Disruption of mitochondrial redox homeostasis by enzymatic activation of a trialkylphosphine probe

Article abstract of DOI:10.1039/d0ob02259d

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.

Full text of DOI:10.1039/d0ob02259d

Volume 19
Number 12
28 March 2021
Pages 2549-2812
Organic &
Biomolecular
Chemistry
rsc.li/obc
ISSN 1477-0520
PAPER
Pablo Rivera-Fuentes et al.
Disruption of mitochondrial redox homeostasis by enzymatic
activation of a trialkylphosphine probe

Organic &
Biomolecular Chemistry
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PAPER
Disruption of mitochondrial redox homeostasis by
enzymatic activation of a trialkylphosphine probe†
Cite this: Org. Biomol. Chem., 2021,
19, 2681
Jade Nguyen,a,b Alina Tirlaa and Pablo Rivera-Fuentes
*
a,b
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 acti-
vated by endogenous nitroreductases, and releases tributylphosphine to trigger redox stress in mitochon-
dria. 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 gluta-
thione, 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.
Received 13th November 2020,
Accepted 5th January 2021
DOI: 10.1039/d0ob02259d
rsc.li/obc
control the ratios of oxidized and reduced nicotinamide
adenine dinucleotide (NAD+/NADH)8 and their phosphate
Introduction
Intracellular reduction–oxidation (redox) balance is primarily derivatives (NADP+/NADPH).9 Small molecules that could
regulated by the relative concentrations of glutathione (GSH) induce reductive stress by directly affecting the ratio of GSH/
and its oxidized, disulfide-bonded dimer (GSSG).1 Multiple GSSG would provide additional information of how cells
physiological processes, ranging from cell signaling to protein respond to reductive stress through modulation of this essen-
folding, depend on redox homeostasis. Consequently, several tial redox buffer.
pathological conditions such as cancer,2 diabetes3 and neuro-
We envisioned that the GSH/GSSG ratio could be manipu-
degenerative diseases4 have been associated with redox imbal- lated by direct reduction of the disulfide bond in GSSG. This
ance. In eukaryotes, this homeostasis is controlled at the level disulfide can be efficiently and rapidly reduced to GSH by
of subcellular compartments and each organelle possesses its trialkylphosphine derivatives, which have no appreciable reac-
own redox environment.5 Being able to modulate the GSH/ tivity against other amino acids.10,11 We hypothesized that we
GSSG ratio in an organelle-specific manner would allow us to could achieve mitochondria-specific reductive stress by taking
investigate the relationship between cellular redox stress advantage of the activity of enzymes that are present only in
responses and the spatial origin of the imbalance, enriching these organelles to trigger the release of tributylphosphine
our understanding of subcellular compartmentalization of from a masked precursor. Here, we report the development of
redox signaling.
such probe, its validation in live human cells, and its appli-
Mitochondria perform multiple essential tasks in the cell cation to characterize the cellular response to mitochondrial
that depend on redox modulation. Disruption of this homeo- reductive stress.
stasis 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
Results and discussion
Design, synthesis and enzymatic activation of a biocompatible
advance in this area was the development of enzymes that can
trialkylphosphine probe
To induce reductive stress selectively in mitochondria, we envi-
sioned a probe that could release the reducing agent tributyl-
phosphine and a reporter fluorophore after activation by
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
endogenous nitroreductases (NTRs) that are naturally over-
6, 1015 Lausanne, Switzerland
expressed in mitochondria.12,13 We developed tributyl-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0ob02259d
phosphonium probe 1 with a nitro group as the enzymatically
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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.
activatable trigger (Scheme 1A). Upon enzymatic reaction, the ditions of all subsequent experiments (incubation of less than
strong electron-withdrawing nitro group is converted to an 2 h), these concentrations did not induce significant toxicity in
amine. This electron-rich substituent donates electron density HEK293 cells (Fig. S6†).
