p53-Mdm2模块和泛素化系统

p53(肿瘤抑制基因)诱导鼠双微粒体蛋白2(Mdm2)的表达,Mdm2反之抑制p53的活性,Mdm2和p53形成了一个自动调整的模块。Mdm2的一个重要的结构标志是一个中心酸性区域,另外的结构标志是在酸结构域下游的一个锌指结构,和一个C端的环指区域。Mdm2的表达是由p53来调节,Mdm2作为E3连接酶使p53泛素化并且驱使p53降解,进而控制p53的功能。对于p53泛素化的结构要求是p53的寡聚化。p53泛素化作用的调整模式是通过蛋白质之间的相互作用。Mdm2中环指区域的作用是通过使p53泛素化来推进p53的降解。泛素化后的酸性结构在Mdm2的降解中起作用。     

Mdmx enhances p53 ubiquitination by altering the substrate preference of the Mdm2 ubiquitin ligase

mdm2 and mdmx oncogenes play essential yet non-redundant roles in synergistic inactivation of the tumor suppressor, p53. While Mdm2 inhibits p53 activity mainly by augmenting its ubiquitination, the functional role of Mdmx on p53 ubiquitination remains obscure. In transfected H1299 cells, Mdmx augmented Mdm2-mediated ubiquitination of p53. In in vitro ubiquitination assays, the Mdmx/Mdm2 heteromeric complex, in comparison to the Mdm2 homomer, showed enhanced ubiquitinase activity toward p53 and the reduced auto-ubiquitination of Mdm2. Alteration of the substrate specificity via binding to Mdmx may contribute to efficient ubiquitination and inactivation of p53 by Mdm2.

Structured summary

MINT-7219995: P53 (uniprotkb:P04637) physically interacts (MI:0914) with Ubiquitin (uniprotkb:P62988) by anti bait coimmunoprecipitation (MI:0006)MINT-7220023: Ubiquitin (uniprotkb:P62988) physically interacts (MI:0914) with P53 (uniprotkb:P04637) by pull down (MI:0096)     

KAP1 dictates p53 response induced by chemotherapeutic agents via Mdm2 interaction

KAP1 recruits many proteins involved in gene silencing and functions as an integral part of co-repressor complex. KAP1 was identified as Mdm2-binding protein and shown to form a complex with Mdm2 and p53 in vivo. We examined the role of KAP1 in p53 activation after the treatment of cells with different types of external stresses. KAP1 reduction markedly enhanced the induction of p21, a product of the p53 target gene, after treatment with actinomycin D or gamma-irradiation, but not with camptothecin. Treatment with actinomycin D, but not with camptothecin, augmented the interaction of p53 with Mdm2 and KAP1. Further, KAP1 reduction in actinomycin D-treated cells facilitated cell cycle arrest and negatively affected clonal cell growth. Thus, the reduction of KAP1 levels promotes p53-dependent p21 induction and inhibits cell proliferation in actinomycin D-treated cells. KAP1 may serve as a therapeutic target against cancer in combination with actinomycin D.     

HAUSP-regulated switch from auto- to p53 ubiquitination by Mdm2 (in silico discovery)

Stability of the 'guardian of the genome' tumor suppressor protein p53 is regulated predominantly through its ubiquitination. The ubiquitin-specific protease HAUSP plays an important role in this process. Recent experiments showed that p53 demonstrates a differential response to changes in HAUSP which nature and significance are not understood yet. Here a data-driven mathematical model of the Mdm2-mediated p53 ubiquitination network is presented which offers an explanation for the cause of such a response. The model predicts existence of the HAUSP-regulated switch from auto- to p53 ubiquitination by Mdm2. This switch suggests a potential role of HAUSP as a downstream target of stress signals in cells. The model accounts for a significant amount of experimental data, makes predictions for some rate constants, and can serve as a building block for the larger model describing a complex dynamic response of p53 to cellular stresses.     

