BACKGROUND
the thiol redox environment of cells is compartmentalized, with disulfide bond formation confined to the lumen of the endoplasmic reticulum  and mitochondrial inter-membrane space in eukaryotes and the periplasmic space in bacteria and largely excluded from the reducing cytosol  <cit> .
together, the tripeptide glutathione and its proteinaceous counterpart, thioredoxin, contribute to a chemical environment that maintains most cytosolic thiols in their reduced state.
the enzymatic machinery for glutathione synthesis, turnover, and reduction is localized to the cytosol, as is the thioredoxin/thioredoxin reductase couple  <cit> .
however, unlike the thioredoxin/thioredoxin reductase system that is largely isolated from the er, several lines of evidence suggest equilibration of glutathione pools between the cytosol and er.
isolated microsomes contain millimolar concentrations of glutathione  <cit> , an estimate buttressed by kinetic measurements  <cit> .
yeast genetics reveals that the kinetic defect in er disulfide bond formation wrought by lack of an important luminal thiol oxidase, ero <dig>  can be ameliorated by attenuated glutathione synthesis in the cytosol  <cit> , whereas deregulated import of glutathione across the plasma membrane into the cytosol compromises oxidative protein folding in the yeast er  <cit> .
import of reduced glutathione into the isolated rat liver microsomal fraction has been observed  <cit> , and in a functional counterpart to these experiments, excessive reduced glutathione on the cytosolic side of the plant cell er membrane compromised disulfide formation  <cit> .
in mammalian cells, experimental mislocalization of the reduced glutathione-degrading enzyme chac <dig> to the er depleted total cellular pools of glutathione  <cit> , arguing for transport of glutathione from its site of synthesis in the cytosol to the er.
despite firm evidence for the existence of a pool of reduced glutathione in the er, its functional role has remained obscure, as depleting er glutathione in cultured fibroblasts affected neither disulfide bond formation nor their reductive reshuffling  <cit> .
the er is an important source of hydrogen peroxide production.
this is partially explained by the activity of ero <dig>  which shuttles electrons from reduced thiols to molecular oxygen, converting the latter to hydrogen peroxide  <cit> .
alternative ero1-independent mechanisms for luminal hydrogen peroxide production also exist  <cit> , yet the fate of this locally generated hydrogen peroxide is not entirely clear.
some is utilized for disulfide bond formation, a process that relies on the er-localized peroxiredoxin  <dig>   <cit>  and possibly other enzymes that function as peroxiredoxins  <cit> .
however, under conditions of hydrogen peroxide hyperproduction , the peroxiredoxins that exploit the pool of reduced protein thiols in the er lumen as electron donors are unable to cope with the excess of hydrogen peroxide, and cells expressing the hyperactive ero <dig> are rendered hypersensitive to concomitant depletion of reduced glutathione  <cit> .
besides, ero <dig> overexpression leads to an increase in cell glutathione content  <cit> .
these findings suggest a role for reduced glutathione in buffering excessive er hydrogen peroxide production.
unfortunately, limitations in methods for measuring changes in the content of er luminal hydrogen peroxide have frustrated efforts to pursue this hypothesis.
here we describe the development of an optical method to track changes in hydrogen peroxide levels in the er lumen.
its application to the study of cells in which the levels of hydrogen peroxide and glutathione were selectively manipulated in the er and cytosol revealed an important role for glutathione in buffering the consequences of excessive er hydrogen peroxide production.
this process appears especially important to insulin-producing β-cells that are encumbered by a heavy burden of er hydrogen peroxide production and a deficiency of the peroxide-degrading calatase.
RESULTS
glutathione depletion exposes the hypersensitivity of pancreatic β-cells to hydrogen peroxide
insulin-producing pancreatic β-cells are relatively deficient in the hydrogen peroxide-degrading enzymes catalase and gpx <dig>  <cit>  and are thus deemed a sensitized experimental system to pursue the hypothesized role of glutathione in er hydrogen peroxide metabolism.
compared with fibroblasts, insulin-producing rinm5f cells  were noted to be hypersensitive to inhibition of glutathione biosynthesis by buthionine sulfoximine .
cytosolic catalase expression reversed this hypersensitivity to bso .fig.
<dig> glutathione depletion sensitizes pancreatic β-cells to endogenous h2o <dig>  a absorbance at 540 nm  by cultures of a β-cell line  or mouse embryonic fibroblasts  that had been exposed to the indicated concentration of buthionine sulfoximine  before fixation and staining with crystal violet.
b plot of in vitro catalase activity, reflected in time-dependent decline in absorbance  of h2o <dig> solution, exposed to lysates of untransfected rinm5f cells  or cells stably transfected with plasmids encoding cytoplasmic  or er-localized catalase .
c as in , comparing untransfected rinm5f cells  or cells stably expressing cytosolic catalase .
d fluorescent photomicrographs of rinm5f cells stably expressing a tetracycline inducible human pro-insulin gene  fixed at the indicated time points post doxycycline  exposure and immunostained for total insulin ; hoechst  <dig> was used to visualize cell nuclei .
e as in , comparing cell mass of uninduced and doxycycline-induced rinm5fteton-ins cells that had or had not been transfected with an expression plasmid encoding er catalase .
shown are mean +/– standard error of the mean , n ≥ 3
induction of pro-insulin biosynthesis via a tetracycline inducible promoter , which burdens the er with disulfide bond formation and promotes the associated production of hydrogen peroxide, contributed to the injurious effects of bso.
but these were partially reversed by the presence of er-localized catalase .
the protective effect of er-localized catalase is likely to reflect the enzymatic degradation of locally produced hydrogen peroxide, as hydrogen peroxide is slow to equilibrate between the cytosol and er  <cit> .
together these findings hint at a role for glutathione in buffering the consequences of excessive production of hydrogen peroxide in the er of pancreatic β-cells.
a probe adapted to detect h2o <dig> in thiol-oxidizing environments
to further explore the role of glutathione in the metabolism of er hydrogen peroxide, we sought to measure the effects of manipulating glutathione availability on the changing levels of er hydrogen peroxide.
exemplified by hyper  <cit> , genetically encoded optical probes responsive to changing levels of hydrogen peroxide have been developed and, via targeted localization, applied to the cytosol, peroxisome, and mitochondrial matrix .
unfortunately, in the thiol-oxidizing environment of the er, the optically sensitive disulfide in hyper  instead forms via oxidized members of the protein disulfide isomerase family , depleting the pool of reduced hyper that can sense hydrogen peroxide  <cit> .
to circumvent this limitation, we sought to develop a probe that would retain responsiveness to hydrogen peroxide in the presence of a high concentration of oxidized pdi.
hyper consists of a circularly permuted yellow fluorescent protein  grafted with the hydrogen peroxide-sensing portion of the bacterial transcription factor oxyr  <cit> .
it possesses two reactive cysteines: a peroxidatic cysteine  that reacts with h2o <dig> to form a sulfenic acid and a resolving cysteine  that attacks the sulfenic acid to form the optically distinct disulfide.
we speculated that introduction of a third cysteine, vicinal to the resolving c <dig>  might permit a rearrangement of the disulfide bonding pattern that could preserve a fraction of the peroxidatic cysteine in its reduced form and thereby preserve a measure of h2o <dig> responsiveness, even in the thiol-oxidizing environment of the er.
replacement of oxyr alanine  <dig>  with cysteine gave rise to a tri-cysteine probe, triper, that retained responsiveness to h2o <dig> in vitro but with an optical readout that was profoundly different from that of hyper: while reduced hyper exhibits a monotonic h2o <dig> and time-dependent increase in its excitability at 488 nm compared to 405 nm , in response to h2o <dig>  the r488/ <dig> of reduced triper increased transiently before settling into a new steady state .
triper’s optical response to h2o <dig> was dependent on the peroxidatic cysteine , as its replacement by serine eliminated all responsiveness .
r <dig> supports the peroxidatic properties of oxyr’s c <dig>  likely by de-protonation of the reactive thiol  <cit> .
the r266a mutation similarly abolished h2o <dig> responsiveness of hyper and triper, indicating a shared catalytic mechanism for oxyr and the two derivative probes .fig.
