A Genetically Encoded FRET Sensor for Intracellular Heme - ACS

Apr 10, 2015 - Peking-Tinghua Center for Life Sciences, Beijing, China. ⊥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceut...
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A Genetically Encoded FRET Sensor for Intracellular Heme Yanqun Song,† Maiyun Yang,† Seraphine V. Wegner,†,‡ Jingyi Zhao,§ Rongfeng Zhu,∥ Yun Wu,⊥ Chuan He,*,†,‡,# and Peng R. Chen*,†,∥,# †

Beijing National Laboratory for Molecular Sciences, Synthetic and Functional Biomolecules Center, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ‡ Department of Chemistry and Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, United States § Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen 518055, China ∥ Peking-Tinghua Center for Life Sciences, Beijing, China ⊥ State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China # Shanghai Universities E-Institute for Chemical Biology, Shanghai, China S Supporting Information *

ABSTRACT: Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.

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play diverse and critical roles, the underlying mechanisms of these cellular processes remain to be clarified. Methods for direct and noninvasive monitoring of intracellular heme could help elucidate how heme participates in various physiological and pathological processes and how intracellular heme levels are regulated. Conventional heme quantification methods are only able to quantify the total amount of heme based on cell extract analysis, precluding the possibility to visualize labile intracellular heme and/or their subcellular distributions in real time.7 Fluorescent proteins (FPs) and small molecule-based probes have been widely used for live-cell visualization and quantification of biological analytes such as metal ions, metabolites, reactive oxidative species (ROS), second messengers, and ATP. In particular, genetically encoded FRET sensors are valuable ratiometric tools in quantification of these molecules within different cellular compartments under resting and stress conditions. Although a green fluorescent protein (GFP)-cytochrome

eme is a prosthetic group essential for a wide range of biological processes. Recent studies also hint at the importance of heme as a signaling messenger participating in a broad spectrum of cellular events, including transcriptional regulation, protein localization/degradation, and iron homeostasis as well as the coupling of circadian rhythms and metabolic pathways.1 Meanwhile, nonprotein-bound free heme is hydrophobic and highly toxic to cells due to its inherent peroxidase activity. The intracellular levels of “labile” heme therefore need to be tightly regulated through heme biosynthesis, transport, and degradation machineries, as well as by the well-controlled heme insertion mechanism in hemoproteins.2 Moreover, there has been an increasing appreciation of labile heme as a sensitizer for directing various cell types to undergo programmed cell death in response to pro-inflammatory stimuli.3,4 In addition, certain pathogens, such as malaria, seem to take advantage of the poisonous effects of heme to invade human hosts, whereas a sharply increased heme degradation capability has been observed as an evolutionary conserved cytoprotective response from host cells.5,6 Although dynamic heme homeostasis and trafficking © XXXX American Chemical Society

Received: November 28, 2014 Accepted: April 10, 2015

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DOI: 10.1021/cb5009734 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology Scheme 1. Design of a Genetically Encoded, FRET-based Heme Sensora

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(a) Heme transfer among members of the Isd system (e.g., IsdX1 and IsdC) for iron acquisition in B. anthracis. (b) Design of a FRET-based heme sensor. NEAT domains of two heme transfer chaperones IsdX1 and IsdC were tethered by a linker as the core heme-sensing component, which was flanked by ECFP and EYFP at the N and C terminus, respectively (n: the number of GGS repeating units in the linker region). Heme-binding induced IsdX1-IsdC heterodimerization triggers an increase in the FRET efficiency between the ECFP-EYFP pair.

