Статті в журналах: "TurboID"

1

Cho, Kelvin F., Tess C. Branon, Sanjana Rajeev, Tanya Svinkina, Namrata D. Udeshi, Themis Thoudam, Chulhwan Kwak, et al. "Split-TurboID enables contact-dependent proximity labeling in cells." Proceedings of the National Academy of Sciences 117, no. 22 (May 18, 2020): 12143–54. http://dx.doi.org/10.1073/pnas.1919528117.

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Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein–protein interaction or membrane–membrane apposition. At endoplasmic reticulum–mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum–mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.

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2

Cho, Kelvin F., Tess C. Branon, Namrata D. Udeshi, Samuel A. Myers, Steven A. Carr, and Alice Y. Ting. "Proximity labeling in mammalian cells with TurboID and split-TurboID." Nature Protocols 15, no. 12 (November 2, 2020): 3971–99. http://dx.doi.org/10.1038/s41596-020-0399-0.

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3

May, Danielle G., Kelsey L. Scott, Alexandre R. Campos, and Kyle J. Roux. "Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation." Cells 9, no. 5 (April 25, 2020): 1070. http://dx.doi.org/10.3390/cells9051070.

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BioID is a well-established method for identifying protein–protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15–18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.

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4

Doerr, Allison. "Proximity labeling with TurboID." Nature Methods 15, no. 10 (October 2018): 764. http://dx.doi.org/10.1038/s41592-018-0158-0.

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5

Garloff, Vera, and Ignacio Rubio. "Schneller, weiter, TurboID – Modulation einer übereifrigen Biotin-Ligase." BIOspektrum 29, no. 3 (May 2023): 273–75. http://dx.doi.org/10.1007/s12268-023-1943-6.

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AbstractProtein-protein interactions are key elements of intracellular signalling and metabolic pathways. These interactions can be revealed with the help of proximity ligation screens, prominently biotinylation screens. This approach has profited from the recent development of the highly active biotin ligase TurboID, which however also led to problems of toxicity related to its high basal activity. We have established a simple protocol to improve TurboID performance and enhance protein functionality.

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6

Makhsatova, S. A., A. B. Kurmanbay, I. A. Akhmetollayev, and A. T. Kulyyassov. "ASSEMBLING THE TURBOID-CONTAINING PLASMID CONSTRUCT FOR INVESTIGATING THE IN VIVO PROTEIN-PROTEIN INTERACTIONS." Eurasian Journal of Applied Biotechnology, no. 3S (September 12, 2024): 47. http://dx.doi.org/10.11134/btp.3s.2024.35.

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In vivo interactions between biomolecules (Proteins, RNA, and DNA) are the basis of cellular functionality including cell cycle, signaling pathways, cellular metabolism, and other biological processes. The traditional methods for detecting protein-protein interactions, such as affinity purification and two-hybrid analysis have limitations for the in-depth study of the cellular proteome. Besides, proteomics of the organelle protein components is still challenging to study, due to the spatial and temporal dynamics of proteins. To address these problems, proximity labeling technology was introduced. This technology not only surpasses all the limitations of traditional methods but also has unique advantages in the qualitative and quantitative analysis of the proteome of living cells. Proximity labeling is one of the most commonly used methods for detecting the functional features and protein composition of target proteins and neighboring ones, through biotin labeling (biotinylation). Biotin is employed due to its high affinity to streptavidin and avidin, which is an efficient procedure to purify and isolate fractions containing biotinylated proteins for further analysis. During the in vivo biotinylation study, we used the mutant biotin ligase TurboID which is the modified enzyme of BirA. BirA is an enzyme found in E. coli bacteria, capable of catalyzing the attachment of biotin to specific lysine residues on a single cellular protein. The development of TurboID involved introducing specific mutations into the BirA sequence to improve its performance. These mutations result in the inability of the TurboID biotin ligase to maintain biotinyl-5'-adenylate in its active form, causing its release from the active center into the surrounding environment. This released substance contains a reactive mixed anhydride bond, enabling it to readily modify lysine residues of nearby proteins within 10nm in the cell. So, the advantage of TurboID-X (X-any protein of interest (POI)) is that it has a high efficiency for in vivo proximity labeling. We use the PTF (pluripotency transcription factors) SOX2, OCT4, and NANOG for X as model systems. In our study, we used genetic engineering methods to obtain recombinant plasmid DNA containing the nucleotide sequence of TurboID and fused protein of interest X. DNA plasmid constructs have next key structural elements: Kozak sequence at the beginning of the fragment, His-Tag, and diglycine sequence at the end, BglII and XhoI restriction sites respectively to replace the wild-type biotin ligase BirA by TurboID. Two rounds of PCR amplification were performed, the first one using terminal primers that amplify only the TurboID ORF. The resulting amplicon of TurboID was applied as a matrix in the second PCR round by using long primers that contain the structural elements mentioned above. Expression of recombinant proteins from the resulting plasmid constructs will be demonstrated in HEK293T (Human embryonic kidney) cells using transient transfection with calcium phosphate method or Lipofectamine 2000. In conclusion, further research will consist of affinity purification, detection of labeled proteins by Western blotting, and their identification by the LC-MS/MS. We hope that our research work will help us to better understand the mechanisms of the early stages of the reprogramming process.

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7

Takano, Tetsuya. "Comprehensive identification of molecules at synapses and non-synaptic cell-adhesion structure." Impact 2023, no. 3 (September 21, 2023): 46–48. http://dx.doi.org/10.21820/23987073.2023.3.46.

