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

描述（由申请人提供）：TP53是一种关键的肿瘤抑制基因，能够诱导细胞周期阻滞、衰老和凋亡。通常，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)直接结合p53反活化结构域，抑制p53活性；2)作为E3泛素连接酶，能够泛素化p53，靶向其核输出和降解。除了同源蛋白Mdm2外，MdmX也参与p53调控，主要是通过结合和阻断p53反活化结构域，机制与Mdm2相似。Mdm2和MdmX基因敲除小鼠都是胚胎致死性的，并且在p53基因缺失的情况下完全获救，这表明它们在p53调控中起着关键作用。研究发现，Mdm2C462A敲入小鼠模型维持Mdm2-p53结合，但破坏Mdm2 E3连接酶活性，导致胚胎致死，同时缺失p53。令人惊讶的是，本研究表明，仅Mdm2-p53结合不足以调节p53，并暗示Mdm2环指结构域在p53调节中起关键作用。除了破坏Mdm2 E3泛素活性外，该突变还破坏Mdm2- mdmx异源二聚化。由于Mdm2C462A突变破坏了RING finger结构域的功能、E3泛素连接酶活性和MdmX结合，因此无法推断出是哪一种变化导致了p53的失调。尽管研究深入，但Mdm2和MdmX如何在体内调节p53的功能仍不清楚。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泛素连接酶的活性，使得这两个Mdm2环指结构域功能分离。通过利用该模型，我们希望进一步阐明

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.