Research

a. Spray-PTM & Super-Resolution Proximity Labeling (SR-PL)

Proximity labeling (PL) is a powerful tool for local proteome mapping in live cells. PL's enzymatic reactions are performed in a spatially restricted manner. However, most current applications of PL use conventional mass analysis, which detects the "non-biotinylated peptides" of streptavidin bead-enriched proteins, often resulting in ambiguous and low-resolution outcomes. To address this, we developed a direct mass analysis workflow (Spot-ID) for the mass detection of "biotinylated" post-translational modification (PTM) sites for APEX (e.g., Y+331 Da) and BioID/TurboID (e.g., K+226 Da), which reflect true biotinylation events at the single amino acid residue level.

Our "super-resolution" proximity labeling (SR-PL) has been applied to reveal highly resolved proteomic information of mitochondrial proteomes (Lee SY et al. JACS, 2017; Kwak et al. PNAS, 2020) and tissue-specific mitochodnrial matrix (Park I et al. Nat. Chem. Biol. 2024) and ER/secreted proteomes (Kim KE et al. Nat. Commun. 2021). We have also demonstrated that our SR-PL method can be used for in vivo identification of drug-binding proteins in live cells (Lee SY et al. ACS Cent. Sci. , 2016; Kwak and Park C et al. Cell Chem Biol, 2022). Additionally, we developed proximal copper-click chemistry for mapping the biomolecules at cell-cell interactions (GEN-Click, Mishra et al. ACS Cent. Sci. 2023).

Our super-resolution PL approach has recently been highlighted in Kang and Rhee, ACR, 2022, and Choi and Rhee, Trends Biotechnol, 2022. In these review articles, we introduced several new terms, such as "spatial biology", "spatiome (spatially localized biomolecules)", and "spatomics (omics technology for spatiome analysis)". Based on the chemical principles of proximity labeling, we recently proposed the chemical mechanism of spray-type post-translational modifications (spray-PTM) in Lee and Rhee, Trends in Biochem Sci, 2024, and our lab is now conducting several projects in this new direction.

b.  Mitoatlas & Mitochondrial Heat Signal

So far ~1,200 different proteins are known to be targeted to the mitochondria in human cells. These proteins are not uniformly distributed throughout the mitochondria, but they are located in highly specific targeted spaces and form diverse protein complexes that conduct a variety of biochemical reactions for cellular metabolism. To map localized protein information in a sub-mitochondrial complex of interest, we employed our own SR-PL (super-resolution proximity labeling) technique to have a sub-mitochondrial proteomic architecture at single-residue level . Using SR-PL, we are now constructing a super-resolution mitochondrial proteome map (Mitoatlas, www.mitoatlas.org, Kang JW and Shin S et al. manuscript in prep) with Prof. Jong-Seo Kim's lab at SNU Biology. 

We recently identified that the heat-generating reaction in mitochondria is the exothermic oxygen reduction reaction (ORR, ΔH°= -286 kJ/mol) and that ORR-driven mitochondrial thermogenesis can trigger the heat shock response in the nucleus, mediated by heat shock factor 1 (Kang MG and Kim HR et al. ACS Cent. Sci., 2024). These findings suggest that mitochondrial thermogenesis itself can be a global signal affecting the structures and functions of proximal proteins in the same cell. Our lab is currently conducting several projects to examine this "mitochondrial heat signal" in other physiological contexts.

c. Fluorescent Reporter development

Genetically encodable fluorescent reporters for visualizing intracellular proteins, calcium ions, pH and ROS level are now essential tools for biological research. However, many biomolecules and biological events still need new optical sensing and actuating systems. Previously, we created an endogenous bilirubin mapping system using UnaG (Park et al. ACS Chem Biol. 2016) and an endogenous ROS recording system using APEX2 (Mishra and Park et al. Anal. Chem. 2022). Additionally, we successfully developed a photo-proximity crosslinking system using a photo-activable fluorescent ligand to identify interacting proteins in live cells (Mishra et al. Chem. Sci. 2022). Recently, we developed a fluorescent signal amplification system using APEX (FLEX) and used it to visualize mitochondria-lysosome contacts with correlative light and electron microscopy (Sharma et al. Cell Chem. Biol. 2024). We believe our new fluorescent system can provide a deeper understanding of dynamic cellular responses under various stress conditions.