Figure 1. Development of a genetically encoded, FRET-based heme sensor. (a) FRET signal change between apo- and holo-CISDY-30 monitored by fluorescence spectroscopy (Ex = 432 nm, Em = 440−650 nm). Apo-CISDY-30 was obtained by adding excess apo-IsdC NEAT to extract heme from holo-CISDY-30 followed by extensive washing and separation. (b) Optimization of the linker length (length = 3 × n amino acids) between IsdX1(NEAT) and IsdC(NEAT) to generate CISDY variants with different FRET ratio change between apo-form and holo-form. (c) FRET signal change of CISDY-9 sensor with and without one equivalent heme. (d) Selectivity of CISDY-9 toward heme as opposed to different metal ions. The FRET ratio change between apo- and holo-CISDY-9, as well as the FRET signal after the readdition of hemin to apo-CISDY-9, are shown. Ten equivalents of metal ions were added to sensor protein for 5 min before the fluorescence was measured. All measurements were carried out with 1 μM protein sensor at pH 7.4 in RT.

b562(cytb562) fused heme sensor has been reported that relied on the direct quenching of GFP fluorescence by heme bound to cytb562 to assess heme concentrations, this probe also responses to other heme catabolites or metabolic intermediates such as bilirubin, biliverdin, or PPIX, rendering it unsuitable for heme imaging in living cells.8 In order to monitor heme levels within a native cellular context and to overcome the intrinsic property of fluorescence quenching by heme, we created a genetically encoded, FRET-

based heme sensor by tethering two bacterial heme transfer chaperones for the sensory module, which is sandwiched by ECFP (enhanced cyan fluorescent protein) and EYFP (enhanced yellow fluorescent protein) at the N and C terminus, respectively (Scheme 1). By systematically varying the linker length between the two heme chaperones, we obtained a heme probe with large ratiometric change upon heme binding. We further targeted this sensor to different subcellular compartments, allowing real-time and quantitative B

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instead of Fe2+) confirmed that this sensor binds one equivalent of hemin (Supporting Figure S4c), which correlated well with our UV−vis analysis (Supporting Figure S2a). Embarking on the CISDY-30 construct, we sought to further improve its FRET efficiency. We reasoned that the length of the (GGS)-repeating linker may affect the sensor’s FRET response and thus could be optimized. A panel of CISDY-30 variants with different numbers of GGS repeating units were constructed, which all exhibited essentially the same stoichiometry for heme and similar heme binding affinity as CISDY-30 (supporting methods and supporting Figures S5, S6a,b). Notably, the FRET ratio change among these new constructs in the presence of one equivalent of heme significantly varied, ranging from 0.15 to 1.20 (Figure 1b). Whereas further increasing the linker length decreased the sensor’s FRET efficiency, a CISDY-30 variant with a much shorter linker length (3 GGS repeating-units, renamed as CISDY-9) exhibited a significantly improved FRET ratio: the (I525nm/I475nm) signal change of CISDY-9 was about 2.7-fold higher than that of CISDY-30 (Figure 1b,c). In addition, the dissociation constant of CISDY-9 (Kd_CISDY‑9) was determined to be 63.5 ± 14.3 nM by direct titration of hemin to apoCISDY-9 with the FRET ratio as the readout (Supporting Figure S7a). Moreover, the binding kinetic of heme to CISDY9 was estimated to be t1/2 = 4 s (Supporting Figure S7b). The purified apo-CISDY-9 also showed higher selectivity for hemin over other metal ions (Figure 1d), whereas the fluorescence of holo-CISDY-9 was not influenced by these metal ions (Supporting Figure S7c). The gradient decrease of protein concentrations exhibited a similar FRET ratio, excluding the possibility of heterodimer formation (Supporting Figure S7d). Further reducing the (GGS) repeating units in the linker region on CISDY-9 rendered the protein complex unexpressed, likely because of the steric hindrance of the two NEAT domains that interfered with protein folding. Next, we created a mutant form of CISDY-9 by mutating Tyr131 to Ala, a key residue for central heme-iron coordination in the heme-binding pocket on IsdC NEAT.15,16 Consistent with previous biochemical data, this mutant, termed CISDY9(Y131A), showed a negligible FRET response in the presence of heme, which can be used as a negative control (Supporting Figure S8). Moreover, the FRET ratio of CISDY-9(Y131A) is similar to the Rmin of CISDY-9 in vitro, making it an excellent control for CISDY-9 in order to exclude the influence from pH variation, fluorescence quenching, or protein abundance (Supporting Figure S9). After several rounds of optimization, we succeeded in developing a genetically encoded, ratiometric heme probe with high selectivity and sensitivity. Subsequently, we evaluated the ratiometric and spatiotemporal response of CISDY-9 in visualizing intracellular heme dynamics. HeLa cells expressing cytosolic residing CISDY-9 were cultured in the heme-deficient medium supplemented with 5 μM succinylacetone (SA) for intracellular heme deprivation for 16 h, after which the cells were fed with 5 μM exogenous hemin. The images in the ECFP and EYFP emission channels were recorded every 10 min (Ex = 405 nm) after the addition of hemin to monitor the dynamic change of heme levels (Figure 2a). As expected, a clear FRET ratio (IEYFP/IECFP) increase was observed within 20 min after the addition of hemin. The FRET ratio maximized at 100 min, followed by a gradual signal decrease to the initial level within 4 h, indicating an intracellular feedback system to maintain heme

