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Guillermina Lozano - One of the best experts on this subject based on the ideXlab platform.
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Loss of digestive organ expansion factor (Diexf) reveals an essential role during murine embryonic development that is independent of p53.
Oncotarget, 2017Co-Authors: Neeraj K. Aryal, Amanda R. Wasylishen, Vinod Pant, Maurisa Riley-croce, Guillermina LozanoAbstract:Increased levels of inhibitors of the p53 tumor suppressor such as Mdm2 and MDM4 drive tumor development and thus serve as targets for therapeutic intervention. Recently, digestive organ expansion factor (Diexf) has been identified as a novel inhibitor of p53 in zebrafish. Here, we address the potential role of Diexf as a regulator of the p53 pathway in mammals by generating Diexf knockout mice. We demonstrate that, similar to Mdm2 and MDM4, homozygous deletion of Diexf is embryonic lethal. However, unlike in Mdm2 and MDM4 mice, loss of p53 does not rescue this phenotype. Moreover, Diexf heterozygous animals are not sensitive to sub-lethal ionizing radiation. Thus, we conclude that Diexf is an essential developmental gene in the mouse, but is not a significant regulator of the p53 pathway during development or in response to ionizing radiation.
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Tumorigenesis promotes MDM4-S overexpression
Oncotarget, 2017Co-Authors: Vinod Pant, Neeraj K. Aryal, Shunbin Xiong, Alfonso Quintás-cardama, Connie A. Larsson, M. James You, Guillermina LozanoAbstract:Disruption of the p53 tumor suppressor pathway is a primary cause of tumorigenesis. In addition to mutation of the p53 gene itself, overexpression of major negative regulators of p53, MDM2 and MDM4, also act as drivers for tumor development. Recent studies suggest that expression of splice variants of Mdm2 and MDM4 may be similarly involved in tumor development. In particular, multiple studies show that expression of a splice variant of MDM4, MDM4-S correlates with tumor aggressiveness and can be used as a prognostic marker in different tumor types. However, in the absence of prospective studies, it is not clear whether expression of MDM4-S in itself is oncogenic or is simply an outcome of tumorigenesis. Here we have examined the role of MDM4-S in tumor development in a transgenic mouse model. Our results suggest that splicing of MDM4 does not promote tumor development and does not cooperate with other oncogenic insults to alter tumor latency or aggressiveness. We conclude that MDM4-S overexpression is a consequence of splicing defects in tumor cells rather than a cause of tumor evolution.
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Molecular pathways: targeting Mdm2 and MDM4 in cancer therapy.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2012Co-Authors: Guillermina LozanoAbstract:The p53 tumor suppressor is activated in response to cellular stresses to induce cell-cycle arrest, cellular senescence, and apoptosis. The p53 gene is inactivated by mutations in more than 50% of human tumors. In addition, tumor cells dampen p53 activities via overexpression of p53-negative regulators, in particular 2 structurally related proteins, Mdm2 and MDM4. And yet, Mdm2 and MDM4 possess p53-independent activities, which also contribute to tumor formation and progression. Given that Mdm2 and MDM4 inhibit p53 activities to promote tumor development, small molecules and peptides were developed to abrogate the inhibition of p53 by Mdm proteins. Antitumor activities of these molecules have already been confirmed in preclinical studies and early-phase clinical trials. These research endeavors and clinical advances constitute the main focus of this review.
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Heterodimerization of Mdm2 and MDM4 is critical for regulating p53 activity during embryogenesis but dispensable for p53 and Mdm2 stability
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Vinod Pant, Shunbin Xiong, Tomoo Iwakuma, Alfonso Quintás-cardama, Guillermina LozanoAbstract:Mdm2 and MDM4 are homologous RING domain-containing proteins that negatively regulate the tumor suppressor p53 under physiological and stress conditions. The RING domain of Mdm2 encodes an E3-ubiquitin ligase that promotes p53 degradation. In addition, Mdm2 and MDM4 interact through their respective RING domains. The in vivo significance of Mdm2-MDM4 heterodimerization in regulation of p53 function is unknown. In this study, we generated an MDM4 conditional allele lacking the RING domain to investigate its role in Mdm2 and p53 regulation. Our results demonstrate that homozygous deletion of the MDM4 RING domain results in prenatal lethality. Mechanistically, Mdm2-MDM4 heterodimerization is critical for inhibiting lethal p53 activation during early embryogenesis. However, Mdm2-MDM4 interaction is dispensable for regulating p53 activity as well as the stability of Mdm2 and p53 at later stages of development. We propose that MDM4 is a key cofactor of Mdm2 that inhibits p53 activity primarily during early embryogenesis but is dispensable for regulating p53 and Mdm2 stability in the adult mouse.
