Kosztyu Pavlína, Slaninová Iva, Valčíková Barbora, Verlande Amandine, Müller Petr, Paleček Jan J, Uldrijan Stjepan
Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czechia.
International Clinical Research Center, St. Anne's University Hospital, Brno, Czechia.
Front Physiol. 2019 Apr 9;10:390. doi: 10.3389/fphys.2019.00390. eCollection 2019.
Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules , suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.
Mdm2和MdmX是相关蛋白，它们以Mdm2同二聚体或Mdm2/MdmX异二聚体的形式作为肿瘤抑制因子p53的E3泛素连接酶发挥作用。这种二聚化对于E3活性是必需的，并且由这两种蛋白中存在的保守RING结构域介导，但只有Mdm2的RING结构域能够有效地形成同二聚体。我们对人Mdm2进行了系统的突变分析，将RING的部分区域与相应的MdmX序列进行交换，以确定这种差异的分子决定因素。Mdm2还可以促进MdmX的降解，我们鉴定出了几个阻断该过程的突变。它们主要位于Mdm2/E2界面，并且没有破坏MdmX与Mdm2之间的相互作用。令人惊讶的是，Mdm2/E2界面的一些突变抑制了由Mdm2/MdmX异二聚体介导的MdmX降解，但不影响由Mdm2同二聚体介导的p53降解。只有一个突变体，即用天冬酰胺取代保守的半胱氨酸449（C449N），破坏了Mdm2与MdmX二聚化的能力。当我们将半胱氨酸残基引入MdmX的相应位点时，RING结构域能够与其他MdmX分子形成二聚体，这表明Mdm2和MdmX的RING结构域中一个保守的氨基酸残基可能是这些结构域形成二聚体的差异能力及其E3活性的决定因素。然而，在免疫沉淀实验中，只有当两个RING结构域中的天冬酰胺残基都被半胱氨酸取代时，才能观察到MdmX的同二聚化。这一结果表明，由一个带有半胱氨酸的突变MdmX RING和一个带有天冬酰胺的野生型MdmX RING组成的异源复合物可能不太稳定，尽管在基于细胞的检测中很容易检测到。此外，Mdm2 C449N阻断了Mdm2-MdmX异二聚化，但没有破坏Mdm2同二聚体促进p53降解的能力，这表明保守的半胱氨酸和天冬酰胺残基对二聚化的影响是上下文特异性的。总的来说，我们的结果表明，Mdm2和MdmX RING结构域之间保守残基的个别交换的影响可能是上下文特异性的，这支持了以下假设：尽管Mdm2 RING同二聚体和Mdm2-MdmX异二聚体表面相似，但它们在结构上可能并不完全等同。
