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At least 19 recordsLinked to original sources

PubMed2026

Antibody-Based Proximity Labeling Reveals Bait-Proximal Proteomes in Paraffin-Embedded and Snap-Frozen Tissue Samples.

Proximity-based labeling approaches have proven highly valuable for uncovering protein-protein interactions, yet their application to primary patient material remains challenging. Here, we present a workflow enabling the use of the antibody-based ProtA-Turbo proximity labeling system in both formalin-fixed paraffin-embedded (FFPE) and snap-frozen tissue specimens. Our method efficiently directs biotinylation to diverse antibody baits across tissues of different origins. Downstream mass spectrometry-based proteomics analyses demonstrate the specificity of the method by profiling the proximal proteome of H3K27ac-marked chromatin, the nuclear lamina-associated protein EMD, and the Ser2-phosphorylated POLR2A subunit of RNA polymerase II. Using this method, we identified cell-type-specific factors and transcriptional regulators in salivary gland carcinoma, healthy testis, and testicular cancer tissue sections. The ability to detect disease-associated complexes directly within their native, spatially resolved cellular context using ProtA-Turbo can provide new insights into the molecular basis of human disease and may reveal novel, potentially actionable factors with translational relevance.

Humans↗

PubMed2025

Unraveling Plant Nuclear Envelope Composition Using Proximity Labeling Proteomics.

The nuclear envelope (NE) defines the eukaryotic cell and functions in a myriad of fundamental cellular processes including but not limited to signal transduction, lipid metabolism, chromatin organization, and nucleocytoplasmic transportation. Although the general structure of the NE is well-conserved across eukaryotic kingdoms, its composition and functions vary substantially between species and remain largely unknown in plants. In this chapter, we describe a proximity-labeling-based proteomic approach to profile novel NE components in the model organism Arabidopsis. This method is generally suitable for the identification of protein components in subcellular compartments or protein complexes that are poorly accessible to traditional mass spectrometry approaches and can be easily applied to other plant species. In addition to giving a step-by-step detailed description of the proximity labeling proteomics procedure in plant samples, we also provide guidelines on the appropriate use of controls and statistical analysis to achieve a highly specific selection of probed candidates.

Proteomics↗

PubMed2025

Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.

m6A-Modified R-loops (m6A-R-loops) play crucial roles in epigenetic regulation and genome stability, yet resolving their spatial distribution and protein interactomes in live cells remains challenging. To address this, we developed m6A-R-loop proximity labeling (m6A-RLPL), a chemically inducible split-APEX2 proximity labeling technology integrating dual-target recognition using the RNA-DNA hybrid binding domain of RNase H1 for R-loop targeting and m6A reader protein's YTH domain for m6A recognition, coupled with an abscisic acid (ABA)-inducible dimerization system for signal amplification. This technology revealed host m6A-R-loops enriched with nucleoli under normal conditions. When applied to herpes simplex virus (HSV) infection, it further demonstrated viral m6A-R-loops undergoing dramatic accumulation within phase-separated granules in replication compartments during late-stage infection. Proximity proteomics identified ZC3H4 and CCDC124 as essential regulators maintaining these structures, which serve as transcription sites for HSV late genes, with disruption selectively impairing viral transcription. m6A-RLPL establishes a generalizable approach for spatially resolved profiling of m6A-R-loop interactomes and organizational dynamics in living systems.

Humans↗

PubMed2026

Proximity labeling puts ZFP36L1 as central hub for posttranscriptional regulation networks in T cells.

Effective T cell responses against pathogens require a rapid yet tightly controlled remodeling of the proteome, and RNA binding proteins (RBPs) are key in this process. For instance, the RBP ZFP36L1 prevents excessive protein production and thereby limits immunopathology. ZFP36L1 is primarily known to mediate mRNA decay, but it can also regulate other processes. How its mode of action relates to its interaction partners is, however, not well-understood. Here, we mapped the ZFP36L1 interactome in primary human T cells. Using proximity labeling, we identified known and new interactors that regulate 3'UTR-mediated RNA degradation, deadenylation, stress granule/p-body formation, as well as 5'UTR-mediated translation repression and mRNA decapping. Snapshot analysis uncovered the ZFP36L1 interactome dynamics and RNA (in)dependency throughout T cell activation. Intriguingly, proximity labeling also uncovered regulators of ZFP36L1 protein expression. This included the helicase UPF1, which not only interacts with ZFP36L1 protein but that may also promote its protein expression. Altogether, this comprehensive interactome map underlines the versatility of interactions with ZFP36L1 and their possible role in cellular function.

