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 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.

Fig. 1.

Sequential elution strategy of Mib1 affinity purification. A, Domain structure of Mib1 and recombinant proteins, depicting full-length Mib1 at 1006 aa, the ND (1–401aa), and MD (384–801aa). Mib1 contains 5 Kelch repeats (gray boxes), 1 ZZ-type zinc finger domain (diagonal lined boxes), 12 ankyrin repeats (dotted boxes), and 3 RING domains (black boxes). B, Gel analysis of total elution from ND and MD. Total elution from ND recovered many more proteins than MD. C, Overview of the purification, sequential elution, TMT labeling, and LC-MS/MS strategy. Rat brain lysate was incubated with GST fusion protein beads, washed and eluted. Eluents were digested and labeled by TMT isobaric tags, mixed, and analyzed by LC-MS/MS. D, Sequential elution from ND. Gel analysis of sequential elutions from ND shows disparate band profiles for each buffer, including protein differences and intensities, as well as overall abundance.

Fig. 2.

Grouping proteins by their sequential elution profiles. A, Intra-elution null comparison and inter-elution experimental comparisons. Null comparison of protein abundances from Medium stringency buffer technical replicates shows a tight distribution around a log₂ value of zero approaching a normal distribution. Meanwhile experimental comparisons between Medium and Low stringency buffers and between Medium and High stringency buffers display widely varied distributions, shifted toward positive values suggesting increased overall abundances in the Medium stringency elution. B, Heat map showing log₂ values for each protein from Low to Medium elutions and Medium to High elutions. Using a log2 value of 0.8 as a threshold, proteins exhibiting increases, decreases, and no change from the Low stringency elution to the Medium stringency elution as well as Medium to High were determined. C, Detailed elution profiles of representative proteins from each group.

Fig. 3.

Mib1 interaction partners participate in several important signaling pathways. Highlight of several important pathways and biological functions enriched in groups 1 through 4 in our data set. Notch signaling showed a modest enrichment, with an isoform of canonical Mib1 interaction partner delta, DLL4, exhibiting strong binding affinity and appearing in group 4. The Wnt pathway, however, was strongly enriched, with several Catenin and Casein kinase family members appearing in group 3. Proteins involved in Endocytosis and Vesicle Transport and Morphogenesis were very strongly enriched across all four elution profile groups, with clathrin-mediated endocytosis regulator Scyl2 and RhoA interaction partner GEFT showing highest affinity binding. The Ubiquitin Proteasome System was also highly enriched across elution profile groups and the Usp9x deubiquitinase displayed one of the strongest elution profiles in addition to high abundance in our analysis. Although many proteins located at Synapses and regulating LTP were recovered in our analysis, none exhibited extremely high binding affinity.

Fig. 4.

Interconnectivity in Mib1 Ubiquitin Proteasome System interactome. Potential Mib1 interaction partners in the UPS pathway show high levels of interconnectivity. Several constituents of the Proteasome itself are included, with Proteasome Subunit Beta 5 showing highest affinity. The deubiquitinating enzyme USP9x is shown to interact with several proteins of interest as well. Edge thickness represents “combined score” from STRING-DB analysis.

Fig. 5.

Mib1 colocalizes with Usp9x (FAM) and 3 members of the catenin family. HEK 293 cells were transfected with HA tagged Mib1, and/or other catenin proteins, followed by immunofluorescence staining. A–C, Immunostaining of recombinant Mib1 and native Usp9x. D–L, Coexpression of HA-Mib1 and different catenin family members and subsequent immunostaining.

Fig. 6.

Mib1 colocalizes with and down-regulates CDKL5. A, Representative MS2 spectrum depicting peptide 826–837 from CDKL5 protein and TMT reporter ion intensities (inset). B–C, Typical HA-CDKL5 localization in HEK293 cells is largely nuclear when expressed alone. D–F, Coexpression of HA-CDKL5 along with GFP-Mib1 led to redistribution of CDKL5 from the nucleus to puncta in cytoplasm. G–H, Western blot analysis of HA-CDLK5 and GFP-Mib1 cotransfected HEK293 cells, dependent on Mib1 ligase activity. C985S is a mutated form of Mib1 with abolished ligase activity. I, Western blot and LC-MS/MS analysis of ubiquitination of CDLK5 by Mib1. J–K, Western blot and immunofluorescence staining analysis show self-association of Mib1 through its C-terminal domain.

Fig. 7.

Mib1 inhibits dendritic spine outgrowth and limits pro-outgrowth effects of CDKL5 in neuronal culture. A, Representative rat hippocampal neuron transfected at DIV7 with EGFP and stained for EGFP at DIV14. B–E, Higher magnification representative images of dendritic spines in control (Ctl, GFP only), Mib1, CDKL5, and Mib1 + CDKL5 transfected neurons, showing decreased outgrowth phenotype with Mib1 and Mib1 + CDKL5, and increased with CDKL5 and C985S + CDKL5. F–G, Quantitative analysis of dendritic spine density from these neurons. Analysis of the spine density showing mean ± S.E. per 10 μm, n = 12 for Ctl, n = 16 for Mib1, n = 14 for CDKL5, n = 11 for Mib1 + CDKL5 in experiment one; n = 11 for Ctl, n = 34 for C985S, n = 27 for CDKL5, n = 12 for C985S + CDKL5 in experiment two (* p ≤ 0.05 from Ctl, *** p ≤ 0.001 from Ctl, # p ≤ 0.05 from CDKL5, t test). H, Classification and analysis of dendritic spine shapes in these neurons. Shape proportion analysis of the same neurons delineated between filopodia-, thinheaded-, and stubby/mushroom-shaped protrusions order of maturation. * p ≤ 0.05 from Ctl, # p ≤ 0.05 from CDKL5, t test). Scale bars, 10 μm.