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The brain is incredibly complex and there is so much we don't know about this organ and its mechanisms. Assistant Professor Tetsuya Takano, School of Medicine, Keio University, Japan, is working to better understand neuroscience. One area of interest is neurons and astrocytes; specifically elucidating the protein component functions in each neural circuit. He and his team are working to shed light on the pathological mechanism of psychiatric and neurological disorders and, in doing so, enabling improved treatments and benefiting patients across the globe. The team has developed spatio-temporal proteome technologies: TurboID-surface and Split-TurboID, that can not only explain the formation and operation principle of neural networks, but also provide essential knowledge for research into psychiatric and neurological diseases. To overcome limitations associated with conventional proteome analysis, Takano and the team recently developed a new in vivo proximal-dependent biotin labelling (BioID) method. Using this, the researchers can label and analyse adjacent proteins with biotin, which enables them to comprehensively analyse local protein components within cells with extremely high spatial resolution. The team has used the BioID method to develop the Split-TurboID method and an innovative spatial proteome technique for searching for molecular groups among heterogeneous cells that makes it possible to comprehensively analyse the protein components in the vicinity of the adhesion site. Using the Split-TurboID method, the team has comprehensively searched for functional molecules between astrocytes and neurons and revealed that astrocytes directly control the formation of inhibitory synapses and neuronal activity in neurons via a novel tripartite synaptic molecule known as NRCAM.

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8

Rabinovich-Ernst, Orna, Clinton Bradfield, SungHwan Yoon, Anthony Armstrong, Samuel Katz, Aleksandra Nita-Lazar, and Iain Fraser. "TurboID biotin-tagging mass spectrometry identifies specific caspase-11-associated proteins regulating non-canonical inflammasome activation." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 15.06. http://dx.doi.org/10.4049/jimmunol.206.supp.15.06.

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Abstract While it has been demonstrated that cytosolic LPS can directly activate caspase11, the cellular processes regulating the non-canonical inflammasome response remain poorly defined. Caspase11 and caspase1 show substantial structural similarity, however, unlike the activation of caspase1 by NLR inflammasomes, there are no sensor or adaptor proteins known to be involved in transmitting cytosolic LPS signal to caspase11. Also, while caspase11 has been shown to associate with LPS, it lacks a characteristic LPS binding domain as observed in many other LPS binding proteins such as MD2 and LBP. Thus, we hypothesized that other effectors may be required to facilitate cytosolic LPS recognition. Moreover, the pathway is likely to be tightly regulated as caspase11 activation leads to highly inflammatory cell death. To identify novel regulators of caspase11, we generated immortalized macrophages expressing a caspase11-TurboID-DHFR chimeric protein. The destabilizing domain was included to avoid cell death induced by caspase11 over-expression. We used a TurboID biotin-tagging MS assay to detect proteins in close proximity to caspase11 pre and post cytosolic LPS introduction. Importantly, the TurboID assay permits recognition of transient interactions, typically missed by traditional IP. To validate relevance of putative hits, we used siRNA knockdown in BMDM. We’ve identified novel regulators specific for cytosolic LPS triggering. Several proteins interact with caspase11 only in the resting state, suggesting negative regulation to prevent pyroptosis. Among the identified regulators are kinases and proteins with pyrin and LRR domains, both common NLR features. This work was supported by the Intramural Research Program of NIAID, NIH.

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9

Kim, Han Byeol, and Kwang-eun Kim. "Precision proteomics with TurboID: mapping the suborganelle landscape." Korean Journal of Physiology & Pharmacology 28, no. 6 (November 1, 2024): 495–501. http://dx.doi.org/10.4196/kjpp.2024.28.6.495.

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10

Gurung, Sadeechya. "Abstract 998: Extracellular proximity labeling (ePL) as a tool to identify protein-protein interactions in the tumor microenvironment." Cancer Research 82, no. 12_Supplement (June 15, 2022): 998. http://dx.doi.org/10.1158/1538-7445.am2022-998.

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Abstract The extracellular matrix (ECM) is a dynamic niche that is extensively reshaped in the development of the tumor microenvironment (TME). Our current understanding of ECM function and dynamics is largely informed by identification of protein-protein interactions (PPIs) using co-immunoprecipitation (co-IP) techniques that may miss transient and weak/unstable interactions. Recent advances in proximity labeling techniques have greatly expanded the interactome networks of numerous intracellular proteins, however these tools have yet to be extended to study PPIs in the ECM. We have recently optimized a systematic approach to identify PPIs in the ECM using fusion constructs of the biotinylating enzymes, BioID2 and TurboID, with the widely expressed matrix regulator TIMP2. BioID2 and TurboID offer differing reaction kinetics that may provide complimentary information on extracellular PPIs (ePPIs). Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) are crucial regulators of ECM structure and composition. TIMPs are widely expressed multifunctional proteins that serve to promote ECM homeostasis that is often perturbed in many cancers and chronic disorders. Although biochemical data suggests that TIMPs are promiscuous proteins, the TIMP interactome is poorly defined. We have optimized a protocol for the identification of ePPIs for the TIMP family of proteins. Fusion constructs equipped with a promiscuous biotin ligase (BioID2/TurboID) fused to the N- or C-terminal of full length TIMP2 were packaged into retroviral vectors for cellular delivery. Cells were exposed to the extracellular proximity labelling (ePL) fusion proteins and processed in our optimized analysis pipeline. ePPIs were identified via streptavidin pulldown and proteomic techniques. We present our optimized ePL pipeline and show that this technique is an effective tool for the identification of novel ePPIs for multiple extracellular targets. Citation Format: Sadeechya Gurung. Extracellular proximity labeling (ePL) as a tool to identify protein-protein interactions in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 998.

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11

Teplova, Anastasia D., Marina V. Serebryakova, Raisa A. Galiullina, Nina V. Chichkova, and Andrey B. Vartapetian. "Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach." International Journal of Molecular Sciences 22, no. 23 (December 4, 2021): 13123. http://dx.doi.org/10.3390/ijms222313123.

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Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.

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12

Holzer, Elisabeth, Cornelia Rumpf-Kienzl, Sebastian Falk, and Alexander Dammermann. "A modified TurboID approach identifies tissue-specific centriolar components in C. elegans." PLOS Genetics 18, no. 4 (April 20, 2022): e1010150. http://dx.doi.org/10.1371/journal.pgen.1010150.

