Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors

Abstract

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.

Enzymatic Assemblies of Thiophosphopeptides Instantly Target Golgi Apparatus and Selectively Kill Cancer Cells**

“…In summary, this work illustrates that rapid dephosphorylation of thiophosphopeptides and the subsequent disulfide bond formation enables instantly targeting of Golgi apparatus and selectively inhibiting the cancer cells. Our observations agree with several known facts: (i) CRPs are enriched in Golgi, [29] (ii) a significant level of oxidation occurs in the Golgi membrane, [30] (iii) ALP, as an “almost perfect” enzyme [31] being anchored on the cell membrane by glycosylphosphatidylinositol (GPI) and overexpressed on certain cancer cell, [16, 32] is known to be sorted as oligomers at the Golgi before secretion [21] . This work also underscored the importance to identify the targets of thiophosphopeptides and the assemblies of thiopeptides for designing different molecules capable of engaging the same pathways.…”

Thiophosphopeptides Instantly Targeting Golgi Apparatus and Selectively Killing Cancer Cells

“…In summary, this work illustrates that rapid dephosphorylation of thiophosphopeptides enables instantly targeting of Golgi apparatus and selectively inhibiting the cancer cells. Our observations agree with several known facts: (i) CRPs are enriched in Golgi, [36][37][38] (ii) a significant level of oxidation occurs in the Golgi membrane, 39 (iii) ALP, as an "almost perfect" enzyme 40 being anchored on the cell membrane by glycosylphosphatidylinositol (GPI) and overexpressed on certain cancer cell, 23,[41][42] is known to be sorted as oligomers at the Golgi before secretion. [28][29] Since thiophosphopeptides [43][44][45] are much less developed than phosphopeptides, replace NBD with other functional motifs (e.g.…”

Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light

“…Overall, CABL was successful in the majority of subcellular environments that were tested, but we did observe significant background oxidation/Diels–Alder reaction in the absence of light in experiments with a ceramide–DHTz derivative. The especially high oxidizing environment of the Golgi, where ceramide is localized, may be responsible for the premature activation of the DHTz in this experiment. We also observed ∼10% background oxidation in the activation of a DHTz–HaloTag conjugate in HeLa cells expressing Halo-mCherry-PDGFR on the extracellular surface (Figure S23A).…”