Posttranslational modification by ubiquitination targets proteins for degradation, recycling, stabilization or altered trafficking, and as such can alter cellular signaling pathways. The substrate specificity of this multistep process is controlled by ubiquitin ligases, including those of the HECT domain-containing NEDD4 family. In the testis, ubiquitination of many proteins contributes to organ development and maturation of spermatozoa and NEDD4 is known to be important in the control of spermatogonial stem cell homeostasis. However, a comprehensive understanding of NEDD4 substrates in testis development is lacking. Here we demonstrate high expression of Nedd4 in somatic cells of the mouse testis and in the murine Leydig cell-like cell line TM3. Immunoprecipitation of NEDD4 tagged with GFP at either the amino or carboxyl terminus was subjected to proteomic analysis for interacting proteins. We identified a substantial list of potential interaction partners, including known NEDD4 substrates, proteins involved in ubiquitination and proteins important for testis development and spermatogenesis. We confirmed the interaction of NEDD4 with a subset of these putative interacting proteins, validating the integrity of the dataset. These potential interactors may be further explored to reveal important roles of NEDD4-mediated ubiquitination in the testis.

Significance

Ubiquitination is important for testis development and function, and NEDD4 is known to ubiquitinate various proteins to affect cellular signaling and development, including those implicated in spermatogenesis. However, substrates of NEDD4 that are important during testis development remain to be identified. Here we report NEDD4 expression in the developing testis and TM3 testicular cell line. This study identifies a substantial list of NEDD4 interacting proteins in the TM3 testicular cell line, with validation of some of these interactions. Hence, this provides novel NEDD4 targets that may contribute to testis development and function that may be further explored.

中文翻译：

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鉴定小鼠睾丸中 NEDD4 泛素连接酶的新型相互作用伙伴。

泛素化的翻译后修饰作用于降解、回收、稳定或改变运输的蛋白质，因此可以改变细胞信号通路。这个多步骤过程的底物特异性由泛素连接酶控制，包括含有 HECT 结构域的 NEDD4 家族的连接酶。在睾丸中，许多蛋白质的泛素化有助于精子的器官发育和成熟，已知 NEDD4 在控制精原干细胞稳态中很重要。然而，缺乏对睾丸发育中 NEDD4 底物的全面了解。在这里，我们展示了Nedd4的高表达在小鼠睾丸的体细胞和鼠 Leydig 细胞样细胞系 TM3 中。对在氨基或羧基末端用 GFP 标记的 NEDD4 进行免疫沉淀，对相互作用的蛋白质进行蛋白质组学分析。我们确定了大量潜在的相互作用伙伴，包括已知的 NEDD4 底物、参与泛素化的蛋白质和对睾丸发育和精子发生很重要的蛋白质。我们证实了 NEDD4 与这些假定的相互作用蛋白的一个子集的相互作用，验证了数据集的完整性。可以进一步探索这些潜在的相互作用物，以揭示 NEDD4 介导的睾丸泛素化的重要作用。

意义

泛素化对睾丸发育和功能很重要，已知 NEDD4 可以泛素化各种蛋白质以影响细胞信号传导和发育，包括那些与精子发生有关的蛋白质。然而，在睾丸发育过程中重要的 NEDD4 底物仍有待确定。在这里，我们报告了发育中的睾丸和 TM3 睾丸细胞系中的 NEDD4 表达。本研究确定了 TM3 睾丸细胞系中大量 NEDD4 相互作用蛋白，并验证了其中一些相互作用。因此，这提供了可能有助于睾丸发育和功能的新的 NEDD4 目标，可以进一步探索。