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Proximity-dependent labeling approaches such as BioID have been a great boon to studies of protein-protein interactions in the context of cytoskeletal structures such as centrosomes which are poorly amenable to traditional biochemical approaches like immunoprecipitation and tandem affinity purification. Yet, these methods have so far not been applied extensively to invertebrate experimental models such as C. elegans given the long labeling times required for the original promiscuous biotin ligase variant BirA*. Here, we show that the recently developed variant TurboID successfully probes the interactomes of both stably associated (SPD-5) and dynamically localized (PLK-1) centrosomal components. We further develop an indirect proximity labeling method employing a GFP nanobody-TurboID fusion, which allows the identification of protein interactors in a tissue-specific manner in the context of the whole animal. Critically, this approach utilizes available endogenous GFP fusions, avoiding the need to generate multiple additional strains for each target protein and the potential complications associated with overexpressing the protein from transgenes. Using this method, we identify homologs of two highly conserved centriolar components, Cep97 and BLD10/Cep135, which are present in various somatic tissues of the worm. Surprisingly, neither protein is expressed in early embryos, likely explaining why these proteins have escaped attention until now. Our work expands the experimental repertoire for C. elegans and opens the door for further studies of tissue-specific variation in centrosome architecture.

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13

Branon, Tess C., Justin A. Bosch, Ariana D. Sanchez, Namrata D. Udeshi, Tanya Svinkina, Steven A. Carr, Jessica L. Feldman, Norbert Perrimon, and Alice Y. Ting. "Efficient proximity labeling in living cells and organisms with TurboID." Nature Biotechnology 36, no. 9 (October 2018): 880–87. http://dx.doi.org/10.1038/nbt.4201.

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14

Peeney, David, Sadeechya Gurung, Josh Rich, Sasha Coates-Park, Yueqin Liu, and William G. Stetler-Stevenson. "Abstract 2348: Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL)." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2348. http://dx.doi.org/10.1158/1538-7445.am2023-2348.

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Abstract Classical methods to investigate 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 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 describe a 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 matrix metalloproteinases (MMPs), the major mediators of ECM breakdown and turnover. TIMP2 is a unique family member, with a broad expression profile that is expressed in both normal and diseased tissues, even in those with minimal metalloproteinase activity. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential to the development of ECM targeted therapeutics. This knowledge may also garner understanding of therapeutic resistance and the failure of conventional and next-generation cancer therapies. Using carboxyl- and amino-terminal fusion peptides of TIMP2 with BioID2 and TurboID, we describe the TIMP2 interactome in unique tissue compartments. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell lines, and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique and comparing our findings with 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. Citation Format: David Peeney, Sadeechya Gurung, Josh Rich, Sasha Coates-Park, Yueqin Liu, William G. Stetler-Stevenson. Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2348.

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15

Artan, Murat, Stephen Barratt, Sean M. Flynn, Farida Begum, Mark Skehel, Armel Nicolas, and Mario de Bono. "Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling." Journal of Biological Chemistry 297, no. 3 (September 2021): 101094. http://dx.doi.org/10.1016/j.jbc.2021.101094.

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16

Smirnova, Evgeniya V., Tatiana V. Rakitina, Rustam H. Ziganshin, George A. Saratov, Georgij P. Arapidi, Alexey A. Belogurov, and Anna A. Kudriaeva. "Identification of Myelin Basic Protein Proximity Interactome Using TurboID Labeling Proteomics." Cells 12, no. 6 (March 20, 2023): 944. http://dx.doi.org/10.3390/cells12060944.

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Myelin basic protein (MBP) is one of the key structural elements of the myelin sheath and has autoantigenic properties in multiple sclerosis (MS). Its intracellular interaction network is still partially deconvoluted due to the unfolded structure, abnormally basic charge, and specific cellular localization. Here we used the fusion protein of MBP with TurboID, an engineered biotin ligase that uses ATP to convert biotin to reactive biotin-AMP that covalently attaches to nearby proteins, to determine MBP interactome. Despite evident benefits, the proximity labeling proteomics technique generates high background noise, especially in the case of proteins tending to semi-specific interactions. In order to recognize unique MBP partners, we additionally mapped protein interaction networks for deaminated MBP variant and cyclin-dependent kinase inhibitor 1 (p21), mimicking MBP in terms of natively unfolded state, size and basic amino acid clusters. We found that in the plasma membrane region, MBP is colocalized with adhesion proteins occludin and myelin protein zero-like protein 1, solute carrier family transporters ZIP6 and SNAT1, Eph receptors ligand Ephrin-B1, and structural components of the vesicle transport machinery—synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3), protein transport protein hSec23B and cytoplasmic dynein 1 heavy chain 1. We also detected that MBP potentially interacts with proteins involved in Fe2+ and lipid metabolism, namely, ganglioside GM2 activator protein, long-chain-fatty-acid-CoA ligase 4 (ACSL4), NADH-cytochrome b5 reductase 1 (CYB5R1) and metalloreductase STEAP3. Assuming the emerging role of ferroptosis and vesicle cargo docking in the development of autoimmune neurodegeneration, MBP may recruit and regulate the activity of these processes, thus, having a more inclusive role in the integrity of the myelin sheath.

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Fujimoto, Shintaro, Shinya Tashiro, and Yasushi Tamura. "Complementation Assay Using Fusion of Split-GFP and TurboID (CsFiND) Enables Simultaneous Visualization and Proximity Labeling of Organelle Contact Sites in Yeast." Contact 6 (January 2023): 251525642311536. http://dx.doi.org/10.1177/25152564231153621.

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Numerous studies have revealed that organelle membrane contact sites (MCSs) play important roles in diverse cellular events, including the transport of lipids and ions between connected organelles. To understand MCS functions, it is essential to uncover proteins that accumulate at MCSs. Here, we develop a complementation assay system termed CsFiND (Complementation assay using Fusion of split-GFP and TurboID) for the simultaneous visualization of MCSs and identification of MCS-localized proteins. We express the CsFiND proteins on the endoplasmic reticulum and mitochondrial outer membrane in yeast to verify the reliability of CsFiND as a tool for identifying MCS-localized proteins.