<dig> triper’s responsiveness to h2o <dig> in vitro.
a–c traces of time-dependent changes to the redox-sensitive excitation ratio of hyper , triper , or the triper mutant lacking its peroxidatic cysteine   in response to increasing concentrations of h2o <dig> or the reducing agent dithiothreitol .
d–g non-reducing and reducing sds-page of recombinant triper , hyper , a triper mutant lacking its peroxidatic cysteine  , and hyperc199s along hyper/triper lacking their resolving cysteine   performed following the incubation in vitro with increasing concentrations of h2o <dig> for 15 min, black arrow denotes the high molecular weight species, exclusive to triper and to hyper/triper lacking their resolving cysteine , emerging as a result of h2o <dig> induced dithiol formation in trans.
shown are representatives of n ≥ 3
the optical response to h2o <dig> of triper correlated in a dose- and time-dependent manner with formation of high molecular weight disulfide-bonded species, detectable on non-reducing sds-page .
these species were not observed in h2o2-exposed hyper, and their presence in triper depended on both the peroxidatic c <dig> and on r <dig> .
furthermore, h2o <dig> promoted such mixed disulfides in probe variants missing the resolving c <dig> or both c <dig> and the triper-specific c <dig> .
the high molecular weight triper species induced by h2o <dig> migrate anomalously on standard sds-page.
however, on neutral ph gradient sds-page their size is consistent with that of a dimer , while their formation was not accompanied by changes in r488/ <dig> in mutants lacking the ability to form c208-c <dig> disulfide .
the observations above indicate that, in the absence of c <dig>  h2o <dig> induced c <dig> sulfenic intermediates are resolved in trans and suggest that formation of the divergent c208-c <dig> pair, unique to triper, favors this alternative route.
to test this prediction we traced the r488/ <dig> of triper under conditions mimicking the oxidizing environment of the er.
triper’s time-dependent biphasic optical response  to h2o <dig> contrasted with the hyperbolic profile of its response to diamide- or pdi-mediated oxidation .
the latter is by far the most abundant er thiol-oxidizing enzyme.
pdi-catalyzed hyper oxidation likewise had a hyperbolic profile but with a noticeably higher r488/ <dig> plateau .
however, whereas triper retained responsiveness to h2o <dig>  even from its pdi-oxidized plateau, pdi-oxidized hyper lost all sensitivity to h2o <dig> .
unlike h2o <dig>  pdi did not promote formation of the disulfide-bonded high molecular weight triper species .fig.
<dig> triper’s responsiveness to h2o <dig> in a thiol-oxidizing environment.
a traces of time-dependent changes to the excitation ratio of hyper , triper , and triper mutant lacking its peroxidatic cysteine  following the introduction of oxidized pdi  or diamide .
b ratiometric traces  of hyper and triper sequentially exposed to oxidized pdi  and h2o <dig> .
c ratiometric traces  of hyper or triper exposed to h2o <dig>  followed by dtt .
the excitation spectra of the reaction phases  are analyzed in additional file 3: figure s3c.
d schema of triper oxidation pathway.
oxidants drive the formation of the optically distinct  cp199-cr <dig>  disulfide, which re-equilibrates with an optically inert  cd187-cr <dig>  disulfide in a redox relay  imposed by the three-cysteine system.
the pool of triper with a reduced peroxidatic cp <dig> thus generated is available to react with h2o <dig>  forming a sulfenic intermediate.
resolution of this intermediate in trans shifts triper to a new, optically indistinct low r488/ <dig> state, depleting the original optically distinct  intermolecular disulfide cp199-cr <dig>  this accounts for the biphasic response of triper to h2o <dig>  and for its residual responsiveness to h2o <dig> after oxidation by pdi
h2o2-driven formation of the optically active c199-c <dig> disulfide in hyper enjoys a considerable kinetic advantage over its reduction by dithiothreitol   <cit> .
this was reflected here in the high r488/ <dig> of the residual plateau of hyper co-exposed to h2o <dig> and dtt .
thus, hyper and triper traces converge at a high ratio point in the presence of h2o <dig> and dtt , a convergence that requires both c <dig> and c <dig> .
in these conditions dtt releases triper’s c <dig> from the divergent disulfide, allowing it to resolve c199-sulfenic in cis, thus confirming c199-c <dig> as the only optically distinct  disulfide.
it is worth noting that the convergence of triper and hyper traces in these conditions confirms that in both probes c199-c <dig> corresponds to the sole high ratio state, consistent with the lack of optical response in all monomeric/dimeric configurations .
thus, triper’s biphasic response to h2o <dig>  which is preserved in the face of pdi-driven oxidation , emerges from the competing h2o2-driven formation of a trans-disulfide, imparting a low r488/ <dig> .
triper detects h2o <dig> in the oxidizing er environment
to test if the promising features of triper observed in vitro enable h2o <dig> sensing in the er, we tagged triper with a signal peptide and confirmed its er localization in transfected cells .
unlike er-localized hyper, whose optical properties remained unchanged in cells exposed to h2o <dig>  er-localized triper responded with a h2o <dig> concentration-dependent decline in the r488/ <dig> .fig.
<dig> er-localized triper responds optically to exogenous h2o <dig>  a fluorescent photomicrographs of live cos <dig> cells, co-expressing triperer and pdi-mcherry as an er marker.
b time course of in vivo fluorescence excitation  ratiometric images  of hyperer or triperer from cells exposed to h2o <dig>  for the indicated time.
the excitation ratio in the images was color coded according to the map shown.
c plot showing the dependence of in vivo fluorescence excitation  ratio of triperer on h2o <dig> concentration in the culture medium.
d bar diagram of excitation ratio of triperer from untreated  cells, cells exposed to h2o <dig> , or cells 15 min after washout  .
e a ratiometric trace of triperer expressed in rinm5f cells exposed to h2o <dig>  followed by bovine catalase for the indicated duration.
f a ratiometric trace of triperer expressed alone or alongside er catalase  in rinm5f cells exposed to increasing concentrations of h2o <dig> .
shown are representatives of n ≥ 3
the h2o2-mediated changes in the optical properties of er-localized triper were readily reversed by washout or by introducing catalase into the culture media, which rapidly eliminated the h2o <dig> .
both the slow rate of diffusion of h2o <dig> into the er  and the inherent delay imposed by the two-step process entailed in triper’s responsiveness to h2o <dig>  contribute to the sluggish temporal profile of the changes observed in triper’s optical properties in cells exposed to h2o <dig>  further evidence that triper was indeed responding to changing h2o <dig> content of the er was provided by the attenuated and delayed response to exogenous h2o <dig> observed in cells expressing an er-localized catalase .
the response of triper to h2o <dig> could be tracked not only by following the changes in its excitation properties  but also by monitoring the fluorophore’s fluorescence lifetime using fluorescent lifetime imaging microscopy  .
exposure of cells expressing er triper to h2o <dig> resulted in highly reproducible increases in the fluorophore’s fluorescence lifetime .
hyper’s fluorescence lifetime was also responsive to h2o <dig>  but only in the reducing environment of the cytoplasm ; the lifetime of er-localized hyper remained unchanged in cells exposed to h2o <dig> .
these findings are consistent with nearly complete oxidation of the c199-c <dig> disulfide under basal conditions in er-localized hyper and highlight the residual h2o <dig> responsiveness of er-localized triper   <cit> .fig.
