,Tumor Suppressor Protein p53 ,Protein Binding ,Developmental Biology - Abstract
Intensive anticancer drug discovery efforts have been made to develop small molecule inhibitors of the p53-MDM2 and p53-MDMX interactions. We present here the structures of the most potent inhibitors bound to MDM2 and MDMX that are based on the new imidazo-indole scaffold. In addition, the structure of the recently reported spiro-oxindole inhibitor bound to MDM2 is described. The structures indicate how the substituents of a small molecule that bind to the three subpockets of the MDM2/X-p53 interaction should be optimized for effective binding to MDM2 and/or MDMX. While the spiro-oxindole inhibitor triggers significant ligand-induced changes in MDM2, the imidazo-indoles share similar binding modes for MDMX and MDM2, but cause only minimal induced-fit changes in the structures of both proteins. Our study includes the first structure of the complex between MDMX and a small molecule and should aid in developing efficient scaffolds for binding to MDMX and/or MDM2.
- Published
- 2010
12. Differential binding of p53 and nutlin to MDM2 and MDMX: Computational studies
- Author
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Thomas Leonard Joseph, Chandra S. Verma, Arumugam Madhumalar, David P. Lane, and Christopher J. Brown
- Subjects
Models, Molecular ,MDMX ,Molecular Sequence Data ,Biology ,Piperazines ,Motion ,chemistry.chemical_compound ,Low affinity ,Humans ,Amino Acid Sequence ,Amino Acids ,neoplasms ,Molecular Biology ,Principal Component Analysis ,Binding Sites ,Imidazoles ,Computational Biology ,Hydrogen Bonding ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Nutlin ,Cell biology ,Gain of function ,chemistry ,Cancer research ,biology.protein ,Thermodynamics ,Mdm2 ,Mutant Proteins ,Tumor Suppressor Protein p53 ,Peptides ,Sequence Alignment ,Signalling pathways ,Function (biology) ,Protein Binding ,Developmental Biology - Abstract
Half of human tumours have mutated p53 while in the other half, defective signalling pathways block its function. One such defect is the overexpression of the MDM2 and MDMX proteins. This has led to an intense effort to develop inhibitors of p53-MDM2/MDMX interactions. Nutlin is the first such compound described to block p53-MDM2 interactions. Molecular dynamics simulations have been used to explore the differences in binding of p53 and nutlin to MDM2/MDMX. Simulations reveal that p53 has a higher affinity for MDM2 than MDMX, driven by stronger electrostatic interactions. p53 is displaced from MDM2 by nutlin because it is more flexible, thus paying a larger entropic penalty upon sequestration by MDM2. The inherent plasticity of MDM2 is higher than that of MDMX, enabling it to bind both p53 and nutlin. The less flexible MDMX interacts with the more mobile p53 because the peptide can adapt conformationally to dock into MDMX, albeit with a reduced affinity; nutlin, however is rigid and hence can only interact with MDMX with low affinity. Evolutionarily, the higher affinity of MDM2 for p53 may enable MDM2 to bind p53 for longer periods as it shuttles it out of the nucleus; in contrast, MDMX only needs to mask the p53 TA domain. This study enables us to hypothesize gain of function mutations or those that have decreased affinity for nutlin. These conclusions provide insight into future drug design for dual inhibitors of MDM2 and MDMX, both of which are oncoproteins found overexpressed in many cancers.
