Jade
Nguyen
ab,
Alina
Tirla
a and
Pablo
Rivera-Fuentes
*ab
aLaboratory of Organic Chemistry, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland. E-mail: pablo.riverafuentes@epfl.ch
bInstitute of Chemical Sciences and Engineering, EPF Lausanne, CH C2 425, Station 6, 1015 Lausanne, Switzerland
First published on 26th February 2021
Redox homeostasis is essential for cell function and its disruption is associated with multiple pathologies. Redox balance is largely regulated by the relative concentrations of reduced and oxidized glutathione. In eukaryotic cells, this ratio is different in each cell compartment, and disruption of the mitochondrial redox balance has been specifically linked to metabolic diseases. Here, we report a probe that is selectively activated by endogenous nitroreductases, and releases tributylphosphine to trigger redox stress in mitochondria. Mechanistic studies revealed that, counterintuitively, release of a reducing agent in mitochondria rapidly induced oxidative stress through accumulation of superoxide. This response is mediated by glutathione, suggesting a link between reductive and oxidative stress. Furthermore, mitochondrial redox stress activates a cellular response orchestrated by transcription factor ATF4, which upregulates genes involved in glutathione catabolism.
Mitochondria perform multiple essential tasks in the cell that depend on redox modulation. Disruption of this homeostasis leads to pathologies such as insulin resistance, obesity and type II diabetes.6 Whereas the effects of oxidative stress in mitochondria have been thoroughly investigated,7 reductive stress has remained significantly underexplored. An important advance in this area was the development of enzymes that can control the ratios of oxidized and reduced nicotinamide adenine dinucleotide (NAD+/NADH)8 and their phosphate derivatives (NADP+/NADPH).9 Small molecules that could induce reductive stress by directly affecting the ratio of GSH/GSSG would provide additional information of how cells respond to reductive stress through modulation of this essential redox buffer.
We envisioned that the GSH/GSSG ratio could be manipulated by direct reduction of the disulfide bond in GSSG. This disulfide can be efficiently and rapidly reduced to GSH by trialkylphosphine derivatives, which have no appreciable reactivity against other amino acids.10,11 We hypothesized that we could achieve mitochondria-specific reductive stress by taking advantage of the activity of enzymes that are present only in these organelles to trigger the release of tributylphosphine from a masked precursor. Here, we report the development of such probe, its validation in live human cells, and its application to characterize the cellular response to mitochondrial reductive stress.
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Scheme 1 (A) Mechanism of enzymatic activation of probe 1 and release of PBu3 and fluorescent reporter 2. (B) Synthesis of probes 1 and 3. |
Probe 1 was synthesized in three steps, and probe 3 in two steps, in overall moderate yields (Scheme 1B). Methylation of 2,3,3-trimethylindolenine (4) by microwave irradiation yielded indoleninium 5. Probe 3 and dye 2 were obtained by Knoevenagel condensation of 5 with 4-nitrobenzaldehyde and 4-aminobenzaldehyde respectively. The phosphonium moiety was introduced by conjugate addition of n-tributyl phosphine to probe 3 to afford probe 1 in high yield.
Probe 1 displayed no significant fluorescence prior to enzymatic activation because the tributylphosphonium moiety interrupts the conjugation of the π-system (Scheme 1A and Table S1†). Reporter dye 2, in contrast, displays strong absorption and fluorescence in the visible range (Scheme 1A, Table S1 and Fig. S1†). To test the efficacy of the design, probe 1 was exposed to a purified bacterial NTR. As expected, this enzyme reduced the nitro group of probe 1, producing reporter 2 with concomitant release of tributylphosphine (Fig. S2†). Probe 3, which is an analogue of probe 1 that lacks tributylphosphine, was also converted to fluorophore 2 by bacterial NTR (Fig. S2†). Probe 3, which cannot release tributylphosphine, is an excellent negative control to study the effects of reductive stress induced by probe 1.
