Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth

Abstract

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 downregulated 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 interac-

Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry

“…As anticipated, pre-MS fractionation reduced sample complexity, leading to low peptide/protein identification per fraction. However, if all fractions were analyzed, the combined number of identifications would be improved 18,32 . Interestingly, the reduction of MS2 isolation window had minor effect on peptide/protein identification, which may be due to high isolation efficiency of quadrupole mass filters that were installed in newly developed MS instruments (e.g.…”

“…The JUMP hybrid algorithm was used to process numerous published large datasets 32,34-36 . RAW files were converted to mzXML format and MS2 spectra were searched against rat and E.coli target-decoy Uniprot databases to estimate false discovery rate (FDR) 37,38 .…”

Structure and function of the Mind bomb E3 ligase in the context of Notch signal transduction

“…Unbiased proteomic studies using immunoprecipitation followed by mass spectrometry and yeast two-hybrid screens have been performed to elucidate a more complete Mib1 interactome. These studies Mib1 associates with diverse targets including membrane trafficking proteins, cell adhesion components, replication/transcription/translation factors and signal transduction proteins [17,18]. …”

“…An important role for Mib1 in centriolar biology is emerging, and suggests direct interaction and regulation of multiple factors including PLK4 (polo-like kinase 4), AZI1 (5-azacytidine induced 1), and PCM1 (pericentriolar material 1) [22,23]. In neuronal cells, Mib1 directly modulates cyclin-dependent kinase 5 (CDK5) and survival of motor neuron (SMN) protein levels independent of its well-studied role in Notch-mediated neurogenesis [17,24,25]. The structural mechanism for these non-canonical interactions is currently unclear, but may utilize binding modes similar to that elucidated for its engagement of Notch ligand substrates [26••].…”

Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry

“…Therefore, each tag generates a unique reporter ion during MS/MS fragmentation, enabling relative quantification of the 10 samples by distinct reporter ions. The TMT strategy is commonly applied in dissecting biological pathways and cellular processes (Churchman et al, 2015; Mertz et al, 2015; Wang et al, 2016). …”