Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome Matthew Fares,† Yinghao Li,† Yu Liu,† Kun Miao,† Zi Gao,‡ Yufeng Zhai,† and Xin Zhang*,†,‡,§ †
Department of Chemistry, ‡Department of Biochemistry and Molecular Biology, and §The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *
ABSTRACT: Cellular stress leads to disruption of protein homeostasis (proteostasis) that is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced proteostasis deficiency remains one of the major technical challenges facing established sensors of this process. Available sensors use solvatochromic fluorophores to detect protein aggregation in forms of soluble oligomers or insoluble aggregates when cells are subjected to severe stress conditions. Misfolded monomers induced by mild stresses, however, remain largely invisible to these sensors. Here, we describe a fluorogenic proteome stress sensor by conjugating a fluorescent molecular rotor with a metastable Halo-tag protein domain that contains a K73T mutation (named AgHalo hereinafter). In nonstressed cells, the AaHalo sensor remains largely folded and the AgHalo•ligand conjugate is fluorescent dark in the folded state. Under various stress conditions, the AgHalo sensor has been established to form both soluble and insoluble aggregates along with metastable proteins of the endogenous cellular proteome. Thus, the AgHalo•ligand conjugate fluoresces strongly when the sensor forms misfolded monomers (a 16-fold increase) or aggregates in both soluble and insoluble forms (a 20-fold increase). Compared to the solvatochromic fluorophore-based sensor, we demonstrate that the molecular rotor-based sensor not only is more effective in detecting mild proteome stress that induces primarily misfolding conformations, but also exhibits a higher fluorescence signal in detecting more severe proteome stress that involves protein aggregates. Thus, the conjugation of a fluorescent molecular rotor to AgHalo further improves the capacity of this sensor to detect conditions of proteome stress. This work highlights the utility of molecular rotor-based fluorophores in direct visualization of the protein aggregation cascade in live cells, providing new methodologies for real-time analyses of cellular proteostasis upon exposure to different types of stress conditions.
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INTRODUCTION Proper protein homeostasis (proteostasis) is essential to all cells.1−3 Exogenous stress conditions (including environmental perturbations, chemical toxins, pathogen invasion, and aging) impair the integrity of proteostasis and cause global misfolding and aggregation of the endogenous proteome, a phenomenon that has been increasingly associated with a growing number of diseases, such as cancer, neurodegeneration, metabolic disorders, cardiovascular disease, and inflammation.4,5 To better understand the molecular mechanisms of these diseases and to advance the development of therapeutic strategies, new methodologies are necessary to sense impairment of cellular proteostasis during stress. Stress-induced protein aggregation is a multiple step process that includes formation of misfolded protein monomers, soluble oligomers, insoluble aggregates, and fibrils (Figure 1a). To detect these aberrant protein conformations in live cells that are subjected to various stresses, protein-based sensors have been developed to report on the aggregation of destabilized client proteins. Most sensors primarily detect the final step of protein aggregation (i.e., insoluble aggregates and fibrils) via formation of punctate fluorescence structure in live © XXXX American Chemical Society
cells. These sensors are exemplified by multiple approaches and client proteins, including destabilized luciferases or split NanoLuc,6,7 fusion of fluorescent protein,8−15 incorporation of fluorescent un-natural amino acids or FlAsH labeling dye,16,17 and destabilized retroaldolase enzyme.18 Although powerful, these sensors often fail to recognize protein conformational changes, such as misfolding or unfolding of monomers, or soluble oligomer formation often found at an early stage of proteome stress. Since these early steps of protein aggregation have been widely associated with various diseases, it is highly desired to develop sensors that can detect formation of these conformations in live cells. Toward this goal, a promising direction is to engineer genetically encoded protein-tag domains and report their stress-induced aggregation using synthetic fluorescent molecules via bioconjugation. Among these technical platforms, Halo-tag is a protein-tag domain that is widely used in multiple applications,19 represented by live cell fluorescence imaging,20,21 analysis of protein dynamics,22 probing cellular redox Received: December 2, 2017 Published: December 18, 2017 A
DOI: 10.1021/acs.bioconjchem.7b00763 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
fluorescent molecular rotor, but not the solvatochromic fluorophore. The unique feature of the fluorescent molecular rotor makes the new generation of the AgHalo proteome sensor more sensitive to misfolded conformations that are primarily induced by mild proteome stress. Further, the new sensor exhibits a higher fluorescence signal when detecting soluble and insoluble protein aggregates that are induced by more severe proteome stress. These data collectively suggest that thermo-labile Halo conjugated with a fluorescent molecular rotor serves as a suitable sensor to detect a wide range of proteostasis deficiency caused by various cellular stresses.