Humans↗

PubMed2025

CRISPR-mediated proximity labeling unveils ABHD14B as a host factor to regulate HBV cccDNA transcriptional activity.

BACKGROUND: The long-term goal of chronic hepatitis B research is a functional cure (HBsAg seroclearance). Although currently used nucleos(t)ide analogs can efficiently inhibit viral replication, they do not reduce viral RNAs or proteins produced from covalently closed circular DNA (cccDNA), and rarely achieve a functional cure. To overcome this situation, revealing the mode of the existence of cccDNA is required, including identifying the interreacting proteins with cccDNA. Here, we aimed to identify novel proteins that interact with cccDNA. METHODS: Using an in vitro HBV infection model and a sequence-specific proximity labelling method consisting of dead Cas9 and biotin identification (BioID2), we comprehensively determined proteins that possibly interact with cccDNA. After identifying the candidate proteins, the HBV RNA transcription levels were examined by knocking out the associated genes. RESULTS: We identified ABHD14B as a protein that interacts with cccDNA and inhibits HBV RNA transcription from cccDNA. ABHD14B decreases the acetylation levels of histone proteins that control the transcription levels of HBV RNA in cccDNA. Moreover, ABHD14B interacts with TFII-I, which binds directly to cccDNA in a sequence-dependent manner. These results suggest that the host protein, ABHD14B, is recruited to cccDNA via the TFII-I protein, inhibiting HBV RNA transcription from cccDNA by deacetylating cccDNA histones. CONCLUSIONS: ABHD14B was newly identified as a suppressor of HBV RNA transcription from cccDNA, which may improve our understanding of the mode of existence of cccDNA, providing a basis for development of a functional cure.

Humans↗

PubMed2026

Mapping subcellular microenvironments using oligonucleotide-directed proximity labeling.

Understanding how cells compartmentalize their biomolecules into discrete structures remains one of the fundamental goals of cell biology. The proliferation of proximity labeling (PL) technologies has been invaluable toward this goal, enabling biochemical "dissection" of compartments that would be intractable to classical biochemical methods. While robust PL approaches have long been established for targeting proteins of interest, targeting nucleic acids-RNAs and genomic loci-has remained significantly more challenging. Here, we review recent advancements in the field that overcome this longstanding roadblock by using programmable DNA oligonucleotides to direct PL enzyme localization. These tools are already revealing new insights into the molecular architecture of cellular compartments that lie at the heart of gene expression. They also provide a foundation for developing a new generation of PL tools that exploit the modularity and programmability of oligonucleotide-based devices to enable precise spatiotemporal control at previously inaccessible targets and in challenging specimen types.

Oligonucleotides↗

PubMed2025

STUPPIT is a proximity labeling tool for labeling intermediary proteins that bridge two non-interacting proteins.

Decoding the complexities of signaling pathways is fundamental for deciphering the mechanisms underlying tissue development, homeostasis, and disease pathogenesis. Proximity labeling tools have been instrumental in identifying upstream or downstream effectors of specific proteins within signaling pathways. However, currently, there are no tools available to directly label and capture intermediary proteins that bridge two non-interacting proteins. Here, we developed Split-TurboID and PUP-IT based Protein Identification Tool (STUPPIT), a novel method combining split-TurboID and PUP-IT to biotinylate intermediary proteins of two non-interacting proteins through a two-step enzymatic reaction. STUPPIT was validated using three well-characterized protein triads, including YAP1/AMOT/β-actin, YAP1/LATS1/MOB1A, and β-catenin/α-catenin/β-actin using HEK293T human cell lines. Combining STUPPIT and proteomics, we identified novel intermediary proteins including ERC1 and USP7, which interacted both with β-catenin and SMAD4, key components of the Wnt and BMP signaling pathways. In conclusion, STUPPIT represents a powerful tool for labeling and capturing intermediary proteins between non-interacting partners, offering new insights into protein-protein interactions and advancing signal transduction research.

Humans↗

PubMed2025

Proximity Labeling of Cell Surface Proteins via Cell Surface Remodeling.

