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, 284 (46), 31532-40

Proximity-based Protein Thiol Oxidation by H2O2-scavenging Peroxidases

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Proximity-based Protein Thiol Oxidation by H2O2-scavenging Peroxidases

Marcus Gutscher et al. J Biol Chem.

Abstract

H(2)O(2) acts as a signaling molecule by oxidizing critical thiol groups on redox-regulated target proteins. To explain the efficiency and selectivity of H(2)O(2)-based signaling, it has been proposed that oxidation of target proteins may be facilitated by H(2)O(2)-scavenging peroxidases. Recently, a peroxidase-based protein oxidation relay has been identified in yeast, namely the oxidation of the transcription factor Yap1 by the peroxidase Orp1. It has remained unclear whether the protein oxidase function of Orp1 is a singular adaptation or whether it may represent a more general principle. Here we show that Orp1 is in fact not restricted to oxidizing Yap1 but can also form a highly efficient redox relay with the oxidant target protein roGFP (redox-sensitive green fluorescent protein) in mammalian cells. Orp1 mediates near quantitative oxidation of roGFP2 by H(2)O(2), and the Orp1-roGFP2 redox relay effectively converts physiological H(2)O(2) signals into measurable fluorescent signals in living cells. Furthermore, the oxidant relay phenomenon is not restricted to Orp1 as the mammalian peroxidase Gpx4 also mediates oxidation of proximal roGFP2 in living cells. Together, these findings support the concept that certain peroxidases harbor an intrinsic and powerful capacity to act as H(2)O(2)-dependent protein thiol oxidases when they are recruited into proximity of oxidizable target proteins.

Figures

FIGURE 1.
FIGURE 1.
Orp1 mediates the oxidation of roGFP2. A, reduced roGFP2 (1 μm) was incubated with either reduced Orp1(wt) or Orp1(CS) (5 μm). H2O2 (1 mm) was injected after 50 s. The response of roGFP2 in the absence of Orp1 was used as a negative control. B, schematic representation of roGFP2-Orp1 fusion proteins. C, response of reduced roGFP2-Orp1(wt) to increasing concentrations of H2O2 (0.1–10 μm). D, response of reduced roGFP2-Orp1(CS) to increasing concentrations of H2O2 (0.1–10 μm). In C and D, dotted horizontal lines indicate the dynamic range as defined by complete reduction (10 mm DTT, lower dotted line, set to 0.1) and oxidation (1 mm diamide, upper dotted line). The ratiometric dynamic range of roGFP2-Orp1(CS) is lower (7-fold rather than 8-fold) because of its susceptibility to overoxidation. In each experiment, the ratio of roGFP2 emissions (510–530 nm) after excitation at 390 and 480 nm was calculated and plotted against time.
FIGURE 2.
FIGURE 2.
roGFP2-Orp1 specifically responds to H2O2. A and B, reduced roGFP2-Orp1(wt) (A) and roGFP2-Orp1(CS) (B) were exposed to the oxidants H2O2, GSSG, cystine (Cys2), hydroxyethyl disulfide (HED), and dehydroascorbic acid (AscOx) (10 μm each) 50 s after starting the measurement (arrow).
FIGURE 3.
FIGURE 3.
Mechanism of Orp1-mediated roGFP2 oxidation. A, dimedone attenuates the response of both roGFP2-Orp1(wt) and roGFP2-Orp1(CS). Reduced fusion proteins were treated with 1 μm H2O2 in the presence or absence of 50 mm dimedone. B, roGFP2-Orp1(CS), but not roGFP2-Orp1(wt), is susceptible to overoxidation (overox.). Reduced fusion proteins were exposed to 5 mm H2O2 for 30 min, desalted, exposed to DTT (10 mm, 20 min), and desalted again. The fusion proteins (1 μm) were then treated with 10 μm H2O2. Untreated fusion proteins served as controls. C, the disulfide form of Orp1 is capable of mediating roGFP2 oxidation. Wild type and mutant Orp1 proteins were oxidized with a 5-fold molar excess of H2O2 or blocked with N-ethyl maleimide (NEM) followed by desalting. 1 μm reduced roGFP2 was incubated with 50 μm pretreated Orp1(wt) or Orp1(CS). D, thioredoxin competes thiol-disulfide exchange between roGFP2 and wild type Orp1. roGFP2-Orp1(wt) was exposed to 1 or 10 μm H2O2 in the presence or absence of a functional TrxS consisting of Trx1, Trx reductase, and NADPH. In A, B, and D, H2O2 was injected after 50 s.
FIGURE 4.
FIGURE 4.
Glutathione interferes with the sulfenic acid but not with the disulfide exchange mechanism. A and B, 1 μm roGFP2-Orp1(wt) (A) or 1 μm roGFP2-Orp1(CS) (B) was exposed to 1 μm H2O2 after 50 s in the presence of increasing GSH concentrations (0, 5, 200, 5000 μm).
FIGURE 5.
FIGURE 5.
roGFP2-Orp1 responds to H2O2 in living cells. A, roGFP2-Orp1(wt), but not roGFP2-Orp1(CS), facilitates monitoring of H2O2 in living cells. HeLa cells were transiently transfected with roGFP2-Orp1(wt), roGFP2-Orp1(CS), or roGFP2 and treated with 50 μm H2O2 after 30 s. Cells were excited with 405- and 488- nm lasers, and the ratio of the emissions (500–554 nm) was calculated. B, roGFP2-Orp1 and HyPer detect H2O2 with similar sensitivity. HeLa cells stably expressing roGFP2-Orp1(wt) or HyPer were exposed to 20 μm H2O2 after 30 s. The ratio (405/488 nm for roGFP2-Orp1(wt) and 488/405 nm for HyPer) of individual cells was plotted against time. C, based on experiments similar to B, the maximal ratiometric change of roGFP2-Orp1(wt)-positive cells was plotted against H2O2 concentration. A value of 1 indicates a fully reduced probe (calibration by treatment with 1 mm DTT). For each H2O2 concentration, measurements on six cells from two different experiments were averaged. Error bars represent S.D.
FIGURE 6.
FIGURE 6.
roGFP2-Orp1 responds to endogenous oxidative changes. HeLa cells expressing roGFP2-Orp1(wt) were cultured for 20 h in medium containing either 0% (solid line) or 10% (dotted line) fetal calf serum (FCS) and analyzed by flow cytometry. A, histogram of one exemplary experiment (10,000 cells). B, quantification of three independent experiments. Error bars represent S.D. C, serum-starved (0% FCS) and non-starved (10% FCS) HeLa cells were loaded with H2DCFDA or left untreated. A histogram of DCF fluorescence (10,000 cells) is shown.
FIGURE 7.
FIGURE 7.
roGFP2-Orp1 visualizes H2O2 signals in primary human T cells. Primary human T cells were isolated, transiently transfected with roGFP2-Orp1(wt), and analyzed by time-resolved flow cytometry. After 200 s (arrow), cells were either stimulated with an anti-CD3 antibody (grey line) or stimulated with an IgG control antibody (black line) together with a cross-linking secondary antibody in both cases. Cells were excited with 405- and 488-nm lasers, and the ratio of the emissions was plotted against time. Approximately 40–60 cells were averaged per second.
FIGURE 8.
FIGURE 8.
Mammalian peroxidase Gpx4, but not Prx6, oxidizes fused roGFP2. roGFP2 fusion proteins of glutathione peroxidase 4 (roGFP2-Gpx4) and peroxiredoxin-6 (Prx6-roGFP2) were transiently expressed in HeLa cells. Oxidation in response to 50 μm H2O2 was monitored by live cell microscopy.

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