Hu Jimin, Huynh Duc T, Dunn Denise E, Wu Jianli, Manriquez-Rodriguez Cindy, Zhang Qianyi E, Hirschkorn Gabrielle A, Hirata Tetsuya, Georgiou George R, Myers Samuel A, Floyd Scott R, Chi Jen-Tsan, Boyce Michael
Department of Biochemistry, Duke University School of Medicine, Box 3711 DUMC, Durham, NC 27710, United States.
Department of Radiation Oncology, Duke University School of Medicine, Box 3085 DUMC, Durham, NC 27710, United States.
Glycobiology. 2025 Aug 11;35(9). doi: 10.1093/glycob/cwaf046.
The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked β-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.
由42个成员组成的kelch样（KLHL）蛋白家族是泛素E3连接酶复合物的衔接子，控制着多种底物的稳定性。KLHL蛋白对于维持多种组织中的蛋白质稳态至关重要，并且在包括癌症、神经退行性疾病和家族性高钾性高血压在内的人类疾病中发生突变。然而，对KLHL蛋白的调控仍未完全了解。此前，我们报道了两个KLHL家族成员KEAP1和巨轴索蛋白受O-连接β-N-乙酰葡糖胺（O-GlcNAc）调控，O-GlcNAc是一种细胞内糖基化形式。有趣的是，KEAP1和巨轴索蛋白的一些泛素化靶点自身也被O-GlcNAc化，这表明这种翻译后修饰的多层次控制可能会影响许多KLHL途径。为了验证这一假设，我们研究了KLHL3，它与无赖氨酸（WNK）激酶发生泛素化反应以调节下游离子通道活性。我们的生化和糖蛋白质组学数据表明，人KLHL3和所有四种WNK激酶（WNK1-4）都被O-GlcNAc化。此外，我们的结果表明，O-GlcNAcylation在渗透压控制和铁死亡中都会影响WNK4的功能，其潜在影响范围从血压调节到神经元健康和存活。这项工作证明了O-GlcNAcylation对KLHL3/WNK轴的功能调节，并支持了O-GlcNAc作为KLHL信号传导和蛋白质稳态的一般调节剂的更广泛模型。