We tested whether release of tributylphosphine from probe 1 increased the concentration of GSH in mitochondria. To assess the redox state in mitochondria, we used a glutathione-specific, mitochondria-targeted Grx1-roGFP2 fusion protein sensor.14 This genetically encoded biosensor reacts rapidly to changes in GSH/GSSG ratio and is ratiometric by excitation. We observed a change towards a more oxidized state after treatment with probe 1 after a 30 min incubation (Fig. S7†), corresponding to an increase in ratio of integrated fluorescence intensities (500–530 nm) measured upon excitation at 405 or 488 nm. The change towards a more oxidized state can be observed using H2O2 (Fig. S8a†). However, the change in 405/488 nm ratio towards a more reduced state can only be observed by treating cells with high concentrations (5 mM) of the strong reducing agent DL-dithiothreitol (DTT) (Fig. S8b†). We hypothesized that the lack of sensitivity towards a more reduced state could prevent the observation of an initial reductive stress response from probe 1. To address this issue and study the glutathione redox state more thoroughly, we developed a mitochondria-targeted fluorescent sensor based on a reported cytosolic probe (Fig. 1B and Fig. S9–S12†).15 We chose this probe because it emits at short wavelengths and does not overlap with the emission of reporter dye 2. In this case, the probe is ratiometric by excitation and emission and an increase in blue/green ratio indicates a shift towards a more reduced state. Cells incubated for 1 h with probe 1 displayed significantly increased mitochondrial GSH/GSSG ratio compared to control probe 3 (Fig. 1B). After 1.5 h, this effect faded, suggesting that redox homeostasis had been restored. Additionally, we did not find a significant difference in GSH/GSSG ratio between cells treated with control probe 3 or DMSO only (Fig. S12†). The good sensitivity toward reduced state but slow kinetics of this sensor could explain that we indeed observe a reductive stress response but with a delayed signal.
With these two redox sensors we could show that activation of probe 1 in mitochondria releases a strong reducing agent capable of transforming GSSG into GSH and despite its initial reductive stress response, it led to oxidative stress after longer incubation times. This response was not a consequence of reduction of the nitro group in probe 1 because control probe 3 did not induce any noticeable change in redox status compared to DMSO (Fig. S12†). So how can a reducing agent induce oxidative stress? Previous studies have reported that an increase in GSH may lead to oxidative stress through accumulation of intracellular superoxide (O2−),16 which would explain our observations. We used a fluorescent indicator of intracellular O2− to test this hypothesis.17
We employed Antimycin-A, an inhibitor of complex III that induces mitochondrial O2− production,17 as positive control and DMSO as negative control. Cells treated with probe 1 displayed significantly higher O2− accumulation than cells treated with probe 3 (Fig. 1C and D). Similar results were obtained even when control probe 3 was used at the same extracellular concentration as probe 1 (15 μM, Fig. S13†), which leads to a much higher intracellular concentration of probe 3 (Fig. S4 and S5†). Moreover, to test whether the increase in O2− induced by probe 1 depends on the total concentration of GSH, we blocked its synthesis by inhibiting γ-glutamylcysteine synthetase (glutamate–cysteine ligase) using buthionine sulfoximine (BSO, Fig. 1E).18 In cells treated with both BSO and probe 1, we observed a significant decrease in O2− levels compared to cells treated only with probe 1 (Fig. 1F). In contrast, BSO did not have an effect on the levels of O2− in cells treated with either control probe 3 or DMSO (Fig. 1F). This observation supports the hypothesis that the observed oxidative stress induced by probe 1 is a consequence of O2− accumulation mediated by excess GSH.
In addition, we explored whether tributylphosphine also reduced disulfide bonds in proteins. Whereas tributylphosphine can reduce GSSG rapidly, breaking disulfide bonds in proteins is greatly hindered by steric bulk.10 If probe 1 was able to break disulfide bonds from proteins, it would increase the amount of free, nucleophilic thiols in the proteome of the treated cells. Using an iodoacetamide alkyne as a general electrophilic probe, and a fluorescent reporter,19 we determined that the proteomes of cells treated with probes 1, 3, or DMSO did not display significantly increased or decreased availability of free, nucleophilic thiols (Fig. S14†), confirming that tributylphosphine reduces the unhindered disulfide bond in GSSG preferentially.
These mitochondrial morphologies are reminiscent of those of depolarized mitochondria, for example, after treatment with the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP, Fig. 2A).20 CCCP is a mitochondrial uncoupler that induces membrane depolarization.20 Depolarized mitochondria leak cationic dyes, such as 2, to the cytosol, which can be observed in cells incubated with CCCP (Fig. 2C). In contrast, cells treated with probe 1 retained most of reporter dye 2 in mitochondria, demonstrating that release of tributylphosphine does not induce significant mitochondrial membrane depolarization (Fig. 2D). Notably, CCCP also induced a much larger decrease in mitochondrial area compared to probe 1 (Fig. S15†), suggesting that redox stress induced by trialkylphosphine triggers morphological changes, but not necessarily fragmentation or degradation.