<dig> fluorescence lifetime reports on the redox status of hyper and triper.
a–c fluorescence intensity-based  and lifetime images  of rinm5f cells expressing triperer , hypercyto , or hyperer before and after exposure to h2o <dig>  .
a histogram of the distribution of lifetimes in the population of cells is provided .
d bar diagram of the fluorescence lifetime peak values from
both ratiometry and flim trace alterations in the fluorophore resulting from c199-c <dig> formation.
however, flim has important advantages over ratiometric measurements of changes in probe excitation, especially when applied to cell imaging: it is a photophysical property of the probe that is relatively independent of the ascertainment platform and indifferent to photobleaching.
therefore, although ratiometric imaging is practical for short-term tracking of single cells, flim is preferable when populations of cells exposed to divergent conditions are compared.
under basal conditions, er-localized triper’s lifetime indicated that it is found in a redox state where the c199-c <dig> pair is nearly half-oxidized , resembling that of pdi-exposed triper in vitro  and validating the use of flim to trace triper’s response to h2o <dig> in vivo.
glutathione depletion leads to h2o <dig> elevation in the er of pancreatic cells
exploiting the responsiveness of er-localized triper to h2o <dig>  we set out to measure the effect of glutathione depletion on the er h2o <dig> signal as reflected in differences in er triper’s fluorescence lifetime.
bso treatment of rinm5f cells increased the fluorescence lifetime of er triper from  <dig> +/– 43 ps at steady state to  <dig> +/– 64 ps .
a corresponding trend was also observed by measuring the changes in er triper’s excitation properties ratiometrically .fig.
<dig> er h2o <dig> increases in glutathione-depleted cells.
a bar diagram of fluorescence lifetime  of triperer expressed in the presence or absence of er catalase  in rinm5f cells containing a tetracycline inducible pro-insulin gene.
where indicated pro-insulin expression was induced by doxycycline  and the cells were exposed to  <dig>  mm bso .
b as in a, but triperer-expressing cells were exposed to  <dig>  mm bso .
c a trace of time-dependent changes in hypercyto or triperer flt in rinm5f cells after exposure to  <dig>  mm bso.
each data point represents the mean ± sd of fluorescence lifetime measured in ≥ <dig> cells.
the ordinate of hypercyto flt was inverted to harmonize the trendlines of the two probes.
d bar diagram of flt of errogfpie, expressed in cells, untreated or exposed to 2 mm dtt, the oxidizing agent  <dig> ′-dipyridyl disulfide , or  <dig>  mm bso .
e bar diagram of flt of an h2o2-unresponsive triper mutant  expressed in the er of untreated or bso-treated cells .
f bar diagram of flt of triperer expressed in the presence or absence of er catalase or an er-localized glutathione-degrading enzyme  or its enzymatically inactive e116q mutant version .
shown are mean values ± sem; *p <  <dig> , **p <  <dig> , ***p <  <dig> , n ≥ 20)
induction of pro-insulin biosynthesis accentuated triper’s response to bso , whereas expression of er catalase counteracted the bso-induced increase of triper’s fluorescence lifetime, both under baseline conditions and following stimulation of pro-insulin production in rinm5ftteton-ins cells .
these observations correlate well with the cytoprotective effect of er catalase in rinm5f cells exposed to bso .
it is noteworthy that the increase in er h2o <dig> signal in bso-treated cells was observed well before the increase in the cytosolic h2o <dig> signal  and also preceded death of the glutathione-depleted cells .
the ability of er catalase to attenuate the optical response of er-localized triper to bso or pro-insulin induction argues for an increase in er h2o <dig> as the underlying event triggering the optical response.
two further findings support this conclusion:  the disulfide state of the er-tuned redox reporter errogfpie  <cit>  remained unaffected by bso.
this argues against the possibility that the observed triper response is a consequence of a more reducing er thiol redox poise induced by glutathione depletion .
triper’s responsiveness to bso and pro-insulin induction was strictly dependent on r <dig>  a residue that does not engage in thiol redox directly, but is required for the peroxidatic activity of triper c <dig> .
in addition, the above h2o <dig> specificity controls of triper response exclude other possible artificial effects on the probe’s fluorophore, such as ph changes.
to further explore the links between glutathione depletion and accumulation of er h2o <dig>  we sought to measure the effects of selective depletion of the er pool of glutathione on the er h2o <dig> signal.
chac <dig> is a mammalian enzyme that cleaves reduced glutathione into 5-oxoproline and cysteinyl-glycine  <cit> .
we have adapted this normally cytosolic enzyme to function in the er lumen and thereby deplete the er pool of glutathione  <cit> .
enforced expression of er-localized chac <dig> in rinm5f cells led to an increase in fluorescence lifetime of er triper, which was attenuated by concomitant expression of er-localized catalase .
cysteinyl-glycine, the product of chac <dig>  has a free thiol, but its ability to balance er h2o <dig> may be affected by other factors such as clearance or protonation status.
given the relative selectivity of er-localized chac <dig> in depleting the luminal pool of glutathione , these observations further support a role for er-localized glutathione in the elimination of luminal h2o <dig>
analysis of the potential for uncatalyzed quenching of h2o <dig> by the er pool of glutathione
two molecules of reduced glutathione  can reduce a single molecule of h2o <dig>  yielding a glutathione disulfide and two molecules of water, eq.
:  <dig> 2gsh+h2o2→gssg+2h2o
however there is no evidence that the er is endowed with enzymes capable of catalyzing this thermodynamically favored reaction.
for while the er possesses two glutathione peroxidases, gpx <dig> and gpx <dig>  both lack key structural determinates for interacting with reduced glutathione and function instead as peroxiredoxins, ferrying electrons from reduced pdi to h2o <dig>  <cit> .
therefore, we revisited the feasibility of a role for the uncatalyzed reaction in h2o <dig> homeostasis in the er.
previous estimates of h2o2’s reactivity with reduced glutathione  yielded a rate constant of 22 m–1s– <dig> for the bimolecular reaction  <cit> .
exploiting the in vitro sensitivity of hyper to h2o <dig> , we revisited this issue at physiologically relevant conditions , obtaining a similar value for the second-order rate constant .fig.
<dig> kinetics of uncatalyzed elimination of h2o <dig> by reduced glutathione..
a traces of time-dependent changes to the excitation ratio  of recombinant hyper introduced into a solution of h2o <dig>  that had been preincubated with gsh  for increasing time periods, as indicated.
note the inverse relationship between hypers oxidation rate and the length of the preceding h2o2-gsh preincubation.
b as in a but following hyper introduction into premixed solutions of h2o <dig>  and reduced glutathione  at the indicated concentrations .
c as in a, but hyper was exposed sequentially to h2o <dig>  and various concentrations of gsh.
d a plot of hyper oxidation rate  as a function of gsh concentration.
the slope  and the y-intercept  of the curve were used to extract the dependence of h2o <dig> consumption rate  on each gsh concentration point  according to eqs.
