Proximity labeling methods using APEX2 have required the use of hydrogen peroxide. Now APEX2 has been shown to catalyze labeling with tetrazine-phenol probes without hydrogen peroxide, enabling spatia...

Here we introduce H2O2-independent APEX2 (Hi-APEX), which uses a clickable tetrazine-phenol (TP) probe, requiring no enzyme engineering.

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Interferon-inducible guanylate binding proteins (GBPs) are large GTPases crucial for the host immune response against Toxoplasma gondii, a globally prevalent intracellular apicomplexan parasite. Upon infection, T. gondii forms a parasitophorous vacuole (PV) that protects the parasite from host intracellular defense mechanisms. Murine GBPs (mGBPs) can recognize these PVs and assemble into supramolecular homo- and heterocomplexes, which is an essential step for PV membrane disruption, ultimately enabling cell-autonomous immune control of vacuole-resident pathogens. Our previous work has shown that mGBP2 is an important factor in the immune control of T. gondii. To further elucidate the functions of mGBP2, we employed an APEX2-based proximity labeling strategy to biotinylate proteins in the vicinity of mGBP2 during T. gondii infection. Mass spectrometry analysis of the biotinylated proteins revealed several novel PV-associated proteins such as Cyclooxygenase 2 (COX2/ Ptgs2), Caspase 12 (Cas12), Annexin 11 (Anxa11), Calnexin (Canx), Galectin 3 (Gal3), and Transgelin (Tagln) and valosin-containing protein (VCP) that co-recruit with mGBP2 at T. gondii PVs. In addition, we investigated their regulation by IFN-{gamma} and determined that inhibition of COX2 severely impairs T. gondii growth, highlighting a critical role for COX2 for parasite survival.

Read the full paper: Directed Evolution Improves the Catalytic Efficiency of APEX2‐Mediated Proximity‐Dependent RNA Labeling

A nontoxic proximity labeling approach is developed using an engineered D-amino acid oxidase and APEX2 fusion to generate H2O2 in situ from D-amino acids. It is applied in vivo in Caenorhabditis elega...

A nontoxic proximity labeling approach is developed using an engineered D-amino acid oxidase and APEX2 fusion to generate H2O2 in situ from D-amino acids. It is applied in vivo in Caenorhabditis elegans, Drosophila, zebrafish and mice and has unique RNA labeling activity to probe RNA structures in vitro and in vivo.

Wang et al. establish an in vivo proteomic labeling (IVPL) system combining the Btn-Ph-3F substrate with APEX2-EGFPf/f Cre crosses to map cell-type-specific proteomes in intact organs and trace patien...

Nature Protocols - This Protocol describes the transcriptome-wide labeling of RNAs in a particular subcellular compartment using proximity biotinylation by localized APEX2 enzyme and subsequent...

Membrane tension controls the dynamic assembly and disassembly of caveolae and their association with the F-actin cortex in migrating cells.