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Mdm2 and MDM4 Loss Regulates Distinct p53 Activities
Molecular cancer research : MCR, 2008Co-Authors: Juan A. Barboza, Tomoo Iwakuma, Tamara Terzian, Adel K. El-naggar, Guillermina LozanoAbstract:Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or MDM4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2 −/− and MDM4 −/− embryos display divergent phenotypes, suggesting that Mdm2 and MDM4 exert distinct control over p53. To explore the interaction between Mdm2 and MDM4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2 , and MDM4 . These mice had identical survival curves and tumor spectrum as p53 −/− mice, substantiating the principal role of Mdm2 and MDM4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, MDM4 , or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V ; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking MDM4 . Cells lacking both Mdm2 and MDM4 had a stable p53 that initiated apoptosis similar to Mdm2 -null cells. Additionally, stabilization of p53 in cells lacking MDM4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and MDM4 in the regulation of p53 activities. (Mol Cancer Res 2008;6(6):947–54)
David P. Lane - One of the best experts on this subject based on the ideXlab platform.
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simultaneous measurement of p53 mdm2 and p53 MDM4 protein protein interactions in whole cells using fluorescence labelled foci
Scientific Reports, 2019Co-Authors: Yuri Frosi, David P. Lane, K Inoue, Siti Radhiah Ramlan, T Watanabe, Christopher J BrownAbstract:In this report we describe the development of a Fluorescent Protein-Protein Interaction-visualization (FLUOPPI) to enable the simultaneous measurement of both Mdm2:p53 and MDM4:p53 interactions in order to assess the relative efficiencies of mimetic molecules of the p53 peptide helix against both PPIs. Mdm2 and MDM4 overexpression frequently leads to the inactivation of non-mutated p53 in human cancers, via inhibition of its transcriptional activity, enhancing its degradation by the proteasome or by preventing its nuclear import. Development of inhibitors to disrupt the binding of one or both of these protein interactions have been the subject of intensive pharmaceutical development for anti-cancer therapies. Using the bimodal FLUOPPI system we have characterised compounds that were either monospecific for Mdm2 or bispecific for both Mdm2 and MDM4. We have also demonstrated that the FLUOPPI assay can reliably differentiate between specific and non-specific disruption of these protein complexes via accurate assessment and normalization to the cell population under measurement. We envision that this methodology will increase the efficiency of identifying compounds that are either specific against a single PPI from a closely related family of interactions or compounds that interact across multiple related PPI pairs, depending on which is more desirable.
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structure activity studies of mdm2 MDM4 binding stapled peptides comprising non natural amino acids
PLOS ONE, 2017Co-Authors: Sharon Chee, David P. Lane, Christopher J Brown, Jantana Wongsantichon, Jiawei Siau, Dawn Thean, Fernando J Ferrer, Robert Robinson, Farid J. GhadessyAbstract:As primary p53 antagonists, Mdm2 and the closely related MDM4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6-125) at 1.66 A resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/MDM4 antagonism and confirms L98 of MDM4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/MDM4 plasticity.
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Anatomy of Mdm2 and MDM4 in evolution.
