Comparing the BAD Protein Interactomes in 2D and 3D Cell Culture Using Proximity Labeling.

Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.

The lipid droplet assembly complex consists of seipin and four accessory factors in budding yeast.

Seipin, a crucial protein for cellular lipid droplet (LD) assembly, oligomerizes at the interface between the endoplasmic reticulum (ER) and LDs to facilitate neutral lipid packaging. Using proximity labeling, we identify four proteins-Ldo45, Ldo16, Tgl4, and Pln1-that are recruited to the vicinity of yeast seipin, the Sei1-Ldb16 complex, exclusively when seipin function is intact, hence termed seipin accessory factors. Localization studies identify Tgl4 at the ER-LD contact site, in contrast to Ldo45, Ldo16 and Pln1 at the LD surface. Cells with compromised seipin function resulted in uneven distribution of these proteins with aberrant LDs, supporting a central role of seipin in orchestrating their association with the LD. Overexpression of any seipin accessory factor causes LD aggregation and affects a subset of LD protein distribution, highlighting the importance of their stoichiometry. Although single factor mutations show minor LD morphology changes, combined mutations have additive effects. Lastly, we present evidence that seipin accessory factors assemble and interact with seipin in the absence of neutral lipids and undergo dynamically rearrangements during LD formation induction, with Ldo45 acting as a central hub recruiting other factors to interact with the seipin complex.

Analysis of Lipid GPCR Molecular Interactions by Proximity Labeling.

G-protein-coupled receptors (GPCRs) are hepta-helical transmembrane proteins that mediate various intracellular signaling events in response to their specific ligands including many lipid mediators. Although analyses of GPCR molecular interactions are pivotal to understanding diverse intracellular signaling events, affinity purification of interacting proteins by a conventional co-immunoprecipitation method is challenging due to the hydrophobic nature of GPCRs and their dynamic molecular interactions. Proximity labeling catalyzed by a TurboID system is a powerful technique for defining the molecular interactions of target proteins in living cells. TurboID and miniTurbo (a modified version of TurboID) are engineered biotin ligases that biotinylate neighboring proteins in a promiscuous manner. When fused with a target protein and expressed in living cells, TurboID or miniTurbo mediates the biotin labeling of the proteins with close proximity to the target protein, allowing efficient purification of the biotinylated proteins followed by a shot-gun proteomic analysis. In this chapter, we describe a step-by-step protocol for the labeling of GPCR neighboring proteins by TurboID or miniTurbo, purification of the biotin-labeled proteins, and subsequent sample preparation for proteomic analysis. We utilized S1PR1 as a model GPCR, a receptor for a bioactive lipid molecule sphingosine 1-phosphate (S1P) that plays various roles in physiological and pathological conditions. This analysis pipeline enables the mapping of interacting proteins of lipid GPCRs in living cells.

TurboID-mediated proximity labeling technologies to identify virus co-receptors.

Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.

Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.

Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.

Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry.

Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.

Pericyte-Specific Secretome Profiling in Hypoxia Using TurboID in a Multicellular in Vitro Spheroid Model.

Cellular communication within the brain is imperative for maintaining homeostasis and mounting effective responses to pathological triggers like hypoxia. However, a comprehensive understanding of the precise composition and dynamic release of secreted molecules has remained elusive, confined primarily to investigations using isolated monocultures. To overcome these limitations, we utilized the potential of TurboID, a non-toxic biotin ligation enzyme, to capture and enrich secreted proteins specifically originating from human brain pericytes in spheroid cocultures with human endothelial cells and astrocytes. This approach allowed us to characterize the pericyte secretome within a more physiologically relevant multicellular setting encompassing the constituents of the blood-brain barrier. Through a combination of mass spectrometry and multiplex immunoassays, we identified a wide spectrum of different secreted proteins by pericytes. Our findings demonstrate that the pericytes secretome is profoundly shaped by their intercellular communication with other blood-brain barrier-residing cells. Moreover, we identified substantial differences in the secretory profiles between hypoxic and normoxic pericytes. Mass spectrometry analysis showed that hypoxic pericytes in coculture increase their release of signals related to protein secretion, mTOR signaling, and the complement system, while hypoxic pericytes in monocultures showed an upregulation in proliferative pathways including G2M checkpoints, E2F-, and Myc-targets. In addition, hypoxic pericytes show an upregulation of proangiogenic proteins such as VEGFA but display downregulation of canonical proinflammatory cytokines such as CXCL1, MCP-1, and CXCL6. Understanding the specific composition of secreted proteins in the multicellular brain microvasculature is crucial for advancing our knowledge of brain homeostasis and the mechanisms underlying pathology. This study has implications for the identification of targeted therapeutic strategies aimed at modulating microvascular signaling in brain pathologies associated with hypoxia.

Synaptic proteomics decode novel molecular landscape in the brain.