to the π-conjugated system, cleaving the weak C–P bond to
release the reducing agent tributylphosphine (PBu3) and fluo- 1 increased the concentration of GSH in mitochondria. To
rescent reporter 2 (Scheme 1A). assess the redox state in mitochondria, we used a glutathione-
We tested whether release of tributylphosphine from probe
Probe 1 was synthesized in three steps, and probe 3 in two specific, mitochondria-targeted Grx1-roGFP2 fusion protein
steps, in overall moderate yields (Scheme 1B). Methylation of sensor.14 This genetically encoded biosensor reacts rapidly to
2,3,3-trimethylindolenine (4) by microwave irradiation yielded changes in GSH/GSSG ratio and is ratiometric by excitation.
indoleninium 5. Probe
3 and dye 2 were obtained by We observed a change towards a more oxidized state after
Knoevenagel condensation of 5 with 4-nitrobenzaldehyde and treatment with probe 1 after a 30 min incubation (Fig. S7†),
4-aminobenzaldehyde respectively. The phosphonium moiety corresponding to an increase in ratio of integrated fluo-
was introduced by conjugate addition of n-tributyl phosphine rescence intensities (500–530 nm) measured upon excitation at
to probe 3 to afford probe 1 in high yield.
405 or 488 nm. The change towards a more oxidized state can
Probe 1 displayed no significant fluorescence prior to enzy- be observed using H2O2 (Fig. S8a†). However, the change in
matic activation because the tributylphosphonium moiety 405/488 nm ratio towards a more reduced state can only be
interrupts the conjugation of the π-system (Scheme 1A and observed by treating cells with high concentrations (5 mM) of
Table S1†). Reporter dye 2, in contrast, displays strong absorp- the strong reducing agent DL-dithiothreitol (DTT) (Fig. S8b†).
tion and fluorescence in the visible range (Scheme 1A, We hypothesized that the lack of sensitivity towards a more
Table S1 and Fig. S1†). To test the efficacy of the design, probe reduced state could prevent the observation of an initial reduc-
1 was exposed to a purified bacterial NTR. As expected, this tive stress response from probe 1. To address this issue and
enzyme reduced the nitro group of probe 1, producing reporter study the glutathione redox state more thoroughly, we devel-
2 with concomitant release of tributylphosphine (Fig. S2†). oped a mitochondria-targeted fluorescent sensor based on a
Probe 3, which is an analogue of probe 1 that lacks tributyl- reported cytosolic probe (Fig. 1B and Fig. S9–S12†).15 We chose
phosphine, was also converted to fluorophore 2 by bacterial this probe because it emits at short wavelengths and does not
NTR (Fig. S2†). Probe 3, which cannot release tributyl- overlap with the emission of reporter dye 2. In this case, the
phosphine, is an excellent negative control to study the effects probe is ratiometric by excitation and emission and an
of reductive stress induced by probe 1.
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 com-
Cellular uptake, organelle specificity and redox modulation
Confocal microscopy experiments employing live HEK293 cells pared to control probe 3 (Fig. 1B). After 1.5 h, this effect faded,
revealed that both probes 1 and 3 are activated exclusively in suggesting that redox homeostasis had been restored.
mitochondria (Fig. 1A, Fig. S3 and Movie S1†). Owing to the Additionally, we did not find a significant difference in GSH/
different cellular uptake of probes 1 and 3 (Fig. S4†), we per- GSSG ratio between cells treated with control probe 3 or DMSO
formed titration experiments to estimate which incubation only (Fig. S12†). The good sensitivity toward reduced state but
conditions produced similar intracellular concentrations of slow kinetics of this sensor could explain that we indeed
dye 2, the enzymatic product of both probe 1 and 3 (Fig. S5†). observe a reductive stress response but with a delayed signal.