Journal of molecular cell biology, 2017Co-Authors: Ban Xiong Tan, Hoe Peng Liew, Joy S. Chua, Farid J. Ghadessy, Yaw Sing Tan, David P. Lane, Cynthia R. CoffillAbstract:Mouse double minute (Mdm) genes span an evolutionary timeframe from the ancient eukaryotic placozoa Trichoplax adhaerens to Homo sapiens, implying a significant and possibly conserved cellular role throughout history. Maintenance of DNA integrity and response to DNA damage involve many key regulatory pathways, including precise control over the tumour suppressor protein p53. In most vertebrates, degradation of p53 through proteasomal targeting is primarily mediated by heterodimers of Mdm2 and the Mdm2-related protein MDM4 (also known as MdmX). Both Mdm2 and MDM4 have p53-binding regions, acidic domains, zinc fingers, and C-terminal RING domains that are conserved throughout evolution. Vertebrates typically have both Mdm2 and MDM4 genes, while analyses of sequenced genomes of invertebrate species have identified single Mdm genes, suggesting that a duplication event occurred prior to emergence of jawless vertebrates about 550-440 million years ago. The functional relationship between Mdm and p53 in T. adhaerens, an organism that has existed for 1 billion years, implies that these two proteins have evolved together to maintain a conserved and regulated function.
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tumor specific signaling to p53 is mimicked by mdm2 inactivation in zebrafish insights from mdm2 and MDM4 mutant zebrafish
Oncogene, 2015Co-Authors: Joy S. Chua, Hoe Peng Liew, L Guo, David P. LaneAbstract:In mice, the deletion of either Mdm2 or MDM4 results in a p53-dependent embryonic lethality. We used zinc-finger nucleases to construct mutations in the mdm2 and MDM4 genes of zebrafish. Although the loss of mdm2 results in a p53-dependent early embryonic lethality, MDM4 mutant fish are viable and grow to adulthood. We also found that an in-frame five-amino acid deletion in mdm2 creates a novel hypomorphic allele. The lethal phenotype observed in the mdm2 mutant fish could be partially rescued by injecting mRNA encoding functional Mdm2, and this required the E3 ligase activity of the protein. Complete rescue was obtained by crossing the mdm2 mutant fish onto a p53M214K mutant background. Although p53 mutant fish on a wild-type mdm2 background were shown to accumulate high levels of p53 protein specifically in tumor tissues, we detected extensive staining of p53 in many normal tissues of the mdm2–p53M214K double-mutant fish. Our results are suggestive of the hypothesis that p53 protein accumulates during tumor formation as a result of tumor-specific inactivation of the Mdm2 pathway.
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abstract 5386 disrupting p53 mdm2 and p53 MDM4 comparative cell based assays for drug screening
Cancer Research, 2014Co-Authors: Larisa Yurlova, Farid J. Ghadessy, David P. Lane, Christopher J Brown, Maarten Derks, Ian Hickson, Eberhard Krausz, Kourosh ZolghadrAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The important interconnected protein-protein interactions between the tumor suppressor p53 and its regulatory binding partners Mdm2 and MDM4 (= MdmX/ HdmX) are implicated in pathogenesis of various cancers. However, until now there is a lack of reversible cell-based assays for concurrent analysis for both PPIs: p53/Mdm2 and p53/MDM4. Our aim was to develop two comparative assays based on the Fluorescent 2-Hybrid (F2H) assay principle, which enable side-by-side analysis of antagonistic compounds. The F2H assay is a fully reversible microscopy-assisted assay for the direct intracellular analysis of PPIs. It offers a fast and straight-forward readout: an interaction-dependent co-localization of two fluorescent signals at a defined spot in the nucleus of mammalian cells. With these assays we analyzed a set of newly developed stapled peptides as potent p53:Mdm2 and/ or p53:MDM4 inhibitors. Live cell data generated by the F2H assays enabled us to discriminate and describe the peptides according to their ability to penetrate into the cells, their efficacy on the PPIs and their cytotoxic side effects in one single assay. In parallel we performed a pilot F2H screen with a sub-set or 20 small molecule compounds (including Nutlin-3) that exhibited activity against one or both interactions at various potencies in ELISAs before. We could identify 5 potent compounds, which were dramatically reducing the number of p53:Mdm2 interactions. However, none of the small molecules, but only the stapled peptides exhibited an intracellular activity on p53:MDM4. Furthermore, we were able to expand these assays and to identify mutants of Mdm2 resistant to Nutlin inhibition. In summary, we show that the p53:Mdm2 and p53:MDM4 F2H assays described here, - enable side-by-side analysis of substances' dual Mdm2-MDM4 activity - are suitable for screening and testing various types of compounds, such as peptidic inhibitors or small molecules - concurrently provide initial data on compound cell-permeability and cytotoxicity - allow real-time visualization of PPI dynamics in living cells. Citation Format: Larisa Yurlova, Christopher J. Brown, Maarten Derks, Farid John Ghadessy, Ian Hickson, David P. Lane, Eberhard Krausz, Kourosh Zolghadr. Disrupting p53:Mdm2 and p53:MDM4 - Comparative cell-based assays for drug screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2014-5386
Jean-christophe Marine - One of the best experts on this subject based on the ideXlab platform.
