A HaloTag-Based Multicolor Fluorogenic Sensor Visualizes and

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A HaloTag-based multi-color fluorogenic sensor visualizes and quantifies proteome stress in live cells using solvatochromic and molecular rotor-based fluorophores Yu Liu, Kun Miao, Yinghao Li, Matthew Fares, Shuyuan Chen, and Xin Zhang Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00135 • Publication Date (Web): 23 Feb 2018 Downloaded from http://pubs.acs.org on February 24, 2018

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Biochemistry

A HaloTag-based multi-color fluorogenic sensor visualizes and quantifies proteome stress in live cells using solvatochromic and molecular rotor-based fluorophores

Yu Liu1, Kun Miao1, Yinghao Li1, Matthew Fares1, Shuyuan Chen1, and Xin Zhang1, 2, 3* 1

Department of Chemistry, 2Department of Biochemistry and Molecular Biology, 3The Huck In-

stitutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802

*Correspondence should be addressed to Xin Zhang ([email protected]).

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ABSTRACT Protein homeostasis, or proteostasis, is essential for cellular fitness and viability. Many environmental factors compromise proteostasis, induce global proteome stress, and cause diseases. Proteome stress sensor is a powerful tool to dissect the mechanism of cellular stress and find therapeutics that ameliorate these diseases. In this work, we present a multi-color HaloTag-based sensor (named AgHalo) to visualize and quantify proteome stresses in live cells. The current AgHalo sensor is equipped with three fluorogenic probes that turn on fluorescence when the sensor forms either soluble oligomers or insoluble aggregates upon exposure to stress conditions, both in vitro and in cellulo. Further, AgHalo probes can be combined with commercially available always-fluorescent HaloTag ligands to enable two-color imaging, allowing for direct visualization of the AgHalo sensor both before and after subjecting cells to stress conditions. Finally, pulse-chase experiments can be carried out to discern changes in cellular proteome in live cells by first forming the AgHalo conjugate and then either applying or removing stress at any desired time point. In summary, the AgHalo sensor can be used to visualize and quantify proteome stress in live cells, a task that is difficult to accomplish using previous always-fluorescent methods. This sensor should be suited to evaluate cellular proteostasis under various exogenous stresses, including chemical toxins, drugs, and environmental factors.

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Table of Contents (TOC)

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INTRODUCTION Protein homeostasis (proteostasis) dynamically adapts to diverse environmental factors and cellular events.1 To achieve an appropriate level of proteostasis, the endogenous proteome has evolved to maintain a specific balance between the folded, misfolded and aggregate states of its protein components. However, exogenous stress conditions (including environmental perturbations, chemical toxins, and pathogen invasion) impair the integrity of proteostasis by shifting the free energy landscape of protein folding and/or inducing chemical or conformational changes in folded proteins.2, 3 Failure to maintain proteostasis during stress leads to global misfolding and aggregation of the endogenous proteome, resulting in a series of aberrant conformations that include misfolded proteins in the form of soluble oligomers, disordered or amorphous aggregates, and fibrils containing ordered hydrogen-bonded β-sheet structures. Formation of these structures often leads to the loss of essential functions and the formation of toxic aggregates. Both of these phenomena have been increasingly associated with a growing number of diseases, such as cancer, neurodegeneration, metabolic disorders, cardiovascular disease and inflammation.4-6 New sensors that can detect and quantify proteostasis loss are important to understand molecular mechanisms and to advance the development of therapeutic strategies for these diseases. Given the complexity of cellular milieu, detection of the misfolding and aggregation of proteome stress sensors in live cell is challenging.7 Fusion of fluorescent protein tags or incorporation of fluorescent probes to the destabilized sensors has been extensively utilized to visualize their aggregation in live cells, exemplified by the pioneering works using destabilized luciferases or split NanoLuc,8, 9 fusion of fluorescent protein,10-17 incorporation of fluorescent un-natural or FlAsH labeling dye,18,

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destabilized retroaldolase enzyme20, and destabilized barnase.21 The

presence of proteome stress was successfully detected by observing the formation of punctate insoluble aggregates of these sensors using microscopes and flow cytometry.22 If stress-induced soluble oligomers of these sensors do not form punctate structures inside cells, these sensors would fall short in detecting these stress conditions. In addition, it is difficult to quantify the proACS Paragon Plus Environment

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Biochemistry

teostasis impairment that occurs in response to stress in live cells and in real time using fluorescence intensity, because samples evaluated by these sensors emit fluorescence both before and after formation of protein aggregates (Figure 1a).

Figure 1: The HaloTag-based multi-color fluorogenic proteome stress sensor. A destabilized HaloTag mutant (K73T) represents the meta-stable population of cellular proteome, remains well folded in unstressed cells, and aggregates in stressed cells. The “AgHalo sensor” refers to the conjugate between Halo K73T and a small molecule ligand. (a) Always-fluorescent methods visualized fluorescent puncta formation to detect AgHalo aggregation in stressed proteome. (b) The new method uses a set of fluorogenic AgHalo probes whose fluorescence is turned on when the AgHalo sensor forms misfolded or unfolded monomers, soluble oligomers, or insoluble aggregates. (c) The fluorogenic AgHalo probes can be combined with class HaloTag ligands to enable a two-color imaging assay.