Synapses play a pivotal role in forming neural circuits, with critical implications for brain functions such as learning, memory, and emotions. Several advances in synaptic research have demonstrated the diversity of synaptic structure and function, which can form thousands of connections depending on the neuronal cell types. Moreover, synapses not only interconnect neurons but also establish connections with glial cells such as astrocytes, which play a key role in the architecture and function of neuronal circuits in the brain. Emerging evidence suggests that dysfunction of synaptic proteins contributes to a variety of neurological and psychiatric disorders. Therefore, it is crucial to determine the molecular networks within synapses in various neuronal cell types to gain a deeper understanding of how the nervous system regulates brain function. Recent advances in synaptic proteome approaches, such as fluorescence-activated synaptosome sorting (FASS) and proximity labeling, have allowed for a detailed and spatial analysis of many cell-type-specific synaptic molecules in vivo. In this brief review, we highlight these novel spatial proteomic approaches and discuss the regulation of synaptic formation and function in the brain. This knowledge of molecular networks provides new insight into the understanding of many neurological and psychiatric disorders.

Astrocyte-secreted neurocan controls inhibitory synapse formation and function.

Astrocytes strongly promote the formation and maturation of synapses by secreted proteins. Several astrocyte-secreted synaptogenic proteins controlling excitatory synapse development were identified; however, those that induce inhibitory synaptogenesis remain elusive. Here, we identify neurocan as an astrocyte-secreted inhibitory synaptogenic protein. After secretion from astrocytes, neurocan is cleaved into N- and C-terminal fragments. We found that these fragments have distinct localizations in the extracellular matrix. The neurocan C-terminal fragment localizes to synapses and controls cortical inhibitory synapse formation and function. Neurocan knockout mice lacking the whole protein or only its C-terminal synaptogenic domain have reduced inhibitory synapse numbers and function. Through super-resolution microscopy, in vivo proximity labeling by secreted TurboID, and astrocyte-specific rescue approaches, we discovered that the synaptogenic domain of neurocan localizes to somatostatin-positive inhibitory synapses and strongly regulates their formation. Together, our results unveil a mechanism through which astrocytes control circuit-specific inhibitory synapse development in the mammalian brain.

eCRUIS captures RNA-protein interaction in vitro and in vivo.

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in various ways. From its synthesis to degradation, RNA is associated with a range of RNA-binding proteins. Therefore, it is necessary to develop innovative methods to study the interaction between RNA and proteins. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The current version allows us to rapidly label RNA-binding proteins on the target RNA within 30 minutes, potentially for in vivo use. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a broader range of in vitro and in vivo applications for studying RNA-protein interactions.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs.

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Study on the interaction protein of transcription factor Smad3 based on TurboID proximity labeling technology.

TurboID is a highly efficient biotin-labelling enzyme, which can be used to explore a number of new intercalating proteins due to the very transient binding and catalytic functions of many proteins. TGF-ß/Smad3 signaling pathway is involved in many diseases, especially in diabetic nephropathy and inflammation. In this paper, a stably cell line transfected with Smad3 were constructed by using lentiviral infection. To further investigate the function of TGF-ß/Smad3, the protein labeling experiment was conducted to find the interacting protein with Smad3 gene. Label-free mass spectrometry analysis was performed to obtain 491 interacting proteins, and the interacting protein hnRNPM was selected for IP and immunofluorescence verification, and it was verified that the Smad3 gene had a certain promoting effect on the expression of hnRNPM gene, and then had an inhibitory effect on IL-6. It lays a foundation for further study of the function of Smad3 gene and its involved regulatory network.

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

Interactions of drosophila cryptochrome.

In this study, we investigate the intricate regulatory mechanisms underlying the circadian clock in Drosophila, focusing on the light-induced conformational changes in the cryptochrome (DmCry). Upon light exposure, DmCry undergoes conformational changes that prompt its binding to Timeless and Jetlag proteins, initiating a cascade crucial for the starting of a new circadian cycle. DmCry is subsequently degraded, contributing to the desensitization of the resetting mechanism. The transient and short-lived nature of DmCry protein-protein interactions (PPIs), leading to DmCry degradation within an hour of light exposure, presents a challenge for comprehensive exploration. To address this, we employed proximity-dependent biotinylation techniques, combining engineered BioID (TurboID) and APEX (APEX2) enzymes with mass spectrometry. This approach enabled the identification of the in vitro DmCry interactome in Drosophila S2 cells, uncovering several novel PPIs associated with DmCry. Validation of these interactions through a novel co-immunoprecipitation technique enhances the reliability of our findings. Importantly, our study suggests the potential of this method to reveal additional circadian clock- or magnetic field-dependent PPIs involving DmCry. This exploration of the DmCry interactome not only advances our understanding of circadian clock regulation but also establishes a versatile framework for future investigations into light- and time-dependent protein interactions in Drosophila.

Spatial proteomics of ER tubules reveals CLMN, an ER-actin tether at focal adhesions that promotes cell migration.

