Targeting the conformational transitions of MDM2 and MDMX: Insights into key residues affecting p53 recognition

The oncogenic proteins MDM2 and MDMX have distinct and critical roles in the control of the activity of the p53 tumor suppressor protein. Recently, we have used spatial coarse graining simulations to analyze the conformational transitions manifest in the p53 recognition of MDM2 and MDMX. These conformational movements are different between MDM2 and MDMX and unveil the presence of conserved and nonconserved interactions in the p53 binding cleft that may be exploited in the design of selective and dual modulators of the oncogenic proteins. In this study, we investigate the conformational profiles of apo‐ and p53‐bound states of MDM2 and MDMX using molecular dynamic simulations along a time scale of 60 ns. The analysis of the trajectories is instrumental to discuss energetical and conformational aspects of p53 recognition and to point out specific key residues whose conformational shifts have crucial roles in affecting the apo‐ and p53‐bound states of MDM2 and MDMX. Among these, in particular, linear discriminant analyses identify diverse conformations of Y99/Y100 (MDMX/MDM2) as markers of the apo‐ and p53‐bound states of the oncogenic proteins. The results of this study shed further light on different p53 recognition in MDM2 and MDMX and may prove useful for the design and identification of new potent and selective synthetic modulators of p53‐MDM2/MDMX interactions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Nucleolar protein GLTSCR2 stabilizes p53 in response to ribosomal stresses

p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus-nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers.     

Cell cycle regulatory functions of the human oncoprotein MDM2

The protein (MDM2) coded by the mouse double minute-2 (mdm2) gene or its human homologue is well known as an oncoprotein. Malignant human tumors particularly breast tumors and soft tissue sarcomas frequently overexpress MDM2. Artificial amplification of mdm2 gene derived from a transformed murine cell line enhances tumorigenic potential of murine cells. Consistent with its tumorigenic property, mouse or human MDM2 can inactivate several functions of the tumor suppressor p53 and can degrade p53. The protein also interacts with other tumor suppressors, and these interactions may contribute to its tumorigenic property. In spite of its oncogenic role, mouse or human MDM2 induces G(1) arrest in normal human or murine cells. Some cell lines bearing known genetic mutations are insensitive to MDM2-mediated growth arrest. This review is aimed to collect available information on the functions of MDM2 that could potentially regulate cell cycle and to discuss how this information may fit in one model that could explain the two apparently opposite G(1) arrest and oncogenic function of MDM2.     

Polymorphisms in p53 and the p53 pathway: roles in cancer susceptibility and response to treatment

The p53 tumour suppressor protein lies at the crossroads of multiple cellular response pathways that control the fate of the cell in response to endogenous or exogenous stresses and inactivation of the p53 tumour suppressor signalling pathway is seen in most human cancers. Such aberrant p53 activity may be caused by mutations in the TP53 gene sequence producing truncated or inactive mutant proteins, or by aberrant production of other proteins that regulate p53 activity, such as gene amplification and overexpression of MDM2 or viral proteins that inhibit or degrade p53. Recent studies have also suggested that inherited genetic polymorphisms in the p53 pathway influence tumour formation, progression and/or response to therapy. In some cases, these variants are clearly associated with clinico-pathological variables or prognosis of cancer, whereas in other cases the evidence is less conclusive. Here, we review the evidence that common polymorphisms in various aspects of p53 biology have important consequences for overall tumour susceptibility, clinico-pathology and prognosis. We also suggest reasons for some of the reported discrepancies in the effects of common polymorphisms on tumourigenesis, which relate to the complexity of effects on tumour formation in combination with other oncogenic changes and other polymorphisms. It is likely that future studies of combinations of polymorphisms in the p53 pathway will be useful for predicting tumour susceptibility in the human population and may serve as predictive biomarkers of tumour response to standard therapies.     

泛素蛋白连接酶MDM2活性及稳定性调控的研究进展

泛素蛋白连接酶MDM2(Murine double minute 2)具有癌基因活性, MDM2高表达会导致抑癌基因p53失活而诱发肿瘤, 但在至少7%的肿瘤中p53基因正常而mdm2异常扩增, 表明MDM2还具有其他底物分子, 以p53不依赖的方式促进肿瘤的发生。鉴于MDM2的重要作用, 文章在基因水平、转录水平、翻译后修饰水平、相互作用分子的调节等方面系统总结了目前对MDM2调控的主要研究机制及其进展。     

p53 Promotes proteasome-dependent degradation of oncogenic protein HBx by transcription of MDM2

Hepatitis B virus X protein (HBx) is closely involved in the development of hepatocellular carcinoma (HCC). Tumor suppressor p53 was reported to induce HBx degradation and repress its oncogenic function recently, but the molecular mechanism is unknown. In this study, we attempted to identify the underlying mechanism. We found that overexpression of p53 protein reduces the level of HBx protein and shortens its half-life, however, in MDM2 knock out cells, p53 has no effects on degradation of HBx, meanwhile, overexpression of MDM2 in absence of p53 can accelerate turnover of HBx protein. These indicate that p53-mediated HBx degradation is MDM2-dependent. MDM2 interacts with HBx in vitro and in vivo but does not promote its ubiquitination. In consistent with the results above, HCC tissue samples with wild-type p53 hardly detect HBx protein, whereas, HBx always accumulate in the tissues with mutant p53. Our data provide a possible mechanism on how p53 regulate HBx stability and also a new clue for the study of p53 mutation and HCC development.     

Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway

The gene encoding p53 mediates a major tumor suppression pathway that is frequently altered in human cancers. p53 function is kept at a low level during normal cell growth and is activated in response to various cellular stresses. The MDM2 oncoprotein plays a key role in negatively regulating p53 activity by either direct repression of p53 transactivation activity in the nucleus or promotion of p53 degradation in the cytoplasm. DNA damage and oncogenic insults, the two best-characterized p53-dependent checkpoint pathways, both activate p53 through inhibition of MDM2. Here we report that the human homologue of MDM2, HDM2, binds to ribosomal protein L11. L11 binds a central region in HDM2 that is distinct from the ARF binding site. We show that the functional consequence of L11-HDM2 association, like that with ARF, results in the prevention of HDM2-mediated p53 ubiquitination and degradation, subsequently restoring p53-mediated transactivation, accumulating p21 protein levels, and inducing a p53-dependent cell cycle arrest by canceling the inhibitory function of HDM2. Interference with ribosomal biogenesis by a low concentration of actinomycin D is associated with an increased L11-HDM2 interaction and subsequent p53 stabilization. We suggest that L11 functions as a negative regulator of HDM2 and that there might exist in vivo an L11-HDM2-p53 pathway for monitoring ribosomal integrity.     

Phosphorylation of MDMX mediated by Akt leads to stabilization and induces 14-3-3 binding

The critical tumor suppressor p53 is mutated or functionally inactivated in nearly all cancers. We have shown previously that the MDM2-MDMX complex functions as an integral unit in targeting p53 for degradation. Here we identify the small protein 14-3-3 as a binding partner of MDMX, which binds at the C terminus (Ser367) in a phosphorylation-dependent manner. Importantly, we demonstrate that the serine/threonine kinase Akt mediates phosphorylation of MDMX at Ser367. This phosphorylation leads to stabilization of MDMX and consequent stabilization of MDM2. Previous studies have shown that Akt phosphorylates and stabilizes MDM2. Our data suggest that stabilization of MDMX by Akt may be an alternative mechanism by which Akt up-regulates MDM2 protein levels and exerts its oncogenic effects on p53 in tumor cells.     

The Regulation of the p53-mediated Stress Response by MDM2 and MDM4

Exquisite control of the activity of p53 is necessary for mammalian survival. Too much p53 is lethal, whereas too little permits tumorigenesis. MDM2 and MDM4 are structurally related proteins critical for the control of p53 activity during development, homeostasis, and the response to stress. These two essential proteins regulate both the activation of p53 in response to stress and the recovery of cells following resolution of the damage, yet both are oncogenic when overexpressed. Thus, multiple regulatory circuits ensure that their activities are fine-tuned to promote tumor-free survival. Numerous diverse stressors activate p53, and much research has gone into trying to find commonalities between them that would explain the mechanism by which p53 becomes active. It is now clear that although these diverse stressors activate p53 by different biochemical pathways, one common feature is the effort they direct, through a variety of means, toward disrupting the functions of both MDM2 and MDM4. This article provides an overview of the relationship between MDM2 and MDM4, features the various biochemical mechanisms by which p53 is activated through inhibition of their functions, and proposes some emerging areas for investigation of the p53-mediated stress response.Regulation of the p53-mediated stress response by the essential inhibitory proteins MDM2 and MDM4 is critical for survival. In response to stressors such as ionizing radiation, p53 induces a number of potentially lethal but tumor-suppressive processes, including cell cycle arrest, senescence, and apoptosis (reviewed by Horn and Vousden 2007). Both MDM2 and MDM4 are critical to surviving the p53-mediated stress response to whole body ionizing irradiation as mice with reduced levels of either protein undergo p53-dependent death after exposure to doses of radiation that are sublethal to wild-type mice (Mendrysa et al. 2003; Terzian et al. 2007). MDM2 and MDM4 are also required to control p53 function during development, as shown by the early embryonic death of mice lacking either MDM2 or MDM4, unless they also lack p53 (Jones et al. 1995; Montes de Oca Luna et al. 1995; Parant et al. 2001; Migliorini et al. 2002).Although both MDM2 and MDM4 are essential for development, they are detrimental to long-term survival when in excess, because both are oncogenic. Both MDM2 and MDM4 confer the tumorigenic phenotype on cultured cells when experimentally overexpressed (Fakharzadeh et al. 1991; Danovi et al. 2004). In addition, targeted expression of MDM2 in the mammary gland results in tumorigenesis (Lundgren et al. 1997). In people, single nucleotide polymorphisms that reduce expression of either of the orthologs of MDM2 or MDM4 (also referred to as Hdm2 and Hdm4) correlate with increased risk for breast cancer (Bond et al. 2004; Atwal et al. 2009). Approximately 10% of human tumors have been found to overexpress either MDM2 or MDM4 and many of these express wild-type p53 (reviewed in Toledo and Wahl 2006). Because the majority of human cancers express mutant forms of p53, overexpression of MDM2 and MDM4 in the subset of tumors expressing wild-type p53 supports the notion that excessive MDM2 and MDM4 promote tumorigenesis, at least in part, by blocking p53 function. Thus, limiting the activities of MDM2 and MDM4 is important to prevent cancer.