The role of Nedd4 family E3 ubiquitin ligases in the regulation of neuronal cell surface receptors

从跨膜细胞表面受体到细胞核转录因子的信号转导受蛋白泛素化在多个水平上调控，泛素化可导致受体表面表达下调、第二信使通路组分降解、转录因子核转运修饰或组蛋白修饰改变转录的表观遗传控制。在非神经元细胞中，单泛素化或k63连接的多泛素化是关键的调控机制，控制胞吞作用和随后的跨膜细胞表面受体下调，从而影响从细胞分化到细胞凋亡的多种细胞生物学过程。然而，尽管神经元的发育和功能受到细胞外信号的严格控制，但对神经细胞表面受体的单泛素化和k63连接的多泛素化的作用知之甚少。在本项目中，我们将研究Nedd4家族的两种hect型E3泛素连接酶Nedd4-1和Nedd4-2在神经细胞中的作用，重点研究它们在控制细胞表面受体方面的功能。Nedd4-1和Nedd4-2介导底物的单泛素化或k63介导的多泛素化，是神经元中最丰富的E3连接酶之一，在神经细胞发育和分化的早期，即在细胞外信号分子如生长因子严格控制的发育阶段，其表达尤其强烈。此外，Nedd4-1和Nedd4-2与多种受体蛋白的控制有关，尽管相应的证据往往仍然不完整。为了准备我们的研究，我们生成了条件Nedd4-1和Nedd4-2 KO小鼠系。我们将使用这些小鼠模型以及生化和细胞生物学方法来鉴定神经元中Nedd4-1和Nedd4-2的跨膜底物蛋白，并检查Nedd4-1 /Nedd4-2介导的泛素化过程对神经元和大脑发育和功能的影响。

Signal transduction from trans-membrane cell surface receptors to nuclear transcription factors is regulated at multiple levels by protein ubiquitination, which can lead to downregulation of receptor surface expression, degradation of components of second messenger pathways, modification of the nuclear transport of transcription factors, or alterations in the epigenetic control of transcription by histone modification. In nonneuronal cells, monoubiquitination or K63-linked polyubiquitination are critical regulatory mechanisms that control the endocytosis and consequent downregulation of trans-membrane cell surface receptors, and thereby affect multiple cell biological processes - from cell differentiation to apoptosis. However, little is known about the role of monoubiquitination and K63-linked polyubiquitination of receptors on the nerve cell surface, although neuronal development and function are tightly controlled by extracellular signals. In the project proposed here, we will study the role of two HECT-type E3 ubiquitin ligases of the Nedd4 family, Nedd4-1 and Nedd4-2, in nerve cells - with a focus on their function in controlling cell surface receptors. Nedd4-1 and Nedd4-2 mediate monoubiquitination or K63-mediated polyubiquitination of substrates, are among the most abundant E3 ligases in neurons, and are particularly strongly expressed during early nerve cell development and differentiation, i.e. at developmental stages that are tightly controlled by extracellular signaling molecules such as growth factors. Moreover, Nedd4-1 and Nedd4-2 have been implicated in the control of multiple receptor proteins, although the corresponding evidence has often remained fragmentary. In preparation of our study, we generated conditional Nedd4-1 and Nedd4-2 KO mouse lines. We will employ these mouse models along with biochemical and cell biological approaches to identify transmembrane substrate proteins of Nedd4-1 and Nedd4-2 in neurons and to examine the consequences of such Nedd4- 1/Nedd4-2-mediated ubiquitination processes for neuron and brain development and function.