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RESULTS AND DISCUSSION Disruption of the folded polypeptide hydrophobic core, causing nonpolar and aromatic residues to be exposed to a hydrophilic environment, is a key feature of protein misfolding. Based on this principle, solvatochromic fluorophores whose fluorescence increase is solely based on the polarity change of its local environment have been used to detect exposed hydrophobic regions of misfolded proteins in vitro.26−28 However, application of these fluorophores to selectively probe misfolding of a specific protein in live cells has been limited by to the high background signal due to nonspecific interactions in a cellular context. Although a solvatochromic fluorophore sulfonyl-benzoxadiazole (SBD; P1 in Figure 1b) is conjugated with the metastable AgHalo sensor to allow for a specific detection of AgHalo in conformations as soluble oligomers and insoluble aggregates in live cells, P1 still cannot detect AgHalo monomers that adopt misfolded conformations in cells. We speculate that the initial misfolded state of AgHalo may not change its dielectric constant sufficiently to turn on P1 fluorescence. To solve this problem, we explored fluorescent molecular rotors whose fluorescence increases when their excited state is rotationally hindered in a viscous or rigid local environment.29−31 AgHalo Aggregation Is Detected by a CCVJ-Based Halo Ligand. Molecular rotors have been applied to detecting insoluble protein aggregates and amyloid fibrils, represented by the PROTEOSTAT assay kit, and have been widely utilized in other studies.32−39 To test whether the misfolded state of AgHalo is able to induce fluorescence increase by hindering rotation of the fluorescent molecular rotor, the 9-(2-carboxy-2cyanovinyl)julolidine (CCVJ) fluorophore was chosen as a proof-of-concept, as it exemplifies the excellent fluorogenicity of molecular rotors.40−48 CCVJ has been the representative fluorescent molecular-rotor, because it contains a julolidine moiety and it has been previously shown that hindering the rotation of this portion of the molecule will result in increasing fluorescence.49,50 This has led to the molecule being used to detect a wide variety of biological phenomenon, including protein degradation,40 peptide−protein interaction,42 and cell membrane viscosity.44 Despite these widespread applications, the ability of fluorescent molecular rotors to detect misfolded protein monomers in live cells has not yet been explored. We first evaluated whether CCVJ can detect AgHalo aggregation by incorporating CCVJ in the Halo-tag ligand, resulting in P2 shown in Figure 1b. To confirm that the fluorophore retained its function as a fluorescent molecular rotor, we increased the viscosity of the solvent to hinder the rotation of the CCVJ moiety. P2 in a mixture of glycerol and water exhibited increasing fluorescence up to 150-fold when the glycerol concentration increased, confirming its viscosity
Figure 1. Fluorogenic approach to detect misfolding protein in a stressed proteome. (a) The present AgHalo sensor only reports on protein aggregates (top panel). In this work, the AgHalo sensor is expanded to visualize misfolded proteins (bottom panel). (b) Structure of the SBD-based P1 and CCVJ-based P2. (c) Fluorescence of P2 in solvents with varying glycerol percentage in H2O and in 1,4dioxane (ex = 440 nm, em = 500 nm). Error bars: standard deviation (n = 3).