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loss of oocytes due to conditional ablation of murine double minute 2 mdm2 gene is p53 dependent and results in female sterility
FEBS Letters, 2016Co-Authors: Gabriel Livera, Jean-christophe Marine, Rustem Uzbekov, Peggy Jarrier, Sophie Fouchecourt, Clotilde Duquenne, Annesimone Parent, Philippe MongetAbstract:Murine double minute 2 and 4 (Mdm2, MDM4) are major p53-negative regulators, preventing thus uncontrolled apoptosis induction in numerous cell types, although their function in the female germ line has received little attention. In the present work, we have generated mice with specific invalidation of Mdm2 and MDM4 genes in the mouse oocyte (Mdm2(Ocko) and MDM4(Ocko) mice), to test their implication in survival of these germ cells. Most of the Mdm2(Ocko) but not MDM4(Ocko) mice were sterile, with a dramatic reduction of the weight of ovaries and genital tract, a strong increase in follicle-stimulating hormone and luteinizing hormone serum levels, and a reduction of anti-mullerian hormone serum levels. Histological analyses revealed an obvious decrease of the number of growing follicles beyond the primary stage in Mdm2(Ocko) ovaries in comparison to controls, with a pronounced increase in the apparition of primary atretic follicles, most being devoid of oocyte. Similar phenotypes were observed with Mdm2(Ocko) MDM4(Ocko) ovaries, with no worsening of the phenotype. However, we failed to detect any increase in p53 level in mutant oocytes, nor any other apoptotic marker, introgression of this targeted invalidation in p53-/- mice restored the fertility of females. This study is the first to show that Mdm2, but not MDM4, has a critical role in oocyte survival and would be involved in premature ovarian insufficiency phenotype.
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mdmx MDM4 a promising target for p53 reactivation therapy and beyond
Cold Spring Harbor Perspectives in Medicine, 2016Co-Authors: Jean-christophe Marine, Aart G. JochemsenAbstract:The MDMX protein was identified as a p53-interacting protein with a strong similarity to MDM2. Like Mdm2, Mdmx expression is essential for curbing p53 activity during embryonic development, indicating nonredundant functions of Mdmx and Mdm2. There is now a large body of evidence indicating that cancers frequently up-regulate MDMX expression as a means to dampen p53 tumor-suppressor function. Importantly, MDMX also shows p53-independent oncogenic functions. These data make MDMX an attractive therapeutic target for cancer therapy. Here, we summarize the mechanisms used by cancer cells to increase MDMX expression and promising pharmacological strategies to target MDMX in cancer-in particular, the recent findings that antisense oligonucleotides (ASOs) can be used to efficiently modulate MDMX messenger RNA (mRNA) splicing.
