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Development of enzyme directed probes for protein-protein interaction and visualization.

Sharma, Nirmali
Kee, Jung-Min
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In the first part of this thesis, we studied peroxidase based multifunctional reactions and their applications in cellular and structural biology. Peroxidase, an enzyme containing heme, plays a crucial role in reducing hydrogen peroxide to water by transferring electrons from aromatic compounds in a stepwise turnover process. During this reaction, a variety of short-lived aromatic radicals, often referred to as "spray" molecules, can be produced. These radicals have the potential to either form covalent bonds with nearby proteins or undergo polymerization through radical-radical coupling reactions. Recent investigations have demonstrated the effectiveness of peroxidase-generated radicals as valuable tools for spatial exploration within biological systems. These applications include studies focused on visualizing the spatial distribution of proteins using electron microscopy, mapping the spatial proteome, and sensing the spatial presence of metabolites like heme and hydrogen peroxide. This review aims to promote the broader adoption of peroxidase-based techniques for advancing spatial discoveries in cellular biology. In the next part we developed a method based on combination of light and electron microscopy known as Correlative light and electron microscopy (CLEM) which is a versatile technique that leverages the strengths of fluorescence microscopy for detecting target biomolecules and understanding their biological roles, while electron microscopy provides contextual information. CLEM results validate dynamic changes in cellular pathways. However, a significant limitation in CLEM is the scarcity of methods for labeling proteins with CLEM reporters. In this study, we have developed a peroxidase-activatable fluorescent probe for signal amplification and its subsequent application in CLEM. Fluorescent tagging of biomolecules enables their sensitive detection during separation and localization within subcellular compartments. To exploit this potential, we introduced a genetically encodable enzymatic fluorescence signal amplification method using APEX(FLEX). This method involves the synthesis of a novel fluorescent probe named Jenfluor triazole (JFT1), which effectively amplifies and confines fluorescence signals under fixed conditions, allowing for fluorescence-based detection of electron-rich metabolites localized within subcellular structures. Moreover, JFT1 consistently displayed stable fluorescence signals even under osmium treatment and polymer embedding, supporting findings from correlative light and electron microscopy (CLEM) using APEX. Consequently, we successfully obtained CLEM images for various APEX-tagged proteins of interest, such as CD63-APEX2 for the multivesicular endosomal structure, APEX2-PLIN2 for lipid droplets, Sec61B-APEX2 targeting the endoplasmic reticulum lumen, and TMEM192-APEX2 targeting lysosomal bodies. Our CLEM method effectively preserved the ultrastructural morphology of these proteins of interest. Furthermore, we extended this technique to visualize dynamic changes occurring between mitochondria and lysosomes under stress conditions. For instance, upon treatment with U18666A, which enhances the retention of lysosomal cholesterol, our FLEX method revealed an increase in lysosome diameter and greater interaction between lysosomes and mitochondria, even under confocal microscopy. In this study, we paired our FLEX method with the well-known CLEM protein mito- mEosEM. Under the U18666A condition, we observed enlarged lysosomes with increased membrane contact with damaged mitochondria, shedding light on the contextual information of lysosomes under U18666A treatment. We also conducted additional experiments with Bafilomycin A1, an inhibitor of lysosomal acidification. Our FLEX approach with mito-mEosEM unveiled damaged mitochondria engulfed in large autophagic vesicles, aligning with existing experiments using Bafilomycin A1. Consequently, our combination of FLEX with mito-mEosEM offers a multicolor CLEM approach suitable for studying protein localization and organelle-organelle interactions. In conclusion, the FLEX technique holds significant promise as a sensitive and versatile system for fluorescently detecting APEX2-proteins of interest in multiscale biological samples. In the next part we developed crosslinking method for studying protein-protein interactions and their biological significance is essential, and chemical crosslinking with small molecules coupled with mass spectrometry is a potent tool. We've developed a proximity crosslinking method called "Spaclick," which utilizes a strain-promoted dibenzocyclooctyne (DBCO) moiety that can react with azidohomoalanine (AHA) residues in newly synthesized proteins. DBCO-HTL, synthesized by conjugating the DBCO moiety with a PEG HaloTag linker (DL1), has been shown to effectively capture protein-protein interactions (PPI), as confirmed using the FKBP-FRB system in the presence of rapamycin. After verifying these results, we explored Spaclick's capability to capture various PPI interactomes in different sub-compartments using HaloTag-conjugated proteins of interest in live cells. Since SpaClick and TurboID can perform orthogonal in-cell chemical reactions on proximal proteins, the mass analysis of dually crosslinked and biotinylated proteins proves to be a high-confidence method for identifying physical interactomes. This combination approach, known as "SpaClick-ID," allowed us to identify the host interactome of the SARS-CoV-2 nucleocapsid (N) protein, which is crucial for viral genome assembly. Mass analysis of the DL1-crosslinked N-HaloTag product in HEK293T cells revealed exclusive enrichment of RNA-binding proteins in stress granules in the cross-linked samples. These findings demonstrate that our method is effective in revealing the interactome of a protein of interest within a short distance in live cells.
Ulsan National Institute of Science and Technology


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