MAGE-A cancer/testis antigens inhibit MDM2 ubiquitylation function and promote increased levels of MDM4

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically

The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells

Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm2 but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4–6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm2, which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutic

Identification of nanoparticle properties for optimal drug delivery across a physiological cell barrier

Nanoparticles (NPs) represent an attractive strategy to overcome difficulties associated with the delivery of therapeutics. Knowing the optimal properties of NPs to address these issues could allow for improved in vivo responses. This work investigated NPs prepared from 5 materials of 3 sizes and 3 concentrations applied to a cell barrier model. The NPs permeability across a cell barrier and their effects on cell barrier integrity and cell viability were evaluated. The properties of these NPs, as determined in water (traditional) vs. media (realistic), were compared to cell responses. It was found that for all cellular activities, NP properties determined in media was the best predictor of the cell response. Notably, ZnO NPs caused significant alterations to cell viability across all 3 cell lines tested. Importantly, we report that the zeta potential of NPs correlates significantly with NP permeability and NP-induced changes in cell viability. NPs with physiological-based zeta potential of −12 mV result in good cell barrier penetration without considerable changes in cell viability.</p

p53-dependent repression of polo-like kinase-1 (PLK1)

PLK1 is a critical mediator of G(2)/M cell cycle transition that is inactivated and depleted as part of the DNA damage-induced G(2)/M checkpoint. Here we show that downregulation of PLK1 expression occurs through a transcriptional repression mechanism and that p53 is both necessary and sufficient to mediate this effect. Repression of PLK1 by p53 occurs independently of p21 and of arrest at G(1)/S where PLK1 levels are normally repressed in a cell cycle-dependent manner through a CDE/CHR element. Chromatin immunoprecipitation analysis indicates that p53 is present on the PLK1 promoter at two distinct sites termed p53RE1 and p53RE2. Recruitment of p53 to p53RE2, but not to p53RE1, is stimulated in response to DNA damage and/or p53 activation and is coincident with repression-associated changes in the chromatin. Downregulation of PLK1 expression by p53 is relieved by the histone deacetylase inhibitor, trichostatin A, and involves recruitment of histone deacetylase to the vicinity of p53RE2, further supporting a transcriptional repression mechanism. Additionally, wild type, but not mutant, p53 represses expression of the PLK1 promoter when fused upstream of a reporter gene. Silencing of PLK1 expression by RNAi interferes with cell cycle progression consistent with a role in the p53-mediated checkpoint. These data establish PLK1 as a direct transcriptional target of p53, independently of p21, that is required for efficient G(2)/M arrest

FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53

AbstractThe p53 tumour suppressor protein is tightly controlled by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), but maintains MDM2 expression as part of a negative feedback loop. We have identified the immunophilin, 25kDa FK506-binding protein (FKBP25), previously shown to be regulated by p53-mediated repression, as an MDM2-interacting partner. We show that FKBP25 stimulates auto-ubiquitylation and proteasomal degradation of MDM2, leading to the induction of p53. Depletion of FKBP25 by siRNA leads to increased levels of MDM2 and a corresponding reduction in p53 and p21 levels. These data are consistent with the idea that FKBP25 contributes to regulation of the p53-MDM2 negative feedback loop.Structured summaryMINT-6823686:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by anti bait coimmunoprecipitation (MI:0006)MINT-6823707, MINT-6823722:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q62446) by pull down (MI:0096)MINT-6823775:P53 (uniprotkb:Q04637) physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by anti bait coimmunoprecipitation (MI:0006)MINT-6823735, MINT-6823749:FKBP25 (uniprotkb:Q62446) binds (MI:0407) to MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823761:Ubiquitin (UNIPROTKB:62988)P physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823669:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by two hybrid (MI:0018

Model depicting the effect of MAGE-A on MDM2 and MDM4.

<p>MDM2 and MDM4 interact through their respective RING finger domains (located at their C-termini). This interaction allows both the activation of MDM2 ubiquitylation function and the ubiquitylation of MDM4 itself leading to MDM4 destruction. MAGE-A interacts preferentially with MDM2 via the N-terminal hydrophobic pocket and the RING finger. The model predicts that this will compete with MDM4 for binding to MDM2, leading to elevated levels of MDM4.</p

Immunohistochemical analysis of MAGE-A and MDM4 in a cohort of 225 human primary breast cancer specimens.

<p>^aThe P value was achieved using Fisher's exact test</p><p>^bThe Odds ratio determines the likelihood of elevated MDM4 staining if the tumor is MAGE-A positive as opposed to MAGE-A negative.</p><p>Immunohistochemical analysis of MAGE-A and MDM4 in a cohort of 225 human primary breast cancer specimens.</p

MAGE-A proteins associate with MDM2 <i>in vitro</i> and in cultured cells.

<p>(A) H1299 cells (left hand panels) or U2OS cells (right hand panels) were lysed and immunoprecipitation was carried out using antibodies against MAGE-A (6C1), MDM2 (SMP14 plus 4B2) or, as control, a non-specific murine IgG. The blots were probed for the presence of MDM2 (top panels) or MAGE-A (bottom panels). The positions of antibody heavy chains (HC) and light chains (LC) are indicated in the lower panels. (B) GST pull-down assays were performed in which ³⁵S-radiolabelled MAGE-A2 was captured on glutathione sepharose 4B beads using GST linked to full length MDM2 or to four mini-proteins (termed MP1, -2, -3 and -4) representing overlapping regions of MDM2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127713#pone.0127713.ref033" target="_blank">33</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127713#pone.0127713.ref034" target="_blank">34</a>]. The co-precipitating MAGE-A2 was detected by SDS-PAGE followed by fluorography (upper panel). The middle panel shows the presence of the GST-MDM2 proteins following binding to the glutathione beads. The bottom panel is a schematic showing full length MDM2 together with the overlapping regions encompassed within the mini-proteins. The data are representative of three independent experiments.</p

Silencing of MAGE-A expression decreases the levels of MDM4 via the proteasome and increase MDM2/MDM4 association.

<p>MAGE-A expression in U2OS cells was silenced by RNAi as previously described or treated with a non-silencing siRNA oligonucleotide as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127713#pone.0127713.ref012" target="_blank">12</a>]. The various proteins were detected by western blotting. (A) Silencing of MAGE-A in U2OS cells. (B) Silencing of MAGE-A was carried out in the absence or presence of the proteasomal inhibitor, bortezomid. The cells were treated with 10 μM bortezomib 6 h prior to harvesting. (C) Co-immunoprecipitation analysis in which MDM2 immunoprecipitates (obtaining using the antibody, 4B2) were probed for MDM4 (8C6) and for MDM2 itself. (D) Cycloheximide-chase analysis of MDM4 in the U2OS cells was carried out following treatment with the non-silencing and MAGE-A-silencing siRNA oligonucleotides. Quantification of the western blots was carried out using ImageJ software. In all cases the data shown are representative of at least three independent experiments.</p