perturbation,23 and induction of autophagy.24 Recently, we have reported a fluorogenic proteome stress sensor AgHalo that comprises a metastable Halo-tag mutant (K73T) and a solvatochromic fluorophore as a Halo-tag ligand.25 The thermodynamic stability of AgHalo is −2.0 kcal/mol (ΔGfolding), comparable to the average stability of the metastable cellular proteome (the bottom 15%). In a previous work, AgHalo was shown to aggregate under a variety of stress conditions that have been established to drive aggregation of metastable proteome. More importantly, the extent of AgHalo aggregation is positively associated with the extent of cellular proteome aggregation. Thus, aggregation of AgHalo flags and represents behavior of metastable proteins under various stress conditions. Using a solvatochromic fluorophore, we have demonstrated that the AgHalo•ligand conjugate exhibits fluorescence intensity increase upon formation of soluble oligomers in stressed cellular proteome, an earlier step of AgHalo aggregation (Figure 1a, top panel). However, misfolded monomers, the initial step of protein aggregation, which likely appear under mild stress conditions still remain largely invisible to this and other sensors. In this work, we describe a new sensor based upon a fluorescent molecular rotor that is conjugated to the K73T Halo mutant. The fluorescent molecular rotor, when conjugated to purified AgHalo to form the proteome stress sensor, is able to report on urea-induced partially unfolded (misfolded) conformations with a higher fluorescent increase than the previously reported solvatochromic fluorophore-based sensor. Heat-induced misfolding is also effectively monitored by the fluorescence change of the sensor that is based on the B
DOI: 10.1021/acs.bioconjchem.7b00763 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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P2 in Conjugation with AgHalo Detects Misfolded AgHalo Monomers Induced by Medium Urea Concentrations. Protein aggregation is a multistep process, including misfolded or unfolded monomers, soluble oligomers, and insoluble aggregates. In this section, we investigated the ability of P2 to detect these conformational species using a series of biophysical experiments. We first tested whether P2 was able to detect unfolded AgHalo monomers. While both the WTHalo and AgHalo adopt folded structures in buffer as measured by the circular dichroism (CD) spectroscopy (Figure S2a), both of them could be incubated in 8 M urea to induce complete denaturation and unfolding (Figure S2b). Denatured AgHalo-P1 and AgHalo-P2 conjugates showed a 1.4- and 2.6fold fluorescence increase, respectively (Figure S2c). The same was true for the fully denatured WT-Halo monomer at 8 M urea (red curve in Figure S2b and d): there was a 2.2-fold fluorescence increase for the unfolded WT-Halo-P2 conjugate and a 50% fluorescence reduction for the unfolded WT-HaloP1 conjugate. These data suggest that both P1 and P2 are incapable of detecting unfolded WT-Halo or AgHalo monomers via a substantial fluorescence increase. Although high urea concentrations could completely denature AgHalo, lower urea concentrations likely result in partially denatured AgHalo that can be considered as misfolded conformations (Figure S2b). To test whether P2 was able to detect misfolded AgHalo conformations, we next incubated the AgHalo-P2 conjugate in 4 M urea to induce its partial denaturation. At this concentration of urea, AgHalo still retained roughly 25% of its secondary structure based on the CD measurement (Figure S2b). Different from unfolded AgHalo, the partially unfolded or misfolded AgHalo with 4 M urea exhibited a 16-fold fluorescence increase (Figures 2d and S2c), nearly equal to what was seen from the AgHalo aggregates (18-fold in Figure 2b). The observed fluorescence change was not due to a direct interaction between urea and P2 as no fluorescence changes were observed in the absence of AgHalo (Figure 2). Although WT-Halo is a more thermodynamically stable variant than AgHalo (Figure S2b), its partially misfolded conformations at 4 M urea (53% remaining secondary structures) could induce a 12-fold fluorescence increase (Figure S2d). These data suggest that P2 likely detects misfolded or partially unfolded structures instead of fully unfolded proteins. To investigate the correlation between fluorescence of AgHalo-P2 and its extent of misfolding, we further carried out a titration experiment wherein the AgHalo-P2 conjugate was incubated in increasing urea concentrations (red curve in Figure S3a). We found that the AgHalo-P2 fluorescence showed a bell-shape as a function of urea concentration, with its maximal intensity being observed at in the range of 3−4 M urea concentrations (red curve in Figure S3a). At urea concentrations lower than 4 M, where CD measurements confirmed there was still a large degree of secondary structure remaining (∼50% at 3 M urea), a prominent florescent signal (14.6 fold at 3 M urea) was observed (red curve in Figure S3a). At concentrations of urea higher than 4 M, CD measurements showed a near complete loss of secondary protein structure (8% remaining at 6 M urea). A decreased, but still significant fluorescent signal from the fluorescent molecular rotor could be seen (8.5-fold at 6 M urea). A similar trend was observed for the more stable WT-Halo (black curve in Figure S3a): the peak of fluorescent intensity was found to be around 4−5 M urea concentrations and the overall
dependence (Figure 1c). The viscosity dependence of the molecule’s fluorescence shows that CCVJ will still become fluorescent when rotationally hindered, and so may become fluorescent upon protein aggregation. In addition to glycerol, we also explored the interaction of the small molecule with sucrose, another viscous solvent. A 68% (w/v) sucrose solution caused a 5-fold increase in fluorescence, with lower concentrations resulting in lower fluorescence (Figure S1a), further confirming the viscosity dependence of P2. Finally, we tested whether P2 would become fluorescent when incubating with a hydrophobic protein, bovine serum albumin (BSA). In the presence of 2 mg/mL BSA, a 60-fold increase in fluorescence was seen (Figure S1b), likely due to nonspecific binding of the small molecule to the hydrophobic surface of BSA. This highlights the necessity of the covalent conjugation of the molecule to the Halo protein and washing out the unbound small molecules in vivo, to limit background signal. It was previously shown that the SBD fluorophore of P1 was able to report on aggregation of the AgHalo-P1 conjugate via an 8-fold fluorescence increase (Figure 2a). To show that P2
Figure 2. Representative fluorescence emission spectra (ex = 440 nm). Fluorescence increase was observed for (a) P1 and (b) P2 when their corresponding AgHalo conjugates were incubated at 59 °C (red curve). In the absence of AgHalo, both (a) P1 and (b) P2 did not exhibit fluorescence change at 59 °C (blue curve). Urea denaturation (c) causes a turn on of fluorescence from both P1 (d) and P2 when it is conjugated to AgHalo, but not from nonconjugated ligands. In the absence of AgHalo, both (c) P1 and (d) P2 did not exhibit fluorescence change in the presence of urea (blue curve).