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18

Artan, Murat, Stephen Barratt, Sean M. Flynn, Farida Begum, Mark Skehel, Armel Nicolas, and Mario de Bono. "Correction: Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling." Journal of Biological Chemistry 298, no. 6 (June 2022): 102081. http://dx.doi.org/10.1016/j.jbc.2022.102081.

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19

Branon, Tess C., Justin A. Bosch, Ariana D. Sanchez, Namrata D. Udeshi, Tanya Svinkina, Steven A. Carr, Jessica L. Feldman, Norbert Perrimon, and Alice Y. Ting. "Author Correction: Efficient proximity labeling in living cells and organisms with TurboID." Nature Biotechnology 38, no. 1 (November 20, 2019): 108. http://dx.doi.org/10.1038/s41587-019-0355-0.

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20

Wang, Chenyu, and Laidong Yu. "TurboID Proximity Labeling of a Protocadherin Protein to Characterize Interacting Protein Complex." American Journal of Molecular Biology 13, no. 04 (2023): 213–26. http://dx.doi.org/10.4236/ajmb.2023.134015.

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21

Wei, Xia-fei, Shan Li, and Jie-li Hu. "A TurboID-based proximity labelling approach for identifying the DNA-binding proteins." STAR Protocols 4, no. 1 (March 2023): 102139. http://dx.doi.org/10.1016/j.xpro.2023.102139.

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22

Schaan Profes, Marcos, Araven Tiroumalechetty, Neel Patel, Stephanie S. Lauar, Simone Sidoli, and Peri T. Kurshan. "Characterization of the intracellular neurexin interactome by in vivo proximity ligation suggests its involvement in presynaptic actin assembly." PLOS Biology 22, no. 1 (January 22, 2024): e3002466. http://dx.doi.org/10.1371/journal.pbio.3002466.

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Neurexins are highly spliced transmembrane cell adhesion molecules that bind an array of partners via their extracellular domains. However, much less is known about the signaling pathways downstream of neurexin’s largely invariant intracellular domain (ICD). Caenorhabditis elegans contains a single neurexin gene that we have previously shown is required for presynaptic assembly and stabilization. To gain insight into the signaling pathways mediating neurexin’s presynaptic functions, we employed a proximity ligation method, endogenously tagging neurexin’s intracellular domain with the promiscuous biotin ligase TurboID, allowing us to isolate adjacent biotinylated proteins by streptavidin pull-down and mass spectrometry. We compared our experimental strain to a control strain in which neurexin, endogenously tagged with TurboID, was dispersed from presynaptic active zones by the deletion of its C-terminal PDZ-binding motif. Selection of this control strain, which differs from the experimental strain only in its synaptic localization, was critical to identifying interactions specifically occurring at synapses. Using this approach, we identified both known and novel intracellular interactors of neurexin, including active zone scaffolds, actin-binding proteins (including almost every member of the Arp2/3 complex), signaling molecules, and mediators of RNA trafficking, protein synthesis and degradation, among others. Characterization of mutants for candidate neurexin interactors revealed that they recapitulate aspects of the nrx-1(-) mutant phenotype, suggesting they may be involved in neurexin signaling. Finally, to investigate a possible role for neurexin in local actin assembly, we endogenously tagged its intracellular domain with actin depolymerizing and sequestering peptides (DeActs) and found that this led to defects in active zone assembly. Together, these results suggest neurexin’s intracellular domain may be involved in presynaptic actin-assembly, and furthermore highlight a novel approach to achieving high specificity for in vivo proteomics experiments.

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Kanzler, Charlotte R., Michael Donohue, Megan E. Dowdle, and Michael D. Sheets. "TurboID functions as an efficient biotin ligase for BioID applications in Xenopus embryos." Developmental Biology 492 (December 2022): 133–38. http://dx.doi.org/10.1016/j.ydbio.2022.10.005.

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24

Holzer, Elisabeth, Cornelia Rumpf-Kienzl, Sebastian Falk, and Alexander Dammermann. "Correction: A modified TurboID approach identifies tissue-specific centriolar components in C. elegans." PLOS Genetics 19, no. 2 (February 13, 2023): e1010645. http://dx.doi.org/10.1371/journal.pgen.1010645.

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25

Larochelle, Marc, Danny Bergeron, Bruno Arcand, and François Bachand. "Proximity-dependent biotinylation mediated by TurboID to identify protein–protein interaction networks in yeast." Journal of Cell Science 132, no. 11 (May 7, 2019): jcs232249. http://dx.doi.org/10.1242/jcs.232249.

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Gottschalk, Robert, Leah Wachsmuth, Dingyin Tao, Sandeep Rana, Tino Sanchez, Yi-Han Lin, Ganesha Rai, Juan Marugan, and Mark Henderson. "Abstract 2657: SNAP-TurboID: A Proximity-based Intracellular Tool for Small Molecule Target Identification." Journal of Biological Chemistry 299, no. 3 (2023): S156. http://dx.doi.org/10.1016/j.jbc.2023.103345.

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Nascari, David, Ryan Eghlimi, Angad Beniwal, Drake Alton, John Fryer, and Nhan L. Tran. "Abstract 5562: Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology." Cancer Research 84, no. 6_Supplement (March 22, 2024): 5562. http://dx.doi.org/10.1158/1538-7445.am2024-5562.