We determined that incubation of cells with probe 1 and 3 at
With these two redox sensors we could show that activation
concentrations of 15 µM and 5 µM, respectively, resulted in of probe 1 in mitochondria releases a strong reducing agent
similar intracellular concentrations of dye 2. Under the con- capable of transforming GSSG into GSH and despite its initial
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pared 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 accumu-
lation of intracellular superoxide (O2−),16 which would explain
our observations. We used a fluorescent indicator of intracellu-
lar 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 dis-
played 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 sul-
foximine (BSO, Fig. 1E).18 In cells treated with both BSO and
Fig. 1 Effect of probe 1 on mitochondrial redox homeostasis in live
HEK293 cells. (A) Co-localization of product of activation of probe 1
(magenta) and mitochondrial marker MitoTracker Deep Red (MDR,
cyan). Scale bar = 10 µm. Pearson’s coefficient = 0.8. Mean is plotted
and error bar represents 95% confidence interval. Measurements were
carried out for N = 12 cells. (B) Structure and response of a mitochon-
dria-targeted fluorescent sensor for measurement of glutathione redox
states upon treatment with probe 1 and 3. Means are plotted and error
bars represent 95% confidence interval. Measurements were carried out
for N > 50 cells from biological triplicates. (C) Superoxide production
−
measured with O2 sensor HK-SOX-1 after treatment with DMSO,
Antimycin-A (Am-A, 10 µM), probe 1, or probe 3. (D) Quantification of
fluorescence intensity compared to DMSO control (intensity = 1) of cells
treated as described in (C) means are plotted and error bars represent
95% confidence interval. Measurements were carried out for N > 20
cells from biological triplicates. (E) Mechanism of BSO inhibition of glu-
Fig. 2 Effect of probe 1 on the morphology and depolarization of mito-
chondria in live HEK293 cells. (A) Three-dimensional confocal
microscopy of cells treated with control probe 3 (5 µM), probe 1 (15 µM)
or CCCP (20 µM). This last group of cells was stained with the dye MDR
(20 nM) for visualization. (B) Quantification of morphological parameters
of cells treated with probes 3 or 1, or with CCCP or DMSO control
stained with MDR. Measurements were carried out in biological tripli-
−
tathione biosynthesis. (F) Quantification of O2 production with
HK-SOX-1 after treatment with DMSO, probe 3 or probe 1, with (inten-
sity = 1) or without prior BSO treatment (250 µM). Means are plotted
and error bars represent 95% confidence interval. Measurements were
carried out for N > 50 cells from biological triplicates. Statistical signifi-
cance was assessed by unpaired, two-tailed, Mann–Whitney test. P
values: **** < 0.0001 and ns = not significant >0.05. Scale bars = 10 µm.
cates and morphological data from
N = 1000 mitochondria were
employed for each condition. Means are plotted and error bars rep-
resent 95% confidence intervals. (C) Mitochondrial membrane depolar-
ization in cells incubated with probe 1 (15 µM), probe 3 (5 µM), or CCCP
(20 µM) co-incubated with probe 3 (1 µM) for 2 h. (D) Quantification of
the ratio of mitochondrial to cytoplasmic area-normalized fluorescence
intensity. Measurements were carried out in biological triplicates and
data from N > 26 cells were employed for each condition. Means are
plotted and error bars represent 95% confidence intervals. In all cases,
statistical significance was assessed by unpaired, two-tailed, Mann–
Whitney test. P values: **** < 0.0001, ** < 0.001, * < 0.05 and ns = not
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 com- significant >0.05. Scale bars = 10 µm.
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−
probe 1, we observed a significant decrease in O2 levels com- ive image analysis (see ESI† for details) and revealed that mito-
pared to cells treated only with probe 1 (Fig. 1F). In contrast, chondria of cells treated with probe 1 displayed shorter length,
−
BSO did not have an effect on the levels of O2 in cells treated greater circularity (Fig. 2B), and smaller aspect ratio
with either control probe 3 or DMSO (Fig. 1F). This observation (Fig. S15†).
supports the hypothesis that the observed oxidative stress
These mitochondrial morphologies are reminiscent of
induced by probe 1 is a consequence of O2 accumulation those of depolarized mitochondria, for example, after treat-
mediated by excess GSH. ment with the protonophore carbonyl cyanide m-chlorophenyl
−
In addition, we explored whether tributylphosphine also hydrazone (CCCP, Fig. 2A).20 CCCP is a mitochondrial uncou-
reduced disulfide bonds in proteins. Whereas tributylphosphine pler that induces membrane depolarization.20 Depolarized
can reduce GSSG rapidly, breaking disulfide bonds in proteins mitochondria leak cationic dyes, such as 2, to the cytosol,
is greatly hindered by steric bulk.10 If probe 1 was able to break which can be observed in cells incubated with CCCP (Fig. 2C).
