The in vivo role of the Mdm2-MdmX interaction in p53 regulation

Mdm2-MdmX 相互作用在 p53 调节中的体内作用

• 批准号: 8299227
• 负责人: YANPING ZHANG
• 金额: $ 30.58万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299227
• 关键词: Acetylation; Address; Affect; Alanine; Amino Acids; Apoptosis; Binding; Biochemical; Cell Cycle Arrest; Cells; Collection; Comparative Study; Development; Embryo; Embryonic Development; Fingers; Gene Targeting; Genetic; Goals; Heterodimerization; Homo; Homologous Gene; Homologous Protein; In Vitro; Investigation; Knock-in Mouse; Knockout Mice; Mediating; Modeling; Mus; Mutant Strains Mice; Mutation; Nuclear Export; Positioning Attribute; Process; Protein p53; Proteins; Regulation; Role; Stress; TP53 gene; Testing; Therapeutic; Therapeutic Agents; Transactivation; Tumor Suppressor Genes; Tyrosine; Ubiquitin; Ubiquitination; biological adaptation to stress; cancer therapy; experience; in vivo; insight; mdm2 protein; mouse model; mutant; novel; senescence; tool; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): TP53 is a critical tumor suppressor gene capable of inducing cell cycle arrest, senescence, and apoptosis. Canonically, the primary negative regulator of the TP53 protein product p53, Mdm2, is considered to regulate p53 through two mechanisms; 1) through direct binding to the p53 transactivation domain, suppressing p53 activity, and 2) through functioning as an E3 ubiquitin ligase capable of ubiquitinating p53, targeting it for nuclear export and degradation. In addition to Mdm2, a homologous protein, MdmX also functions in p53 regulation, primarily through binding and blocking the p53 transactivation domain in a similar mechanism to Mdm2. Both Mdm2 and MdmX knockout mice are embryonically lethal, and rescued completely with concomitant deletion of p53, indicative of their critical role in p53 regulation. The development of an Mdm2C462A knock-in mouse model that maintains Mdm2-p53 binding, but disrupts Mdm2 E3 ligase activity, was found to result in embryonic lethality, rescued with simultaneous deletion of p53. Surprisingly, this study suggests that Mdm2-p53 binding alone is not sufficient for p53 regulation, and implicates the Mdm2 RING finger domain as critical in p53 regulation. Along with disrupting Mdm2 E3 ubiquitin activity, the mutation also disrupts Mdm2-MdmX heterodimerization. Because the Mdm2C462A mutation disrupts both functions of the RING finger domain, the E3 ubiquitin ligase activity and the MdmX binding, it cannot be deduced which of these changes is causing the observed misregulation of p53. Despite intensive study, much remains unknown about how Mdm2 and MdmX function in vivo to regulate p53. In vitro this binding has been demonstrated to amplify or rescue Mdm2 E3 ligase activity towards p53, but its role in vivo is not yet clear. Recent development of an Mdm2Y487A knock-in mouse, which maintains the ability to bind to MdmX and p53, but has disrupted E3 ubiquitin ligase activity has allowed for the separation of these two Mdm2 RING finger domain functions. Through utilizing this model, we hope to further elucidate the function of the Mdm2 RING finger domain in p53 regulation, as further understanding p53 regulation is critical in the development of effective therapeutics. PUBLIC HEALTH RELEVANCE: We have very recently generated mice with a single-amino acid residue substitution of tyrosine to alanine at the Mdm2-487 residue (Y487A). In vitro this substitution was characterized to result in an Mdm2 protein fully capable of binding to p53 and MdmX, but devoid of E3 ubiquitin ligase activity. To our surprise, despite lacking E3 activity the Mdm2Y487A mice are viable and are overtly phenotipically normal. Preliminary study has demonstrated lack of E3 function and retain of MdmX binding of the in vivo Mdm2Y487A mutant protein, indicating that the Mdm2-MdmX binding, but not the Mdm2 E3 ligase function, is critical for p53 control, at least during embryogenesis and early development. Characterization thus far of the Mdm2Y487A mutant mice has provided further insight into the role of the Mdm2 RING finger domain in p53 regulation. A better understanding of both the function and mechanism of the Mdm2 E3 ligase and the Mdm2-MdmX interaction in p53 regulation, a goal of this investigation, will help to identify novel targets for the treatment of cancer by enhancing the efficacy of p53-activating therapeutic agents.

