TY - JOUR
AU - Chen, Peng, R.
AB - Abstract A ratiometric and reversible organic hydroperoxide (OHP) sensor, rOHSer, was developed with high sensitivity and selectivity for subcellular OHP visualization. Through targeting rOHSer to the nucleus, we demonstrated that high levels of d-glucose cause elevated OHP production in this compartment. Further utilization of rOHSer probe may allow more elucidation of unique roles of OHPs in diverse biological processes. Insight, innovation, integration Organic hydroperoxide (OHP) is a highly reactive form of reactive oxygen species (ROS) that is widely distributed in biology and plays critical roles in biological signaling and cellular processes, often with mechanisms distinct to H2O2. The differentiation of OHP over other forms of ROS may give insight into various cellular events, which is hindered by the lack of selective probes for OHP. This manuscript describes the optimization-based development of a ratiometric and reversible OHP sensor, named rOHSer, which exhibits high sensitivity and selectivity for intracellular OHP visualization. We also targeted rOHSer into various subcellular compartments and demonstrated its applicability for monitoring localized OHP homeostasis by detecting glucose-induced endogenous OHP production in the cell nucleus with nuclear-targeting rOHSer. Our rOHSer probe may provide a valuable tool for monitoring subcellular OHP dynamics and therefore facilitate our understanding of the unique roles of OHPs in diverse biological processes. Introduction The development of novel fluorescent indicators has allowed for the dynamic monitoring of various cellular metabolites and/or signaling molecules within living systems.1–8 Reactive oxygen species (ROS) have long been associated with aging as well as a number of degenerative and chronic diseases,9–11 while the maintenance of physiological concentrations of ROS is crucial for mediating various biological activities such as cell proliferation, migration, differentiation, and apoptosis.12–14 Organic hydroperoxide (OHP) is a frequently encountered form of ROS found in diverse organisms, ranging from molecules with simple alkyl groups to more complex products from lipid peroxidation. They have been shown to cause DNA damage, cell apoptosis, fast lipid peroxidation and various chronic diseases15,16 Recent studies suggest that instead of being freely diffused and thus functioning in the entire cellular space, ROS molecules including OHPs are produced in a highly localized fashion in distinct subcellular compartments or structures including endosomes, endoplasmic reticulum, mitochondria, nucleus, lipid rafts, and lamellipodium.17,18 The spatially confined production of ROS ensures that the corresponding redox-sensitive targets are located within close proximity, thereby facilitating the activation of specific redox signaling events without much loss-of-signal due to the decomposition of ROS molecules. Precise control of ROS production is particularly critical to the nucleus because ROS are known to influence the expression of diverse genes and signal transduction pathways while excess ROS inside the nucleus is extremely toxic to the cell.14,19,20 Therefore, the development of probes for monitoring subcellular ROS dynamics is highly desired. Since their recent emergence, genetically encoded fluorescent ROS indicators have become a powerful tool for the selective detection of these highly reactive species in living organisms.21,22 Different from small-molecule based fluorescent indicators, many of these protein-based probes can be intracellularly expressed, easily targeted to various subcellular compartments, and are compatible with multiple rounds of the oxidation–reduction cycle without much loss of activity. Specifically, the insertion of a circularly permutated yellow fluorescent protein (cpYFP) into the H2O2-sensitive protein OxyR generated a genetically encoded sensor for H2O2, a highly important and commonly encountered form of ROS within cells. This indicator, named Hyper, shows high selectivity as well as excellent reversibility and ratiometric property, which allows for quantitative measurement of in situ generated H2O2 in various types of cells, and even within plants and animals. Moreover, by using signal peptides or retention sequence, this powerful probe has been targeted to various subcellular compartments, thus permitting organelle-specific detection of H2O2 inside cells. Inspired by this strategy, we have recently constructed an OHP specific sensor by inserting a circularly permutated Venus protein (cpVenus) into the OHP-sensitive protein OhrR, which is one of the MarR family transcription regulators with a reactive