The endoplasmic reticulum (ER) is structurally and functionally diverse, yet how its functions are organized within morphological subdomains is incompletely understood. Utilizing TurboID-based proximity labeling and CRISPR knock-in technologies, here we map the proteomic landscape of the human ER and nuclear envelope. Spatial proteomics reveals enrichments of proteins into ER tubules, sheets, and nuclear envelope. We uncover an ER-enriched actin-binding protein, Calmin (CLMN), and define it as an ER-actin tether that localizes to focal adhesions adjacent to ER tubules. CLMN depletion perturbs focal adhesion disassembly, actin dynamics, and cell movement. Mechanistically, CLMN-depleted cells also exhibit defects in calcium signaling near ER-actin interfaces, suggesting CLMN promotes calcium signaling near adhesions to facilitate their disassembly. Collectively, we map the sub-organelle proteome landscape of the ER, identify CLMN as an ER-actin tether, and describe a non-canonical mechanism by which ER tubules engage actin to regulate cell migration.

Proximity labeling reveals dynamic changes in the SQSTM1 protein network.

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network, and these interactions are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA and KIR. Technological advances in cell biology continue to expand our knowledge of the SQSTM1 protein network and of the relationship of the actions of the SQSTM1 protein network in cellular physiology and disease states. Here we apply proximity profile labeling to investigate the SQSTM1 protein interaction network by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners, and capture of novel SQSTM1 protein interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. We demonstrate the importance of the PB1 and/or UBA domains for binding network members, including the K18 domain of tau. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

Streamlined Biotinylation, Enrichment and Analysis for Enhanced Plasma Membrane Protein Identification Using TurboID and TurboID-Start Biotin Ligases.

Plasma membrane proteins (PMPs) play pivotal roles in various cellular events and are crucial in disease pathogenesis, making their comprehensive characterization vital for biomedical research. However, the hydrophobic nature and low expression levels of PMPs pose challenges for conventional enrichment methods, hindering their identification and functional profiling. In this study, we presented a novel TurboID-based enrichment approach for PMPs that helped overcoming some of the existing limitations. We evaluated the efficacy of TurboID and its modified form, TurboID-START, in PMP enrichment, achieving efficient and targeted labelling of PMPs without the need for stable cell line generation. This approach resulted reduction in non-specific biotinylation events, leading to improved PMP enrichment and enabled assessment of the subcellular proteome associated with the plasma membrane. Our findings paved the way for studies targeting the dynamic nature of the plasma membrane proteome and aiming to capture transient associations of proteins with the plasma membrane. The novel TurboID-based enrichment approach presented here offers promising prospects for in-depth investigations into PMPs and their roles in cellular processes.

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics.

The nucleus controls cell growth and division through coordinated interactions between nuclear proteins and chromatin. Mutations that impair nuclear protein association with chromatin are implicated in numerous diseases. Covalent ligands are a promising strategy to pharmacologically target nuclear proteins, such as transcription factors, which lack ordered small-molecule binding pockets. To identify nuclear cysteines that are susceptible to covalent liganding, we couple proximity labeling (PL), using a histone H3.3-TurboID (His-TID) construct, with chemoproteomics. Using covalent scout fragments, KB02 and KB05, we identified ligandable cysteines on proteins involved in spindle assembly, DNA repair, and transcriptional regulation, such as Cys101 of histone acetyltransferase 1 (HAT1). Furthermore, we show that covalent fragments can affect the abundance, localization, and chromatin association of nuclear proteins. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and chromatin association upon KB02 modification at Cys106. Together, this platform provides insights into targeting nuclear cysteines with covalent ligands.

Highly efficient identification of nucleocytoplasmic O-glycosylation by the TurboID-based proximity labeling method in living cells.

Glycosylation is a ubiquitous posttranslational modification and plays an important role in many processes, such as protein stability, folding, processing, and trafficking. Among glycosylation types, O-glycosylation is difficult to analyze due to the complex glycan composition, low abundance and lack of glycosidases to remove the O-glycans. Many methods have been applied to analyze the O-glycosylation of membrane glycoproteins and secreted glycoproteins since the synthesis of O-glycosylation occurred in the Golgi apparatus. In recent years, some O-glycosylation has been reported in the nucleus. In this work, we present a proximity labeling strategy based on TurboID by combining core 1 ß1-3 galactosyltransferase (C1GalT1), which has been reported in the nucleus, to characterize nucleocytoplasmic O-glycosylation in living HeLa cells. The O-glycosylated protein C1GalT1 was biotinylated by the proximity labeling method in living HeLa cells overexpressing C1GalT1 fused by TurboID and enriched by streptavidin-coated beads. Following digestion with trypsin and mass spectrometry analysis, 68 high-confidence and 298 putative O-glycosylated sites were identified on 366 peptides mapped to 267 proteins. These results indicated that the proximity labeling method is a highly efficient technique to identify O-glycosylation. Furthermore, the finding of abundant O-glycosylation from nucleocytoplasmic proteins indicates a new pathway of O-glycosylation synthesis in cells.