- Published
- 2010
13. Mechanism of p53 stabilization by ATM after DNA damage
- Author
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Jiandong Chen and Qian Cheng
- Subjects
MDMX ,DNA damage ,Ubiquitin-Protein Ligases ,Molecular Sequence Data ,Cell Cycle Proteins ,Ataxia Telangiectasia Mutated Proteins ,Protein Serine-Threonine Kinases ,Biology ,Models, Biological ,Article ,Mice ,Proto-Oncogene Proteins c-mdm2 ,Ubiquitin ,Animals ,Amino Acid Sequence ,Phosphorylation ,Protein Structure, Quaternary ,Molecular Biology ,Protein Stability ,Tumor Suppressor Proteins ,Ubiquitination ,Cell Biology ,Cell biology ,Ubiquitin ligase ,DNA-Binding Proteins ,biology.protein ,Cancer research ,Mdm2 ,Tumor Suppressor Protein p53 ,DNA Damage ,Developmental Biology - Abstract
p53 suppresses tumor development by responding to unauthorized cell proliferation, growth factor or nutrient deprivation, and DNA damage. Distinct pathways have been identified that cause p53 activation, including ARF-dependent response to oncogene activation, ribosomal protein-mediated response to abnormal rRNA synthesis, and ATM-dependent response to DNA damage. Elucidating the mechanisms of these signaling events are critical for understanding tumor suppression by p53 and development of novel cancer therapeutics. More than a decade of research has established the ATM kinase as a key molecule that activates p53 after DNA damage. Our recent study revealed that ATM phosphorylation of MDM2 is likely to be the key step in causing p53 stabilization. Upon activation by ionizing irradiation, ATM phosphorylates MDM2 on multiple sites near its RING domain. These modifications inhibit the ability of MDM2 to poly-ubiquitinate p53, thus leading to its stabilization. MDM2 phosphorylation does not inactivate its E3 ligase activity per se, since MDM2 self-ubiquitination and MDMX ubiquitination functions are retained. The selective inhibition of p53 poly-ubiquitination is accomplished through disrupting MDM2 oligomerization that may provide a scaffold for processive elongation of poly ubiquitin chains. These findings suggest a novel model of p53 activation and a general mechanism of E3 ligase regulation by phosphorylation.
- Published
- 2010
14. MdmX regulates transformation and chromosomal stability in p53-deficient cells
- Author
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Anna Krzywicka-Racka, Greenfield Sluder, Zdenka Matijasevic, and Stephen N. Jones
- Subjects
MDMX ,Cell division ,Mitosis ,Video microscopy ,medicine.disease_cause ,Article ,Mice ,Chromosomal Instability ,medicine ,Animals ,Humans ,Molecular Biology ,Centrosome ,biology ,Cell growth ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Embryonic stem cell ,Cell biology ,Cell Transformation, Neoplastic ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,Carcinogenesis ,Cell Division ,Developmental Biology - Abstract
The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.
- Published
- 2008
15. Elevated MDM2 boosts the apoptotic activity of p53-MDM2 binding inhibitors by facilitating MDMX degradation
- Author
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Baoying Huang, David C. Heimbrook, Christian Tovar, Mingxuan Xia, Lyubomir T. Vassilev, and Dejan Knezevic
- Subjects
MDMX ,Cell cycle checkpoint ,DNA damage ,Immunoblotting ,Apoptosis ,Cell Cycle Proteins ,Biology ,Piperazines ,law.invention ,chemistry.chemical_compound ,law ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Humans ,RNA, Small Interfering ,Molecular Biology ,DNA Primers ,Reverse Transcriptase Polymerase Chain Reaction ,Imidazoles ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Nutlin ,Ubiquitin ligase ,Gene Expression Regulation, Neoplastic ,chemistry ,Doxorubicin ,Cancer cell ,biology.protein ,Cancer research ,Suppressor ,Mdm2 ,Tumor Suppressor Protein p53 ,Signal Transduction ,Developmental Biology - Abstract
The p53 tumor suppressor is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in human cancer. Many tumors overproduce its negative regulator MDM2, a specific p53 ubiquitin ligase and transcriptional inhibitor, to disable p53 function. Therefore, p53 activation by inhibiting MDM2 has been proposed as a novel strategy for cancer therapy in tumors expressing wild-type p53. Recently developed small-molecule p53-MDM2 binding inhibitors, the nutlins, selectively activate p53 function and induce cell cycle arrest and apoptosis in cancer cells. By stabilizing p53, nutlins also elevate the cellular level of its transcriptional target MDM2. Here, we present evidence that nutlin-induced MDM2 retains its ubiquitin ligase activity and contributes to the anti-tumor activity of p53-MDM2 binding inhibitors by facilitating the degradation of another p53 inhibitor, MDMX. MDM2 and MDMX levels were analyzed in a panel of 12 randomly selected solid tumor cell lines. In the presence of nutlin-3, MDM2 increased in all and MDMX decreased in most of the cell lines. MDMX was resistant to nutlin-induced degradation in 2/12 cell lines. In these cells, MDMX appears to be a major suppressor of the apoptotic response to p53 activation although this effect was only partially p53-dependent. Doxorubicin facilitated MDMX degradation through DNA damage response pathways and restored their sensitivity to nutlin, suggesting that combination therapy may be an effective way to overcome nutlin resistance in cancers with MDMX aberrations.