描述（申请人提供）：TP53是一种关键的肿瘤抑制基因，能够诱导细胞周期停滞、衰老和凋亡。典型地，TP53蛋白产物p53的主要负调节因子Mdm2被认为通过两种机制调节p53： 1) 通过直接结合 p53 反式激活结构域，抑制 p53 活性，2) 通过作为能够泛素化 p53 的 E3 泛素连接酶，将其靶向核输出和降解。除了同源蛋白 Mdm2 之外，MdmX 也在 p53 调节中发挥作用，主要通过与 Mdm2 类似的机制结合并阻断 p53 反式激活结构域。 Mdm2 和 MdmX 敲除小鼠均具有胚胎致死性，并通过同时删除 p53 来完全获救，这表明它们在 p53 调节中发挥着关键作用。 Mdm2C462A 敲入小鼠模型的开发可维持 Mdm2-p53 结合，但破坏 Mdm2 E3 连接酶活性，被发现会导致胚胎致死，并可通过同时删除 p53 来挽救。令人惊讶的是，这项研究表明仅 Mdm2-p53 结合不足以调节 p53，并表明 Mdm2 RING Finger 结构域在 p53 调节中至关重要。除了破坏 Mdm2 E3 泛素活性外，该突变还会破坏 Mdm2-MdmX 异二聚化。由于 Mdm2C462A 突变破坏了环指结构域、E3 泛素连接酶活性和 MdmX 结合的两种功能，因此无法推断出这些变化中的哪一个导致了观察到的 p53 的错误调节。尽管进行了大量研究，但关于 Mdm2 和 MdmX 在体内如何调节 p53 的功能仍然未知。在体外，这种结合已被证明可以增强或挽救 Mdm2 E3 连接酶对 p53 的活性，但其在体内的作用尚不清楚。最近开发的 Mdm2Y487A 敲入小鼠保持了与 MdmX 和 p53 结合的能力，但破坏了 E3 泛素连接酶活性，从而可以分离这两个 Mdm2 RING 指结构域功能。通过利用该模型，我们希望进一步阐明 Mdm2 RING Finger 结构域在 p53 调节中的作用，因为进一步了解 p53 调节对于开发有效的治疗方法至关重要。 公共卫生相关性：我们最近培育出了 Mdm2-487 残基 (Y487A) 处的酪氨酸单氨基酸残基替换为丙氨酸的小鼠。在体外，这种取代的特征是产生完全能够结合 p53 和 MdmX 的 Mdm2 蛋白，但缺乏 E3 泛素连接酶活性。令我们惊讶的是，尽管缺乏 E3 活性，Mdm2Y487A 小鼠仍能存活，并且表型明显正常。初步研究表明体内 Mdm2Y487A 突变蛋白缺乏 E3 功能并保留 MdmX 结合，表明 Mdm2-MdmX 结合（而不是 Mdm2 E3 连接酶功能）对于 p53 控制至关重要，至少在胚胎发生和早期发育期间。迄今为止，Mdm2Y487A 突变小鼠的表征进一步深入了解了 Mdm2 RING Finger 结构域在 p53 调节中的作用。本研究的目标之一是更好地了解 Mdm2 E3 连接酶的功能和机制以及 Mdm2-MdmX 在 p53 调节中的相互作用，这将有助于通过增强 p53 激活疗法的功效来确定癌症治疗的新靶点代理。