Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei, Taiwan; Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan.
J Biol Chem. 2021 Jan-Jun;296:100484. doi: 10.1016/j.jbc.2021.100484. Epub 2021 Feb 27.
Mutations in the human gene encoding the neuron-specific Eag1 voltage-gated K channel are associated with neurodevelopmental diseases, indicating an important role of Eag1 during brain development. A disease-causing Eag1 mutation is linked to decreased protein stability that involves enhanced protein degradation by the E3 ubiquitin ligase cullin 7 (CUL7). The general mechanisms governing protein homeostasis of plasma membrane- and endoplasmic reticulum (ER)-localized Eag1 K channels, however, remain unclear. By using yeast two-hybrid screening, we identified another E3 ubiquitin ligase, makorin ring finger protein 1 (MKRN1), as a novel binding partner primarily interacting with the carboxyl-terminal region of Eag1. MKRN1 mainly interacts with ER-localized immature core-glycosylated, as well as nascent nonglycosylated, Eag1 proteins. MKRN1 promotes polyubiquitination and ER-associated proteasomal degradation of immature Eag1 proteins. Although both CUL7 and MKRN1 contribute to ER quality control of immature core-glycosylated Eag1 proteins, MKRN1, but not CUL7, associates with and promotes degradation of nascent, nonglycosylated Eag1 proteins at the ER. In direct contrast to the role of CUL7 in regulating both ER and peripheral quality controls of Eag1, MKRN1 is exclusively responsible for the early stage of Eag1 maturation at the ER. We further demonstrated that both CUL7 and MKRN1 contribute to protein quality control of additional disease-causing Eag1 mutants associated with defective protein homeostasis. Our data suggest that the presence of this dual ubiquitination system differentially maintains Eag1 protein homeostasis and may ensure efficient removal of disease-associated misfolded Eag1 mutant channels.
人类基因编码神经元特异性 Eag1 电压门控钾通道的突变与神经发育疾病有关，表明 Eag1 在大脑发育过程中起着重要作用。一种致病的 Eag1 突变与蛋白质稳定性降低有关，涉及到 E3 泛素连接酶 cullin 7 (CUL7) 增强的蛋白质降解。然而，与质膜和内质网 (ER) 定位的 Eag1 K 通道的蛋白质整体稳态相关的一般机制仍不清楚。通过使用酵母双杂交筛选，我们鉴定了另一种 E3 泛素连接酶，makorin 环指蛋白 1 (MKRN1)，作为一种与 Eag1 羧基末端区域主要相互作用的新型结合伴侣。MKRN1 主要与 ER 定位的未成熟核心糖基化以及新生非糖基化的 Eag1 蛋白相互作用。MKRN1 促进未成熟 Eag1 蛋白的多泛素化和 ER 相关的蛋白酶体降解。虽然 CUL7 和 MKRN1 都有助于未成熟核心糖基化 Eag1 蛋白的 ER 质量控制，但 MKRN1 而不是 CUL7，与 ER 中新生的非糖基化 Eag1 蛋白结合并促进其降解。与 CUL7 调节 Eag1 的 ER 和外周质量控制的作用直接相反，MKRN1 专门负责 Eag1 在 ER 中的早期成熟阶段。我们进一步证明，CUL7 和 MKRN1 都有助于与蛋白质稳态缺陷相关的其他致病 Eag1 突变体的蛋白质质量控制。我们的数据表明，这种双重泛素化系统的存在差异地维持了 Eag1 蛋白质稳态，并可能确保有效去除与疾病相关的错误折叠的 Eag1 突变体通道。