- Published
- 2008
16. CARPs enhance p53 turnover by degrading 14-3-3σ and stabilizing MDM2
- Author
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Jiandong Chen, Wafik S. El-Deiry, David T. Dicker, and Wensheng Yang
- Subjects
Exonucleases ,Programmed cell death ,MDMX ,Regulator ,Cell Cycle Proteins ,Nerve Tissue Proteins ,Plasma protein binding ,Mice ,Proto-Oncogene Proteins c-mdm2 ,Cell Line, Tumor ,Biomarkers, Tumor ,Animals ,Humans ,Cell Cycle Protein ,Carp ,neoplasms ,Molecular Biology ,biology ,Cell Biology ,biology.organism_classification ,Neoplasm Proteins ,Cell biology ,enzymes and coenzymes (carbohydrates) ,14-3-3 Proteins ,Exoribonucleases ,Cancer research ,biology.protein ,Thermodynamics ,Mdm2 ,sense organs ,Tumor Suppressor Protein p53 ,Apoptosis Regulatory Proteins ,Carrier Proteins ,Protein Processing, Post-Translational ,Protein Binding ,Developmental Biology - Abstract
CARP1 and CARP2 proteins (CARPs) are E3 ligases that target p53 as well as phospho-p53 for degradation. Because MDM2 is a critical regulator of p53 turnover, we investigated and found that CARPs associate with MDM2. We provide evidence that CARPs stabilize MDM2 by inhibiting MDM2 self-ubiquitination. CARPs together with MDM2 enhance p53 degradation, thereby inhibiting p53-mediated cell death. CARP protein levels correlate with MDM2 levels including under hypoxia where both are reduced. CARP2 was found to target 14-3-3sigma for degradation, leading to MDM2 stabilization. MDMX, a homolog of MDM2, is not absolutely required for MDM2 stabilization by CARPs, although overexpression of CARP2 enhances MDM2/MDMX interaction. Taken together, our study identifies novel mechanisms by which CARP proteins regulate the p53 signaling pathway.
- Published
- 2008
17. Distinct Roles of MDMX in the Regulation of p53 Response to Ribosomal Stress
- Author
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Jiandong Chen and Daniele M. Gilkes
- Subjects
MDMX ,Cell cycle checkpoint ,biology ,Regulator ,Ribosome biogenesis ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Ribosomal RNA ,Molecular biology ,Cell biology ,Oxidative Stress ,Ribosomal protein ,biology.protein ,Animals ,Humans ,Mdm2 ,Tumor Suppressor Protein p53 ,Ribosomes ,Molecular Biology ,Biogenesis ,Developmental Biology - Abstract
Approximately 50% of protein and RNA synthesis in proliferating cells are devoted to ribosomal biogenesis. Coordination between ribosome biogenesis, growth, and proliferation is critical for maintenance of homeostasis and tumor suppression. Aberrant rRNA expression and processing is sensed by p53. Ribosomal stress increases the binding between MDM2 and ribosomal proteins L5, L11, and L23, resulting in p53 stabilization. Our recent study showed that p53 activation by ribosomal stress also involves degradation of MDMX in an MDM2-dependent fashion. Failure to eliminate MDMX due to overexpression results in the sequestration of p53 into inactive complexes, severely impairing p53-dependent cell cycle arrest during ribosomal stress. Furthermore, MDMX overexpression promotes resistance to the chemotherapeutic agent 5-FU, which at low concentrations activates p53 by inhibiting RNA metabolism. Therefore, MDMX is an important regulator of p53 response to ribosomal stress. MDMX overexpression in tumors may significantly influence response to chemotherapy agents that target rRNA biogenesis.