measurement of intracellular heme distributions under living conditions. By reporting the FRET efficiency change between a pair of FP variants (e.g., CFP-YFP pair), the genetically encoded FRET sensors operate via conformational changes of their sensory modules, which in many cases only produce limited fluorescence responses.9,10 Detection of small organic molecules, cofactors, or inorganic ions is especially challenging when the sensory module is a single domain protein with negligible conformational changes upon substrate binding. In order to increase the detection sensitivity in response to local conformational change, circularly permuted FPs (e.g., cpYFP) have been developed to allow for their incorporation with the sensory region in close proximity. However, such cpFP variants are typically nonratiometric and are often sensitive to intracellular pH.11 We envisioned using heme-transfer proteins as the sensory component, which can undergo heme-induced protein heterodimerization and may thus cause a larger conformational change with a detectable FRET signal.12 In recent years, a family of cell-wall-attached Iron-regulated surface determinant (Isd) proteins have been discovered in B. anthracis for capturing the essential iron nutrients from heme molecules in hosts.13 An essential step in the Isd-mediated heme acquisition process on the cell surface of B. anthracis is the transfer of heme between the IsdX1 and IsdC hemoproteins through the interaction of NEAr iron Transporter (NEAT) domains (Scheme 1a). A transient but specific IsdX1(NEAT):heme:IsdC(NEAT) transfer complex has been observed and was shown to promote the passage of heme 70 000 times faster than the rate of transfer in the absence of the heterohemoprotein complex formation. We reasoned that this heme-induced heterodimerization between two heme transfer chaperones can be coupled with the ECFP−EYFP FRET pair to afford a genetically encoded fluorescent indicator for intracellular heme detection. We started to construct the FRET-based heme sensor by combining the two NEAT domains from IsdX1 and IsdC via a flexible linker containing multiple (GGS) repeating units. This tethered core sensory component was flanked by ECFP and EYFP at the protein’s N and C terminus, respectively (Scheme 1b), generating the ECFP-IsdX1(NEAT)-Linker(30 amino acids)IsdC(NEAT)-EYFP construct that we named CISDY-30. The heme-bound form of the sensor (holo-CISDY-30) was overexpressed in E. coli cells and purified by affinity chromatography (Supporting Figure S1 and S2b). To obtain the apo form of the sensor without denaturing the fluorescent proteins, we used a noninvasive method by adding an excess amount of apo-IsdC NEAT to extract heme from holo-CISDY30 followed by separating the mixture by size-exclusion chromatography (Supporting Figure S1b and S2b). A significant difference in FRET signal (R = I525nm/I475nm) was observed for the purified apo- and holo-CISDY-30 (Figure 1a and Supporting Figure S4a). Interestingly, substitution of the NEAT domains with the entire heme transfer chaperones or replacing ECFP-EYFP with the previously reported FRET pair from the eCALWY model in CISDY-30 both led to a reduced FRET response than the original CISDY-30 (Supporting Figure S2 and S3).12,14 Therefore, we focused on the characterization of the CISDY-30 heme sensor. FRET measurement with CISDY-30 showed high selectivity for heme over a wide range of physiologically relevant metal ions as well as the protoporphrin (Supporting Figure S4b,c). Moreover, titration of apo-CISDY-30 with hemin (heme that is coordinated to Fe3+ C