disulfide bonds from proteins, it would increase the amount of In contrast, cells treated with probe 1 retained most of reporter
free, nucleophilic thiols in the proteome of the treated cells. dye 2 in mitochondria, demonstrating that release of tributyl-
Using an iodoacetamide alkyne as a general electrophilic probe, phosphine does not induce significant mitochondrial mem-
and a fluorescent reporter,19 we determined that the proteomes brane depolarization (Fig. 2D). Notably, CCCP also induced a
of cells treated with probes 1, 3, or DMSO did not display signifi- much larger decrease in mitochondrial area compared to
cantly increased or decreased availability of free, nucleophilic probe 1 (Fig. S15†), suggesting that redox stress induced by
thiols (Fig. S14†), confirming that tributylphosphine reduces trialkylphosphine triggers morphological changes, but not
the unhindered disulfide bond in GSSG preferentially.
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
Morphological changes, mitochondrial damage and
mitophagy
untargeted
trimethyl
3,3′,3″-phosphanetriyltripropionate
Three-dimensional confocal microscopy revealed that probe 1,
but not 3, induced drastic changes in the morphology of mito-
chondria (Fig. 2A). These changes were evaluated by quantitat-
(tmTCEP) did not show any significant changes in mitochon-
drial length compared to the control probe 3 (Fig. S16†). A
Fig. 3 Tributylphosphine-induced redox stress does not trigger mitophagy in live HEK293 cells. (A) Mechanism of parkin-mediated mitophagy. (B)
Confocal imaging of cells expressing mTurquoise2-parkin and treated with probe 1 (15 µM), DMSO, or CCCP (20 µM) for 2–3 h. Arrows indicate
examples of parkin recruited to damaged mitochondria. Quantification of number of Parkin punctae per cell under the conditions mentioned above.
Measurements were carried out for N > 25 cells from biological duplicates. (C) Confocal imaging of cells expressing mTurquoise2-LC3 and treated
with probe 1 (15 µM), DMSO, or rapamycin (rapa, 1 µM) for 2–3 h. Quantification of number of LC3 punctae that co-localized with mitochondria per
cell under the conditions mentioned above. Measurements were carried out for N > 50 cells from biological triplicates. (B and C) Means are plotted
and error bars represent 95% confidence interval. Statistical significance was assessed by unpaired, two-tailed, Mann–Whitney test. P values: **** <
0.0001, *** < 0.001; ** < 0.01 and ns = not significant >0.05. Scale bars = 10 µm.
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slight increase in circularity was observed after treatment with played more co-localized punctae (Fig. 3C).24 These obser-
tmTCEP compared to probe 3, but this change was not as pro- vations indicate that mitochondrial redox stress induced by tri-
nounced as with the mitochondrial stressors probe 1 and butylphosphine does not trigger mitophagy, suggesting that a
CCCP (Fig. S16†). The fact that neither probe 3 nor tmTCEP cellular stress response might mitigate the effects of this dis-
led to morphological changes indicates that this phenotype is ruption of redox homeostasis.
caused by trialkylphosphine generated within mitochondria.
Transcriptional response
Severely damaged mitochondria undergo degradation and
recycling through a selective autophagic process known as
mitophagy.21 This process involves recruitment of the ubiqui-
tin ligase parkin to the membrane of depolarized mitochon-
dria,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 (autophago-
somes) 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 differ-
ence between cells treated with probe 1 or DMSO, whereas
cells treated with rapamycin, which triggers autophagy, dis-
To gain further understanding of how the cell reacts to mito-
chondrial redox stress, we performed global mRNA sequencing
and differential gene expression analysis. This study revealed
that cells treated with probe 1 displayed significant upregula-
tion of several genes, particularly γ-glutamyl cyclotransferase
(CHAC1), compared to DMSO-treated cells (Fig. 4A). On the
other hand, probe 3 induced the upregulation of fewer genes
compared to DMSO treatment (Fig. 4B) and the products of
these genes had very different cellular and molecular functions
compared to probe 1 (Fig. S18†). This comparison confirms
that redox stress induced by tributylphosphine is responsible
for the upregulation of genes observed with probe 1. CHAC1
regulates redox homeostasis through degradation of GSH,25
suggesting that the cell responds to redox stress by promoting
the depletion of excess GSH produced by tributylphosphine.