- Published
- 2007
18. Glycogen Synthase Kinase 3-Dependent Phosphorylation of Mdm2 Regulates p53 Abundance
- Author
-
Karen A. Boehme, R. Kulikov, and Christine Blattner
- Subjects
Time Factors ,MDMX ,Cell Cycle Proteins ,Serine ,Glycogen Synthase Kinase 3 ,Mice ,GSK-3 ,Radiation, Ionizing ,Phosphorylation ,Nuclear protein ,Glutathione Transferase ,Alanine ,Reverse Transcriptase Polymerase Chain Reaction ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Biochemistry ,COS Cells ,Electrophoresis, Polyacrylamide Gel ,Plasmids ,Protein Binding ,Signal Transduction ,Cyclin-Dependent Kinase Inhibitor p21 ,Proteasome Endopeptidase Complex ,Immunoprecipitation ,Blotting, Western ,Molecular Sequence Data ,Protein domain ,macromolecular substances ,Biology ,Transfection ,Cell Line ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Animals ,Humans ,Amino Acid Sequence ,neoplasms ,Molecular Biology ,Ubiquitin ,Cell Biology ,Blotting, Northern ,Protein Structure, Tertiary ,enzymes and coenzymes (carbohydrates) ,Gene Expression Regulation ,Microscopy, Fluorescence ,Mutation ,RNA ,Tumor Suppressor Protein p53 ,Peptides - Abstract
The Mdm2 oncoprotein regulates abundance and activity of the p53 tumor suppressor protein. For efficient degradation of p53, Mdm2 needs to be phosphorylated at several contiguous residues within the central conserved domain. We show that glycogen synthase kinase 3 (GSK-3) phosphorylated the Mdm2 protein in vitro and in vivo in the central domain. Inhibition of GSK-3 rescued p53 from degradation in an Mdm2-dependent manner while its association with Mdm2 was not affected. Likewise, inhibition of GSK-3 did not alter localization of p53 and Mdm2 or the interaction of Mdm2 and MdmX. Ionizing radiation, which leads to p53 accumulation, directed phosphorylation of GSK-3 at serine 9, which preceded and overlapped with the increase in p53 levels. Moreover, expression of a GSK-3 mutant where serine 9 was replaced with an alanine reduced the accumulation of p53 and induction of its target p21(WAF-1). We therefore conclude that inhibition of GSK-3 contributes to hypophosphorylation of Mdm2 in response to ionizing rays, and in consequence to p53 stabilization.
- Published
- 2005
19. A New Twist in the Feedback Loop: Stress-Activated MDM2 Destabilization is Required for p53 Activation
- Author
-
Geoffrey M. Wahl and Jayne M. Stommel
- Subjects
Transcriptional Activation ,MDMX ,DNA Repair ,DNA repair ,DNA damage ,Cell Line ,Transactivation ,Proto-Oncogene Proteins ,Animals ,Humans ,Enzyme Inhibitors ,Phosphorylation ,neoplasms ,Molecular Biology ,Transcription factor ,Feedback, Physiological ,biology ,Cell Cycle ,Proto-Oncogene Proteins c-mdm2 ,DNA ,Cell Biology ,Cell cycle ,Protein Structure, Tertiary ,Ubiquitin ligase ,Cell biology ,Enzyme Activation ,Gene Expression Regulation, Neoplastic ,enzymes and coenzymes (carbohydrates) ,Gene Expression Regulation ,biology.protein ,Cancer research ,Mdm2 ,Tumor Suppressor Protein p53 ,Transcription Factors ,Developmental Biology - Abstract
The p53 tumor suppressor is a transcription factor that is activated by diverse genotoxic and cytotoxic stresses. Upon activation, p53 prevents the proliferation of genetically unstable cells by regulating the expression of genes that initiate cell cycle arrest, apoptosis, and DNA repair. Consequently, p53 must be kept inactive in unstressed cells as its inappropriate activation can cause premature senescence and death. p53 inhibition occurs primarily through the E3 ubiquitin ligase, MDM2. Because MDM2 is also a p53 target gene, stresses paradoxically activate p53 while simultaneously increasing MDM2 expression. Therefore, a challenge has been to explain how the abundant MDM2 is prevented from inhibiting p53, thus ensuring that p53 can execute an appropriate stress response. Here we discuss a new mechanism for p53 activation involving DNA damage-induced auto-degradation of MDM2. Our data reveal that DNA damage leads to the destabilization of MDM2, which correlates with p53 stabilization and target gene induction. Conversely, p53 levels and activity decrease when MDM2 returns to a more stable state later in the stress response. The destabilization of MDM2 is required for p53 activation, as blocking MDM2 degradation via proteasome inhibition prevents p53 transactivation in DNA-damaged cells by enabling MDM2 to bind and inhibit p53. MDM2 destabilization is controlled by DNA damage-activated post-translational modifications and by its own RING domain, implying a possible role for the RING domain-interacting protein, MDMX, in regulating MDM2 stability. We propose that accelerated degradation of MDM2 limits its binding to p53 during a stress response and enables p53 to accumulate and remain active, even as p53 transcriptionally activates more MDM2. Thus, the induction of MDM2 RNA by activated p53 may create a reserve of MDM2 that can inactivate p53 once the DNA damage stimulus has abated and MDM2 is restabilized. As many tumors inactivate wild type p53 through MDM2 overexpression, exploiting the pathways that trigger MDM2 auto-degradation may be an important new strategy for chemotherapeutic intervention.
