College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
Nat Chem. 2022 Jul;14(7):831-840. doi: 10.1038/s41557-022-00946-9. Epub 2022 May 30.
Liquid-liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O-GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex. We identified multiple O-GlcNAc modification sites for the endogenous SynGAP isolated from rat brain and the recombinantly expressed protein. Protein semisynthesis was used to generate site-specifically O-GlcNAcylated forms of SynGAP, and in vitro and cell-based LLPS assays demonstrated that T1306 O-GlcNAc of SynGAP blocks the interaction with PSD-95, thus inhibiting LLPS. Furthermore, O-GlcNAcylation suppresses SynGAP/PSD-95 LLPS in a dominant-negative manner, enabling sub-stoichiometric O-GlcNAcylation to exert effective regulation. We also showed that O-GlcNAc-dependent LLPS is reversibly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). These findings demonstrate that OGT- and OGA-catalysed O-GlcNAc cycling may serve as an LLPS-regulating post-translational modification.
液-液相分离 (LLPS) 的 SynGAP 和 PSD-95，两种在神经元突触后密度 (PSD) 中相互作用的丰富蛋白，已被牵连到调节兴奋性突触中 SynGAP 的 PSD 富集。然而，潜在的调节机制仍然是个谜。在这里，我们报告说，SynGAP 的 O-GlcNAcylation 作为 SynGAP/PSD-95 复合物 LLPS 的抑制剂。我们鉴定了从大鼠脑中分离的内源性 SynGAP 和重组表达蛋白的多个 O-GlcNAc 修饰位点。使用蛋白半合成生成了 SynGAP 的特异性 O-GlcNAc 化形式，并进行了体外和基于细胞的 LLPS 测定，结果表明 SynGAP 的 T1306 O-GlcNAc 阻断了与 PSD-95 的相互作用，从而抑制了 LLPS。此外，O-GlcNAcylation 以显性负性方式抑制 SynGAP/PSD-95 LLPS，使亚化学计量的 O-GlcNAcylation 能够发挥有效的调节作用。我们还表明，O-GlcNAc 依赖性 LLPS 可被 O-GlcNAc 转移酶 (OGT) 和 O-GlcNAcase (OGA) 可逆调节。这些发现表明，OGT 和 OGA 催化的 O-GlcNAc 循环可能作为一种 LLPS 调节的翻译后修饰。
