Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine, CRSA, 75012, Paris, France.
Cell Polarity, Migration and Cancer Unit, Institut Pasteur, UMR3691 CNRS, Université Paris Cité, F-75015, Paris, France.
BMC Biol. 2023 Apr 7;21(1):73. doi: 10.1186/s12915-023-01576-4.
E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-β signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain.
We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-β signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding.
Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.
E3 泛素连接酶通过诱导关键成分的泛素化，在调节细胞信号通路中起着关键作用。RNF111/Arkadia 是一种 RING E3 泛素连接酶，通过诱导转录抑制因子 SKIL/SnoN 的泛素化和蛋白酶体降解来激活 TGF-β 信号。在这项研究中，我们试图通过寻找与 RNF111 的 RING 结构域特异性相互作用的蛋白质来鉴定 RNF111 的 E3 泛素连接酶活性的新调节剂。
我们发现 UBXN7，一种 UBA-UBX 家族的成员，直接与 RNF111 的 RING 结构域或与其具有高度序列同源性的相关 E3 RNF165/ARK2C 相互作用。我们表明，UBXN7 通过其 UAS 硫氧还蛋白样结构域与 RNF111 或 RNF165 RING 结构域对接。UBXN7 的过表达增加内源性 RNF111，而缺乏 UAS 结构域的 UBXN7 突变体则没有影响。相反，UBXN7 的耗尽会降低 RNF111 蛋白水平。因此，我们发现 UBXN7 可以调节 TGF-β 信号响应中 RNF111 底物 SKIL 的降解。我们通过表明 UBXN7 的 UAS 结构域的对接抑制了 RNF111 的泛素化，从而阻止了 RING 结构域与 E2 连接酶的相互作用，进一步揭示了这种调节机制。通过分析 UBXN7 的 UAS 结构域的互作组，我们发现它还与 E3 TOPORS 的 RING 结构域相互作用，并通过损害 E2 结合同样调节其 E3 泛素连接酶活性。
总之，我们的结果表明 UBXN7 是 RNF111、RNF165 和 TOPORS 的 E3 泛素连接酶的直接调节剂，并揭示了一种硫氧还蛋白样结构域可以与特定的 RING 结构域对接，以调节它们的 E3 泛素连接酶活性。