OHP-sensing cysteine. The resulting probe, named OHSer, enables intracellular visualization of OHPs.22 One possible shortcoming of genetically encoded indicators is that the difference in cellular expression levels might interfere with measurements. This problem can be overcome by creating ratiometric indicators that exhibit a change in the emission fluorescence ratio between two excitation wavelengths upon recognition of the target molecules.21,23,24 The induced structural change of ratiometric fluorescent protein favors protonation of the chromophore, which shifts the excitation maxima to a shorter wavelength instead of showing a typical quenching effect. In addition to normalizing the variable expression levels of the indicator, the ratiometric signal can also help eliminate unnecessary artifacts during the imaging experiment. Our initial OHSer probe exhibited higher pH stability than Hyper, but lacked the ratiometric feature. Herein we report the development and characterization of a ratiometric OHP-sensor, termed rOHSer, for ratiometric and reversible monitoring of OHP dynamics in living cells (Fig. 1). Fig. 1 Open in new tabDownload slide Characterization of rOHSer in vitro. (A) Excitation and emission spectra for fully oxidized and reduced rOHSer. The spectra showed an emission peak at 520 nm and a dual excitation peak at 420 nm and 500 nm, respectively. (B) The response curves of rOHSer incubated with different concentrations of TBHP for 20 minutes. The emission wavelength is fixed at 520 nm. (C) Fluorescence response of rOHSer to 100 μM of H2O2, TBHP or CHP for 30 min. (D) Reversibility of rOHSer. The rOHSer probe was treated with two rounds of oxidation (with 10 μM TBHP) and reduction (with 10 mM NAC) cycle. All measurements were repeated three times. Error bars represent standard deviation (SD). Fig. 1 Open in new tabDownload slide Characterization of rOHSer in vitro. (A) Excitation and emission spectra for fully oxidized and reduced rOHSer. The spectra showed an emission peak at 520 nm and a dual excitation peak at 420 nm and 500 nm, respectively. (B) The response curves of rOHSer incubated with different concentrations of TBHP for 20 minutes. The emission wavelength is fixed at 520 nm. (C) Fluorescence response of rOHSer to 100 μM of H2O2, TBHP or CHP for 30 min. (D) Reversibility of rOHSer. The rOHSer probe was treated with two rounds of oxidation (with 10 μM TBHP) and reduction (with 10 mM NAC) cycle. All measurements were repeated three times. Error bars represent standard deviation (SD). We took two different approaches towards the development of rOHSer. First we performed successive point mutations based on the sequence analysis between Venus (from OHSer) and YFP (from Hyper; Fig. S1, ESI†). A series of OHSer variants with distinct pH profiles were generated, but none exhibited notable ratiometric properties (Fig. S2, ESI†). Next, we turned our attention to the direct substitution of the entire cpVenus domain on OHSer by different YFP variants including mVenus, SYFP2, Ypet, and YFP.25 The circularly permuted version of these fluorescent proteins were created and used to replace the cpVenus domain on OHSer. The resulting constructs show different fluorescence properties in terms of brightness, photostability, and pH stability. Among them, the cpYFP-replaced OHSer variant exhibits the best ratiometric property: upon full oxidation with 100 μM OHP overnight, a >4-fold increased fluorescence at 500 nm (peak 1) and a constant fluorescence response at 420 nm (peak 2) were observed simultaneously with the wavelength fixed at 520 nm (Fig. 1A). This cpYFP-inserted OhrR protein construct was thus named ratiometric OHSer (rOHSer) and used in the following study. We next characterized rOHSer in vitro. Through the use of a spectrofluorometer the sensitivity of rOHSer was evaluated using excitation-spectrum scanning with the addition of a series of different concentration of tert-butyl hydroperoxide (TBHP) ranging from 0 to 200 μM. As shown in Fig. 1B, the excitation peak at 420 nm remained steady in all of the spectra, while the excitation peak at 500 nm increased promptly with the treatment of TBHP as low as 1 μM. When the TBHP concentration was higher than 50 μM, the peak at 500 nm decreased slightly, probably a result of oxidation damage to the probe. Verification of the selectivity of rOHSer toward organic hydroperoxides over H2O2 was then achieved through the treatment of rOHSer with 100 μM of H2O2, cumene hydroperoxide (CHP) or TBHP for 20 min. As shown in Fig. 1C, while H2O2 only induced a less than 10% increase in the relative emission ratio (F500nm/F420nm: the fluorescence emission at 520 nm with excitation at 500 nm divided by