Importantly, the effects of probe 1 on mitochondria could not be reproduced using an untargeted trialkyphosphine. For example, treatment of cells with the membrane-permeant but untargeted trimethyl 3,3′,3′′-phosphanetriyltripropionate (tmTCEP) did not show any significant changes in mitochondrial length compared to the control probe 3 (Fig. S16†). A slight increase in circularity was observed after treatment with tmTCEP compared to probe 3, but this change was not as pronounced as with the mitochondrial stressors probe 1 and CCCP (Fig. S16†). The fact that neither probe 3 nor tmTCEP led to morphological changes indicates that this phenotype is caused by trialkylphosphine generated within mitochondria.
Severely damaged mitochondria undergo degradation and recycling through a selective autophagic process known as mitophagy.21 This process involves recruitment of the ubiquitin ligase parkin to the membrane of depolarized mitochondria,22 ubiquitination, engulfment by the autophagosome, and degradation of the organelle (Fig. 3A).23 To assess whether reductive stress induced by probe 1 triggers mitophagy, we transfected cells with either parkin or the autophagosomal marker LC3 fused to the bright fluorescent protein mTurquoise2 (Fig. S17 and Table S2†). No recruitment of parkin to the mitochondrial membrane was observed in cells treated with probe 1. In contrast, cells treated with CCCP recruited parkin efficiently (Fig. 3B).
We also analyzed the number of LC3 punctae (autophagosomes) that co-localized with mitochondria in cells under various treatments using a custom-made algorithm for image analysis (Fig. 3C and ESI†). We did not find a significant difference between cells treated with probe 1 or DMSO, whereas cells treated with rapamycin, which triggers autophagy, displayed more co-localized punctae (Fig. 3C).24 These observations indicate that mitochondrial redox stress induced by tributylphosphine does not trigger mitophagy, suggesting that a cellular stress response might mitigate the effects of this disruption of redox homeostasis.
The UPR in mitochondria (UPRmt) can be activated by employing the mitochondria-specific HSP90 inhibitor gamitrinib-triphenylphosphonium (GTPP), which leads to upregulation of genes HSPD1 and HSPE1.27 Activation of tributylphosphine in mitochondria did not alter transcription of either of these genes (Fig. 4C). Additionally, GTPP induces parkin-mediated mitophagy,28 whereas probe 1 does not (Fig. 3B). These results suggest that redox stress induced by trialkylphosphine does not trigger the UPRmt, or at least not in the way that chaperone inhibitors such as GTPP do.
Our mRNA sequencing results revealed that transcription factors ATF4, ATF3 and CHOP were upregulated by probe 1 (Fig. 4D). These observations are consistent with a recent multi-omics characterization of the stress response to inhibitors of mitochondrial import, translation, membrane potential, or oxidative phosphorylation.29 These inhibitors triggered the integrated stress response (ISR), which is regulated by ATF4 and activates CHAC1.29 This study, however, also reported significant upregulation of genes involved in amino acid metabolism, such as asparagine synthetase (ASNS) and phosphoserine phosphatase (PSPH).29 These genes were only mildly upregulated by redox stress induced by probe 1 (Fig. 4D). Therefore, even though tributylphosphine shares some common features with various mitochondrial inhibitors, for example increased production of O2− (Fig. 1C and D),16 the cellular response that it elicits is unique and seems to address the GSH/GSSG imbalance through CHAC1 upregulation (Fig. 4E).
Our results indicate that trialkylphosphines, a broad family of compounds that have been largely neglected in chemical biology, can expand the chemical space of small molecules that are used to modulate redox biology, with potential impact in the development of new therapies. For example, CHAC1 upregulation has been reported to deplete GSH levels in triple-negative breast cancer cells, making them more susceptible to necroptosis and ferroptosis during cystine starvation.30 In this work, we demonstrated that even though trialkylphosphines are highly reducing and often water-insoluble compounds, they can be transformed into chemical probes for biological use by developing strategies to tune their reactivity, mask their reducing power, target them to specific organelles, and release them selectively.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0ob02259d |
This journal is © The Royal Society of Chemistry 2021 |