Pirh2 is an E3 ligase belonging to the RING-H2 family and shown to bind, ubiquitinate and downregulate p73 tumor suppressor function without altering p73 protein levels. AIP4, an E3 ligase belonging to the HECT domain family, has been reported to be a negative regulatory protein that promotes p73 ubiquitination and degradation. Herein, we found that Pirh2 is a key regulator of AIP4 that inhibits p73 function. Pirh2 physically interacts with AIP4 and significantly downregulates AIP4 expression. This downregulation is shown to involve the ubiquitination of AIP4 by Pirh2. Importantly, we demonstrated that the ectopic expression of Pirh2 inhibits the AIP4-p73 negative regulatory pathway, which was restored when depleting endogenous Pirh2 utilizing Pirh2-siRNAs. We further observed that Pirh2 decreases AIP4-mediated p73 ubiquitination. At the translational level and specifically regarding p73 cell cycle arrest function, Pirh2 still ensures the arrest of p73-mediated G1 despite AIP4 expression. Our study reveals a novel link between two E3 ligases previously thought to be unrelated in regulating the same effector substrate, p73. These findings open a gateway to explain how E3 ligases differentiate between regulating multiple substrates that may belong to the same family of proteins, as it is the case for the p53 and p73 proteins.

中文翻译：

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Pirh2 是一种 E3 连接酶，通过调节 AIP4 表达和泛素化来调节 AIP4-p73 调节途径。

Pirh2 是属于 RING-H2 家族的 E3 连接酶，可结合、泛素化和下调 p73 肿瘤抑制功能，而不改变 p73 蛋白水平。AIP4 是一种属于 HECT 结构域家族的 E3 连接酶，据报道是一种促进 p73 泛素化和降解的负调节蛋白。在此，我们发现 Pirh2 是抑制 p73 功能的 AIP4 的关键调节因子。Pirh2 与 AIP4 物理相互作用并显着下调 AIP4 表达。这种下调显示出涉及 Pirh2 对 AIP4 的泛素化。重要的是，我们证明了 Pirh2 的异位表达抑制了 AIP4-p73 负调控途径，当利用 Pirh2-siRNA 消耗内源性 Pirh2 时，该途径得以恢复。我们进一步观察到 Pirh2 降低了 AIP4 介导的 p73 泛素化。在翻译水平，特别是关于 p73 细胞周期阻滞功能，尽管有 AIP4 表达，Pirh2 仍确保 p73 介导的 G1 阻滞。我们的研究揭示了两种 E3 连接酶之间的新联系，这些连接酶以前被认为与调节相同的效应底物 p73 无关。这些发现为解释 E3 连接酶如何区分调节可能属于同一蛋白质家族的多种底物打开了大门，就像 p53 和 p73 蛋白一样。