From the Department of Neuroscience, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458 and.
the Harriet L. Wilkes Honors College, Florida Atlantic University, Jupiter, Florida 33458.
J Biol Chem. 2019 Apr 26;294(17):6843-6856. doi: 10.1074/jbc.RA119.007406. Epub 2019 Mar 11.
Inhibitory GABAergic transmission is required for proper circuit function in the nervous system. However, our understanding of molecular mechanisms that preferentially influence GABAergic transmission, particularly presynaptic mechanisms, remains limited. We previously reported that the ubiquitin ligase EEL-1 preferentially regulates GABAergic presynaptic transmission. To further explore how EEL-1 functions, here we performed affinity purification proteomics using and identified the -GlcNAc transferase OGT-1 as an EEL-1 binding protein. This observation was intriguing, as we know little about how OGT-1 affects neuron function. Using biochemistry, we confirmed that the OGT-1/EEL-1 complex forms in neurons and showed that the human orthologs, OGT and HUWE1, also bind in cell culture. We observed that, like EEL-1, OGT-1 is expressed in GABAergic motor neurons, localizes to GABAergic presynaptic terminals, and functions cell-autonomously to regulate GABA neuron function. Results with catalytically inactive point mutants indicated that OGT-1 glycosyltransferase activity is dispensable for GABA neuron function. Consistent with OGT-1 and EEL-1 forming a complex, genetic results using automated, behavioral pharmacology assays showed that and act in parallel to regulate GABA neuron function. These findings demonstrate that OGT-1 and EEL-1 form a conserved signaling complex and function together to affect GABA neuron function.
抑制性 GABA 能传递对于神经系统中正常的回路功能是必需的。然而，我们对于优先影响 GABA 能传递的分子机制，尤其是突触前机制的理解仍然有限。我们之前报道过，泛素连接酶 EEL-1 优先调节 GABA 能突触前传递。为了进一步探索 EEL-1 的作用机制，我们在这里使用 进行了亲和纯化蛋白质组学研究，并鉴定出 -GlcNAc 转移酶 OGT-1 是 EEL-1 的结合蛋白。这一观察结果很有趣，因为我们对 OGT-1 如何影响神经元功能知之甚少。通过 生物化学实验，我们证实了 OGT-1/EEL-1 复合物在神经元中形成，并且表明人类同源物 OGT 和 HUWE1 在细胞培养中也结合。我们观察到，像 EEL-1 一样，OGT-1 在 GABA 能运动神经元中表达，定位于 GABA 能突触前末梢，并以细胞自主的方式发挥作用，调节 GABA 神经元功能。催化失活点突变体的结果表明，OGT-1 糖基转移酶活性对于 GABA 神经元功能不是必需的。与 OGT-1 和 EEL-1 形成复合物的结果一致，使用自动化、行为药理学测定的遗传结果表明， 和 平行作用以调节 GABA 神经元功能。这些发现表明，OGT-1 和 EEL-1 形成一个保守的信号复合物，并共同作用以影响 GABA 神经元功能。
