Department of Molecular and Integrative Physiology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Neuroscience Program, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
J Neurochem. 2019 Nov;151(3):289-300. doi: 10.1111/jnc.14840. Epub 2019 Aug 25.
Neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2) is an epilepsy-associated gene, which encodes a ubiquitin E3 ligase that is highly expressed in the brain. Nedd4-2's substrates include many ion channels and receptors because its N-terminal C2 domain guides Nedd4-2 to the cell membrane. We previously found that Nedd4-2 ubiquitinates the glutamate receptor subunit 1 (GluA1) subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, which leads to reduction of neuronal excitability and seizures in mice. However, despite awareness of a Nedd4-2 isoform with no C2 domain, the functions of this isoform remain elusive. In this study, we showed that the C2-lacking Nedd4-2 has reduced membrane distribution and exhibits reduced affinity toward ubiquitinating GluA1. However, when expressed in primary cortical neurons, we found that the C2-lacking Nedd4-2 exhibits a similar activity toward reducing excitatory synaptic strength as does the C2-containing Nedd4-2. In an attempt to identify novel Nedd4-2 substrates that could mediate excitatory synaptic strength, we used unbiased proteomic screening and found multiple synaptic regulators that were up-regulated in the brain of conditional Nedd4-2 knockout mice, including protein phosphatase 3 catalytic subunit-α (PPP3CA; alternately called calcineurin A-α). We confirmed PPP3CA as a substrate of the C2-lacking Nedd4-2 and showed that all three epilepsy-associated missense mutations of Nedd4-2 disrupted PPP3CA ubiquitination. Altogether, our results revealed novel potential Nedd4-2 substrates and suggest that the C2-lacking Nedd4-2 represses excitatory synaptic strength most likely through GluA1 ubiquitination-independent mechanisms. These findings provide novel information to further our knowledge about Nedd4-2-dependent neuronal excitability homeostasis and pathological hyperexcitability when Nedd4-2 is compromised.
神经前体细胞表达的发育下调基因 4 样蛋白（Nedd4-2）是一种与癫痫相关的基因，编码一种高度表达于大脑中的泛素 E3 连接酶。Nedd4-2 的底物包括许多离子通道和受体，因为其 N 端 C2 结构域指导 Nedd4-2 到达细胞膜。我们之前发现，Nedd4-2 泛素化α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体的谷氨酸受体亚基 1（GluA1）亚基，导致小鼠神经元兴奋性降低和癫痫发作。然而，尽管人们已经意识到存在一种没有 C2 结构域的 Nedd4-2 同工型，但这种同工型的功能仍然难以捉摸。在这项研究中，我们表明，缺乏 C2 结构域的 Nedd4-2 膜分布减少，对泛素化 GluA1 的亲和力降低。然而，当在原代皮质神经元中表达时，我们发现缺乏 C2 结构域的 Nedd4-2 对降低兴奋性突触强度的活性与含有 C2 结构域的 Nedd4-2 相似。为了鉴定可能介导兴奋性突触强度的新的 Nedd4-2 底物，我们使用无偏蛋白组筛选发现了多个在条件性 Nedd4-2 敲除小鼠脑中上调的突触调节因子，包括蛋白磷酸酶 3 催化亚基-α（PPP3CA；也称为钙调神经磷酸酶 A-α）。我们证实 PPP3CA 是缺乏 C2 结构域的 Nedd4-2 的底物，并表明 Nedd4-2 的三种与癫痫相关的错义突变均破坏了 PPP3CA 的泛素化。总的来说，我们的研究结果揭示了新的潜在的 Nedd4-2 底物，并表明缺乏 C2 结构域的 Nedd4-2 可能通过 GluA1 泛素化非依赖机制抑制兴奋性突触强度。这些发现为进一步了解 Nedd4-2 依赖的神经元兴奋性稳态和 Nedd4-2 受损时病理性过度兴奋提供了新的信息。