–.
the bimolecular rate constant was calculated by division of the slope of the resulting curve  by the initial
considering a scenario whereby oxidative folding of pro-insulin  proceeds by the enzymatic transfer of two electrons from di-thiols to o <dig> , and given three disulfides in pro-insulin, a maximal production rate of 6*10- <dig> fmol pro-insulin/min/cell  <cit> , and an er volume of 280 fl  <cit> , the resultant maximal generation rate of h2o <dig> has the potential to elevate its concentration by  <dig>  μm s– <dig>  given the rate constant of 29 m–1s– <dig> for the bimolecular reaction of h2o <dig> with reduced glutathione and an estimated er concentration of 15 mm gsh  <cit> , at this production rate the concentration of h2o <dig> would stabilize at  <dig>  μm, based solely on uncatalyzed reduction by gsh.
parallel processes that consume h2o <dig> to generate disulfide bonds would tend to push this concentration even lower  <cit> ; nonetheless, this calculation indicates that gsh can play an important role in the uncatalyzed elimination of h2o <dig> from the er.
discussion
the sensitivity of β-cells to glutathione depletion, the accentuation of this toxic effect by pro-insulin synthesis, and the ability of er catalase to counteract these challenges all hinted at a role for glutathione in coping with the burden of h2o <dig> produced in the er.
however, without means to track h2o <dig> in the er of living cells, this would have remained an untested idea.
triper has revealed that er hydrogen peroxide levels increase with increased production of disulfide bonds in secreted proteins, providing direct evidence that the oxidative machinery of the er does indeed produce h2o <dig> as a product.
less anticipated has been the increased h2o <dig> in the er of glutathione-depleted cells.
the contribution of glutathione to a reducing milieu in the nucleus and cytoplasm is well established, but it has not been easy to rationalize its presence in the oxidizing er, especially as glutathione appears dispensable for the reductive step of disulfide isomerization in oxidative protein folding  <cit> .
consistently, the er thiol redox poise resisted glutathione depletion.
triper has thus pointed to a role for glutathione in buffering er h2o <dig> production, providing a plausible benefit from the presence of a glutathione pool in the er lumen.
triper’s ability to sense h2o <dig> in a thiol-oxidizing environment relies on the presence of an additional cysteine residue  near the resolving c <dig> in the oxyr segment.
the presence of this additional cysteine attenuates the optical responsiveness of the probe to oxidation by pdi by creating a diversionary disulfide involving c <dig>  importantly, this diversionary disulfide, which forms at the expense of the  c199-c <dig> disulfide, preserves a fraction of the peroxidatic c <dig> in its reduced form.
thus, even in the thiol-oxidizing environment of the er, a fraction of c <dig> thiolate is free to form a reversible h2o2-driven sulfenic intermediate that is resolved by disulfide formation in trans.
furthermore, by preserving a fraction of c <dig> in its reduced form, the diversionary c187-c <dig> disulfide also maintains a pool of c <dig> to resolve the c <dig> sulfenic to form a trans-disulfide bond.
thus, the h2o2-induced formation of the trans disulfide is a feature unique to triper, and it too is a consequence of the diversionary disulfide, which eliminates the strongly competing reaction of the resolving c <dig> in cis with the sulfenic acid at c <dig>
the ability of er-expressed triper to alter its optical properties in response to h2o <dig> is enabled through its semioxidized steady state, dictated by kinetically/quantitatively dominant pdi.
in vitro this stage can be reached by oxidants such as diamide or pdi, while the second oxidation phase, with lowering r488/ <dig>  is exclusive to h2o <dig>  the net result of the presence of a diversionary thiol at triper residue  <dig>  is to render the probe optically sensitive to h2o <dig> even in the presence of high concentrations of oxidized pdi, conditions in which the precursor probe, hyper, loses all optical responsiveness.
the aforementioned theoretical arguments for triper’s direct responsiveness to h2o <dig> are further supported by empirical observations: the peroxidatic potential  of the probe’s c <dig> is enabled through a finely balance charge distribution in its vicinity, of which r <dig> is a crucial determinant  <cit> .
eliminating this charge yielded a probe variant with its intact cysteine system, but it was unresponsive to h2o <dig>  further, the ability of er catalase to reverse the changes in triper’s disposition argues that these are initiated by changes in h2o <dig> concentration.
both hyper and triper react with elements of the prevailing er thiol redox buffering system .
in the case of hyper, this reactivity is ruinous, but even in the case of er triper, which retains a modicum of sensitivity to h2o <dig>  the elements of the complex kinetic regime that drive its redox state are not understood in quantitative terms.
thus, it is impossible to fully deconvolute the potential impact on triper of changes in the er thiol redox milieu from changes in h2o <dig> concentration wrought by a given physiological perturbation — triper is sensitive to both.
however, it is noteworthy that oxidation of triper by h2o <dig>  leading to a mixed disulfide state, shifts the optical readout towards lower r488/ <dig> and shorter fluorescent lifetimes.
although such shifts are also consistent with a surge in thiol reductive activity, it seems unlikely that exposure of cells to h2o <dig> results in a more thiol-reducing er.
similar considerations apply to the state of the er in cells depleted of glutathione, as it is hard to imagine how this would lead to a more thiol-reducing er.
indeed the h2o2-insensitive redox probe rogfpie, which is known to equilibrate with er-localized pdi, was unaffected by glutathione depletion.
thus, while we cannot formally exclude that glutathione depletion also affects triper’s redox status independently of changes in h2o <dig> concentration, the bulk of the evidence favors a role for triper in tracking the latter and in reporting on an increase in er h2o <dig> in glutathione-depleted cells.
triper has been instrumental in flagging glutathione’s role in buffering er luminal h2o <dig>  this raises the question as to whether the thermodynamically favored reduction of h2o <dig> by gsh is accelerated by er-localized enzymes or proceeds by uncatalyzed mass action.
the cytoplasm and mitochondria possess peroxide-consuming enzymes that are fueled by reduced glutathione  <cit> .
however, the er lacks known counterparts.
such enzymes may be discovered in the future, as well as possible pathways of gsh-mediated pdrx <dig> modulation.
but meanwhile it is notable that the kinetic properties of the uncatalyzed reduction of h2o <dig> by gsh are also consistent with a contribution to keeping h2o <dig> concentration at bay.
an er that eschews catalyzed reduction of h2o <dig> by gsh and relies instead on the slower uncatalyzed reaction may acquire certain advantages.
at low concentrations of h2o <dig>  an er organized along these lines would be flexible to deploy a kinetically favored prdx4-mediated peroxidation reaction to exploit h2o <dig> generated by ero <dig> for further disulfide bond formation  <cit> .
at higher concentrations of h2o <dig>  prdx <dig> inactivation  <cit>  limits the utility of a prdx4-based coping strategy  <cit> .
however, the concentration-dependent reduction of h2o <dig> by gsh is poised to counteract a build-up of er h2o <dig>  although uncertainty regarding the rate of h2o <dig> transported across the er membrane exists  <cit> , we favor a model whereby such transport is comparatively slow  <cit> , which is consistent with the observed delay between the increase in er and cytosolic h2o <dig> when glutathione synthesis was inhibited.
sequestration of h2o <dig> in the lumen of the er protects the genome from this potentially harmful metabolite and enables the higher concentrations needed for the uncatalyzed reaction to progress at a reasonable pace.
the implementation of a probe that detects h2o <dig> in thiol-oxidizing environments has revealed a remarkably simple mechanism to defend the cytosol and nucleus from a product of oxidative protein folding in the er.
this mechanism is especially important in secretory pancreatic β-cells that are poorly equipped with catalase/peroxidase.
CONCLUSIONS
here we report on the development and mechanistic characterization of an optical probe, triper, that circumvents the limitations of previous sensors by retaining specific responsiveness to h2o <dig> in thiol-oxidizing environments.
application of this tool to the er of an insulin-producing pancreatic β-cells model system revealed that er glutathione antagonizes locally produced and potentially cytotoxic h2o <dig>  resulting from the oxidative folding of pro-insulin.
the redox biochemistry concept developed here sets a precedent for exploiting a tri-cysteine relay system to discriminate between various oxidative reactants in complex redox milieux.