- Published
- 2005
20. MdmX Inhibits ARF Mediated Mdm2 Sumoylation
- Author
-
Sarah Woodruff, Richard F. Kefford, and Helen Rizos
- Subjects
MDMX ,biology ,SUMO protein ,Cell Biology ,Plasma protein binding ,Ubiquitin ligase ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Transactivation ,Ubiquitin ,p14arf ,biology.protein ,Cancer research ,Mdm2 ,neoplasms ,Molecular Biology ,Developmental Biology - Abstract
Mdm2, by virtue of an intrinsic E3 ubiquitin ligase activity, is capable of autoubiquitination and the ubiquitination of the p53 tumor suppressor protein. Additionally, Hdm2 has been reported to undergo a p14ARF-dependent sumoylation with concurrent Hdm2 stabilization. In this present work, we report that MdmX can undergo ARF-mediated sumoylation similar to that reported for Mdm2. When coexpressed, MdmX overexpression results in a dose-dependent inhibition of Mdm2 sumoylation and a concurrent increase in Mdm2 ubiquitination. This switch from Mdm2 sumoylation to Mdm2 ubiquitination may explain the destablization of Mdm2 previously observed in cells overexpressing both ARF and MdmX. Given that MdmX can heterodimerize with Mdm2 and separately associate with ARF we employed a series of MdmX mutants to examine how MdmX blocks Mdm2 sumoylation. A MdmX miniprotein capable of binding to ARF, but not p53 or Mdm2 was able to competitively inhibit Mdm2 sumoylation and reverse ARF mediated activation of p53 transactivation. Taken together, these results demonstrate that MdmX can affect post-translational modification and stability of Mdm2 and p53 activity through interaction with ARF.
- Published
- 2005
21. Amplification of Mdmx (or Mdm4) Directly Contributes to Tumor Formation by Inhibiting p53 Tumor Suppressor Activity
- Author
-
Pier Giuseppe Pelicci, Ruth Frenk, Erik Meulmeester, Domenico Migliorini, Maria Capra, Alberto Gobbi, Patrizia Gasparini, Diego Pasini, Jean-Christophe Marine, Aart G. Jochemsen, Davide Danovi, Sarah Francoz, Kristian Helin, and Petra de Graaf
- Subjects
Mutation ,MDMX ,biology ,Biologie moléculaire ,Cell Biology ,Transfection ,medicine.disease_cause ,Gene dosage ,Molecular biology ,Cancer research ,biology.protein ,medicine ,Mdm2 ,Biologie cellulaire ,Neoplastic transformation ,HRAS ,Nuclear protein ,Cell Growth and Development ,Molecular Biology - Abstract
Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14ARF or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRasV12. Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy., SCOPUS: ar.j, info:eu-repo/semantics/published
- Published
- 2004
22. Dynamics in the p53-Mdm2 Ubiquitination Pathway
- Author
-
Christopher L. Brooks and Wei Gu
- Subjects
MDMX ,biology ,Cell Biology ,P53 mdm2 ,law.invention ,Cell biology ,Ubiquitin ligase ,Biochemistry ,p14arf ,Ubiquitin ,law ,biology.protein ,Suppressor ,Mdm2 ,neoplasms ,Molecular Biology ,Developmental Biology ,Deubiquitination - Abstract
The tumor suppressor p53 is highly regulated under various states of cellular stress. p53 stability is predominantly regulated through the ubiquitin-proteasomal pathway by the E3 ligase Mdm2. p53 ubiquitination is a dynamic process with Mdm2 capable of catalyzing both mono- and polyubiquitination. Additionally, deubiquitination is an important step occurring in p53 and Mdm2 stabilities. Factors such as HAUSP, p14ARF, and MdmX play important regulatory roles in p53 ubiquitination/deubiquitination and their interplay with Mdm2 and p53 compound layers of complexity for regulating this important pathway.