that with excitation at 420 nm). The samples that were treated with CHP or TBHP both demonstrated a nearly 2-fold increase in their relative emission ratio. These results confirmed that our new rOHSer probe exhibits high selectivity towards OHPs. Reversibility represents another major advantage for protein-based probes over small molecule-based indicators. Two rounds of oxidation–reduction cycle to the same batch of sample confirmed the reversible response of rOHSer. As shown in Fig. 1D, reduced rOHSer was treated with 10 μM TBHP, which gave a nearly 2-fold increase in the relative emission ratio. The oxidants were then removed. Subsequently the same sample was treated with 10 mM N-acetyl cysteine (NAC), which reduced the relative emission ratio back to the basal level. Next, the reducing agents were removed and the sample was treated again with 10 μM TBHP, which promoted the relative emission ratio over 1.6-fold. Finally, a second reduction following oxidation effectively reduced the ratio back to near basal levels in a similar fashion. These results indicate that rOHSer could withstand multiple rounds of oxidation–reduction cycle without significant loss of integrity and sensitivity. In order to apply rOHSer to live-cell imaging of OHPs, particularly within different subcellular compartments, we first expressed rOHSer in mammalian cytosol using pCMV-Tag1, a mammalian expressing vector. In addition, we targeted rOHSer into nucleus, mitochondrion, and the cytoplasmic membrane of mammalian cells by fusing different signaling peptides.24,26,27 Use of confocal laser microscopy verified the localization of these subcellular-specific rOHSer variants (Fig. 2A, Fig. S3 and S4, ESI†). We then focused on the nucleus-targeted rOHSer, named nu-rOHSer, in order to compare the OHP production and homeostasis in this compartment as opposed to cytosol. HeLa cells expressing rOHSer were first treated with 10 mM NAC or 100 μM TBHP in order to generate rOHSer spectra resembling the fully reduced and fully oxidized conditions, respectively. HeLa cells expressing rOHSer or nu-rOHSer under basal levels were then imaged using the same parameters as the aforementioned experiments. Amounts of oxidized and reduced forms of rOHSer under normal conditions were calculated by quantifying the change of relative emission ratio under oxidizing and reducing conditions. Results showed that in both cytosol and nucleus, less than 10% of rOHSer was present in the oxidized form (Fig. 2B), thus indicating an overall reducing environment in these two spaces. In addition, consistent with previous observations, the nucleus is slightly more reduced than the cytosol.28 Considering fluorescent proteins are generally sensitive to pH change, we also constructed an inactive mutant of rOHSer (rOHSer-mut) by mutation of the active cysteines and used it as a pH control to exclude the possibility of false positive signals caused by pH fluctuation. Upon the same treatment with TBHP, cells expressing rOHSer-mut did not exhibit notable change in fluorescence comparing to the basal level (Fig. S5, ESI†), thus verifying that the signal increase of rOHSer was indeed from TBHP detection. Fig. 2 Open in new tabDownload slide In vivo characterization of rOHSer. (A) Intracellular localization of rOHSer targeted to the nucleus of HeLa cells. From left to right: nucleus-located rOHSer, Hoechst 33433 staining of nucleus, outer membrane stained with CellMask Deep Red, and merged images of all three channels. (B) Percentage of oxidized rOHSer in cytosol and nucleus. To estimate the percentage, the fluorescence of HeLa cell expressing rOHSer and n-rOHSer under reducing (10 mM NAC, 4 h) and oxidizing (100 μM TBHP, 30 min) conditions was recorded. (C) In vivo selectivity of rOHSer. HeLa cells expressing rOHSer in cytosol and nucleus were treated with 10 mM NAC, 100 μM H2O2, 100 μM TBHP or without the treatment (basal condition). Below are the pseudocolor ratiometric fluorescence images of cells generated from the pixel-by-pixel ratios of the 488 nm excitation images by the 405 nm excitation images on the same cell. All error bars represent standard error of the mean (SEM). All scale bars represent 10 μm. Fig. 2 Open in new tabDownload slide In vivo characterization of rOHSer. (A) Intracellular localization of rOHSer targeted to the nucleus of HeLa cells. From left to right: nucleus-located rOHSer, Hoechst 33433 staining of nucleus, outer membrane stained with CellMask Deep Red, and merged images of all three channels. (B) Percentage of oxidized rOHSer in cytosol and nucleus. To estimate the percentage, the fluorescence of HeLa cell expressing rOHSer and n-rOHSer under reducing (10 mM NAC, 4 h) and oxidizing (100 μM TBHP, 30 min) conditions was recorded. (C) In vivo selectivity of rOHSer. HeLa cells expressing rOHSer in cytosol and nucleus were treated with 10 mM NAC, 100 μM H2O2, 100 μM TBHP or without the treatment (basal condition). Below are the pseudocolor ratiometric fluorescence images of cells generated from the pixel-by-pixel ratios of the 488 nm excitation images by the 405 nm excitation images on the same cell. All error bars represent standard error of the mean (SEM). All scale bars represent 10 μm. In order to quantitatively examine the selectivity of rOHSer in subcellular compartments, living HeLa cells were further imaged using a Leica SP2 confocal microscope with a fixed emission range (500–560 nm) and sequential dual excitations at 488 nm and 405 nm, respectively. The relative emission ratio was then calculated, with the basal value as the standard. As shown in Fig. 2C, upon treatment with 100 μM exogenous TBHP for 30 min, prompt increases in relative emission ratio were observed in both the cytosol and the nucleus. Cytosol appeared to be more sensitive than the nucleus, possibly due to the blockage of exogenous OHP molecules by the nuclear membrane. In contrast, control groups that were treated with the same concentrations of H2O2 produced a negligible response, thus indicating that the selectivity of rOHSer was retained when expressed in subcellular compartments such as the nucleus. Next, we investigated the reversibility of rOHSer inside cells. The enhanced relative emission ratio of the rOHSer-expressing HeLa cells under oxidizing conditions (100 μM TBHP for 30 min) was largely decreased once shifted to the reducing environment (10 mM NAC for 4 h, Fig. S6, ESI†). Therefore, the oxidized form of rOHSer can be readily converted back to its reduced form upon reduction. To further demonstrate its applications, we employed nu-rOHSer to investigate the glucose-induced ROS production within the nucleus. High levels of d-glucose, caused by chronic diseases such as hyperglycemia, could impair diverse cell functions that may eventually lead to cell death.29–32 Through the use of a GFP-fused p53 reporter (p53-GFP),33 we found that treatment of 30 mM d-glucose caused the translocation of p53 from nucleus to cytosol (Fig. S7, ESI†), resulting in apoptosis.34 As elevated ROS production is often associated with hyperglycemia, excessive amounts of d-glucose may cause apoptosis by triggering the production of endogenous OHPs, especially within close proximity to the genetic materials in the nucleus. To test this hypothesis, HeLa cells expressing rOHSer or nu-rOHSer were treated with high concentrations of d-glucose (30 mM or 60 mM d-glucose) for 24 h before visuals produced using confocal microscopy. The results indicate that excessive amounts of d-glucose could elevate the relative emission ratio of rOHSer in both spaces, with the change in nucleus more significant than that in cytosol (Fig. 3A). The calculation also shows a higher percentage of rOHSer in its oxidized form within the nucleus (Fig. 3B). Therefore, these observations suggest that the production of endogenous OHPs may be the result of high amount d-glucose treatment inside cells, especially within the nucleus. Fig. 3 Open in new tabDownload slide Monitoring OHP production in HeLa cells upon the treatment of d-glucose. (A) Comparison of the sensitivity of rOHSer and nu-rOHSer to excessive d-glucose. HeLa cells expressing these rOHSer variants were treated with 10 mM NAC, 30 mM d-glucose, 60 mM d-glucose or under the basal condition. Below are the pseudocolor ratiometric fluorescence images of representative cells in each group. (B) Percentage of oxidized rOHSer in cytosol and nucleus upon the treatment of excessive d-glucose. Similar calculation methods were used as in Fig. 2B. All error bars represent SEM. All scale bars represent 10 μm. Fig. 3 Open in new tabDownload slide Monitoring OHP production in HeLa cells upon the treatment of d-glucose. (A) Comparison of the sensitivity of rOHSer and nu-rOHSer to excessive d-glucose. HeLa cells expressing these rOHSer variants were treated with 10 mM NAC, 30 mM d-glucose, 60 mM d-glucose or under the basal condition. Below are the pseudocolor ratiometric fluorescence images of representative cells in each group. (B) Percentage of oxidized rOHSer in cytosol and nucleus upon the treatment of excessive d-glucose. Similar calculation methods were used as in Fig. 2B. All error bars represent SEM. All scale bars represent 10 μm. Finally, time-lapse imaging was performed to monitor glucose-induced dynamic OHP production. HeLa cells expressing nu-rOHSer were treated with an extremely high concentration of d-glucose (200 mM) in order to