Orthogonal interactome profiling of RNF41

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins such as ErbB3 receptors, BRUCE and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK-2 associated cytokine receptors by ubiquitinating and suppressing USP8 which in turn destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: Affinity Purification-Mass Spectrometry, BioID and Virotrap. We combined these results with known datasets for RNF41obtained with microarray MAPPIT and Y2H screens. By combining all these datasets , we provide a comprehensive high resolution interactome network. To further remove potential methodological artifacts from this network, we further distilled the data into a high confidence interactome map by retaining protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.

环指蛋白41（Ring finger protein 41，RNF41）是一类E3泛素连接酶，参与ErbB3受体、BRUCE以及帕金蛋白等多种蛋白质的泛素化修饰与降解过程。此外，RNF41还可通过泛素化并抑制USP8，调控部分JAK-2相关细胞因子受体的细胞内转运过程，而USP8的抑制会进一步导致ESCRT-0复合物的稳定性下降。为进一步阐明RNF41的生物学功能，本研究采用多种正交实验手段解析RNF41蛋白复合物，所用技术包括亲和纯化-质谱联用技术（Affinity Purification-Mass Spectrometry）、BioID以及Virotrap。我们将上述实验结果与此前通过微阵列MAPPIT及Y2H筛选获得的RNF41相关已知数据集进行整合，构建了一套完整的高分辨率相互作用组网络。为进一步去除该网络中潜在的方法学伪影，我们通过保留至少在两种及以上正交实验方法中被鉴定到的蛋白质命中靶点，对数据进行精炼筛选，得到高置信度相互作用组图谱。AP2S1作为一种新型RNF41相互作用伴侣蛋白，从该高置信度相互作用组图谱中被筛选出来用于后续功能验证实验。本研究揭示了AP2S1在瘦素（leptin）及LIF（Leukemia Inhibitory Factor）受体信号通路中的功能，并证实RNF41能够稳定AP2S1并使其发生亚细胞定位的改变。