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Loss of oocytes due to conditional ablation of Murine double minute 2(Mdm2) gene is p53-dependent and results in female sterility
FEBS Letters, 2016Co-Authors: Gabriel Livera, Jean-christophe Marine, Rustem Uzbekov, Sophie Fouchecourt, Clotilde Duquenne, Annesimone Parent, Peggy Jarrier-gaillard, Philippe MongetAbstract:Murine double minute 2 and 4 (Mdm2, MDM4) are major p53-negative regulators, preventing thus uncontrolled apoptosis induction in numerous cell types, though their function in the female germ line has received little attention. In the present work, we have generated mice with specific invalidation of Mdm2 and MDM4 genes in the mouse oocyte (Mdm2 Ocko and MDM4 Ocko mice), to test their implication in survival of these germ cells. Most of the Mdm2 Ocko but not MDM4 Ocko mice were sterile, with a dramatic reduction of the weight of ovaries and genital tract, a strong increase in FSH and LH serum levels, and a reduction of AMH serum levels. Histological analyses revealed an obvious decrease of the number of growing follicles beyond the primary stage in Mdm2Ocko ovaries in comparison to controls, with a pronounced increase in the apparition of primary atretic follicles, most being devoid of oocyte. Similar phenotypes were observed with Mdm2Ocko MDM4Ocko ovaries, with no worsening of the phenotype. Whereas we failed to detect any increase of p53 level in mutant oocytes, nor any other apoptotic marker, introgression of this targeted invalidation in p53-/- mice restored the fertility of females. The present study is the first to show that Mdm2 but not MDM4 has a critical role in oocyte survival and would be involved in premature ovarian insufficiency phenotype.
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Widespread overexpression of epitope-tagged MDM4 does not accelerate tumor formation in vivo.
Molecular and cellular biology, 2010Co-Authors: Sarah De Clercq, Marion M. Maetens, Agnieszka Gembarska, Geertrui Denecker, Michael Naessens, Katharina Haigh, Jody J. Haigh, Jean-christophe MarineAbstract:Mdm2 and MDM4 are critical negative regulators of p53. A large body of evidence indicates that elevated expression of either Mdm2 or MDM4 may favor tumor formation by inhibiting p53 tumor suppression function. To explore this possibility in vivo, we generated conditional Mdm2 and MDM4 transgenic mice. We show that although both transgenes are designed to be expressed ubiquitously and at comparable levels, only the MDM4 transgenic protein is produced at high levels in vivo. In contrast, exogenous Mdm2 is constitutively degraded in a proteasome-dependent manner, indicating that cells are equipped with efficient mechanisms that prevent Mdm2 accumulation in vivo. Mice that are homozygous for the MDM4 transgene die during embryogenesis owing to severe vascular maturation defects. Importantly, this lethality is not rescued on a p53-null background, indicating that high levels of MDM4 impact on a pathway(s) other than p53 that controls vascular and embryonic development. Mice expressing a single copy of the MDM4 transgene are viable and, surprisingly, are not prone to spontaneous, radiation-induced or Eμ-myc-induced tumor formation. The findings have clear implications for cancer etiology as well as for cancer therapy.
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Distinct roles of Mdm2 and MDM4 in red cell production
Blood, 2006Co-Authors: Marion M. Maetens, Guillermina Lozano, Sarah De Clercq, Gilles Doumont, Sarah Francoz, Pascal Froment, Eric Bellefroid, Ursula Klingmüller, Jean-christophe MarineAbstract:Mdm2 and MDM4 are critical negative regulators of the p53 tumor suppressor. MDM4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of MDM4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. Here, we further explore the tissue-specific functions of Mdm2 and MDM4 in the erythroid lineage by intercrossing conditional MDM4 and Mdm2 alleles to an erythroid-specific Cre (Er-GFP-Cre) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, MDM4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, MDM4 only contributes to p53 regulation at a specific phase of the differentiation program.
Farid J. Ghadessy - One of the best experts on this subject based on the ideXlab platform.
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structure activity studies of mdm2 MDM4 binding stapled peptides comprising non natural amino acids
PLOS ONE, 2017Co-Authors: Sharon Chee, David P. Lane, Christopher J Brown, Jantana Wongsantichon, Jiawei Siau, Dawn Thean, Fernando J Ferrer, Robert Robinson, Farid J. GhadessyAbstract:As primary p53 antagonists, Mdm2 and the closely related MDM4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6-125) at 1.66 A resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/MDM4 antagonism and confirms L98 of MDM4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/MDM4 plasticity.
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Anatomy of Mdm2 and MDM4 in evolution.
