Apply Proximity Labeling and Multi-Omics to Dissect Drug Targets and Cellular Trafficking

Abstract

Proximity labeling (PL) is a widely used technique to explore biomolecule interactions. It involves the introduction of a genetically encoded tag, such as ascorbate peroxidase, biotin ligase, or a small molecule, which can be activated to label proteins and RNAs in its vicinity. The tagged molecules are subsequently enriched and analyzed by mass spectrometry or RNA sequencing (RNA-Seq). So, it offers opportunities to study the dynamic protein-protein and protein-RNA interactions that occur in living systems, providing insights into cellular processes, signaling pathways, and molecular networks. The integration of proximity labeling with modern multi-omics holds great potential for providing new and deep insights into biological processes. This thesis research leveraged different proximity labeling technologies with proteomics and transcriptomics and revealed novel mechanisms of a therapeutic compound and cellular transportation.

Photodynamic therapy (PDT) has been proposed for the treatment of cancers and can induce cell death. However, it remains challenging to define the precise localization of PDT reagents and the mechanisms triggering cell death. To address this, we established a pipeline that integrates chemical biology with multi-omics approaches to investigate the cellular targets and responses of an iridium(III) photosensitizer (PS).

In this study, we investigated the PDT mechanisms of [Ir(pqe)₂(pic)], a cyclometallated iridium complex, under 450nm light irradiation. We monitored cell viability, apoptosis ratio, and transcriptome changes in a time series analysis comprising six time points.
 Our results revealed significant apoptosis and necrosis in cells following light irradiation. Next, Nanopore-seq was applied to investigate the effects of the photosensitizer on the cell transcriptome. We saw the downregulation of genes associated with endoplasmic reticulum (ER)-related pathways and upregulation of genes involved in the mitochondrial cellular compartment.

As a new-generation method of RNA-seq, nanopore sequencing has the advantage of generating long reads and can detect various base modifications. We then analyzed the transcriptome changes in RNA read length and observed a decrease in average read length with increasing irradiation time. Genes showing a correlation between decreased read length and irradiation time were enriched in pathways related to the respiratory electron transport chain and mitochondrial ATP synthesis, suggesting mitochondrial DNA (mtDNA)-encoded mRNAs and rRNAs as PDT targets. Furthermore, analysis of mutants revealed an increase in G>T mutations, indicating oxidative modifications on 8-oxoguanine in RNA (o8G), resulting from guanine oxidation by reactive oxygen species. GO analysis showed that the o8G genes were enriched in ribosomal subunits and mitochondrial complexes involved in peptide translation, oxidative phosphorylation, and ATP synthesis in mitochondria.

To further validate the PDT targets, we employed photocatalytic proximity labeling. The total enriched biotinylated proteins and RNAs were analyzed by liquid chromatography-mass spectrometry (LC-MS) and nanopore sequencing, respectively. The MS results showed that over 57% of enriched proteins were located in mitochondria or the ER. Moreover, translation, cellular respiration, and protein folding pathways were enriched. The RNA-seq results showed that most mtDNA-encoded RNAs were enriched, which aligns with proteomics results.

Then, we compared transcriptome and proteome data and confirmed that the mitochondrial ribosome complexes were enriched, including 2 rRNAs (12S and 16S) and 21 ribosomal proteins out of 80 (26%), which means that the ribosome complexes and the translation process were targets of the Ir(III) complex. Immunofluorescence experiments confirmed the colocalization of the photocatalytic biotinylation with the ER-Tracker and Mito-Tracker, indicating an affinity of the iridium complex for the ER and mitochondria. These results revealed that mitochondrial genes, protein processing in the ER, and ribonucleoprotein biogenesis are targets of PDT reagent [Ir(pqe)₂(pic)].

The Kinesin-1 protein family encompasses three subtypes: KIF5A, KIF5B, and KIF5C. These proteins are crucial for the intracellular transport of various cargoes, such as organelles, proteins, and RNAs. They play significant roles in cellular processes, particularly in neuronal functions. While KIF5B is ubiquitously expressed, KIF5A and KIF5C are specific to neurons. The precise cargoes and mechanisms of these proteins are still under investigation. In the second part of the thesis, APEX2 proximity labeling was employed to discern the cargo selectivity of Kinesin-1 isoforms. We attached the ascorbate peroxidase APEX2 to the three Kinesin-1 proteins and performed APEX-MS and APEX-seq in 293T cells and neurons. Subsequently, we enriched the proximal proteins and RNAs, comparing the interactomes of the three Kinesin-1 proteins. Our findings indicated that while the three Kinesin-1 proteins share cargoes like mRNAs for axogenesis, they also possess unique cargoes.

This thesis established a pipeline integrating proximity labeling with multi-omics analysis to analyze drug targets and molecular cargo. The findings offer new understanding of the molecular and cellular processes involving the iridium complex as a PDT agent. In addition, this work also sheds light on the cargo selectivity of Kinesin-1 motor proteins in cellular transportation.

Date of Award	4 Nov 2025
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Liang ZHANG (Supervisor)