High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-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 data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 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）是一种E3泛素连接酶（E3 ubiquitin ligase），参与包括ErbB3受体、BIRC6以及parkin在内的多种蛋白质的泛素化与降解过程。除此之外，RNF41还可通过泛素化并抑制USP8，调控部分JAK2相关细胞因子受体的胞内运输，而USP8的失活会进一步导致ESCRT-0复合物不稳定。为进一步阐明RNF41的功能，我们采用多种正交实验方法解析其蛋白质复合物：包括亲和纯化-质谱联用（affinity purification–mass spectrometry）、BioID以及Virotrap技术。我们将这些实验结果与此前通过微阵列MAPPIT（microarray MAPPIT）与Y2H筛选（Y2H screens）获得的RNF41相关已知数据集进行整合，由此构建出包含175个候选蛋白质互作伴侣的高分辨率综合互作组网络。为去除该网络中潜在的方法学伪影，我们通过保留在至少两种正交实验方法中均被鉴定到的19个蛋白质命中物，将原始数据提炼为高可信度互作组图谱。其中AP2S1作为新发现的RNF41互作伴侣，从该高可信度互作组中被选出用于后续功能验证。我们证实了AP2S1在瘦素（leptin）与LIF受体信号通路中的作用，并证明RNF41能够稳定并重新定位AP2S1。