Journal of molecular cell biology, 2017Co-Authors: Ban Xiong Tan, Hoe Peng Liew, Joy S. Chua, Farid J. Ghadessy, Yaw Sing Tan, David P. Lane, Cynthia R. CoffillAbstract:Mouse double minute (Mdm) genes span an evolutionary timeframe from the ancient eukaryotic placozoa Trichoplax adhaerens to Homo sapiens, implying a significant and possibly conserved cellular role throughout history. Maintenance of DNA integrity and response to DNA damage involve many key regulatory pathways, including precise control over the tumour suppressor protein p53. In most vertebrates, degradation of p53 through proteasomal targeting is primarily mediated by heterodimers of Mdm2 and the Mdm2-related protein MDM4 (also known as MdmX). Both Mdm2 and MDM4 have p53-binding regions, acidic domains, zinc fingers, and C-terminal RING domains that are conserved throughout evolution. Vertebrates typically have both Mdm2 and MDM4 genes, while analyses of sequenced genomes of invertebrate species have identified single Mdm genes, suggesting that a duplication event occurred prior to emergence of jawless vertebrates about 550-440 million years ago. The functional relationship between Mdm and p53 in T. adhaerens, an organism that has existed for 1 billion years, implies that these two proteins have evolved together to maintain a conserved and regulated function.
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abstract 5386 disrupting p53 mdm2 and p53 MDM4 comparative cell based assays for drug screening
Cancer Research, 2014Co-Authors: Larisa Yurlova, Farid J. Ghadessy, David P. Lane, Christopher J Brown, Maarten Derks, Ian Hickson, Eberhard Krausz, Kourosh ZolghadrAbstract:Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The important interconnected protein-protein interactions between the tumor suppressor p53 and its regulatory binding partners Mdm2 and MDM4 (= MdmX/ HdmX) are implicated in pathogenesis of various cancers. However, until now there is a lack of reversible cell-based assays for concurrent analysis for both PPIs: p53/Mdm2 and p53/MDM4. Our aim was to develop two comparative assays based on the Fluorescent 2-Hybrid (F2H) assay principle, which enable side-by-side analysis of antagonistic compounds. The F2H assay is a fully reversible microscopy-assisted assay for the direct intracellular analysis of PPIs. It offers a fast and straight-forward readout: an interaction-dependent co-localization of two fluorescent signals at a defined spot in the nucleus of mammalian cells. With these assays we analyzed a set of newly developed stapled peptides as potent p53:Mdm2 and/ or p53:MDM4 inhibitors. Live cell data generated by the F2H assays enabled us to discriminate and describe the peptides according to their ability to penetrate into the cells, their efficacy on the PPIs and their cytotoxic side effects in one single assay. In parallel we performed a pilot F2H screen with a sub-set or 20 small molecule compounds (including Nutlin-3) that exhibited activity against one or both interactions at various potencies in ELISAs before. We could identify 5 potent compounds, which were dramatically reducing the number of p53:Mdm2 interactions. However, none of the small molecules, but only the stapled peptides exhibited an intracellular activity on p53:MDM4. Furthermore, we were able to expand these assays and to identify mutants of Mdm2 resistant to Nutlin inhibition. In summary, we show that the p53:Mdm2 and p53:MDM4 F2H assays described here, - enable side-by-side analysis of substances' dual Mdm2-MDM4 activity - are suitable for screening and testing various types of compounds, such as peptidic inhibitors or small molecules - concurrently provide initial data on compound cell-permeability and cytotoxicity - allow real-time visualization of PPI dynamics in living cells. Citation Format: Larisa Yurlova, Christopher J. Brown, Maarten Derks, Farid John Ghadessy, Ian Hickson, David P. Lane, Eberhard Krausz, Kourosh Zolghadr. Disrupting p53:Mdm2 and p53:MDM4 - Comparative cell-based assays for drug screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2014-5386
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the fluorescent two hybrid assay to screen for protein protein interaction inhibitors in live cells targeting the interaction of p53 with mdm2 and MDM4
Journal of Biomolecular Screening, 2014Co-Authors: Larisa Yurlova, Farid J. Ghadessy, Christopher J Brown, Maarten Derks, Ian Hickson, Andrea Buchfellner, M Janssen, Denise Morrison, Ian Stansfield, David P. LaneAbstract:Protein-protein interactions (PPIs) are attractive but challenging targets for drug discovery. To overcome numerous limitations of the currently available cell-based PPI assays, we have recently established a fully reversible microscopy-assisted fluorescent two-hybrid (F2H) assay. The F2H assay offers a fast and straightforward readout: an interaction-dependent co-localization of two distinguishable fluorescent signals at a defined spot in the nucleus of mammalian cells. We developed two reversible F2H assays for the interactions between the tumor suppressor p53 and its negative regulators, Mdm2 and MDM4. We then performed a pilot F2H screen with a subset of compounds, including small molecules (such as Nutlin-3) and stapled peptides. We identified five cell-penetrating compounds as potent p53-Mdm2 inhibitors. However, none exhibited intracellular activity on p53-MDM4. Live cell data generated by the F2H assays enable the characterization of stapled peptides based on their ability to penetrate cells and disrupt p53-Mdm2 interaction as well as p53-MDM4 interaction. Here, we show that the F2H assays enable side-by-side analysis of substances' dual Mdm2-MDM4 activity. In addition, they are suitable for testing various types of compounds (e.g., small molecules and peptidic inhibitors) and concurrently provide initial data on cellular toxicity. Furthermore, F2H assays readily allow real-time visualization of PPI dynamics in living cells.