Within the complex interplay of proteins, lipids and carbohydrates at the cell surface is the surfaceome, a dense layer of proteins and their posttranslationally modified counterparts that serves as a hub for cell signaling and signal transduction. The surfaceome plays crucial roles in mediating interactions between cells and the extracellular environment, which combined with their availability at the cell surface make it an attractive therapeutic target. Despite its importance, the development of technologies to selectively target cell surface proteins for empirical identification is challenged by their structural complexity. Here, we describe a proximity labeling-based technique to covalently label proteins at the cell surface with a biotin handle, enabling downstream streptavidin-based enrichment and manipulation in a variety of modalities, including fluorescence imaging, western blotting, and mass spectrometry-based proteomics.

Membrane Proteins↗

PubMed2024

Induced proximity labeling and editing for epigenetic research.

Epigenetic regulation plays a pivotal role in various biological and disease processes. Two key lines of investigation have been pursued that aim to unravel endogenous epigenetic events at particular genes (probing) and artificially manipulate the epigenetic landscape (editing). The concept of induced proximity has inspired the development of powerful tools for epigenetic research. Induced proximity strategies involve bringing molecular effectors into spatial proximity with specific genomic regions to achieve the probing or manipulation of local epigenetic environments with increased proximity. In this review, we detail the development of induced proximity methods and applications in shedding light on the intricacies of epigenetic regulation.

Epigenesis, Genetic↗

PubMed2025

Mapping the FOXA1 Interactome in ER+ Breast Cancer Cells Using Proximity Labeling Reveals Novel Interactions with the Orphan Nuclear Receptor NR2C2.

UNLABELLED: FOXA1 is a pioneer transcription factor essential for chromatin accessibility and transcriptional regulation in hormone-driven cancers. In breast cancer, FOXA1 plays a central role in facilitating nuclear receptor binding, reprogramming enhancer landscapes, and promoting transcriptional changes associated with therapy resistance. Whereas FOXA1's function has been primarily studied in the context of estrogen receptor-α (ER), its broader protein interaction network remains incompletely defined. In this study, we systematically map FOXA1-interacting proteins in ER-positive breast cancer cells using proximity-dependent biotin labeling (miniTurbo) combined with quantitative LC-MS/MS proteomics. We engineered MCF-7 cell lines stably expressing miniTurbo-tagged FOXA1 at either the N-terminus or C-terminus to ensure comprehensive coverage of interaction interfaces. This approach recovered known FOXA1 partners, including AR, MLL3, YAP1, and GATA3, and identified 157 previously unreported FOXA1 interactors. Notably, 42 of these novel partners, including NR2C2, were significantly associated with poor relapse-free survival in patients with ER-positive breast cancer. To demonstrate the utility of this resource, we characterized the FOXA1-NR2C2 interaction in depth. Integrating chromatin immunoprecipitation sequencing and RNA sequencing, we show that FOXA1 and NR2C2 co-occupy a subset of genomic regions and drive co-regulated transcriptional programs involved in tumor progression. Our study reveals an expanded FOXA1 interactome and new insights into its functional network in breast cancer, providing candidate proteins for further exploration as biomarkers or therapeutic targets. IMPLICATIONS: These findings expand the FOXA1 interactome in breast cancer and uncover new candidate proteins with potential as biomarkers and therapeutic targets in hormone-driven tumors.

Humans↗

PubMed2026

A Spatiotemporal Atlas of the Androgen Receptor Proximal Interactome.

Androgen receptor-interacting proteins (AR-IPs) number close to 1,000, yet their organization across subcellular space and time remains uncharted. Proximity labeling identifies direct partners and neighboring proteins, thereby expanding AR-IPs to AR-proximal interacting proteins (AR-PIPs). Using proximity labeling quantitative mass spectrometry (PL-qMS), we construct a spatiotemporal atlas of the cytosolic, microsomal, and nuclear compartments in LNCaP prostate tumor cells. PL-qMS recovered 82.2% of the known AR-interactome in extranuclear compartments and 84.2% in the nucleus, identifying 4,751 AR-PIPs that remodel across an androgen time course. The retromer formed an androgen-sensitive AR-proximal interaction network (AR-PIN) verified by proximity ligation assays (PLAs). Moreover, partial VPS26A disruption attenuated androgen-regulated transcription and mislocalized the AR coactivator TMF1, defining a retromer-AR-TMF1 axis. In the nucleus, AR-PINs recover 100% of the Launonen 2021 ChIP-SICAP chromatome and reveal a PLA-verified translation-to-transcription handoff involving eIF4G and 4E-BP1. This spatiotemporal atlas provides a proximal framework for probing AR function in cells.

