Mertz Joseph, Tan Haiyan, Pagala Vishwajeeth, Bai Bing, Chen Ping-Chung, Li Yuxin, Cho Ji-Hoon, Shaw Timothy, Wang Xusheng, Peng Junmin
From the ‡Departments of Structural Biology and Developmental Neurobiology.
§St. Jude Proteomics Facility.
Mol Cell Proteomics. 2015 Jul;14(7):1898-910. doi: 10.1074/mcp.M114.045898. Epub 2015 Apr 30.
The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development.
mind bomb 1（Mib1）泛素连接酶对于通过Notch信号通路以及可能的Wnt通路控制后生动物发育至关重要。它也在有丝分裂后的神经元中表达，并通过很大程度上未知的机制调节神经元形态发生和突触活动。我们试图全面表征Mib1相互作用组，并利用一种用于亲和纯化的新型顺序洗脱策略研究其在神经元发育中的潜在功能，在该策略中，Mib1结合蛋白在不同的严格条件下洗脱，然后通过等压标记法进行定量。该策略以深度覆盖以及区分高亲和力伙伴与低亲和力伙伴的能力鉴定了Mib1相互作用组。在Mib1亲和纯化过程中总共鉴定出817种蛋白质，包括56个高亲和力伙伴和335个低亲和力伙伴，而其余426种蛋白质可能是共纯化的污染物或结合力极弱的蛋白质。该分析检测到了所有先前已知的与Mib1相互作用的蛋白质，并揭示了大量参与Notch和Wnt通路、内吞作用和囊泡运输、泛素 - 蛋白酶体系统、细胞形态发生以及突触活动的新成分。免疫荧光研究进一步表明Mib1与五种选定的蛋白质共定位：Usp9x（FAM）去泛素化酶、α - 、β - 和δ - 连环蛋白以及CDKL5。CDKL5的突变与早期婴儿癫痫性脑病2型（EIEE2）相关，EIEE2是一种严重的智力发育迟缓形式。我们发现Mib1的表达通过泛素化下调CDKL5的蛋白质水平，并在树突棘形成过程中拮抗CDKL5的功能。因此，顺序洗脱策略能够对蛋白质相互作用组进行生化表征；而对Mib1的分析为研究其在信号网络和神经元发育中的作用提供了一个全面的相互作用组。