- Published
- 2004
23. Mdmx and Mdm2: Brothers in Arms?
- Author
-
Aart G. Jochemsen and Jean-Christophe Marine
- Subjects
Regulation of gene expression ,MDMX ,Effector ,Wild type ,Cell Biology ,Biology ,Gene mutation ,medicine.disease_cause ,medicine ,Cancer research ,biology.protein ,Mdm2 ,Carcinogenesis ,Molecular Biology ,Gene ,Developmental Biology - Abstract
The p53 tumor suppressor pathway is inactivated in most if not all human tumors. In about 50% of the cases this is accomplished directly by gene mutations. The tumors that retain wild type p53 frequently show defects either in effector target genes, or in the expression of p53 regulatory proteins. The Mdm2 protein is generally considered THE master regulator of the p53 tumor suppressor activity. Recently, however, the Mdm2-related protein Mdmx is taking the stage in the p53-Mdm2-Mdmx play. We summarize here observations unambiguously assigning a critical role for the Mdmx protein in the regulation of p53 function during development and tumor formation.
- Published
- 2004
24. MdmX Represses E2F1 Transactivation
- Author
-
Karen Weghorst, Mark Wunderlich, Steven J. Berberich, and Mithua Ghosh
- Subjects
Transcriptional Activation ,MDMX ,Cellular differentiation ,Blotting, Western ,Cell Cycle Proteins ,Biology ,Transfection ,Retinoblastoma Protein ,Transactivation ,Proto-Oncogene Proteins c-mdm2 ,p14arf ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Humans ,E2F1 ,Luciferases ,Molecular Biology ,Dose-Response Relationship, Drug ,Nuclear Proteins ,Cell Differentiation ,Cell Biology ,beta-Galactosidase ,Precipitin Tests ,E2F Transcription Factors ,Protein Structure, Tertiary ,DNA-Binding Proteins ,Microscopy, Fluorescence ,Cancer research ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,biological phenomena, cell phenomena, and immunity ,Signal transduction ,Dimerization ,Cell Division ,E2F1 Transcription Factor ,Gene Deletion ,Plasmids ,Signal Transduction ,Transcription Factors ,Developmental Biology - Abstract
Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.
- Published
- 2004
25. Critical Contribution of the MDM2 Acidic Domain to p53 Ubiquitination
- Author
-
Zhi-Min Yuan, Hidehiko Kawai, and Dmitri Wiederschain
- Subjects
MDMX ,Ubiquitin-Protein Ligases ,Ligases ,Mice ,Ubiquitin ,Proto-Oncogene Proteins c-mdm2 ,Proto-Oncogene Proteins ,Animals ,Nuclear protein ,Nuclear export signal ,Cell Growth and Development ,neoplasms ,Molecular Biology ,Cells, Cultured ,Cell Nucleus ,Binding Sites ,biology ,Nuclear Proteins ,Cell Biology ,Fibroblasts ,Fusion protein ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Ubiquitin ligase ,Cell biology ,enzymes and coenzymes (carbohydrates) ,Protein Transport ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 - Abstract
MDM2 is an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Recent studies have shown, however, that the ring-finger domain (RFD) of MDM2, where the ubiquitin E3 ligase activity resides, is necessary but not sufficient for p53 ubiquitination, suggesting that an additional activity of MDM2 might be required. To test this possibility, we generated a series of MDM2/MDMX chimeric proteins to assess the contribution of each domain of MDM2 to the ubiquitination process. MDMX is a close structural homolog of MDM2 that nevertheless lacks the E3 ligase activity in vivo. We demonstrate here that MDMX gains self-ubiquitination activity and becomes extremely unstable upon introduction of the MDM2 RFD, indicating that the RFD is essential for self-ubiquitination. This MDMX chimeric protein, however, is unable to ubiquitinate p53 in vivo despite its E3 ligase activity and binding to p53, separating the self-ubiquitination activity of MDM2 from its ability to ubiquitinate p53. Significantly, fusion of the central acidic domain (AD) of MDM2 to the MDMX chimeric protein renders the protein fully capable of ubiquitinating p53, and p53 ubiquitination is associated with p53 degradation and nuclear export. Moreover, the AD mini protein expressed in trans can functionally rescue the AD-lacking MDM2 mutant, further supporting a critical role for the AD in MDM2-mediated p53 ubiquitination.