Journal Article↗

PubMed2026

Resolving cellular signaling in space and time: From organelle proteomics to spatial phosphoproteomics.

Cellular signaling is inherently organized in space and time, requiring coordinated control of protein localization, molecular interactions, and enzymatic activity across subcellular compartments. Recent advances in chemical biology, protein engineering, and quantitative proteomics have made it possible to interrogate these dimensions in an integrated manner. Here, we highlight emerging strategies to resolve signaling organization across three interconnected dimensions: organelle-resolved proteome mapping to define spatial context, proximity labeling to capture local protein interaction networks, and spatially resolved phosphoproteomics to quantify signaling outputs. Developments in proximity labeling, including split, conditionally activated and light-gated enzymes, enable temporally controlled, context-dependent profiling of transient protein assemblies in living cells. Advances in high-throughput and low-input phosphoproteomics, together with improved computational frameworks for kinase activity inference and subcellular enrichment strategies, are enabling spatially resolved measurement of signaling activity. Together, these approaches are shifting the field from static localization maps toward dynamic models of signaling networks.

Proteomics↗

PubMed2026

Dual proximity-based interactome mapping of FKBP51 and FKBP52 uncovers shared metabolic networks.

The 51 kDa FK506-binding protein (FKBP51) has been studied for its involvement in regulating multiple biological systems, particularly as a regulator of steroid hormone receptors, but roles in metabolism, pain response, cell survival, protein turnover, autophagy, immune response, and insulin signaling have also been described. Genetic variants of FKBP51 are associated with various stress-related mental disorders. While recent research has clarified aspects of these processes, the complete range of FKBP51 interactions remains undetermined. FKBP52, a closely related homolog, also affects similar pathways. Recent studies have identified new protein partners for FKBP51 and FKBP52, suggesting an even broader interactome with transient associations. To further characterize interactions, TurboID-based proximity labeling was performed in HeLa cells. Proteomic analysis confirmed known FKBP51 and FKBP52 interactions, while also identifying additional shared and unique binding partners with strong enrichment in metabolic pathways, amino acid biosynthesis, and carbon metabolism. Although FKBP51 and FKBP52 proximal proteins were primarily cytosolic, FKBP51 showed additional associations with exosomal proteins while FKBP52 engaged with additional nuclear proteins. These findings highlight the overlapping roles in metabolic signaling and differentiate pathway-specific partners.

Tacrolimus Binding Proteins↗

PubMed2025

Proximity Proteomics to Profile Ebola Virus Protein Interactome in Its Functional Context.

Proximity labeling-based proteomics (proximity proteomics) has emerged as a popular and versatile approach to illuminate the molecular interactions between viruses and their hosts. In this approach, a proximity labeling enzyme tag is fused to a bait protein and labels neighboring proteins with a chemical handle such as biotin, allowing for downstream affinity purification. Compared to another widely used technique, affinity purification coupled mass spectrometry, proximity proteomics enables the detection of low affinity or transient interactors that might have important functions in the viral life cycle. Further, proximity proteomics can identify interactors of a labile bait protein, of which affinity purification is technically challenging. Here, we describe a proximity proteomic protocol to identify cellular interactors of the Ebola virus polymerase. A similar strategy is readily applicable to elucidate the virus-host interactions for Marburg virus.

Ebolavirus↗

PubMed2025

Integrative proximal-ubiquitomics profiling for deubiquitinase substrate discovery applied to USP30.

The growing interest in deubiquitinases (DUBs) as drug targets for modulating critical molecular pathways in disease is fueled by the discovery of their specific cellular roles. A crucial aspect of this fact is the identification of DUB substrates. While mass spectrometry-based proteomic methods can be used to study global changes in cellular ubiquitination following DUB activity perturbation, these datasets often include indirect and downstream ubiquitination events. To enrich for the direct substrates of DUB enzymes, we have developed a proximal-ubiquitome workflow that combines proximity labeling methodology (ascorbate peroxidase-2 [APEX2]) with subsequent ubiquitination enrichment based on the K-ε-GG motif. We applied this technology to identify altered ubiquitination events in the vicinity of the DUB ubiquitin-specific protease 30 (USP30) upon its inhibition. Our findings reveal ubiquitination events previously associated with USP30 on TOMM20 and FKBP8, as well as the candidate substrate LETM1, which is deubiquitinated in a USP30-dependent manner.

