Localization and Dynamics of Disulfide Reduction and Their Key Redox Regulators during Internalization in Endolysosomal Compartments.

Abstract

Subcellular differences in redox potential are essential for intracellular protein dynamics, cellular entry by many viruses and intracellular pathogens, and the targeted delivery of macromolecular therapeutics. This study focuses on evaluation of disulfide reduction in the endocytic pathway in the context of macromolecules internalized as particles. Recently, bioconjugation employing disulfide reduction has been exploited in drug delivery and those conjugates are being used more frequently in protein and oligonucleotide systems. Disulfide-based macromolecular therapeutic agents are membrane-impermeant, thus typically internalized into cells via endocytosis, and apparently reduced at some point in endocytic compartments en route to the lysosomal compartments. However, little is known about the spatiotemporal dynamics of disulfide bond reduction at the subcellular level, especially within endolysosomal compartments. Direct analysis of intracellular redox conditions is limited by current redox indicators, which either lack the necessary sensitivity or perturb the normal subcellular physiology. The probe in this study was designed to address some of these challenges. A genetically engineered redox-sensitive fusion protein, consisting of monomeric enhanced cyan fluorescent protein (mECFP) and monomeric Citrine (mCit), joined by an intervening disulfide-bonded and protease-sensitive linker, were expressed and characterized in vitro and used to measure redox potential following endocytosis by living cells. FRET microscopy revealed that disulfide bond reduction began in the early endosome and continued throughout endolysosomal maturation. Phagocytic oxidase activity slowed reduction, while expression of gamma-interferon inducible lysosomal thiol reductase (GILT) accelerated reduction, indicating at least one mechanism of regulation of reduction in endocytic compartments. The information obtained from this study demonstrated not only the potential utility of this reporter for the design of targeted delivery systems, but also for studying cell type-dependent variations in the disufide reduction mechanism of endocytosed macromolecules and cellular factors modulating the reduction processes. There were differential rates of disulfide reduction by BMDCs, fibroblasts, cancer cells and BMMs, suggesting that further investigation in other cell types in various states will provide critical information that can be used to investigate the impact of novel treatments, as well as informing the design of targeted pharmaceutical agents that rely on disulfide bonds.