Eukaryotic intracellular membranes serve as platforms for numerous biochemical processes, mediated by membrane proteins and lipids. These components are vulnerable to oxidative damage from reactive oxygen species (ROS), potentially disrupting organellar function and triggering programmed cell death, implicated in various diseases. While lipid peroxidation by ROS has been studied, the cellular responses to oxidative stress specifically targeting intracellular membrane proteins remain unclear. Oxidative photocatalysis offers a promising tool for spatiotemporally controlling oxidative stress within cells. Previous studies have explored organelle-targeted oxidative stress using photocatalysts, but a photocatalyst capable of targeting all intracellular membranes simultaneously has been lacking. This research addresses this gap by developing a novel amphiphilic photocatalyst for investigating cellular responses to membrane protein oxidation across various organelles.

Existing literature highlights the susceptibility of intracellular membranes to oxidative damage caused by endogenous and exogenous ROS. This damage can disrupt organellar function and contribute to various diseases. Studies have demonstrated that lipid peroxidation can be initiated by ROS generated through cytosolic iron-induced hydroxyl radical (•OH) generation. While the effects of ROS on membrane lipids are known, the precise cellular responses to ROS-mediated oxidation of membrane proteins, especially within the complex environment of intracellular membranes, have not been fully elucidated. Previous work has used photocatalysts to induce oxidative stress in specific organelles, but a universal approach for studying membrane protein oxidation across organelles has been lacking. The challenge lies in creating a photocatalyst lipophilic enough to embed in the membranes but hydrophilic enough to cross the plasma membrane.

This study synthesized an amphiphilic photocatalyst, BTP, designed for intracellular membrane localization. BTP’s photocatalytic cycle was characterized through various techniques, including fluorescence quenching assays, H2O2 generation assays using peroxidase and DPD, •OH generation assays using HPF, and EPR spectroscopy with BMPO. The subcellular localization of BTP was determined using confocal and structured illumination microscopy (SIM). The effect of BTP photocatalysis on membrane protein folding stability was investigated using an in vitro assay with *E. coli* rhomboid protease GlpG and single-molecule magnetic tweezers. Label-free quantitative mass spectrometry was employed to identify oxidized proteins in HeLa cells following BTP photocatalysis. Cellular responses, including cation mobilization (Ca2+ and K+), mitochondrial membrane potential, and cell death morphology, were analyzed using various assays including flow cytometry and imaging techniques. The type of cell death was investigated using inhibitors of ferroptosis and caspases, and western blotting was used to analyze caspase activation and GSDMD cleavage. Control experiments were conducted by omitting BTP and/or light exposure. Specific methods include cyclic voltammetry, the HPF assay for hydroxyl radical detection, EPR spectroscopy with BMPO for ROS detection, cell culture of various cell lines (HeLa, Panc-1, MiaPaca-2), membrane protein expression and purification of GlpG, membrane protein stability assays, single-molecule tweezer assays, preparation of tryptic peptides for LC-MS/MS, high-pH reversed-phase chromatography for peptide fractionation, LC-MS/MS analysis and data processing, LDH and ATP release assays, and western blotting.

BTP effectively localizes to intracellular membranes. Its photocatalysis generates hydroxyl radicals and hydrogen peroxide, with hypoxia accelerating H2O2 production. BTP photocatalysis destabilizes membrane protein folding, as demonstrated by GlpG unfolding assays. Proteomic analysis revealed that membrane proteins related to protein quality control (PQC) are primarily oxidized by BTP photocatalysis. Specifically, proteins within the endoplasmic reticulum (ER), Golgi apparatus (GA), and mitochondria were significantly affected. This extensive oxidation of PQC proteins leads to the accumulation of misfolded proteins, triggering unfolded protein response (UPR) and consequent ER and mitochondrial stress. BTP photocatalysis causes Ca2+ mobilization and K+ efflux. Importantly, BTP photocatalysis triggers non-canonical pyroptosis, characterized by lytic cell death, GSDMD-NT release, and activation of caspase-4/5, but not caspase-1. The study conclusively demonstrates that light-induced membrane protein oxidation activates a caspase-dependent pathway of pyroptosis, rather than ferroptosis.

This research provides compelling evidence that intracellular membrane protein oxidation, induced by photocatalysis, initiates a cascade of events leading to non-canonical pyroptosis. The findings address the knowledge gap concerning the cellular responses to membrane protein oxidation. The identification of BTP as an effective tool for inducing this type of cell death opens new avenues for studying the mechanism of pyroptosis. The observation that the process occurs even under hypoxic conditions is particularly significant, given the prevalence of hypoxia in cancer. The ability to spatiotemporally control pyroptosis using light offers exciting therapeutic potential for cancer and other diseases. While the precise interplay between all the observed phenomena requires further investigation, this study has established a clear link between membrane oxidation, protein quality control dysfunction, and pyroptosis activation.

This study demonstrates that oxidation of intracellular membrane proteins, induced by the amphiphilic photocatalyst BTP, triggers non-canonical pyroptosis via caspase-4/5 activation and GSDMD cleavage. This research provides a novel approach for studying pyroptosis and has significant implications for developing targeted therapeutic strategies. Future research should focus on elucidating the detailed molecular mechanisms and exploring the therapeutic potential of this approach in various disease models.

The study primarily used HeLa cells. While other cell lines (Panc-1, MiaPaca-2) were tested for cytotoxicity, a broader range of cell types would strengthen the generalizability of the findings. Although proteomic analysis identified numerous oxidized proteins, further investigation is needed to clarify the specific roles of each oxidized protein in the pathway leading to pyroptosis. The exact mechanism by which the observed ER stress and mitochondrial dysfunction trigger caspase-4/5 activation warrants additional research. The study focused on in vitro and in cell experiments. Future in vivo studies are crucial to assess the clinical relevance of these findings.