Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):12001-6. doi: 10.1073/pnas.1102309108. Epub 2011 Jul 5.
There are currently two distinct models proposed to explain why both MDM2 and MDMX are required in p53 control, with a key difference centered on whether these two p53 inhibitors work together or independently. To test these two competing models, we generated knockin mice expressing a point mutation MDMX mutant (C462A) that is defective in MDM2 binding. This approach allowed a targeted disassociation of the MDM2/MDMX heterocomplex without affecting the ability of MDMX to bind to p53, and while leaving the MDM2 protein itself completely untouched. Significantly, Mdmx(C462A/C462A) homozygous mice died at approximately day 9.5 of embryonic development, as the result of a combination of apoptosis and decreased cell proliferation, as shown by TUNEL and BrdU incorporation assays, respectively. Interestingly, even though the MDMX mutant protein abundance was found slightly elevated in the Mdmx(C462A/C462A) homozygous embryos, both the abundance and activity of p53 were markedly increased. A p53-dependent death was demonstrated by the finding that concomitant deletion of p53 completely rescued the embryonic lethality in Mdmx(C462A/C462A) homozygous mice. Our data demonstrate that MDM2 and MDMX function as an integral complex in p53 control, providing insights into the nonredundant nature of the function of MDM2 and MDMX.
目前有两种截然不同的模型被提出，用以解释为什么 MDM2 和 MDMX 都需要参与 p53 调控，其关键区别在于这两种 p53 抑制剂是协同作用还是独立作用。为了验证这两种相互竞争的模型，我们构建了表达点突变 MDMX 突变体（C462A）的 knockin 小鼠，该突变体与 MDM2 结合缺陷。这种方法允许靶向分离 MDM2/MDMX 异源二聚体，而不影响 MDMX 结合 p53 的能力，同时使 MDM2 蛋白本身完全不受影响。重要的是，Mdmx(C462A/C462A)纯合子小鼠在胚胎发育的第 9.5 天左右死亡，这是由于凋亡和细胞增殖减少的共同作用，分别通过 TUNEL 和 BrdU 掺入测定来证明。有趣的是，尽管在 Mdmx(C462A/C462A)纯合子胚胎中发现 MDMX 突变体蛋白的丰度略有升高，但 p53 的丰度和活性均显著增加。通过发现同时缺失 p53 可完全挽救 Mdmx(C462A/C462A)纯合子小鼠的胚胎致死性，证明了 p53 依赖性死亡。我们的数据表明，MDM2 和 MDMX 作为 p53 调控的一个整体复合物发挥作用，为 MDM2 和 MDMX 功能的非冗余性质提供了新的见解。