- Published
- 2003
26. DNA Damage Induces MDMX Nuclear Translocation by p53-Dependent and -Independent Mechanisms
- Author
-
Lihong Chen, Changgong Li, and Jiandong Chen
- Subjects
Cytoplasm ,MDMX ,DNA damage ,Blotting, Western ,Active Transport, Cell Nucleus ,Regulator ,Transfection ,Polymerase Chain Reaction ,Adenoviridae ,Cell Line ,Proto-Oncogene Proteins ,Tumor Cells, Cultured ,Humans ,Nuclear protein ,Cell Growth and Development ,Molecular Biology ,Models, Genetic ,biology ,Gene Expression Regulation, Developmental ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Precipitin Tests ,Chromatin ,Cell biology ,Protein Transport ,Microscopy, Fluorescence ,Cell culture ,biology.protein ,Cancer research ,Mdm2 ,Tumor Suppressor Protein p53 ,DNA Damage ,Protein Binding - Abstract
The MDM2 homolog MDMX is an important regulator of p53 activity during embryonic development. MDMX inactivation in mice results in embryonic lethality in a p53-dependent fashion. The expression level of MDMX is not induced by DNA damage, and its role in stress response is unclear. We show here that ectopically expressed MDMX is mainly localized in the cytoplasm. DNA damage promotes nuclear translocation of MDMX in cells with or without p53. Coexpression of MDM2 or p53 is sufficient to induce MDMX nuclear translocation, suggesting that activation of p53 and induction of MDM2 expression can contribute to this process. Stable transfection of MDMX into U2OS cells does not alter p53 level but results in reduced p53 DNA-binding activity and reduced MDM2 expression. The ability of ARF (alternate reading frame of INK4a) to activate p53 is also significantly inhibited by expression of MDMX. These results suggest that MDMX function may be regulated by DNA damage. Furthermore, MDMX may complement MDM2 in regulating p53 during embryonic development due to its ability to inhibit p53 in the presence of ARF.
- Published
- 2002
27. The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation
- Author
-
Michael J. Sank, Maureen E. Murphy, Jack T. Zilfou, William H. Hoffman, and Donna L. George
- Subjects
Proteasome Endopeptidase Complex ,MDMX ,Proline ,Repressor ,Biology ,Models, Biological ,Cell Line ,Transactivation ,Drug Stability ,Proto-Oncogene Proteins c-mdm2 ,Multienzyme Complexes ,Proto-Oncogene Proteins ,Humans ,Point Mutation ,Nuclear protein ,Molecular Biology ,DNA Primers ,Sequence Deletion ,Transrepression ,Transcriptional Regulation ,Regulation of gene expression ,Binding Sites ,Base Sequence ,Nuclear Proteins ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,Repressor Proteins ,Cysteine Endopeptidases ,Sin3 Histone Deacetylase and Corepressor Complex ,Biochemistry ,Tumor Suppressor Protein p53 ,Protein Processing, Post-Translational ,Corepressor ,DNA Damage - Abstract
While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14(ARF), Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.
- Published
- 2001
28. MDM2/MDMX: Master negative regulators for p53 and RB
- Author
-
Johann Bergholz, Haibo Zhang, Linshan Hu, Shengnan Sun, and Zhi-Xiong Jim Xiao
- Subjects
0301 basic medicine ,Cancer Research ,MDMX ,biology ,Chemistry ,Retinoblastoma protein ,Molecular biology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Commentary ,biology.protein ,Molecular Medicine ,Gene silencing ,Mdm2 ,Tumor growth ,Tumor xenograft ,Protein p53 - Abstract
MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.
- Published
- 2016
29. Novel insights about the MDM2/MDM4 heterodimer
- Author
-
Fabiola Moretti
- Subjects
p53 ,0301 basic medicine ,Cancer Research ,MDMX ,DNA damage ,Apoptosis ,Biology ,Molecular biology ,Cell biology ,MDM4 ,03 medical and health sciences ,DDB1 ,030104 developmental biology ,MDM2 ,heterodimer ,Transcriptional repression ,biology.protein ,Molecular Medicine ,Mdm2 ,Phosphorylation ,Protein kinase A ,Author's View - Abstract
MDM2 (mouse double minute 2 homolog) and MDM4 (double minute 4 human homolog, also known as MDMX) inhibit the activity of tumor protein p53 (TP53, best known as p53) through their heterodimerization. New evidence indicates that under stress conditions the heterodimer is modified, leading to different activities of the single molecules. In particular, following lethal DNA damage, MDM2 and MDM4 dissociate and MDM4 promotes the stabilization of homeodomain-interacting protein kinase 2 (HIPK2) and the phosphorylation of p53, resulting in transcriptional repression of antiapoptotic targets of p53/HIPK2.
