Project description:Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.
Project description:The gene BIN1 is the second-largest genetic risk factor for late-onset Alzheimer’s disease (LOAD). BIN1 is expressed in neurons and glia in the brain as multiple isoforms, including neuron-specific and ubiquitously expressed isoforms. BIN1 is an adaptor protein that regulates membrane dynamics in many cell types. Previously, we reported that BIN1 predominantly localizes to presynaptic terminals in neurons and regulates presynaptic vesicular release. However, the function of neuronal BIN1 as it relates to LOAD is not fully understood. A fundamental gap in the field is an unbiased characterization of neuronal BIN1-interacting proteins and proximal neighbors. To fill this void and help define neuronal BIN1’s functions in the brain, we applied TurboID-based proximity labeling to identify proteins biotinylated by neuronal BIN1 isoform 1-TurboID fusion protein (BIN1iso1-TID) in cultured mouse neuroblastoma (N2a) cells in vitro and in adult mouse brain neurons in vivo. Label-free proteomics identification of BIN1iso1-TID biotinylated proteins resulted in the discovery of 361 proteins from the N2a cells and 897 proteins in mouse brain neurons as BIN1iso1-associated (proximal) or interacting proteins. A total of 92 proteins were common in the two datasets, indicative that these are high-confidence BIN1-interacting or proximity proteins. SynapticGO analysis of the mouse brain dataset showed that BIN1iso1-TurboID labeled 159 synaptic proteins, with 60 corresponding to the synaptic vesicle cycle. Based on a phosphopeptide analysis of the neuronal BIN1iso1-TID interactome and kinase prediction, we chose to validate AAK1, CDK16, SYNJ1, PP2BA, and RANG through immunostaining and proximity ligation assays as members of the BIN1 interactome in the mouse brain. By identifying a number of previously unknown BIN1 proximal and potential interacting proteins, this study lays a foundation for further investigations on neuronal BIN1 function.
Project description:Different brain cell types play distinct roles in brain development and disease via cellular mechanisms. Molecular characterization of these cellular mechanisms using cell type-specific approaches, particularly at the protein (proteomic) level, can provide biological and therapeutic insights. Conventional approaches to investigate cell type-specific proteomes from brain pose several technical barriers. To overcome these, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using TurboID with a Nuclear Export Sequence (TurboID-NES), coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy to sample cell type-specific proteomes in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID-NES approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. We generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing TurboID-NES to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-NES expression and biotinylation did not significantly impact homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration of BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. TurboID-NES mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under resting conditions. Acute LPS treatment significantly altered microglial proteomes, but not N2A proteomes, and the LPS effect was partly captured by analysis of the TurboID-NES-labeled proteome of BV2 cells.
Project description:In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. While physiological expression of the labelling enzyme is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for the rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. Endogenous tagging of the μ subunit of the AP-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. We used the KI-strategy to compare the interactome of the different adaptor protein (AP) complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.
Project description:We report the application of enyzme-based 4C-Seq technique for exploring Pou5f1 enhancer interactome in mouse ES cells. We explored the interactome of Pou5f1 upstream enhancer in mouse ES cells by using an enzyme digestion based 4C-Seq protocol. The interactome is involved in gene active regulation.
Project description:BRD9 was identified in a genome-wide screen for genes regulating the response to interferon (IFN) in a A549 based reporter cell line. Subsequent experiments determined an involvement of BRD9 in the transcriptional regulation of Interferon-stimulated genes (ISGs) expression following stimulation with IFN-a2. The aim of this proximity-labelling experiments was to gain a more mechanistic understanding of BRD9 recruitment during the IFN signal transduction using A549 cells stably transduced with BRD9-TurboID and mCherry-TurboID fusion proteins. The BRD9 interactome in the absence of IFN- a2 was determined. We found that following IFN-a2 treatment, STAT2 significantly associates with BRD9-TurboID.
Project description:In Alzheimer's disease (AD) and other tauopathies, tau dissociates from microtubules and forms toxic aggregates that contribute to neurodegeneration. Although some of the pathological interactions of tau have been identified from postmortem brain tissue, these studies are limited by their inability to capture transient interactions. To investigate the interactome of aggregate-prone fragments of tau, we applied an in vitro proximity labeling technique using split TurboID fused with the tau microtubule repeat domain (TauRD), a core region implicated in tau aggregation. We characterized this split TurboID TauRD displaying the requirement for both ligase fragment co-expression for robust enzyme activity and nuclear and cytoplasmic localization of the recombinant proteins. Following enrichment of biotinylated proteins and mass spectrometry, we identified over 700 TauRD interactors. Gene ontology analysis of enriched TauRD interactors highlighted processes often dysregulated in tauopathies, including spliceosome complexes, RNA-binding proteins (RBPs), and nuclear speckles. These in vitro results were further supported by integrating the TauRD interactome data with human AD tau interactome datasets and protein co-expression networks from human AD and related tauopathies. This revealed an overlap with the in vitro TauRD interactome and several modules enriched with RBPs and increased in AD and PSP. These findings emphasize the importance of nuclear pathways in tau pathology, such as RNA splicing and nuclear-cytoplasmic transport and establishes the sTurbo TauRD system as a valuable tool for exploring the tau interactome.
Project description:We employed the TurboID proximity labeling approach to map the interactome of SREBP2 with mass spectrometry-based proteomic analysis.