was able to report on AgHalo aggregation, we incubated AgHalo-P2 conjugate at 59 °C to induce aggregation and observed an 18-fold fluorescence increase of CCVJ in P2 (Figure 2b), roughly twice the signal change of P1 (Figure 2a). By contrast, heating P2 ligand at 59 °C without AgHalo did not result in noticeable fluorescence increase (Figure 2b). Thus, the CCVJ moiety in P2 not only reliably reports on aggregation of the AgHalo-P2 conjugate but also yields a higher fluorescence enhancement than P1. C
DOI: 10.1021/acs.bioconjchem.7b00763 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry fluorescence increase was less prominent than that of the AgHalo-P2 conjugate. These data collectively suggest that P2 is able to report on the misfolding of the AgHalo protein in a fluorogenic manner. Its fluorescence turn-on requires a certain extent of secondary structures remaining in the misfolded conformations, because the completely denatured AgHalo-P2 conjugate at 8 M urea barely showed any fluorescence increase. We speculate that the molecular rotor-based P2 fluorophore needs to interact with an exposed and structured region to effectively hinder its rotation to turn on fluorescence. The unfolded structure, however, cannot efficiently engage this integration and cannot be reported by this class of fluorophores. The same experiments were carried out using the AgHalo-P1 conjugate. Interestingly, the AgHalo-P1 conjugate showed a 5.6-fold fluorescence increase upon misfolding with 4 M urea (Figure 2b and red curve in Figure S3b). In the urea titration experiment, the AgHalo-P1 conjugate exhibited very similar behaviors to those of the AgHalo-P2 conjugate in terms of its bell-shaped fluorescence intensity as a function of urea concentration (red curve in Figure S3b). However, the fluorescence increase from P1 was at most 5.6-fold at 4 M urea and thus P2 is able to detect misfolded AgHalo monomers with a substantially more potent fluorescence increase. When we tested the WT-Halo-P1 conjugate in these experiments (Figure S 2d and black curve in Figure S3b), the overall fluorescence turn-on was much less substantial than that of the WT-Halo-P2 conjugate. These data strongly suggest that P2 has a higher potency to detect misfolded structures of both WT-Halo and AgHalo than P2 does. P2 Detects Misfolded AgHalo Monomers Induced by Heat Stress. While urea denaturation reveals the detection of misfolded AgHalo by P2, it is not the natural misfolding condition that AgHalo encounters in a biological context. Thus, we used heat stress, a well-established cellular stress, to further demonstrate that P2 can report on the formation of misfolded AgHalo monomers. The CD spectrum showed that AgHalo underwent significant secondary structural changes at temperatures between 37 and 50 °C (blue box in Figure 3a), indicating misfolding of AgHalo. In contrast to the signal seen from misfolding induced by medium urea concentrations, P1 only exhibited minimal (98% based on SDS-PAGE. General Synthetic and Chromatography Methods. Detailed information is provided in Supporting Information.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00763.
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Figures S1−S11, general synthetic and chromatographic methods, and supporting reference (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yu Liu: 0000-0002-0779-1488 Kun Miao: 0000-0001-6567-3650 Xin Zhang: 0000-0001-6686-1645 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface, Paul Berg Early Career Professorship, and Lloyd and Dottie Huck Early Career Award. We thank the Penn State Microscopy and Cytometry Facility and Biophysical Facility for assistance in data acquisition. H
DOI: 10.1021/acs.bioconjchem.7b00763 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.7b00763 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.bioconjchem.7b00763 Bioconjugate Chem. XXXX, XXX, XXX−XXX