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verified the subcellular targeting of these CISDY-9 variants (Supporting Figures S10−S12). Moreover, these organellespecific probes were shown to be functional within these compartments (Supporting Figure S13). Interestingly, dualemission ratio imaging of these organelle-specific CISDY-9 variants revealed that the heme content in ER was significantly lower than that in other tested organelles in HeLa cells (Figure 2b). These results indicated that the intracellular heme level is spatially controlled, likely due to the cellular regulation of heme distribution. To examine whether this is the case for other cell types, we tested six additional cell lines: Mouse melanoma B16 cell, human HCT116 colon cancer cell, human MCF-7 cell (human breast adenocarcinoma cell line), human PANC-1 cell (human pancreatic carcinoma, epithelial-like cell line), hamster kidney fibroblast BHK-21 cell, and CHO (Chinese Hamster Ovary) cell. Emission ratios of organelle-specific CISDY-9 variants were measured (n > 30) and calibrated with the control indicator CISDY-9(Y131A) (Supporting Figure S14 and Figure 2c). However, in contrast to our observations in HeLa cells, no significant variations of free heme were observed in ER as opposed to other organelles in these cell lines. The unique heme distribution in HeLa cells might be due to the high expression level of heme oxygenase 1 (HO-1) that is primarily localized on the ER membrane in certain cancer cells.19,20 To test this hypothesis, we next established a stable HeLa cell line constitutively expressing CISDY-9 in its Cytosol (this cell line was designated as HeLa-C9C hereafter) and monitored the change in free heme levels. Flow cytometric analysis was first performed on HeLa-C9C cells precultured with heme-deficient medium, which showed a decreased cytosolic heme level and thus verified the proper function of our heme reporter in this cell line (Supporting Figure S15). Treatment of doxycycline (2 μg/mL, 12 h) was able to upregulate the expression level of HO-1 in HeLa-C9C cells (Supporting Figure S16b). Notably, we found that the heme level was decreased after doxycycline treatment (Figure 3a Supporting Figure S16a). Finally, we measured the concentration of labile heme in cell cytosol, which is difficult to determine using conventional methods. According to the previous procedure developed by Tsien et al. (Formula 1),21 we set the FRET signal of the control mutant CISDY-9(Y131A) as Rmin, which was calculated to be 2.03 ± 0.25 (n = 65, Figure 2c and Supporting Figure S14). We measured the emission ratio of CISDY-9 in the cytosol to be 3.04 ± 0.27 and defined this as Rresting (n = 75, Figure 2c and Supporting Figure S14). We then obtained Rmax by adding excessive hemin to the cell in the presence of digitonin (20 μM), which can permeablize the cell membrane for complete hemin entry (Figure 3b). The signal in HeLa cells was fully saturated within 30 min and Rmax was calculated as 5.5 ± 0.5 (n = 6). By combining these parameters, the concentration of free heme in cytosol was estimated to be 25.6 ± 5.5 nM (formula 1), which falls within the same range as the previous estimation.22 Because it is technically difficult to obtain the Rmax values for each subcellular organelle, we assumed that Rmax − Rmin is kept constant in different organelles as that in cytosol, and the labile heme concentrations for Mt, ER, and Nu were subsequently determined to be 23.3 ± 4.9 nM, 5.4 ± 1.4 nM, and 31.0 ± 7.0 nM, respectively (these numbers are subject to variation according to the organellespecific Rmax).

