Dataset Information

Sequential elution interactome analysis of Mind bomb 1 and a novel role in dendritic spine outgrowth

ABSTRACT: Mind bomb 1 (Mib1) is an E3 ubiquitin-ligase that is essential for overall metazoan development, including multiple stages of neuronal development. It is located in puncta throughout neurons, including near post synaptic densities (PSD), and has been shown to participate in several signaling pathways via its E3 ligase catalytic activity. The most well-characterized of these is the Notch signaling pathway, in which Mib1 facilitates Delta/Serrate/LAG-2 (DSL) ligand endocytosis and activation, allowing cell-cell communication to determine neuronal versus glial cell fate. This, however, is not the limit of Mib1 activity in the developing nervous system, as it also contributes to cell polarity, neurite outgrowth, and long-term potentiation (LTP), but it has not been demonstrated to affect dendritic spine development. We therefore sought to comprehensively characterize the Mib1 interactome and study its potential function in dendritic spine morphogenesis. We utilized a novel sequential elution method from Mib1 affinity purification to recover Mib1 binding proteins with both deep coverage and the ability to distinguish between high affinity binding partners from low affinity binding partners. This procedure revealed 837 potential binding partners, distinguished 72 from these as very-high confidence, and a further 387 as high confidence of interaction. Included in these were many proteins previously demonstrated to interact with Mib1, as well as 5 proteins of particular interest to us: Usp9x, a deubiquitinase; alpha-, beta-, and delta-catenins; regulators of Wnt signaling; and CDKL5, which is mutated in EIEE2, a severe form of mental retardation. We demonstrated that Mib1 downregulates CDKL5, limits its effects on dendritic spine outgrowth, and inhibits spine outgrowth itself. These data further elaborate upon the signaling networks and biological functions influenced by this critical protein and expand our understanding of the signaling networks involved in neuronal development.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

SUBMITTER: xusheng wang

LAB HEAD: Junmin Peng

PROVIDER: PXD001255 | Pride | 2016-07-06

REPOSITORIES: Pride

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Publications

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

Mertz Joseph J Tan Haiyan H Pagala Vishwajeeth V Bai Bing B Chen Ping-Chung PC Li Yuxin Y Cho Ji-Hoon JH Shaw Timothy T Wang Xusheng X Peng Junmin J

Molecular & cellular proteomics : MCP 20150430 7

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 prote ...[more]

PMID: 25931508

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Project description:Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, cognitive deficits, and seizures, as well as potentially driving glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via ‘Ribotag’, morphometric analysis, and intravital imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden on excitatory neuronal pathophysiology as well as the impact of mTOR inhibition on these neuronal alterations. The Ribotag analysis of peritumoral excitatory neurons showed an upregulation in transcripts encoding for F-actin binding and other dendritic spine-enriched proteins. Light and electron microscopy analyses revealed marked decreases in dendritic spine density in peritumoral neurons. Intravital two-photon imaging in peritumoral excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single neuron level, throughout tumor growth. Intravital two-photon imaging also revealed altered stimulus-evoked somatic calcium activity, both in rate and temporal alignment, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a mTORC1 and mTORC2 inhibitor reversed the translational effect of glioma on neurons, increased dendritic spine density, and functional neuronal alterations. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma – manifested by alterations in ribosome-bound mRNA, dendritic spine morphology, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that peritumoral neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing foundational knowledge for developing therapies targeting neuronal signaling in glioma.

Dataset Information

Sequential elution interactome analysis of Mind bomb 1 and a novel role in dendritic spine outgrowth

Publications

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

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