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21^Cip1 and p27^Kip1. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Regulation of the Mdm2–p53 pathway by the ubiquitin E3 ligase MARCH7

The tumor suppressor p53 plays a prominent role in the protection against cancer. The activity of p53 is mainly controlled by the ubiquitin E3 ligase Mdm2, which targets p53 for proteasomal degradation. However, the regulation of Mdm2 remains not well understood. Here, we show that MARCH7, a RING domain‐containing ubiquitin E3 ligase, physically interacts with Mdm2 and is essential for maintaining the stability of Mdm2. MARCH7 catalyzes Lys⁶³‐linked polyubiquitination of Mdm2, which impedes Mdm2 autoubiquitination and degradation, thereby leading to the stabilization of Mdm2. MARCH7 also promotes Mdm2‐dependent polyubiquitination and degradation of p53. Furthermore, MARCH7 is able to regulate cell proliferation, DNA damage‐induced apoptosis, and tumorigenesis via a p53‐dependent mechanism. These findings uncover a novel mechanism for the regulation of Mdm2 and reveal MARCH7 as an important regulator of the Mdm2–p53 pathway.     

The p53 isoforms are differentially modified by Mdm2

The discovery that the single p53 gene encodes several different p53 protein isoforms has initiated a flurry of research into the function and regulation of these novel p53 proteins. Full-length p53 protein level is primarily regulated by the E3-ligase Mdm2, which promotes p53 ubiquitination and degradation. Here, we report that all of the novel p53 isoforms are ubiquitinated and degraded to varying degrees in an Mdm2-dependent and -independent manner, and that high-risk human papillomavirus can degrade some but not all of the novel isoforms, demonstrating that full-length p53 and the p53 isoforms are differentially regulated. In addition, we provide the first evidence that Mdm2 promotes the NEDDylation of p53β. Altogether, our data indicates that Mdm2 can distinguish between the p53 isoforms and modify them differently.     

Mdmx as an essential regulator of p53 activity

The murine double minute 2 (Mdm2) is a critical negative regulator of the p53 tumor suppressor. Almost 10 years ago, a search for new p53-interactors revealed the existence of an Mdm2-structurally related protein, Mdmx (or Mdm4). Since then a large body of biochemical data has accumulated on the functions of Mdmx, often leading to conflicting molecular models. Nevertheless, virtually all these data pointed toward a critical role for Mdmx in the regulation of the p53-Mdm2 network. A view that was recently confirmed by genetic studies. This review is a summary of our current understanding of this molecule, its structure and biological functions, as well as its relationship to its known binding partners.     

Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.     

Novel isatin-derived molecules activate p53 via interference with Mdm2 to promote apoptosis

The p53 protein is a key tumor suppressor in mammals. In response to various forms of genotoxic stress p53 stimulates expression of genes whose products induce cell cycle arrest and/or apoptosis. An E3-ubiquitin ligase, Mdm2 (mouse-double-minute 2) and its human ortholog Hdm2, physically interact with the amino-terminus of p53 to mediate its ubiquitin-mediated degradation via the proteasome. Thus, pharmacological inhibition of the p53-Mdm2 interaction leads to overall stabilization of p53 and stimulation of its anti-tumorigenic activity. In this study we characterize the biological effects of a novel class of non-genotoxic isatin Schiff and Mannich base derivatives (ISMBDs) that stabilize p53 on the protein level. The likely mechanism behind their positive effect on p53 is mediated via the competitive interaction with Mdm2. Importantly, unlike Nutlin, these compounds selectively promoted p53-mediated cell death. These novel pharmacological activators of p53 can serve as valuable molecular tools for probing p53-positive tumors and set up the stage for development of new anti-cancer drugs.     

MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.     

The can and can't dos of p53 RNA

The p53 protein, like any other protein, cannot be made in the cell without RNA. And even once made, the p53 protein will be more rapidly degraded without the p53 RNA. Furthermore, the p53 RNA helps deciding which p53 isoform should be produced and under which cell conditions. Mutant p53 mRNA codes for an unstable and inactive protein. These matters are discussed in this article as well as the recent reports on p53 RNA mutations, interacting-proteins, 3′ processing and 5′–3′ loop.     

p53 downregulates Down syndrome-associated DYRK1A through miR-1246

Several microRNAs mediate the functions of p53 family members. Here we characterize miR-1246 as a new target of this family. In response to DNA damage, p53 induces the expression of miR-1246 which, in turn, reduces the level of DYRK1A, a Down syndrome-associated protein kinase. Knockdown of p53 has the opposite effect. Overexpression of miR-1246 reduces DYRK1A levels and leads to the nuclear retention of NFATc1, a protein substrate of DYRK1A, and the induction of apoptosis, whereas a miR-1246-specific inhibitor prevented the nuclear import of NFATc1. Together, these results indicate that p53 inhibits DYRK1A expression through the induction of miR-1246.