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Abstract Glioblastoma (GBM) and Alzheimer’s disease (AD) are two devastating central nervous system diagnoses with no known cure. Both diseases are associated with advanced age. The majority of GBM tumors form in the frontal and temporal lobes, two of the brain regions most impacted by amyloid and tau pathology in AD. Females have a higher risk of AD, while males have a higher risk of GBM. Several large case series have established epidemiological evidence that the two diseases are inversely correlated. Nevertheless, it remains unknown whether presence of subclinical amyloid or tau pathology antagonizes the establishment or progression of GBM. The present study sought to characterize GBM growth, progression, and immune response in the setting of concomitant AD pathology using the syngeneic murine GL261 tumor model. To further understand the tumor cell changes that occur in the presence or absence of AD pathology, we engineered GL261 with a dual-tagged vector consisting of RiboTag (to allow for full-length mRNA profiling) and TurboID (to allow for proteomic profiling). GL261 cells were injected intracranially into APPNL-G-F/NL-G-F - MAPThuman/human, APPNL-G-F/+ - MAPThuman/+, and wildtype C57BL/6J mice. Tumors in both wildtype and AD conditions contained ~50% intratumoral myeloid cells (Iba+, P2RY12-). Differential activation of intratumoral myeloid cells will be assessed with immunohistochemistry. Microgliosis (IBA1, P2RY12) and astrogliosis (GFAP) around the tumor in the setting of Alzheimer’s pathology versus normal conditions will be compared. Invasiveness, proliferation, DNA damage (pH2AX), and apoptosis (cleaved PARP, cleaved Caspase 3) will be compared. Tumor cell-specific changes unique to tumors in the AD microenvironment will be assessed transcriptionally with RiboTag profiling as well as proteomically with TurboID purifications. Our preliminary data suggests that the presence of concomitant AD pathology affects GBM growth and progression as well as the immune response to the tumor, and warrants further investigation. Citation Format: David Nascari, Ryan Eghlimi, Angad Beniwal, Drake Alton, John Fryer, Nhan L. Tran. Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5562.

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Kalkan, Batuhan, Can Ozcan, Enes Cicek, and Ceyda Acilan. "Nek2A Prevents Centrosome Clustering and Induces Cell Death in Cancer Cells Via KIF2C Interaction." JCO Global Oncology 10, Supplement_1 (July 2024): 133. http://dx.doi.org/10.1200/go-24-10800.

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PURPOSE Modern chemotherapeutics frequently suffer from selective targeting of cancer cells. In contrast to normal cells, cancer cells frequently exhibit extra centrosomes, which tend to form multipolar spindles (MPS), triggering cell death. Nevertheless, cancer cells can divide successfully by coalescing their extra centrosomes into two poles. Nek2 is a mitotic kinase regulating several mitotic processes. Our preliminary data showed that Nek2 overexpression unclusters extra centrosomes while its disruption favors centrosome clustering. Thus, revealing the molecular mechanism and targeting Nek2 in cells with extra centrosomes may be an alternative strategy for selective targeting for cancer cells. METHODS IF stainings, cell viability, apoptosis and competition assays were performed to investigate the effect of Nek2 overexpression to induce MPS. To investigate how Nek2 regulates centrosome clustering, we tested known Nek2 targets with relevant function (C-NAP1, Rootlein, Trf1, Hec1, Gas2L1) and assessed their involvement in centrosome clustering by knock out or siRNA mediated silencing. Next, we identified the interactome of Nek2 using TurboID proximity labelling and validated our results with co-IP. RESULTS Cells harboring supernumerary centrosomes were depleted from the population upon Nek2 overexpression. Known Nek2 targets exhibited no effect on centrosome clustering. Furthermore, the effect of Nek2 overexpression on MPS was additive with other known unclustering factors such as HSET, suggesting an independent mechanism. We identified several potential targets of Nek2 using TurboID proximity labelling. NuMA - also previously known to uncluster centrosomes, was significantly enriched in our samples. Interestingly, while NuMA did not co-IP with Nek2, its knockdown reverted the unclustering activity. Another significantly enriched motor protein, Kif2C not only reverted Nek2 activity, but also co-IP'ed with Nek2. Moreover, we identified MAPRE3 a regulator of microtubule dynamics, as interaction partners of both Nek2 and Kif2C. Our data suggests that Nek2 partners with Kif2C and MAPRE3, regulating centrosome clustering. CONCLUSION We assigned a novel function for Nek2 in centrosome clustering. We are currently elucidating the detailed mechanism of action for how target proteins interact with Nek2 regulating its centrosome unclustering activity in cancer cells. Understanding the mechanism will provide new translational approaches for cancer-specific treatment.

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Li, Haorong, Ashley M. Frankenfield, Ryan Houston, Shiori Sekine, and Ling Hao. "Thiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics." Journal of the American Society for Mass Spectrometry 32, no. 9 (April 28, 2021): 2358–65. http://dx.doi.org/10.1021/jasms.1c00079.

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Yan, Biao, Ting Zeng, Xiaoshan Liu, Yuanyuan Guo, Hongguang Chen, Shuang Guo, and Wu Liu. "Study on the interaction protein of transcription factor Smad3 based on TurboID proximity labeling technology." Genomics 116, no. 3 (May 2024): 110839. http://dx.doi.org/10.1016/j.ygeno.2024.110839.

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Chevalier, Benoît, Nesrine Baatallah, Matthieu Najm, Solène Castanier, Vincent Jung, Iwona Pranke, Anita Golec, et al. "Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters." International Journal of Molecular Sciences 23, no. 16 (August 11, 2022): 8937. http://dx.doi.org/10.3390/ijms23168937.

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Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.

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Ciesla, Jessica, Kai-Lieh Huang, Eric J. Wagner, and Joshua Munger. "A UL26-PIAS1 complex antagonizes anti-viral gene expression during Human Cytomegalovirus infection." PLOS Pathogens 20, no. 5 (May 20, 2024): e1012058. http://dx.doi.org/10.1371/journal.ppat.1012058.

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Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

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Shioya, Ryouhei, Kohdai Yamada, Kohki Kido, Hirotaka Takahashi, Akira Nozawa, Hidetaka Kosako, and Tatsuya Sawasaki. "A simple method for labeling proteins and antibodies with biotin using the proximity biotinylation enzyme TurboID." Biochemical and Biophysical Research Communications 592 (February 2022): 54–59. http://dx.doi.org/10.1016/j.bbrc.2021.12.109.

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Hu, Yaofang, Changsheng Jiang, Yueqiao Zhao, Hua Cao, Jingping Ren, Wei Zeng, Mengjia Zhang, Yongtao Li, Qigai He, and Wentao Li. "TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages." Veterinary Research 54, no. 1 (July 20, 2023). http://dx.doi.org/10.1186/s13567-023-01194-6.