 the thiol redox environment of cells is compartmentalized, with disulfide bond formation confined to the lumen of the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasmic space in bacteria and largely excluded from the reducing cytosol. together, the tripeptide glutathione and its proteinaceous counterpart, thioredoxin, contribute to a chemical environment that maintains most cytosolic thiols in their reduced state. the enzymatic machinery for glutathione synthesis, turnover, and reduction is localized to the cytosol, as is the thioredoxin/thioredoxin reductase couple. however, unlike the thioredoxin/thioredoxin reductase system that is largely isolated from the er, several lines of evidence suggest equilibration of glutathione pools between the cytosol and er. isolated microsomes contain millimolar concentrations of glutathione, an estimate buttressed by kinetic measurements. yeast genetics reveals that the kinetic defect in er disulfide bond formation wrought by lack of an important luminal thiol oxidase, ero can be ameliorated by attenuated glutathione synthesis in the cytosol, whereas deregulated import of glutathione across the plasma membrane into the cytosol compromises oxidative protein folding in the yeast er. import of reduced glutathione into the isolated rat liver microsomal fraction has been observed, and in a functional counterpart to these experiments, excessive reduced glutathione on the cytosolic side of the plant cell er membrane compromised disulfide formation. in mammalian cells, experimental mislocalization of the reduced glutathione-degrading enzyme chac to the er depleted total cellular pools of glutathione, arguing for transport of glutathione from its site of synthesis in the cytosol to the er. despite firm evidence for the existence of a pool of reduced glutathione in the er, its functional role has remained obscure, as depleting er glutathione in cultured fibroblasts affected neither disulfide bond formation nor their reductive reshuffling. the er is an important source of hydrogen peroxide production. this is partially explained by the activity of ero which shuttles electrons from reduced thiols to molecular oxygen, converting the latter to hydrogen peroxide. alternative ero1-independent mechanisms for luminal hydrogen peroxide production also exist, yet the fate of this locally generated hydrogen peroxide is not entirely clear. some is utilized for disulfide bond formation, a process that relies on the er-localized peroxiredoxin and possibly other enzymes that function as peroxiredoxins. however, under conditions of hydrogen peroxide hyperproduction, the peroxiredoxins that exploit the pool of reduced protein thiols in the er lumen as electron donors are unable to cope with the excess of hydrogen peroxide, and cells expressing the hyperactive ero are rendered hypersensitive to concomitant depletion of reduced glutathione. besides, ero overexpression leads to an increase in cell glutathione content. these findings suggest a role for reduced glutathione in buffering excessive er hydrogen peroxide production. unfortunately, limitations in methods for measuring changes in the content of er luminal hydrogen peroxide have frustrated efforts to pursue this hypothesis. here we describe the development of an optical method to track changes in hydrogen peroxide levels in the er lumen. its application to the study of cells in which the levels of hydrogen peroxide and glutathione were selectively manipulated in the er and cytosol revealed an important role for glutathione in buffering the consequences of excessive er hydrogen peroxide production. this process appears especially important to insulin-producing β-cells that are encumbered by a heavy burden of er hydrogen peroxide production and a deficiency of the peroxide-degrading calatase.  glutathione depletion exposes the hypersensitivity of pancreatic β-cells to hydrogen peroxide insulin-producing pancreatic β-cells are relatively deficient in the hydrogen peroxide-degrading enzymes catalase and gpx and are thus deemed a sensitized experimental system to pursue the hypothesized role of glutathione in er hydrogen peroxide metabolism. compared with fibroblasts, insulin-producing rinm5f cells were noted to be hypersensitive to inhibition of glutathione biosynthesis by buthionine sulfoximine. cytosolic catalase expression reversed this hypersensitivity to bso.fig. glutathione depletion sensitizes pancreatic β-cells to endogenous h2o a absorbance at 540 nm by cultures of a β-cell line or mouse embryonic fibroblasts that had been exposed to the indicated concentration of buthionine sulfoximine before fixation and staining with crystal violet. b plot of in vitro catalase activity, reflected in time-dependent decline in absorbance of h2o solution, exposed to lysates of untransfected rinm5f cells or cells stably transfected with plasmids encoding cytoplasmic or er-localized catalase. c as in, comparing untransfected rinm5f cells or cells stably expressing cytosolic catalase. d fluorescent photomicrographs of rinm5f cells stably expressing a tetracycline inducible human pro-insulin gene fixed at the indicated time points post doxycycline exposure and immunostained for total insulin; hoechst was used to visualize cell nuclei. e as in, comparing cell mass of uninduced and doxycycline-induced rinm5fteton-ins cells that had or had not been transfected with an expression plasmid encoding er catalase. shown are mean +/– standard error of the mean, n ≥ 3 induction of pro-insulin biosynthesis via a tetracycline inducible promoter, which burdens the er with disulfide bond formation and promotes the associated production of hydrogen peroxide, contributed to the injurious effects of bso. but these were partially reversed by the presence of er-localized catalase. the protective effect of er-localized catalase is likely to reflect the enzymatic degradation of locally produced hydrogen peroxide, as hydrogen peroxide is slow to equilibrate between the cytosol and er. together these findings hint at a role for glutathione in buffering the consequences of excessive production of hydrogen peroxide in the er of pancreatic β-cells. a probe adapted to detect h2o in thiol-oxidizing environments to further explore the role of glutathione in the metabolism of er hydrogen peroxide, we sought to measure the effects of manipulating glutathione availability on the changing levels of er hydrogen peroxide. exemplified by hyper, genetically encoded optical probes responsive to changing levels of hydrogen peroxide have been developed and, via targeted localization, applied to the cytosol, peroxisome, and mitochondrial matrix. unfortunately, in the thiol-oxidizing environment of the er, the optically sensitive disulfide in hyper instead forms via oxidized members of the protein disulfide isomerase family, depleting the pool of reduced hyper that can sense hydrogen peroxide. to circumvent this limitation, we sought to develop a probe that would retain responsiveness to hydrogen peroxide in the presence of a high concentration of oxidized pdi. hyper consists of a circularly permuted yellow fluorescent protein grafted with the hydrogen peroxide-sensing portion of the bacterial transcription factor oxyr. it possesses two reactive cysteines: a peroxidatic cysteine that reacts with h2o to form a sulfenic acid and a resolving cysteine that attacks the sulfenic acid to form the optically distinct disulfide. we speculated that introduction of a third cysteine, vicinal to the resolving c might permit a rearrangement of the disulfide bonding pattern that could preserve a fraction of the peroxidatic cysteine in its reduced form and thereby preserve a measure of h2o responsiveness, even in the thiol-oxidizing environment of the er. replacement of oxyr alanine with cysteine gave rise to a tri-cysteine probe, triper, that retained responsiveness to h2o in vitro but with an optical readout that was profoundly different from that of hyper: while reduced hyper exhibits a monotonic h2o and time-dependent increase in its excitability at 488 nm compared to 405 nm, in response to h2o the r488/ of reduced triper increased transiently before settling into a new steady state. triper’s optical response to h2o was dependent on the peroxidatic cysteine, as its replacement by serine eliminated all responsiveness. r supports the peroxidatic properties of oxyr’s c likely by de-protonation of the reactive thiol. the r266a mutation similarly abolished h2o responsiveness of hyper and triper, indicating a shared catalytic mechanism for oxyr and the two derivative probes.fig. triper’s responsiveness to h2o in vitro. a–c traces of time-dependent changes to the redox-sensitive excitation ratio of hyper, triper, or the triper mutant lacking its peroxidatic cysteine in response to increasing concentrations of h2o or the reducing agent