Yan Sheng, Yuan Aiyun, Shao Guangcan, Zhou Wen, Xu Xin, Dong Meng-Qiu, Liu Xiaoqian, Li Jing
Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing, China.
National Institute of Biological Sciences, Beijing, China.
J Biol Chem. 2025 May 29;301(7):110314. doi: 10.1016/j.jbc.2025.110314.
O-GlcNAcase (OGA) is the sole eraser for the intracellular O-GlcNAc. OGA has many roles in distinct biological processes, such as cancer and embryonic stem cells, but its precise regulatory mechanism is far from being understood. Herein, we studied the small ubiquitin-like modifier (SUMO) modification of OGA and found that OGA is SUMOylated at K358. SUMOylation targets OGA to the chaperone-mediated autophagy (CMA) pathway, which shunts client proteins to the lysosome for degradation. We demonstrate that SUMOylation increases the association between OGA and the heat shock cognate protein 70 (HSC70), the CMA chaperone, and facilitates OGA further degradation. We further mapped a SUMO-interacting motif (SIM) (VLIFD, aa. 195-199) on HSC70. Notably, HSC70-SIM is essential for affinity with other CMA client proteins, such as pyruvate kinase M2. We thus posit that the SIM of HSC70 binds SUMOylated client proteins in a lock-and-key manner to confer substrate selectivity during CMA. To further test our hypothesis, we used label-free quantitative mass spectrometry to study the HSC70-SIM mutant interactome and generated a proteome-wide SUMO-mediated CMA client pool. We then validated this model by studying YEATS domain-containing two from the protein pool and demonstrated that YEATS domain-containing two is SUMOylated at K592, targeting it to CMA. Our work uncovers the SUMO-SIM interaction as a fundamental mechanism governing CMA substrate selectivity and identifies a potential CMA client proteome to deepen our understanding of its pathophysiological relevance.
O-连接的N-乙酰葡糖胺酶（OGA）是细胞内O-连接的N-乙酰葡糖胺（O-GlcNAc）的唯一去除酶。OGA在不同的生物学过程中发挥多种作用，如癌症和胚胎干细胞，但对其精确的调控机制仍知之甚少。在此，我们研究了OGA的小泛素样修饰物（SUMO）修饰，发现OGA在K358位点发生SUMO化修饰。SUMO化修饰将OGA靶向伴侣介导的自噬（CMA）途径，该途径将底物蛋白转运至溶酶体进行降解。我们证明SUMO化修饰增加了OGA与热休克同源蛋白70（HSC70）（CMA伴侣蛋白）之间的相互作用，并促进OGA的进一步降解。我们进一步在HSC70上定位了一个SUMO相互作用基序（SIM）（VLIFD，第195 - 199位氨基酸）。值得注意的是，HSC70-SIM对于与其他CMA底物蛋白（如丙酮酸激酶M2）的亲和力至关重要。因此，我们推测HSC70的SIM以锁钥方式结合SUMO化修饰的底物蛋白，从而在CMA过程中赋予底物选择性。为了进一步验证我们的假设，我们使用无标记定量质谱法研究了HSC70-SIM突变体的相互作用组，并生成了全蛋白质组范围的SUMO介导的CMA底物库。然后，我们通过研究底物库中的含YEATS结构域蛋白2验证了该模型，并证明含YEATS结构域蛋白2在K592位点发生SUMO化修饰，使其靶向CMA。我们的工作揭示了SUMO-SIM相互作用是控制CMA底物选择性的基本机制，并确定了一个潜在的CMA底物蛋白质组，以加深我们对其病理生理相关性的理解。
