Tribbles homolog 2 (TRIB2) is known to boost liver tumorigenesis via regulating Ubiquitin (Ub) proteasome system (UPS). At least two ways are involved, i.e., acts as an adaptor protein to modulate ubiquitination functions of certain ubiquitin E3 ligases (E3s) and reduces global Ub levels via increasing the proteolysis activity of proteasome. Recently, we have identified the role of TRIB2 to relieve oxidative damage via reducing the availability of Ub that is essential for the ubiquitination and subsequent degradation of Glutathione peroxidase 4 (GPX4). Although GPX4 is a critical antioxidant factor to protect against ferroptosis, the exact evidence showing that TRIB2 desensitizes ferroptosis is lacking. Also, whether such function is via E3 remains unclear. Here, we demonstrated that deletion of TRIB2 sensitized ferroptosis via lifting labile iron in liver cancer cells. By contrast, overexpression of TRIB2 led to the opposite outcome. We further demonstrated that transferrin receptor (TFRC) was required for TRIB2 to desensitize the cells to ferroptosis. Without TFRC, the labile iron pool could not be reduced by overexpressing TRIB2. We also found that beta-transducin repeat containing E3 ubiqutin protein ligase (βTrCP) was a genuine E3 for the ubiquitination of TFRC, and TRIB2 was unable to decline labile iron level once upon βTrCP was knocked out. In addition, we confirmed that the opposite effects on ferroptosis and ferroptosis-associated lipid reactive oxygen species (ROS) generation resulted from knockout and overexpression of TRIB2 were all indispensible of TFRC and βTrCP. Finally, we demonstrated that TRIB2 exclusively manipulated RSL3- and erastin-induced-ferroptosis independent of GPX4 and glutathione (GSH). In conclusion, we elucidated a novel role of TRIB2 to desensitize ferroptosis via E3 βTrCP, by which facilitates TFRC ubiquitiation and finally decreases labile iron in liver cancer cells.

已知 Tribbles 同源物 2 (TRIB2) 通过调节泛素 (Ub) 蛋白酶体系统 (UPS) 促进肝脏肿瘤发生。至少涉及两种方式，即作为衔接蛋白调节某些泛素 E3 连接酶 (E3s) 的泛素化功能，并通过增加蛋白酶体的蛋白水解活性降低整体 Ub 水平。最近，我们已经确定了 TRIB2 通过降低 Ub 的可用性来减轻氧化损伤的作用，Ub 对于泛素化和随后的谷胱甘肽过氧化物酶 4 (GPX4) 的降解至关重要。尽管 GPX4 是防止铁死亡的关键抗氧化因子，但缺乏表明 TRIB2 使铁死亡脱敏的确切证据。此外，此类功能是否通过 E3 尚不清楚。这里，我们证明了 TRIB2 的缺失通过提升肝癌细胞中的不稳定铁而对铁死亡敏感。相比之下，TRIB2 的过度表达导致相反的结果。我们进一步证明了转铁蛋白受体 (TFRC) 是 TRIB2 使细胞对铁死亡脱敏所必需的。如果没有 TFRC，过表达 TRIB2 就无法减少不稳定的铁池。我们还发现含有 E3 泛素蛋白连接酶 (βTrCP) 的 β-转导重复序列是用于 TFRC 泛素化的真正 E3，一旦 βTrCP 被敲除，TRIB2 就无法降低不稳定铁水平。此外，我们证实TRIB2的敲除和过表达对铁死亡和铁死亡相关脂质活性氧（ROS）产生的相反影响都是TFRC和βTrCP不可或缺的。最后，我们证明了 TRIB2 独立于 GPX4 和谷胱甘肽 (GSH) 专门操纵 RSL3 和erastin 诱导的铁死亡。总之，我们阐明了 TRIB2 通过 E3 βTrCP 使铁死亡脱敏的新作用，通过它促进 TFRC 泛素化并最终减少肝癌细胞中的不稳定铁。