Figure 2. Spatial-temporal imaging of heme distribution using CISDY9 in living cells. (a) Time-course imaging of CISDY-9’s response to the extracellularly added heme in live HeLa cells. Pseudocolored ratiometric images of HeLa cells expressing CISDY-9 were obtained at different time spots (up to 250 min) after the addition of 5 μM hemin. Cells were precultured in serum-free DMEM containing 5 μM SA to deprive the intracellular heme before being imaged by two fluorescence channels (ECFP: 450−510 nm; EYFP: 520−700 nm) under confocol fluorescence microscopy. Scale bars: 5 μm. (b) Pseudocolored ratiometric images of HeLa cells expressing subcellular targeted CISDY-9 variants. Scale bars: 5 μm. (c) Comparison of the emission ratio of CISDY-9 in various cellular compartments of different cell lines including HeLa cells, mouse B16 cells, human HCT116 cells, human MCF-7 cells, human PANC-1 cells, hamster BHK-21 cells, and hamster CHO cells. All the emission ratios of CISDY-9 were normalized with that of CISDY-9(Y131A). Emission ratios of over 30 cells were quantified and averaged for each experiment. All images were taken by confocal microscopy.

homeostasis. Therefore, our probe can report the labile heme dynamics in living cells. Although the pathway and regulation of heme biosynthesis have been extensively studied,1,17,18 intracellular heme trafficking and distribution among different membrane-enclosed organelles remains elusive. To test whether free heme levels vary in different subcellular compartments in HeLa cells, we specifically targeted CISDY-9 to the mitochondrial matrix (Mt), the endoplasmic reticulum (ER), and the nucleus (Nu) by introducing organelle-specific signal peptides to CISDY-9. Colocalization of organelle-specific biomarkers with CISDY-9 D

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ASSOCIATED CONTENT

S Supporting Information *

General considerations, supporting methods, and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank J. Karpus for advice on fluorescent imaging and T. Shpigel for editing the manuscript. This work was supported by research grants from the National Natural Science Foundation of China (21225206, 91313301, and 21432002 to P.R.C.), the National Key Basic Research Foundation of China (2011CB809103 to C.H.), and National Science Foundation (CHE-1213598 for C.H.). C.H. is an investigator supported by Howard Hughes Medical Institute.

■ Figure 3. Measurement of heme concentration by CISDY-9 in HeLa cells. (a) Doxycycline induces a great decrease in the heme level in vivo as measured by flow cytometry. Stable cell line (HeLa-C9C cell) was treated with 2 μg/mL doxycycline for 12 h, which caused significantly decreased FRET ratio (Ex: 405 nm; Em: 450/50 nm for ECFP, 525/ 50 nm for EYFP) than control cells. (b) Determination of labile heme concentration in HeLa cells by using CISDY-9. Y131A mutant was used as a control and set as Rmin. Rmax was acquired after the addition of 10 μM hemin and 20 μM digitonin for 30 min. Labile heme in cytosol was calculated to be 6.5 ± 0.4 nM (Formula 1; mean ± s.d.; n = 6) in HeLa cells. All images were taken by confocal microscopy.

[heme] = Kd ×

R resting − R min R max − R resting

(1)

In summary, we developed, to our knowledge, the first genetically encoded heme sensor with high sensitivity and selectivity. This FRET-based probe exhibited a heme binding affinity at a similar range to that of the intracellular levels of labile heme, which allowed for spatial and temporal visualization of heme dynamics under living conditions. Using this probe, we found that free heme levels vary in certain subcellular compartments such as ER within specific cancer cells. In addition, the labile heme levels within cell cytosol, as well as in different organelles, were measured. Taken together, our probe provides a powerful tool for studying the physiological and pathological roles of heme in diverse biological processes such as heme-mediated signal transduction and iron homeostasis.

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REFERENCES

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METHOD

Experimental procedures are described in detail in the Supporting Information. E

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