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

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的主要负调控因子MDM2被认为通过两种机制调节P53：1)直接与P53反式激活域结合，抑制P53活性；2)作为E3泛素连接酶，能够泛化P53，靶向于核输出和降解。除了MDM2(一种同源蛋白)外，MDMX还参与p53的调节，主要是通过与MDM2类似的机制结合和阻断p53的反式激活结构域。MDM2和MDMX基因敲除小鼠都是胚胎致死的，并且伴随着p53的缺失而被完全挽救，这表明它们在p53调控中发挥了关键作用。研究发现，维持MDM2-P53结合，但破坏MDM2 E3连接酶活性的Mdm2C462a敲入小鼠模型的发展会导致胚胎死亡，同时P53的缺失得以挽救。令人惊讶的是，这项研究表明MDM2-P53单独结合不足以调节P53，并暗示MDM2环指结构域在P53调节中起关键作用。在破坏MDM2 E3泛素活性的同时，该突变还破坏了MDM2-MDMX的异二聚化。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，因此无法推断这些变化中的哪一种导致了观察到的p53调控错误。尽管进行了深入的研究，但关于MDM2和MDMX在体内如何调节p53的作用仍不清楚。在体外，这种结合已被证明能放大或挽救MDM2 E3连接酶对p53的活性，但其在体内的作用尚不清楚。最近发展的Mdm2Y487A敲入小鼠，保持了与MDMX和P53结合的能力，但扰乱了E3泛素连接酶的活性，使得这两个MDM2环指结构域功能得以分离。通过利用这一模型，我们希望进一步阐明其功能。 MDM2环指结构域在p53调控中的作用，因为进一步了解p53调控对于开发有效的治疗方法至关重要。

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.