CHAC1 is a component of the PERK branch of the unfolded
protein response in the endoplasmic reticulum (UPRER).26 In
our case, however, upregulation of CHAC1 is not mediated by
Fig. 4 Transcriptional response to mitochondrial reductive stress induced by tributylphosphine. (A and B) Volcano plots of differential gene
expression in cells treated with probe 1 (15 µM) (A) or with probe 3 (5 µM) (B) and compared against cells treated with DMSO. Genes and number of
genes that are significantly upregulated are depicted in blue, those that are significantly downregulated depicted in red, using thresholds of P <
0.001 and log2(probe/DMSO) >0.5 or <−0.5 obtained from biological triplicates (N = 3). (C and D) Differential regulation of genes involved in the
UPRER or UPRmt (C) or ISR (D) upon treatment of cells with probe 1 (15 µM) or probe 3 (5 µM). (E) Proposed mechanism of action of tributylphosphine
in mitochondria.
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the UPRER, because probe 1 did not trigger upregulation of the biology, can expand the chemical space of small molecules that
ER stress marker BiP (Fig. 4C), which is involved in all three are used to modulate redox biology, with potential impact in the
branches of the UPRER pathway.26 In addition, other classical development of new therapies. For example, CHAC1 upregulation
activators of the UPRER such as ATF6 or IRE1α, or target genes has been reported to deplete GSH levels in triple-negative breast
of this response, such as chaperone GRP94, phosphatase cancer cells, making them more susceptible to necroptosis and
GADD34, or disulfide isomerase PDIA6 were not affected by ferroptosis during cystine starvation.30 In this work, we demon-
either probe 1 or 3 (Fig. 4C). These results indicate that upre- strated that even though trialkylphosphines are highly reducing
gulation of CHAC1 is not triggered by the UPRER, further con- and often water-insoluble compounds, they can be transformed
firming the organelle selectivity of probe 1.
into chemical probes for biological use by developing strategies
The UPR in mitochondria (UPRmt) can be activated by to tune their reactivity, mask their reducing power, target them to
employing the mitochondria-specific HSP90 inhibitor gamitri- specific organelles, and release them selectively.
nib-triphenylphosphonium (GTPP), which leads to upregula-
tion of genes HSPD1 and HSPE1.27 Activation of tributyl-
phosphine in mitochondria did not alter transcription of
Conflicts of interest
either of these genes (Fig. 4C). Additionally, GTPP induces
parkin-mediated mitophagy,28 whereas probe 1 does not The authors have no conflicts to declare.
(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.
Acknowledgements
Our mRNA sequencing results revealed that transcription
factors ATF4, ATF3 and CHOP were upregulated by probe 1 We thank Elias Halabi (ETH Zurich) for providing the
(Fig. 4D). These observations are consistent with a recent pmTurquoise2-LAMP1 plasmid, Zacharias Thiel (ETH Zurich
multi-omics characterization of the stress response to inhibi- and EPF Lausanne) for providing the superoxide sensor probe
tors of mitochondrial import, translation, membrane potential, HKSOX-1 and Bertran Rubi (ETH Zurich) for assistance with
or oxidative phosphorylation.29 These inhibitors triggered the the LC-MS SIM method. We thank the Scientific Center for
integrated stress response (ISR), which is regulated by ATF4 and Optical and Electron Microscopy (ScopeM) at ETH Zurich and
activates CHAC1.29 This study, however, also reported significant the Bioimaging and Optics Platform (BIOP) of EPF Lausanne
upregulation of genes involved in amino acid metabolism, such for access to confocal microscopes. mRNA library generation,
as asparagine synthetase (ASNS) and phosphoserine phospha- sequencing, and preliminary bioinformatic analyses were
tase (PSPH).29 These genes were only mildly upregulated by carried out at the Functional Genomics Center Zurich. This
redox stress induced by probe 1 (Fig. 4D). Therefore, even work was supported by ETH Zurich, EPF Lausanne and the
though tributylphosphine shares some common features with Swiss
National
Science
Foundation
(SNSF
grants
various mitochondrial inhibitors, for example increased pro- 200021_165551 and PCEGP2_186862).
−
duction 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).
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