Francesca Mancini - One of the best experts on this subject based on the ideXlab platform.
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MDM4 hipk2 p53 cytoplasmic assembly uncovers coordinated repression of molecules with anti apoptotic activity during early dna damage response
Oncogene, 2016Co-Authors: Francesca Mancini, Luisa Pieroni, Valentina Monteleone, Rossella Luca, Laura Fici, Emilia Luca, Andrea UrbaniAbstract:The p53 inhibitor, MDM4 (MDMX) is a cytoplasmic protein with p53-activating function under DNA damage conditions. Particularly, MDM4 promotes phosphorylation of p53 at Ser46, a modification that precedes different p53 activities. We investigated the mechanism by which MDM4 promotes this p53 modification and its consequences in untransformed mammary epithelial cells and tissues. In response to severe DNA damage, MDM4 stimulates p53Ser46P by binding and stabilizing serine–threonine kinase HIPK2. Under these conditions, the p53-inhibitory complex, MDM4/MDM2, dissociates and this allows MDM4 to promote p53/HIPK2 functional interaction. Comparative proteomic analysis of DNA damage-treated cells versus -untreated cells evidenced a diffuse downregulation of proteins with anti-apoptotic activity, some of which were targets of p53Ser46P/HIPK2 repressive activity. Importantly, MDM4 depletion abolishes the downregulation of these proteins indicating the requirement of MDM4 to promote p53-mediated transcriptional repression. Consistently, MDM4-mediated HIPK2/p53 activation precedes HIPK2/p53 nuclear translocation and activity. Noteworthy, repression of these proteins was evident also in mammary glands of mice subjected to γ-irradiation and was significantly enhanced in transgenic mice overexpressing MDM4. This study evidences the flexibility of MDM2/MDM4 heterodimer, which allows the development of a positive activity of cytoplasmic MDM4 towards p53-mediated transcriptional function. Noteworthy, this activity uncovers coordinated repression of molecules with shared anti-apoptotic function which precedes active cell apoptosis and that are frequently overexpressed and/or markers of tumour phenotype in human cancer.
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targeting the mdm2 MDM4 interaction interface as a promising approach for p53 reactivation therapy
Cancer Research, 2015Co-Authors: Marsha Pellegrino, Francesca Mancini, Rossella Luca, Alice Coletti, Nicola Giacchè, Isabella Manni, Ivan Arisi, Fulvio Florenzano, Emanuela Teveroni, Marianna ButtarelliAbstract:Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function.