dithiothreitol. d–g non-reducing and reducing sds-page of recombinant triper, hyper, a triper mutant lacking its peroxidatic cysteine, and hyperc199s along hyper/triper lacking their resolving cysteine performed following the incubation in vitro with increasing concentrations of h2o for 15 min, black arrow denotes the high molecular weight species, exclusive to triper and to hyper/triper lacking their resolving cysteine, emerging as a result of h2o induced dithiol formation in trans. shown are representatives of n ≥ 3 the optical response to h2o of triper correlated in a dose- and time-dependent manner with formation of high molecular weight disulfide-bonded species, detectable on non-reducing sds-page. these species were not observed in h2o2-exposed hyper, and their presence in triper depended on both the peroxidatic c and on r. furthermore, h2o promoted such mixed disulfides in probe variants missing the resolving c or both c and the triper-specific c. the high molecular weight triper species induced by h2o migrate anomalously on standard sds-page. however, on neutral ph gradient sds-page their size is consistent with that of a dimer, while their formation was not accompanied by changes in r488/ in mutants lacking the ability to form c208-c disulfide. the observations above indicate that, in the absence of c h2o induced c sulfenic intermediates are resolved in trans and suggest that formation of the divergent c208-c pair, unique to triper, favors this alternative route. to test this prediction we traced the r488/ of triper under conditions mimicking the oxidizing environment of the er. triper’s time-dependent biphasic optical response to h2o contrasted with the hyperbolic profile of its response to diamide- or pdi-mediated oxidation. the latter is by far the most abundant er thiol-oxidizing enzyme. pdi-catalyzed hyper oxidation likewise had a hyperbolic profile but with a noticeably higher r488/ plateau. however, whereas triper retained responsiveness to h2o even from its pdi-oxidized plateau, pdi-oxidized hyper lost all sensitivity to h2o. unlike h2o pdi did not promote formation of the disulfide-bonded high molecular weight triper species.fig. triper’s responsiveness to h2o in a thiol-oxidizing environment. a traces of time-dependent changes to the excitation ratio of hyper, triper, and triper mutant lacking its peroxidatic cysteine following the introduction of oxidized pdi or diamide. b ratiometric traces of hyper and triper sequentially exposed to oxidized pdi and h2o. c ratiometric traces of hyper or triper exposed to h2o followed by dtt. the excitation spectra of the reaction phases are analyzed in additional file 3: figure s3c. d schema of triper oxidation pathway. oxidants drive the formation of the optically distinct cp199-cr disulfide, which re-equilibrates with an optically inert cd187-cr disulfide in a redox relay imposed by the three-cysteine system. the pool of triper with a reduced peroxidatic cp thus generated is available to react with h2o forming a sulfenic intermediate. resolution of this intermediate in trans shifts triper to a new, optically indistinct low r488/ state, depleting the original optically distinct intermolecular disulfide cp199-cr this accounts for the biphasic response of triper to h2o and for its residual responsiveness to h2o after oxidation by pdi h2o2-driven formation of the optically active c199-c disulfide in hyper enjoys a considerable kinetic advantage over its reduction by dithiothreitol. this was reflected here in the high r488/ of the residual plateau of hyper co-exposed to h2o and dtt. thus, hyper and triper traces converge at a high ratio point in the presence of h2o and dtt, a convergence that requires both c and c. in these conditions dtt releases triper’s c from the divergent disulfide, allowing it to resolve c199-sulfenic in cis, thus confirming c199-c as the only optically distinct disulfide. it is worth noting that the convergence of triper and hyper traces in these conditions confirms that in both probes c199-c corresponds to the sole high ratio state, consistent with the lack of optical response in all monomeric/dimeric configurations. thus, triper’s biphasic response to h2o which is preserved in the face of pdi-driven oxidation, emerges from the competing h2o2-driven formation of a trans-disulfide, imparting a low r488/. triper detects h2o in the oxidizing er environment to test if the promising features of triper observed in vitro enable h2o sensing in the er, we tagged triper with a signal peptide and confirmed its er localization in transfected cells. unlike er-localized hyper, whose optical properties remained unchanged in cells exposed to h2o er-localized triper responded with a h2o concentration-dependent decline in the r488/.fig. er-localized triper responds optically to exogenous h2o a fluorescent photomicrographs of live cos cells, co-expressing triperer and pdi-mcherry as an er marker. b time course of in vivo fluorescence excitation ratiometric images of hyperer or triperer from cells exposed to h2o for the indicated time. the excitation ratio in the images was color coded according to the map shown. c plot showing the dependence of in vivo fluorescence excitation ratio of triperer on h2o concentration in the culture medium. d bar diagram of excitation ratio of triperer from untreated cells, cells exposed to h2o, or cells 15 min after washout. e a ratiometric trace of triperer expressed in rinm5f cells exposed to h2o followed by bovine catalase for the indicated duration. f a ratiometric trace of triperer expressed alone or alongside er catalase in rinm5f cells exposed to increasing concentrations of h2o. shown are representatives of n ≥ 3 the h2o2-mediated changes in the optical properties of er-localized triper were readily reversed by washout or by introducing catalase into the culture media, which rapidly eliminated the h2o. both the slow rate of diffusion of h2o into the er and the inherent delay imposed by the two-step process entailed in triper’s responsiveness to h2o contribute to the sluggish temporal profile of the changes observed in triper’s optical properties in cells exposed to h2o further evidence that triper was indeed responding to changing h2o content of the er was provided by the attenuated and delayed response to exogenous h2o observed in cells expressing an er-localized catalase. the response of triper to h2o could be tracked not only by following the changes in its excitation properties but also by monitoring the fluorophore’s fluorescence lifetime using fluorescent lifetime imaging microscopy. exposure of cells expressing er triper to h2o resulted in highly reproducible increases in the fluorophore’s fluorescence lifetime. hyper’s fluorescence lifetime was also responsive to h2o but only in the reducing environment of the cytoplasm; the lifetime of er-localized hyper remained unchanged in cells exposed to h2o. these findings are consistent with nearly complete oxidation of the c199-c disulfide under basal conditions in er-localized hyper and highlight the residual h2o responsiveness of er-localized triper.fig. fluorescence lifetime reports on the redox status of hyper and triper. a–c fluorescence intensity-based and lifetime images of rinm5f cells expressing triperer, hypercyto, or hyperer before and after exposure to h2o. a histogram of the distribution of lifetimes in the population of cells is provided. d bar diagram of the fluorescence lifetime peak values from both ratiometry and flim trace alterations in the fluorophore resulting from c199-c formation. however, flim has important advantages over ratiometric measurements of changes in probe excitation, especially when applied to cell imaging: it is a photophysical property of the probe that is relatively independent of the ascertainment platform and indifferent to photobleaching. therefore, although ratiometric imaging is practical for short-term tracking of single cells, flim is preferable when populations of cells exposed to divergent conditions are compared. under basal conditions, er-localized triper’s lifetime indicated that it is found in a redox state where the c199-c pair is nearly half-oxidized, resembling that of pdi-exposed triper in vitro and validating the use of flim to trace triper’s response to h2o in vivo. glutathione depletion leads to h2o elevation in the er of pancreatic cells exploiting the responsiveness of er-localized triper to h2o we set out to measure the effect of glutathione depletion on the er h2o signal as reflected in differences in er triper’s fluorescence lifetime. bso treatment of rinm5f cells increased the fluorescence lifetime of er triper from +/– 43 ps at steady state to +/– 64 ps. a corresponding trend was also observed by measuring the changes in er triper’s excitation properties ratiometrically.fig. er h2o increases in glutathione-depleted cells. a bar