Research Projects

Rationale: An equilibrium between reduction and oxidation—redox homeostasis—is critical for normal cellular function, while its disruption is linked to diseases including aging, cancer, neurodegeneration, and cardiovascular conditions. Cells regulate redox balance by spatially compartmentalizing the production and consumption of reactive oxygen species (ROS) within organelles such as mitochondria, endoplasmic reticulum (ER), and the nucleus. Each organelle maintains a distinct redox environment that contributes differently to signalling, metabolism, and stress responses. Current biochemical and mass spectrometry approaches provide valuable information on bulk cellular ROS pools, and both genetic and chemical tools exist for manipulating ROS globally. However, modulation of ROS at the organelle level remains a challenge, as most existing tools affect ROS across the entire cell, masking the contribution of specific compartments. To address this gap, we will develop a genetically directed chemical toolkit for organelle-specific ROS scavenging. We will express HaloTag fusion proteins that localize to chosen organelles and bind covalently to small-molecule ROS scavengers bearing HaloTag ligands. This hybrid approach combines genetic localization with pharmacological precision, enabling localized ROS depletion at subcellular resolution. Ultimately, this strategy will help construct a spatial map of ROS and clarify the role of organelle-specific redox signals in cellular physiology and disease. Hypothesis: Selective scavenging of ROS at the organelle level will reveal distinct spatial distributions and localized roles of ROS that are obscured by global ROS depletion, enabling an accurate understanding of compartment-specific redox dynamics in cellular processes. Objectives: This project has two broad objectives. Synthesis of HaloTag ligand (HTL) conjugated ROS scavengers for genetically precise scavenging of superoxide, peroxide, and hypochlorite. Another objective is the comparative benchmarking of the synthesized HaloTag ligands against conventional, whole-cell-targeted commercial antioxidants for ROS scavenging, both in test tubes and in mammalian cells. This will allow us to infer relative ROS levels across organelles by comparing the effects of localized versus global scavenging, providing insights into spatial distribution of ROS species. Significance: This will be the first hybrid method to precisely manipulate subcellular ROS pools. The ROSmap toolkit that we create will enable targeted redox modulation, overcoming the limitations of traditional ROS scavengers. It offers a platform to map compartment-specific ROS to potentially uncover how local ROS levels shape physiology and disease. To maximise impact, we will release ROSmap ligands as a freely accessible resource for the redox-biology scientific community through our OpenReagents (https://dyecraftlab.com/openreagent) platform.