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1. Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions

   https://doi.org/10.1242/jcs.261430  

   Shafraz, Omer; Davis, Carolyn Marie Orduno; Sivasankar, Sanjeevi (September 2023, Journal of Cell Science)

   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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2. Uncovering the proximal proteome of CTR1 through TurboID‐mediated proximity labeling

   https://doi.org/10.1002/pmic.202300212  

   Chien, Yuan‐Chi; Reyes, Andres; Park, Hye_Lin; Xu, Shou‐Ling; Yoon, Gyeong_Mee (October 2023, PROTEOMICS)

   Abstract Protein‐protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene‐Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID‐based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 inNicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using theN. benthamianatransient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes. 

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3. Metabolic labeling and targeted modulation of adipocytes

   https://doi.org/10.1039/d4bm01352b  

   Wang, Yueji; Bo, Yang; Liu, Yusheng; Zhou, Jiadiao; Nguyen, Daniel; Baskaran, Dhyanesh; Liu, Yuan; Wang, Hua (January 2025, Biomaterials Science)

   Here, we report metabolic glycan labeling of adipocytes and targeted modulation via click chemistry, offering a novel platform to manipulate adipocyte interactions with other cells. 

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4. Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR

   https://doi.org/10.1002/chem.202403160  

   Hunter, Hannah_R; Kankati, Shashank; Hasanbasri, Zikri; Saxena, Sunil (November 2024, Chemistry – A European Journal)

   Abstract In‐cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in‐cell. However, the need to spin label proteins ex‐situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in‐cell spin labeling. We quantify the amount of Cu(II)‐NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat‐shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)‐NTA intoE. coli. Notably, over six repeats we accomplish a bulk average of 57 μM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community. 

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5. Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

   https://doi.org/10.1101/2024.10.29.620861  

   Lee, Song-Yi; Roh, Heegwang; Gonzalez-Perez, David; Mackey, Mason R; Kim, Keun-Young; Hoces, Daniel; McLaughlin, Colleen N; Adams, Stephen R; Nguyen, Khanh; Luginbuhl, David J; et al (October 2024, bioRxiv)

   Abstract Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H₂O₂. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O₂instead of toxic H₂O₂, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID. 

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