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Targeting the MDM2/MDM4 Interaction Interface as a Promising Approach for p53 Reactivation Therapy
Cancer research, 2015Co-Authors: Marsha Pellegrino, Francesca Mancini, Rossella Luca, Alice Coletti, Nicola Giacchè, Isabella Manni, Ivan Arisi, Fulvio Florenzano, Emanuela Teveroni, Marianna ButtarelliAbstract:Restoration of wild-type p53 tumor suppressor function has emerged as an attractive anticancer strategy. Therapeutics targeting the two p53-negative regulators, MDM2 and MDM4, have been developed, but most agents selectively target the ability of only one of these molecules to interact with p53, leaving the other free to operate. Therefore, we developed a method that targets the activity of MDM2 and MDM4 simultaneously based on recent studies indicating that formation of MDM2/MDM4 heterodimer complexes are required for efficient inactivation of p53 function. Using computational and mutagenesis analyses of the heterodimer binding interface, we identified a peptide that mimics the MDM4 C-terminus, competes with endogenous MDM4 for MDM2 binding, and activates p53 function. This peptide induces p53-dependent apoptosis in vitro and reduces tumor growth in vivo. Interestingly, interfering with the MDM2/MDM4 heterodimer specifically activates a p53-dependent oxidative stress response. Consistently, distinct subcellular pools of MDM2/MDM4 complexes were differentially sensitive to the peptide; nuclear MDM2/MDM4 complexes were particularly highly susceptible to the peptide-displacement activity. Taken together, these data identify the MDM2/MDM4 interaction interface as a valuable molecular target for therapeutic reactivation of p53 oncosuppressive function.
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MDM4 enhances p53 stability by promoting an active conformation of the protein upon DNA damage.
Cell cycle (Georgetown Tex.), 2012Co-Authors: Giusy Di Conza, Francesca Mancini, Marianna Buttarelli, Alfredo Pontecorvi, Francesco Trimarchi, Fabiola MorettiAbstract:Stabilization of p53 protein is an important step in the activation of its function. p53 levels are regulated by ubiquitin-dependent and -independent degradation pathways. MDM4 (MDMX) is an important regulator of p53, able to both stimulate and antagonize p53 degradation. Both of these activities have been attributed to the ability of MDM4 to potentiate or antagonize the function of MDM2, the main ubiquitin ligase of p53, depending on their relative levels. Here, we have investigated the stabilizing function of endogenous MDM4 using genetic models of knockout MEFs and RNA interference in human non-transformed cell lines. Our data demonstrate that MDM4 is able to stabilize p53, protecting it from proteasome-mediated degradation in a MDM2- and ubiquitin-independent manner. Upon DNA damage, MDM4 is associated to p53 independently of MDM2 and promotes a conformational change of the protein toward an active form. This correlates with a decreased association of p53 to the proteasome and increased protein levels...
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Regulation of MDM4 (MDMX) function by p76 MDM2 : a new facet in the control of p53 activity
Oncogene, 2010Co-Authors: Simona Giglio, Francesca Mancini, Alfredo Pontecorvi, Marsha Pellegrino, G Di Conza, Efisio Puxeddu, Ada Sacchi, Fabiola MorettiAbstract:Under basal growth conditions, p53 function is tightly controlled by the members of MDM family, MDM2 and MDM4. The Mdm2 gene codes, in addition to the full-length p90(MDM2), for a short protein, p76(MDM2) that lacks the p53-binding domain. Despite this property and at variance with p90(MDM2), this protein acts positively toward p53, although the molecular mechanism remains elusive. Here, we report that p76(MDM2) antagonizes MDM4 inhibitory function. We show that p76(MDM2) possesses intrinsic ubiquitinating and degrading activity, and through these activities controls MDM4 levels. Furthermore, the presence of p76(MDM2) decreases the association of MDM4 with p53 and p90(MDM2), and antagonizes p53 degradation by the heterodimer MDM4/p90(MDM2). The p76(MDM2)-mediated regulation of MDM4 occurs in the cytoplasm, under basal growth conditions. Conversely, upon DNA damage, phosphorylation of MDM4Ser403 dissociates p76(MDM2) and prevents MDM4 degradation. The overall negative control of MDM4 by p76(MDM2) reflects on p53 function as p76(MDM2) impairs MDM4-mediated inhibition of p53 activity. In agreement with the positive role of p76(MDM2) toward p53, the p76(MDM2)/p90(MDM2) ratio significantly decreases in a group of thyroid tumor samples compared with normal counterparts. Overall, these findings reveal a new mechanism in the control of p53 basal activity that may account for the distinct sensitivity of tissues to stress signals depending on the balance among MDM proteins. Moreover, these data suggest an oncosuppressive function for a product of the Mdm2 gene.