370 Heritage Medical Research Center, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
Department of Laboratory Medicine and Pathology (5B4. 09), University of Alberta, Edmonton, Alberta, Canada.
Carcinogenesis. 2021 Apr 30;42(4):650-662. doi: 10.1093/carcin/bgab009.
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.
Pirh2 是一种 E3 连接酶，属于 RING-H2 家族，已被证明能够结合、泛素化和下调 p73 肿瘤抑制子的功能，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，已被报道为一种负调控蛋白，可促进 p73 的泛素化和降解。在此，我们发现 Pirh2 是 AIP4 的关键调节因子，可抑制 p73 功能。Pirh2 与 AIP4 发生物理相互作用，并显著下调 AIP4 的表达。这种下调涉及 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-siRNAs 耗尽内源性 Pirh2 时，该途径得到恢复。我们进一步观察到 Pirh2 减少了 AIP4 介导的 p73 泛素化。在翻译水平上，特别是关于 p73 细胞周期阻滞功能，即使表达 AIP4，Pirh2 仍能确保 p73 介导的 G1 期阻滞。我们的研究揭示了先前认为在调节同一效应子底物 p73 方面无关的两种 E3 连接酶之间的新联系。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家族的多个底物打开了一扇门，就像 p53 和 p73 蛋白一样。