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AbstractActinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

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Wang, Bo, Fan Yang, Wuqian Wang, Fei Zhao, and Xiaofang Sun. "TurboID-mediated proximity labeling technologies to identify virus co-receptors." Frontiers in Cellular and Infection Microbiology 14 (June 27, 2024). http://dx.doi.org/10.3389/fcimb.2024.1371837.

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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.

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Mair, Andrea, Shou-Ling Xu, Tess C. Branon, Alice Y. Ting, and Dominique C. Bergmann. "Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID." eLife 8 (September 19, 2019). http://dx.doi.org/10.7554/elife.47864.

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Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

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Shafraz, Omer, Carolyn Marie Orduno Davis, and Sanjeevi Sivasankar. "Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions." Journal of Cell Science, September 27, 2023. http://dx.doi.org/10.1242/jcs.261430.

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Proximity labeling with genetically encoded enzymes are widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called Light Activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID, and initiate biotinylation. Turning off the light dissociates CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin binding partners with higher accuracy and significantly fewer false positives compared to TurboID.

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Kushner, Jared S., Aaron Rodriques, Sergey Zakharov, Alexander Katchman, STAVROS FANOURAKIS, and Steven Marx. "Abstract 12045: Mapping the CaV1.2 Interactome in Rat Heart in vivo." Circulation 146, Suppl_1 (November 8, 2022). http://dx.doi.org/10.1161/circ.146.suppl_1.12045.

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Introduction: The Ca2+ channel CaV1.2 is an essential part of excitation contraction coupling, neurotransmission and vascular tone. Identifying protein partners of CaV1.2 is critical to make mechanistic insights into these fundamental processes. Prior CaV1.2 proximity proteomes used APEX labeling, which labels other amino acids, introduces oxidative stress and is catalyzed ex vivo. Hypothesis: α1C-TurboID knockin rats can generate tissue specific interactomes in vivo, in cells with intact cell and matrix contacts, which may differ substantially from interactomes made from ex vivo peroxidase catalyzed labeling. Methods: CRISPR/Cas9 homology directed repair (HDR) was applied to rat spermatogonial stem cells with a targeting vector that includes cacna1c exon 44, coding the C-terminus of CaV1.2, and birA*, which codes TurboID, cloned 3’ to the stop codon (Fig A). Figures were made with Biorender. Results: Streptavidin blotting of brain and heart lysates demonstrated robust biotinylation in knockin rats (Fig B). In paced heart myocytes, α1C-TurboID produces Ca2+ transients and responds appropriately to adrenergic stimulation (Fig C), indicating proper localization to the sarcolemma. Expression of α1C-TurboID does not alter systolic function (Fig D). Mass spectrometry of heart lysates affinity purified on streptavidin beads identified 629 proteins, of which 454 were significantly enriched in knockin rats. These include known sarcolemmal proteins and interactors of CaV1.2 (Fig E), which was confirmed by antibody detection (Fig F). 73% of these 454 proteins were not significantly enriched in the APEX-α1C proteome. Conclusion: TurboID fusion to CaV1.2 generates a reliable and substantially different proteome compared to other techniques. Successful in vivo characterization of the channel interactome confirms α1C-TurboID rats will be a powerful tool for identifying changing protein networks across developmental stages, tissues, and disease models.

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Zhang, Bo, Yuanbing Zhang, and Ji-Long Liu. "Highly effective proximate labeling in Drosophila." G3 Genes|Genomes|Genetics 11, no. 5 (March 16, 2021). http://dx.doi.org/10.1093/g3journal/jkab077.

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Abstract The protein–protein interaction (PPI) is a basic strategy for life to operate. The analysis of PPIs in multicellular organisms is very important but extremely challenging because PPIs are particularly dynamic and variable among different development stages, tissues, cells, and even organelles. Therefore, understanding PPI needs a good resolution of time and space. More importantly, understanding in vivo PPI needs to be realized in situ. Proximity-based biotinylation combined with mass spectrometry (MS) has emerged as a powerful approach to study PPI networks and protein subcellular compartmentation. TurboID, the newly engineered promiscuous ligase, has been reported to label proximate proteins effectively in various species. In Drosophila, we systematically apply TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and demonstrate the feasibility of TurboID-mediated labeling system in desired cell types. For a proof-of-principle, we use the TurboID-mediated biotinylation coupled with MS to distinguish CTP synthase with or without the ability to form filamentous cytoophidia, retrieving two distinct sets of proximate proteomes. Therefore, this makes it possible to map PPIs in vivo and in situ at a defined spatiotemporal resolution, and demonstrates a referable resource for cytoophidium proteome in Drosophila.

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Su, Yanting, Yuanyuan Guo, Jieyu Guo, Ting Zeng, Ting Wang, and Wu Liu. "Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology." BMC Genomics 24, no. 1 (March 24, 2023). http://dx.doi.org/10.1186/s12864-023-09238-z.

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Abstract Background Protein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes. Results The FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification. Conclusion The TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.

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Szczesniak, Laura M., Caden G. Bonzerato, and Richard J. H. Wojcikiewicz. "Identification of the Bok Interactome Using Proximity Labeling." Frontiers in Cell and Developmental Biology 9 (May 31, 2021). http://dx.doi.org/10.3389/fcell.2021.689951.

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The function of the Bcl-2 family member Bok is currently enigmatic, with various disparate roles reported, including mediation of apoptosis, regulation of mitochondrial morphology, binding to inositol 1,4,5-trisphosphate receptors, and regulation of uridine metabolism. To better define the roles of Bok, we examined its interactome using TurboID-mediated proximity labeling in HeLa cells, in which Bok knock-out leads to mitochondrial fragmentation and Bok overexpression leads to apoptosis. Labeling with TurboID-Bok revealed that Bok was proximal to a wide array of proteins, particularly those involved in mitochondrial fission (e.g., Drp1), endoplasmic reticulum-plasma membrane junctions (e.g., Stim1), and surprisingly among the Bcl-2 family members, just Mcl-1. Comparison with TurboID-Mcl-1 and TurboID-Bak revealed that the three Bcl-2 family member interactomes were largely independent, but with some overlap that likely identifies key interactors. Interestingly, when overexpressed, Mcl-1 and Bok interact physically and functionally, in a manner that depends upon the transmembrane domain of Bok. Overall, this work shows that the Bok interactome is different from those of Mcl-1 and Bak, identifies novel proximities and potential interaction points for Bcl-2 family members, and suggests that Bok may regulate mitochondrial fission via Mcl-1 and Drp1.

