Protein Persulfide Detection Protocol reveals vital roles for thioredoxin and glutathione systems in maintaining sulfane sulfur homeostasis in cells and in vivo.

Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H 2 O 2 ) production. Understanding the significance of this response is limited by the fact that the source of EGF-induced H 2 O 2 production is unknown. Here we show that EGF-induced H 2 O 2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1).Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H 2 O 2 production in both cell lines. We also demonstrate that H 2 O 2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1.

An energy transfer-based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue-excitable coumarins to yellow/red emitting cyanine frames is presented. Suchr elay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes-shifts allowing lower background fluorescence even in no-wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells.These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three-color imaging of intracellular targets using one single excitation source with separate emission windows.S ubdiffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopyis also presented.

Two sets of bioorthogonally applicable, double fluorogenic probes, capable of sensing DNA–protein interactions, were prepared by installing an azide or tetrazine motif onto structurally fluorogenic, DNA sensitive frames. Installation of these bioorthogonal functions onto DNA intercalating dyes furnished these scaffolds with reactivity based fluorogenicity, rendering these probes double-fluorogenic, AND-type logic switches that require the simultaneous occurrence of a bioorthogonal reaction and interaction with DNA to trigger high intensity fluorescence. The probes were evaluated for double fluorogenic behavior in the presence/absence of DNA and a complementary bioorthogonal function. Our studies revealed that azide and tetrazine appending thiazole orange frames show remarkable double fluorogenic features. One of these probes, a membrane permeable tetrazine modified thiazole orange derivative was further tested in live cell labeling studies. Cells expressing bioorthogonalized DNA-binding proteins showed intensive fluorescence characteristics of the localization of the proteins upon treatment with our double fluorogenic probe. On the contrary, labeling similarly bioorthogonalized cytosolic proteins did not result in the appearance of the fluorescence signal. These studies suggest that such double-fluorogenic probes are indeed capable of sensing DNA–protein interactions in cells.