diagram of fluorescence lifetime of triperer expressed in the presence or absence of er catalase in rinm5f cells containing a tetracycline inducible pro-insulin gene. where indicated pro-insulin expression was induced by doxycycline and the cells were exposed to  mm bso. b as in a, but triperer-expressing cells were exposed to  mm bso. c a trace of time-dependent changes in hypercyto or triperer flt in rinm5f cells after exposure to  mm bso. each data point represents the mean ± sd of fluorescence lifetime measured in ≥ cells. the ordinate of hypercyto flt was inverted to harmonize the trendlines of the two probes. d bar diagram of flt of errogfpie, expressed in cells, untreated or exposed to 2 mm dtt, the oxidizing agent ′-dipyridyl disulfide, or  mm bso. e bar diagram of flt of an h2o2-unresponsive triper mutant expressed in the er of untreated or bso-treated cells. f bar diagram of flt of triperer expressed in the presence or absence of er catalase or an er-localized glutathione-degrading enzyme or its enzymatically inactive e116q mutant version. shown are mean values ± sem; *p < , **p < , ***p < , n ≥ 20) induction of pro-insulin biosynthesis accentuated triper’s response to bso, whereas expression of er catalase counteracted the bso-induced increase of triper’s fluorescence lifetime, both under baseline conditions and following stimulation of pro-insulin production in rinm5ftteton-ins cells. these observations correlate well with the cytoprotective effect of er catalase in rinm5f cells exposed to bso. it is noteworthy that the increase in er h2o signal in bso-treated cells was observed well before the increase in the cytosolic h2o signal and also preceded death of the glutathione-depleted cells. the ability of er catalase to attenuate the optical response of er-localized triper to bso or pro-insulin induction argues for an increase in er h2o as the underlying event triggering the optical response. two further findings support this conclusion: the disulfide state of the er-tuned redox reporter errogfpie remained unaffected by bso. this argues against the possibility that the observed triper response is a consequence of a more reducing er thiol redox poise induced by glutathione depletion. triper’s responsiveness to bso and pro-insulin induction was strictly dependent on r a residue that does not engage in thiol redox directly, but is required for the peroxidatic activity of triper c. in addition, the above h2o specificity controls of triper response exclude other possible artificial effects on the probe’s fluorophore, such as ph changes. to further explore the links between glutathione depletion and accumulation of er h2o we sought to measure the effects of selective depletion of the er pool of glutathione on the er h2o signal. chac is a mammalian enzyme that cleaves reduced glutathione into 5-oxoproline and cysteinyl-glycine. we have adapted this normally cytosolic enzyme to function in the er lumen and thereby deplete the er pool of glutathione. enforced expression of er-localized chac in rinm5f cells led to an increase in fluorescence lifetime of er triper, which was attenuated by concomitant expression of er-localized catalase. cysteinyl-glycine, the product of chac has a free thiol, but its ability to balance er h2o may be affected by other factors such as clearance or protonation status. given the relative selectivity of er-localized chac in depleting the luminal pool of glutathione, these observations further support a role for er-localized glutathione in the elimination of luminal h2o analysis of the potential for uncatalyzed quenching of h2o by the er pool of glutathione two molecules of reduced glutathione can reduce a single molecule of h2o yielding a glutathione disulfide and two molecules of water, eq.: 2gsh+h2o2→gssg+2h2o however there is no evidence that the er is endowed with enzymes capable of catalyzing this thermodynamically favored reaction. for while the er possesses two glutathione peroxidases, gpx and gpx both lack key structural determinates for interacting with reduced glutathione and function instead as peroxiredoxins, ferrying electrons from reduced pdi to h2o. therefore, we revisited the feasibility of a role for the uncatalyzed reaction in h2o homeostasis in the er. previous estimates of h2o2’s reactivity with reduced glutathione yielded a rate constant of 22 m–1s– for the bimolecular reaction. exploiting the in vitro sensitivity of hyper to h2o, we revisited this issue at physiologically relevant conditions, obtaining a similar value for the second-order rate constant.fig. kinetics of uncatalyzed elimination of h2o by reduced glutathione.. a traces of time-dependent changes to the excitation ratio of recombinant hyper introduced into a solution of h2o that had been preincubated with gsh for increasing time periods, as indicated. note the inverse relationship between hypers oxidation rate and the length of the preceding h2o2-gsh preincubation. b as in a but following hyper introduction into premixed solutions of h2o and reduced glutathione at the indicated concentrations. c as in a, but hyper was exposed sequentially to h2o and various concentrations of gsh. d a plot of hyper oxidation rate as a function of gsh concentration. the slope and the y-intercept of the curve were used to extract the dependence of h2o consumption rate on each gsh concentration point according to eqs. –. the bimolecular rate constant was calculated by division of the slope of the resulting curve by the initial considering a scenario whereby oxidative folding of pro-insulin proceeds by the enzymatic transfer of two electrons from di-thiols to o, and given three disulfides in pro-insulin, a maximal production rate of 6*10- fmol pro-insulin/min/cell, and an er volume of 280 fl, the resultant maximal generation rate of h2o has the potential to elevate its concentration by  μm s– given the rate constant of 29 m–1s– for the bimolecular reaction of h2o with reduced glutathione and an estimated er concentration of 15 mm gsh, at this production rate the concentration of h2o would stabilize at  μm, based solely on uncatalyzed reduction by gsh. parallel processes that consume h2o to generate disulfide bonds would tend to push this concentration even lower; nonetheless, this calculation indicates that gsh can play an important role in the uncatalyzed elimination of h2o from the er. discussion the sensitivity of β-cells to glutathione depletion, the accentuation of this toxic effect by pro-insulin synthesis, and the ability of er catalase to counteract these challenges all hinted at a role for glutathione in coping with the burden of h2o produced in the er. however, without means to track h2o in the er of living cells, this would have remained an untested idea. triper has revealed that er hydrogen peroxide levels increase with increased production of disulfide bonds in secreted proteins, providing direct evidence that the oxidative machinery of the er does indeed produce h2o as a product. less anticipated has been the increased h2o in the er of glutathione-depleted cells. the contribution of glutathione to a reducing milieu in the nucleus and cytoplasm is well established, but it has not been easy to rationalize its presence in the oxidizing er, especially as glutathione appears dispensable for the reductive step of disulfide isomerization in oxidative protein folding. consistently, the er thiol redox poise resisted glutathione depletion. triper has thus pointed to a role for glutathione in buffering er h2o production, providing a plausible benefit from the presence of a glutathione pool in the er lumen. triper’s ability to sense h2o in a thiol-oxidizing environment relies on the presence of an additional cysteine residue near the resolving c in the oxyr segment. the presence of this additional cysteine attenuates the optical responsiveness of the probe to oxidation by pdi by creating a diversionary disulfide involving c importantly, this diversionary disulfide, which forms at the expense of the c199-c disulfide, preserves a fraction of the peroxidatic c in its reduced form. thus, even in the thiol-oxidizing environment of the er, a fraction of c thiolate is free to form a reversible h2o2-driven sulfenic intermediate that is resolved by disulfide formation in trans. furthermore, by preserving a fraction of c in its reduced form, the diversionary c187-c disulfide also maintains a pool of c to resolve the c sulfenic to form a trans-disulfide bond. thus, the h2o2-induced formation of the trans disulfide is a feature unique to triper, and it too is a consequence of the diversionary disulfide, which eliminates the strongly competing reaction of the resolving c in cis with the sulfenic acid at c the ability of er-expressed triper to alter its optical properties in response to h2o is enabled through its semioxidized steady state, dictated by