- Published
- 2015
30. Length matters
- Author
-
Masha V. Poyurovsky
- Subjects
MDMX ,biology ,Mechanism (biology) ,Cell Biology ,Processivity ,Ubiquitin ligase ,Cell biology ,Ubiquitin ,Proto-Oncogene Proteins c-mdm2 ,Biochemistry ,biology.protein ,Mdm2 ,Molecular Biology ,Function (biology) ,Developmental Biology - Abstract
Mechanisms controlling the p53 regulatory network remain the focus of numerous investigations in hopes of identifying more robust cancer therapies. Both Mdm2 and MdmX are found overexpressed in tumors with wild-type p53 and represent a key molecular device modulating p53 function. Thus, examining the interplay between these three proteins becomes highly relevant in the search for new pharmacological interventions in oncology. Mdm2 is a RING-type E3 ubiquitin ligase capable of forming homo-oligomers and hetero-oligomerization with MdmX via the extreme C termini of their RING domains. Since its discovery 15 years ago, MdmX has been assigned many roles in the regulation of p53, either on its own or in concert with Mdm2. While clearly an essential negative regulator or p53 in development, its lack of intrinsic ubiquitin ligase activity has made the mechanism of p53 regulation more elusive than in the case of Mdm2. The capacity of MdmX to stimulate Mdm2-mediated p53 ubiquitination was first reported in 2003.1 Subsequent biochemical comparisons of the activity of Mdm2–MdmX complexes showed that not only does the presence of MdmX in the complex alter the substrate specificity of the holo-enzyme, it also allows for poly-ubiquitin chain formation on p53 (modification required for nuclear exclusion and degradation of p53).2-4 In vitro observations describing the importance of the MdmX RING domain in regulation of p53 turnover have now gained in vivo experimental support from the two knock-in animal models.5,6 Consistent with the notion that MdmX is an essential component of p53 polyubiquitination/proteasomal degradation pathway, mice expressing either a point mutant in the MdmX RING domain or a RING domain deletion mutant succumbed to a p53-dependent embryonic lethality. These data implicate the RING domain of MdmX as the sole region of importance in the ability of MdmX to regulate p53 and, by extension, the Mdm2-MdmX complex (and not the Mdm2 homodimer), as the principle negative regulator of p53 activity during development. The growing body of evidence describing the presence of MdmX in the complex as crucial for target selectivity as well as the processivity of the holoezyme somewhat flies in the face of the existing structural data. Two published structures of the Mdm2 homodimer and Mdm2/MdmX heterodimer indicate virtually no difference in the complexes.7,8 In the absence of structural differences, how then are such significant differences in function accomplished? A hypothesis unifying structural and functional data is brought forth by a very intriguing study from the Uldrijan group, which systematically looks at the differences between complex formation and activity of Mdm2 and MdmX.9 Phylogenetic analysis showed that the last cystein of the RING domain is followed by exactly 13 amino acids in all Mdm orthologs of vertebrate origin. Based on this, the authors hypothesized that not only the sequence of the C-terminal tails, but also their exact length are of central importance to the function of the complexes. Subsequent investigation of the ability of Mdm2 and MdmX proteins, which have been extended at the C terminus by 5, 14 or 18 amino acids, was designed to test the importance of the length of the C-terminal extensions. To the researchers surprise, when examined based on their ability to hetero-oligomerize and ubiquitinate p53, Mdm2 proteins behaved differently depending on whether the oligomeric partner was Mdm2 or MdmX. Dolezelova et al. present unexpected experimental evidence for the heterocomplex being structurally and functionally distinct from the Mdm2 homodimer, while providing a mechanism for the observed in vivo functional differences between the complexes. Although the work casts slight doubt on the complete accuracy of the existing structures, it nicely aligns with the above-mentioned results, showing the singular importance of the MdmX RING domain in the activity of the holoenzyme. In light of these results, additional structural studies that will take in to account reported differences between the complexes will undoubtedly be informative and contribute to our understanding of the biochemistry of RING-type ubiquitin ligases and the mechanisms regulating p53 in cells.
- Published
- 2012
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