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Lau, Chun Sing, Adam Dowle, Gavin H. Thomas, Philipp Girr, and Luke C. M. Mackinder. "A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii." Plant Cell, May 17, 2023. http://dx.doi.org/10.1093/plcell/koad131.

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Abstract Phase separation underpins many biologically important cellular events such as RNA metabolism, signaling and CO2 fixation. However, determining the composition of a phase-separated organelle is often challenging due to its sensitivity to environmental conditions, which limits the application of traditional proteomics techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in which proximal proteins in Chlamydomonas chloroplasts are labeled by biotin radicals generated from the TurboID-tagged protein. By fusing two core pyrenoid components with the TurboID tag, we generated a high-confidence pyrenoid proxiome that contains most known pyrenoid proteins, in addition to new pyrenoid candidates. Fluorescence protein tagging of seven previously uncharacterized TurboID-identified proteins showed that six localized to a range of sub-pyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox-sensitive iron-sulfur cluster metabolism. This developed pipeline can be used to investigate a broad range of biological processes in Chlamydomonas, especially at a temporally resolved sub-organellar resolution.

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Li, Xiaofang, Yanping Wei, Qili Fei, Guilin Fu, Yu Gan, and Chuanlin Shi. "TurboID‐mediated proximity labeling for screening interacting proteins of FIP37 in Arabidopsis." Plant Direct 7, no. 12 (December 2023). http://dx.doi.org/10.1002/pld3.555.

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AbstractProximity labeling was recently developed to detect protein–protein interactions and members of subcellular multiprotein structures in living cells. Proximity labeling is conducted by fusing an engineered enzyme with catalytic activity, such as biotin ligase, to a protein of interest (bait protein) to biotinylate adjacent proteins. The biotinylated protein can be purified by streptavidin beads, and identified by mass spectrometry (MS). TurboID is an engineered biotin ligase with high catalytic efficiency, which is used for proximity labeling. Although TurboID‐based proximity labeling technology has been successfully established in mammals, its application in plant systems is limited. Here, we report the usage of TurboID for proximity labeling of FIP37, a core member of m6A methyltransferase complex, to identify FIP37 interacting proteins in Arabidopsis thaliana. By analyzing the MS data, we found 214 proteins biotinylated by GFP‐TurboID‐FIP37 fusion, including five components of m6A methyltransferase complex that have been previously confirmed. Therefore, the identified proteins may include potential proteins directly involved in the m6A pathway or functionally related to m6A‐coupled mRNA processing due to spatial proximity. Moreover, we demonstrated the feasibility of proximity labeling technology in plant epitranscriptomics study, thereby expanding the application of this technology to more subjects of plant research.

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Yheskel, Matanel, Simone Sidoli, and Julie Secombe. "Proximity labeling reveals a new in vivo network of interactors for the histone demethylase KDM5." Epigenetics & Chromatin 16, no. 1 (February 18, 2023). http://dx.doi.org/10.1186/s13072-023-00481-y.

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Abstract Background KDM5 family proteins are multi-domain regulators of transcription that when dysregulated contribute to cancer and intellectual disability. KDM5 proteins can regulate transcription through their histone demethylase activity in addition to demethylase-independent gene regulatory functions that remain less characterized. To expand our understanding of the mechanisms that contribute to KDM5-mediated transcription regulation, we used TurboID proximity labeling to identify KDM5-interacting proteins. Results Using Drosophila melanogaster, we enriched for biotinylated proteins from KDM5-TurboID-expressing adult heads using a newly generated control for DNA-adjacent background in the form of dCas9:TurboID. Mass spectrometry analyses of biotinylated proteins identified both known and novel candidate KDM5 interactors, including members of the SWI/SNF and NURF chromatin remodeling complexes, the NSL complex, Mediator, and several insulator proteins. Conclusions Combined, our data shed new light on potential demethylase-independent activities of KDM5. In the context of KDM5 dysregulation, these interactions may play key roles in the alteration of evolutionarily conserved transcriptional programs implicated in human disorders.

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Haidar-Ahmad, Nathaline, Kyle Tomaro, Mathieu Lavallée-Adam, and François-Xavier Campbell-Valois. "The promiscuous biotin ligase TurboID reveals the proxisome of the T3SS chaperone IpgC in Shigella flexneri." mSphere, October 31, 2024. http://dx.doi.org/10.1128/msphere.00553-24.

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ABSTRACT Promiscuous biotin ligases derived from the bacterial enzyme BirA are used to identify proteins vicinal to a bait protein, thereby defining its proxisome. Despite the popularity of this approach, surprisingly little is known about its use in prokaryotes. Here, we compared the activity of four widely used promiscuous biotin ligases in the cytoplasm of Shigella flexneri , a pathogenic subgroup of Escherichia coli . Our data indicate that the kinetics of TurboID’s biotinylating activity is the highest of those tested. In addition, TurboID showed reduced interaction with the natural BirA binding partners, BccP and the biotin operator, when compared to its ancestor BioID. We therefore evaluated the ability of TurboID to probe the proxisome of the type III secretion system (T3SS) chaperone IpgC and the transcriptional activator MxiE. When the T3SS is inactive (off-state), these proteins are inhibited by forming complexes with the T3SS substrates OspD1 and IpaBC, respectively. In contrast, when the T3SS is active (on-state), OspD1 and IpaBC are secreted allowing MxiE and IpgC to interact together and activate their target genes. The results obtained with the IpgC and TurboID fusions capture a good fraction of these known interactions. It also suggests that the availability of IpgC increases in the on-state, resulting in a greater number of proteins detected in its vicinity. Among these is the T3SS ATPase SpaL (also known as Spa47 or SctN), further supporting the notion that chaperones escort their substrate to the T3SS. Interestingly, a specific subset of proteins conserved in E. coli completes the IpgC proxisome in the on-state. IMPORTANCE Promiscuous biotin ligases are widely used to study protein function in eukaryotes. Strikingly, their use in prokaryotes has been rare. Indeed, the small volume and the cytoplasmic location of the biotin ligase’s natural binding partners in these organisms pose unique challenges that can interfere with the study of the proxisome of proteins of interest. Here, we evaluated four of the most common promiscuous biotin ligases and found TurboID was best suited for use in the cytoplasm of Shigella flexneri . Using this method, we extended the proxisome of IpgC beyond its known direct binding partners involved in the regulation of the type III secretion system (T3SS) signaling cascade. Of particular interest for further study are transcription factors and housekeeping proteins that are enriched around IpgC when the T3SS is active. We propose a model in which the increased availability of IpgC in the on-state may allow cross-talk of the T3SS with other cellular processes.