kinetically/quantitatively dominant pdi. in vitro this stage can be reached by oxidants such as diamide or pdi, while the second oxidation phase, with lowering r488/ is exclusive to h2o the net result of the presence of a diversionary thiol at triper residue is to render the probe optically sensitive to h2o even in the presence of high concentrations of oxidized pdi, conditions in which the precursor probe, hyper, loses all optical responsiveness. the aforementioned theoretical arguments for triper’s direct responsiveness to h2o are further supported by empirical observations: the peroxidatic potential of the probe’s c is enabled through a finely balance charge distribution in its vicinity, of which r is a crucial determinant. eliminating this charge yielded a probe variant with its intact cysteine system, but it was unresponsive to h2o further, the ability of er catalase to reverse the changes in triper’s disposition argues that these are initiated by changes in h2o concentration. both hyper and triper react with elements of the prevailing er thiol redox buffering system. in the case of hyper, this reactivity is ruinous, but even in the case of er triper, which retains a modicum of sensitivity to h2o the elements of the complex kinetic regime that drive its redox state are not understood in quantitative terms. thus, it is impossible to fully deconvolute the potential impact on triper of changes in the er thiol redox milieu from changes in h2o concentration wrought by a given physiological perturbation — triper is sensitive to both. however, it is noteworthy that oxidation of triper by h2o leading to a mixed disulfide state, shifts the optical readout towards lower r488/ and shorter fluorescent lifetimes. although such shifts are also consistent with a surge in thiol reductive activity, it seems unlikely that exposure of cells to h2o results in a more thiol-reducing er. similar considerations apply to the state of the er in cells depleted of glutathione, as it is hard to imagine how this would lead to a more thiol-reducing er. indeed the h2o2-insensitive redox probe rogfpie, which is known to equilibrate with er-localized pdi, was unaffected by glutathione depletion. thus, while we cannot formally exclude that glutathione depletion also affects triper’s redox status independently of changes in h2o concentration, the bulk of the evidence favors a role for triper in tracking the latter and in reporting on an increase in er h2o in glutathione-depleted cells. triper has been instrumental in flagging glutathione’s role in buffering er luminal h2o this raises the question as to whether the thermodynamically favored reduction of h2o by gsh is accelerated by er-localized enzymes or proceeds by uncatalyzed mass action. the cytoplasm and mitochondria possess peroxide-consuming enzymes that are fueled by reduced glutathione. however, the er lacks known counterparts. such enzymes may be discovered in the future, as well as possible pathways of gsh-mediated pdrx modulation. but meanwhile it is notable that the kinetic properties of the uncatalyzed reduction of h2o by gsh are also consistent with a contribution to keeping h2o concentration at bay. an er that eschews catalyzed reduction of h2o by gsh and relies instead on the slower uncatalyzed reaction may acquire certain advantages. at low concentrations of h2o an er organized along these lines would be flexible to deploy a kinetically favored prdx4-mediated peroxidation reaction to exploit h2o generated by ero for further disulfide bond formation. at higher concentrations of h2o prdx inactivation limits the utility of a prdx4-based coping strategy. however, the concentration-dependent reduction of h2o by gsh is poised to counteract a build-up of er h2o although uncertainty regarding the rate of h2o transported across the er membrane exists, we favor a model whereby such transport is comparatively slow, which is consistent with the observed delay between the increase in er and cytosolic h2o when glutathione synthesis was inhibited. sequestration of h2o in the lumen of the er protects the genome from this potentially harmful metabolite and enables the higher concentrations needed for the uncatalyzed reaction to progress at a reasonable pace. the implementation of a probe that detects h2o in thiol-oxidizing environments has revealed a remarkably simple mechanism to defend the cytosol and nucleus from a product of oxidative protein folding in the er. this mechanism is especially important in secretory pancreatic β-cells that are poorly equipped with catalase/peroxidase.  here we report on the development and mechanistic characterization of an optical probe, triper, that circumvents the limitations of previous sensors by retaining specific responsiveness to h2o in thiol-oxidizing environments. application of this tool to the er of an insulin-producing pancreatic β-cells model system revealed that er glutathione antagonizes locally produced and potentially cytotoxic h2o resulting from the oxidative folding of pro-insulin. the redox biochemistry concept developed here sets a precedent for exploiting a tri-cysteine relay system to discriminate between various oxidative reactants in complex redox milieux. 
interfering with glutathione production in the cytosol by buthionine sulfoximine or enhancing its localized destruction by expression of the glutathione-degrading enzyme chac in the lumen of the er further enhanced the luminal h2o signal and eroded β-cell viability. over the years hints have suggested that glutathione, puzzlingly abundant in the er lumen, might have a role in reducing the heavy burden of h2o produced by the luminal enzymatic machinery for disulfide bond formation. consistent with the hypothesized contribution of oxidative protein folding to h2o production, er-localized triper detected an increase in the luminal h2o signal upon induction of pro-insulin, which was attenuated by the ectopic expression of catalase in the er lumen. the fate of hydrogen peroxide in the endoplasmic reticulum has been inferred indirectly from the activity of er-localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. endoplasmic reticulumredoxh2o probehydrogen peroxideglutathionefluorescent protein sensorfluorescence lifetime imaginglive cell imagingpancreatic β-cellswellcome trust 200848/z/16/zron david fundação para a ciência e a tecnologia ptdc/qui/biq/119677/2010uid/bim/04773/2013-cbmrmelo eduardo pinho european commission eu fp beta-bat no: 277713ron david http://dx.doi.org/ /100004440wellcome trustuns18966kaminski clemens f. engineering and physical sciences research council 1503478kaminski clemens f. medical research council mr/k015850/1kaminski clemens f. issue-copyright-statement© the author 2017
 the fate of hydrogen peroxide in the endoplasmic reticulum has been inferred indirectly from the activity of er-localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. over the years hints have suggested that glutathione, puzzlingly abundant in the er lumen, might have a role in reducing the heavy burden of h2o produced by the luminal enzymatic machinery for disulfide bond formation. however, limitations in existing organelle-targeted h2o probes have rendered them inert in the thiol-oxidizing er, precluding experimental follow-up of glutathione’s role in er h2o metabolism.  here we report on the development of triper, a vital optical probe sensitive to changes in the concentration of h2o in the thiol-oxidizing environment of the er. consistent with the hypothesized contribution of oxidative protein folding to h2o production, er-localized triper detected an increase in the luminal h2o signal upon induction of pro-insulin, which was attenuated by the ectopic expression of catalase in the er lumen. interfering with glutathione production in the cytosol by buthionine sulfoximine or enhancing its localized destruction by expression of the glutathione-degrading enzyme chac in the lumen of the er further enhanced the luminal h2o signal and eroded β-cell viability.  a tri-cysteine system with a single peroxidatic thiol enables h2o detection in oxidizing milieux such as that of the er. tracking er h2o in live pancreatic β-cells points to a role for glutathione in h2o turnover. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords endoplasmic reticulumredoxh2o probehydrogen peroxideglutathionefluorescent protein sensorfluorescence lifetime imaginglive cell imagingpancreatic β-cellswellcome trust 200848/z/16/zron david fundação para a ciência e a tecnologia ptdc/qui/biq/119677/2010uid/bim/04773/2013-cbmrmelo eduardo pinho european commission eu fp beta-bat no: 277713ron david http://dx.doi.org/ /100004440wellcome trustuns18966kaminski clemens f. engineering and physical sciences research council 1503478kaminski clemens f. medical research council mr/k015850/1kaminski clemens f. issue-copyright-statement© the author 2017 