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 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.
Highlights
From the ‡Departments of Structural Biology and Developmental Neurobiology, §St
Mind bomb 1 (Mib1) was found to mediate the degradation of survival motor neuron 1 (SMN1), which contributes to spinal muscular atrophy [17]
We found that Mib1 acts to down-regulate CDKL5 and inhibits its promotion of dendritic spine outgrowth
Summary
Mind bomb 1; DSL, Delta/ Serrate/lag-2; PSD, post-synaptic density; LTP, long-term potentiation; LC-MS/MS, liquid chromatography tandem mass spectrometry; AP-MS, affinity purification mass spectrometry; GST, glutathione Stransferase; TMT, tandem mass tag; FDR, false discovery rate; ppm, parts per million. The primary advantage of this technique, has proven to be its greatest weakness: without stringent washes and data filtering, a vast number of false positives are included in the resulting data sets [23] Methods such as tandem-affinity purification [24] have been developed to remove nonspecific contaminants, but two-step purification requires large quantities of starting materials and reduces sensitivity to loosely bound proteins. In our sequential elution strategy, Mib interaction partners were bound to affinity resins coated with GST-Mib domains, eluted in three sequential buffers of increasing stringency Proteins in these three eluents were identified and quantified by an isobaric labeling Tandem Mass Tag (TMT) method [15]. We found that Mib acts to down-regulate CDKL5 and inhibits its promotion of dendritic spine outgrowth
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