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Zhang, Kaixin, Yinyin Li, Tengbo Huang, and Ziwei Li. "Potential application of TurboID-based proximity labeling in studying the protein interaction network in plant response to abiotic stress." Frontiers in Plant Science 13 (August 16, 2022). http://dx.doi.org/10.3389/fpls.2022.974598.

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Abiotic stresses are major environmental conditions that reduce plant growth, productivity and quality. Protein-protein interaction (PPI) approaches can be used to screen stress-responsive proteins and reveal the mechanisms of protein response to various abiotic stresses. Biotin-based proximity labeling (PL) is a recently developed technique to label proximal proteins of a target protein. TurboID, a biotin ligase produced by directed evolution, has the advantages of non-toxicity, time-saving and high catalytic efficiency compared to other classic protein-labeling enzymes. TurboID-based PL has been successfully applied in animal, microorganism and plant systems, particularly to screen transient or weak protein interactions, and detect spatially or temporally restricted local proteomes in living cells. This review concludes classic PPI approaches in plant response to abiotic stresses and their limitations for identifying complex network of regulatory proteins of plant abiotic stresses, and introduces the working mechanism of TurboID-based PL, as well as its feasibility and advantages in plant abiotic stress research. We hope the information summarized in this article can serve as technical references for further understanding the regulation of plant adaptation to abiotic stress at the protein level.

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Zhang, Qianshen, Zhiyan Wen, Xin Zhang, Jiajie She, Xiaoling Wang, Zongyu Gao, Ruiqi Wang, et al. "RETICULON-LIKE PROTEIN B2 is a pro-viral factor co-opted for the biogenesis of viral replication organelles in plants." Plant Cell, May 22, 2023. http://dx.doi.org/10.1093/plcell/koad146.

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Abstract Endomembrane remodeling to form a viral replication complex (VRC) is crucial for a virus to establish infection in a host. Although the composition and function of VRCs have been intensively studied, host factors involved in the assembly of VRCs for plant RNA viruses have not been fully explored. TurboID-based proximity labeling (PL) has emerged as a robust tool for probing molecular interactions in planta. However, few studies have employed the TurboID-based PL technique for investigating plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum (ER)-replicating virus, as a model, and systematically investigated the composition of BBSV VRCs in Nicotiana benthamiana by fusing the TurboID enzyme to viral replication protein p23. Among the 185 identified p23-proximal proteins, the reticulon family of proteins showed high reproducibility in the mass spectrometry datasets. We focused on RETICULON-LIKE PROTEIN B2 (RTNLB2) and demonstrated its pro-viral functions in BBSV replication. We showed that RTNLB2 binds to p23, induces ER membrane curvature, and constricts ER tubules to facilitate the assembly of BBSV VRCs. Our comprehensive proximal interactome analysis of BBSV VRCs provides a resource for understanding plant viral replication and offers additional insights into the formation of membrane scaffolds for viral RNA synthesis.

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Park, Sohyeon, Xiaorong Wang, Yajin Mo, Sicheng Zhang, Xiangpeng Li, Katie C. Fong, Clinton Yu, et al. "Proximity Labeling Expansion Microscopy (PL-ExM) Evaluates Interactome Labeling Techniques." Journal of Materials Chemistry B, 2024. http://dx.doi.org/10.1039/d4tb00516c.

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Understanding protein-protein interactions (PPIs) through proximity labeling has revolutionized our comprehension of cellular mechanisms and pathology. Various proximity labeling techniques, such as HRP, APEX, BioID, TurboID, and µMap, have been...

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Chen, Rui, Ningxia Zhang, Yubin Zhou, and Ji Jing. "Optical Sensors and Actuators for Probing Proximity-Dependent Biotinylation in Living Cells." Frontiers in Cellular Neuroscience 16 (February 16, 2022). http://dx.doi.org/10.3389/fncel.2022.801644.

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Proximity-dependent biotinylation techniques have been gaining wide applications in the systematic analysis of protein-protein interactions (PPIs) on a proteome-wide scale in living cells. The engineered biotin ligase TurboID is among the most widely adopted given its enhanced biotinylation efficiency, but it faces the background biotinylation complication that might confound proteomic data interpretation. To address this issue, we report herein a set of split TurboID variants that can be reversibly assembled by using light (designated “OptoID”), which enable optogenetic control of biotinylation based proximity labeling in living cells. OptoID could be further coupled with an engineered monomeric streptavidin that permits real-time monitoring of biotinylation with high temporal precision. These optical actuators and sensors will likely find broad applications in precise proximity proteomics and rapid detection of biotinylation in living cells.

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Kreis, Elena, Katharina König, Melissa Misir, Justus Niemeyer, Frederik Sommer, and Michael Schroda. "TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response." Plant Physiology, June 13, 2023. http://dx.doi.org/10.1093/plphys/kiad335.

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Abstract In Chlamydomonas (Chlamydomonas reinhardtii) the VESICLE-INDUCING PROTEINS IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiome can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL1-11). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

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