induce rapid OHP production. Indeed, we observed an elevated relative emission ratio within approximately 4 min after the addition of d-glucose, which was steadily increased further by ∼50% in 30 min (Fig. 4). In order to exclude the possibilities that environmental variations such as pH fluctuation may be induced by d-glucose and thus give a false positive signal, we also performed control experiments with inactive rOHSer-mut-expressing cells. Upon treatment with the same level of d-glucose, these cells exhibited a fairly stable fluorescence signal (Fig. S8, ESI†), indicating that our previously observed signals were not a result of the interference of the fluorescent protein domain on these indicators. Taken together, we have demonstrated that addition of high concentrations of d-glucose inside cells rapidly stimulates endogenous OHP production. Fig. 4 Open in new tabDownload slide Time lapse imaging of HeLa cells treated with d-glucose. (A) HeLa cells expressing nu-rOHSer were treated with 200 mM d-glucose and the images with excitation at 405 nm or 488 nm were recorded with a 1 min interval over a 30 minute period. The fluorescence ratios of the same cell excited in two different wavelengths were calculated and plotted. Error bars represent SEM. (B) The pseudocolor ratiometric fluorescence images of representative cells expressing nu-rOHSer, treated with d-glucose. Scale bars represent 10 μm. Fig. 4 Open in new tabDownload slide Time lapse imaging of HeLa cells treated with d-glucose. (A) HeLa cells expressing nu-rOHSer were treated with 200 mM d-glucose and the images with excitation at 405 nm or 488 nm were recorded with a 1 min interval over a 30 minute period. The fluorescence ratios of the same cell excited in two different wavelengths were calculated and plotted. Error bars represent SEM. (B) The pseudocolor ratiometric fluorescence images of representative cells expressing nu-rOHSer, treated with d-glucose. Scale bars represent 10 μm. Conclusions In summary, we present the development and application of a protein-based, ratiometric, and reversible OHP indicator suitable for cellular and subcellular measurements. These features enable the resulting probe-rOHSer for quantitative visualization of the intracellular OHP levels in a highly localized and compartment-specific manner. We have further demonstrated the utility of rOHSer for monitoring the glucose-induced OHP production within the nucleus, which reveals OHP as a possible cause for cell apoptosis under excessive d-glucose treatment.29 The lipophilic OHPs possess a longer half time and tend to induce cellular stress more prolonged than H2O2.35 In addition, OHPs have been implicated in DNA damage, fast lipid peroxidation, mitochondrial function disruption, and cardiovascular disease-related vasoconstriction, with mechanisms distinct from H2O2.16,36 Nevertheless, we consider this research as positioned at the beginning of our understanding of the underlining mechanisms of these lipidic and non-polar oxidants. Our rOHSer indicator may provide a valuable tool for expanding our knowledge of the unique roles of OHPs in diverse physiological and pathological processes. Acknowledgements We thank S.F. Reichard, MA for editing the manuscript and Christine Labno for confocal imaging. This work was supported by the National Key Basic Research Foundation of China (2010CB912300 and 2012CB917301), National Natural Science Foundation of China (21001010 and 21225206) and National Science Foundation (CHE-1213598 for C.H.) Notes and references 1 B. N. G. Giepmans , S. R. Adams, M. H. Ellisman and R. Y. Tsien, Science , 2006 , 312 , 217 – 224 . Crossref Search ADS PubMed 2 L. E. McQuade and S. J. Lippard, Curr. Opin. Chem. Biol. , 2010 , 14 , 43 – 49 . Crossref Search ADS PubMed 3 J. Wang , J. Karpus, B. S. Zhao, Z. Luo, P. R. Chen and C. He, Angew. Chem., Int. Ed. , 2012 , 51 , 9652 – 9656 . Crossref Search ADS 4 J. Chan , S. C. Dodani and C. J. Chang, Nat. Chem. , 2012 , 4 , 973 – 984 . Crossref Search ADS PubMed 5 Y. Qian , J. Karpus, O. Kabil, S. Y. Zhang, H. L. Zhu, R. Banerjee, J. Zhao and C. He, Nat. Commun. , 2011 , 2 , 495 . Crossref Search ADS PubMed 6 M. Gutscher , A. L. Pauleau, L. Marty, T. Brach, G. H. Wabnitz, Y. Samstag, A. J. Meyer and T. P. Dick, Nat. 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TI - Probing subcellular organic hydroperoxide formation via a genetically encoded ratiometric and reversible fluorescent indicator
JF - Integrative Biology
DO - 10.1039/c3ib40209f
DA - 2013-12-18
UR - https://www.deepdyve.com/lp/oxford-university-press/probing-subcellular-organic-hydroperoxide-formation-via-a-genetically-tJMXjuEj1h
SP - 1485
VL - 5
IS - 12
DP - DeepDyve
ER -