Humans↗

PubMed2026

From stem cells to somites: Revealing genetic and exogenous factors of human embryogenesis.

Stem-cell-based human embryo models offer an ethically tractable platform for studying early human development. This study employs somitoids, three-dimensional models of human somitogenesis, to investigate how transcriptional programs and culture conditions influence somite formation and segmentation. We show that pre-differentiation culture medium impacts the developmental potential of induced pluripotent stem cells (iPSCs), with StemFit medium and Matrigel embedding outperforming mTeSR Plus medium in generating robust somite-like structures. Strikingly, these differences arise despite only subtle changes in transcriptomic and time-resolved proteomic profiles. P300-based proximity labeling also reveals a largely overlapping set of chromatin-associated regulators across iPSC conditions. In somitoids, enhancer-associated profiling highlights factors linked to somitogenesis, including MESP2 and TBX6. Knockout of three identified regulators, BPTF, RBPJ, and CITED2, demonstrate their essential roles in somite formation. Together, these findings highlight how culture conditions and enhancer-associated networks influence early human development and demonstrate somitoids as a scalable system for functional genomics.

Humans↗

PubMed2026

ChromID: A Protocol for Mapping Protein Chromatin Interactions in Living Cells.

Chromatin modifications regulate genome function by recruiting proteins that control transcription, genome organization, and DNA repair. Identifying the proteins associated with specific chromatin modifications is therefore essential for understanding how these regulatory processes operate. Traditional approaches, including chromatin immunoprecipitation and affinity purification coupled to mass spectrometry, have uncovered many chromatin-associated proteins. However, they often rely on crosslinking and chromatin fragmentation, which can disrupt native chromatin architecture and limit the detection of transient interactions. Here, we describe a proximity-labeling protocol for identifying the chromatin-dependent protein interactome associated with specific chromatin marks, termed ChromID. ChromID uses engineered chromatin readers (eCRs) fused to a promiscuous biotin ligase, which labels proteins in the immediate vicinity of the targeted chromatin mark. The protocol includes in vivo biotin labeling, nuclear extract preparation, streptavidin-based enrichment, and tryptic digestion for downstream LC-MS/MS analysis. The protocol has been validated across multiple cell types and chromatin contexts and can be extended to other chromatin-associated proteins, providing a versatile approach to profile chromatin-associated proteomes within their native cellular environment. Key features • Maps proteins associated with different chromatin modifications in living cells using engineered chromatin readers fused to TurboID, BASU, or other promiscuous biotin ligases. • Preserves native chromatin organization and captures transient chromatin-associated interactions that are often lost during conventional affinity purification workflows. • Validated across multiple chromatin contexts, including histone modifications, DNA methylation, transcription factors, RNA polymerase II, and DNA damage-associated chromatin states. • Applicable to diverse cell types and organisms and adaptable to other chromatin-associated proteins, including transcription factors and chromatin regulators.

Biotin proximity labeling↗

PubMed2025

The proxiome of a plant viral protein with dual targeting to mitochondria and chloroplasts revealed MAPK cascade and splicing components as proviral factors.

The coat protein (CP) of the melon necrotic spot virus (MNSV) is a multifunctional factor localized in the chloroplast, mitochondria, and cytoplasm, playing a critical role in overcoming plant defenses such as RNA silencing (RNAi) and the necrotic hypersensitive response. However, the molecular mechanisms through which CP interferes with plant defenses remain unclear. Identifying viral-host interactors can reveal how viruses exploit fundamental cellular processes and help elucidate viral survival strategies. Here, we employed a TurboID-based proximity labeling approach to identify interactors of both the wild-type MNSV CP and a cytoplasmic CP mutant lacking the dual transit peptide (ΔNtCP). Of the interactors, eight were selected for silencing. Notably, silencing MAP4K SIK1 and NbMAP3Kε1 kinases, and a splicing factor homolog NbSMU2 significantly reduced MNSV accumulation, suggesting a proviral role for these proteins in plants. Yeast two-hybrid and bimolecular fluorescence complementation assays confirmed the CP and ΔNtCP interaction with NbSMU2 and NbMAP3Kε1 but not with NbSIK1, which interacted with NbMAP3Kε1. These findings open up new possibilities for exploring how MNSV CP might modulate gene expression and MAPK, thereby facilitating MNSV infection.

Chloroplasts↗