To overcome these limitations, we recently developed a fluorescence turn-on (fluorogenic) proteome stress sensor that is only fluorescent upon its misfolding and aggregation in live cells (Figure 1b). This sensor comprises a protein component and a small molecule component. The protein component is a destabilized HaloTag mutant (K73T, ∆Gfolding = -2.06 kcal/mol) hereinafter named AgHalo, which undergoes misfolding and aggregation in cells that are subjected to a variety of cellular stress conditions. The small molecule component is a set of environmentACS Paragon Plus Environment

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sensitive fluorophores that are covalently conjugated to AgHalo and exhibits significant fluorescence increase upon aggregation of AgHalo.23, 24 Using two environment-sensitive fluorophores (Figure 2, AgHalo 495 and 550), we have demonstrated their ability to exhibit fluorescence increase (~8-18 fold) in response to the misfolding or aggregation of AgHalo. 22 In this work, we have enriched the AgHalo sensor system by developing a new fluorogenic probe (Figure 2, AgHalo 564) of an improved fold-of-change in fluorescence increase (57.6-fold) upon aggregation of AgHalo in vitro. Thus, the AgHalo sensor is equipped with three different fluorophores: AgHalo 495, 550, and 564. We applied the AgHalo sensor to detect proteostasis deficiency induced by a variety of exogenous stress conditions, including thermal, oxidative, osmotic, and chemical factors. Compared to previous always-fluorescent methods wherein fluorescent punctate structures are used to identify insoluble protein aggregates, the AgHalo sensor uses fluorogenic signal to detect existence of both soluble and insoluble aggregates. Thus, this system is more sensitive to detect abovementioned proteome stresses. More importantly, we further show that a dual-probe labeling strategy allows for a simultaneous visualization of AgHalo in both folded and aggregated structures using different fluorescent channels (Figure 1c), a resolution that has never been achieved before. This attractive goal is easily realized by combining fluorogenic AgHalo probes with commercially available HaloTag ligands (Figure 1c). Lastly, the flexibility of using small molecule probes to regulate fluorescence signal enabled pulse-chase experiments to monitor recovery of proteome after stress conditions without the need of translational attenuation.8 Owing to its unique fluorogenicity, we believe that the AgHalo sensor presented in this work is suited to quantify and detect proteome stress through direct fluorescence intensity readout and microscopic fluorescent imaging. This method can serve as a powerful tool to evaluate the impact of nanoparticles, biomaterials, and chemical toxins on cellular proteostasis. EXPERIMENTAL PROCEDURE ACS Paragon Plus Environment

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1. Plasmids, probes, chemicals, and materials. Plasmid encoding AgHalo protein sensor (Halo K73T mutant) was previously constructed based on pHTN HaloTag® CMV-neo vector from Promega (G7721).23 Plasmid is available upon request addressed to XZ. AgHalo probes are available upon request addressed to XZ. Details on the synthesis of AgHalo probes can be found in the supporting information and previously reported literatures.23 Halo K73T was expressed and purified as previously described. 22 Chemical stressors: •

MG132: Selleckchem, Catalog No. S2619

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Sodium (meta)arsenite (CAS: 7784-46-5): Sigma Aldrich, Catalog No. S7400-100G.

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D-Sorbitol (CAS: 50-70-4): Sigma Aldrich, BioXtra, Catalog No. S7547-250G.

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Nilotinib: Selleckchem, Catalog No. S1033

Tissue culture materials: •

MatTek poly-D-lysine coated 35 mm glass bottom dishes (MatTek Corporation): Part No. P35GC-1.5-14-C.

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Dulbecco's Modified Eagle Medium (DMEM): ThermoFisher, Catalog No. 11995065.

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Opti-MEM® Reduced-Serum Medium: Catalog No. 31985062.

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TrpLE Express: ThermoFisher, Catalog No. 12605028.

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Fetal Bovine Serum (FBS): ThermoFisher, Catalog No. 26140079.

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Penicillin-Streptomycin-Glutamine (PSQ, 100X): ThermoFisher, Catalog No. 10378016.

2. Cell culture and in-situ labeling. The HEK293T cells were seeded at 25% confluency (8×105 cells/chamber) 24 h prior to transfection in poly-D-lysine coated 35 mm glass bottom dishes (MatTek Corporation) for fluorescence confocal microscopy and a 12-well or 96-well plate for fluorescence intensity measurement. Cells were grown in DMEM media supplemented with 10% FBS and PenicillinStreptomycin antibiotics until they reached 50-60% confluency. For in situ probe labeling, probes in DMSO stock (2 mM) were diluted into fresh DMEM media as 10X stock solution (10 ACS Paragon Plus Environment

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µM) and introduced to the media to a final concentration of 1 µM. Previous studies show that AgHalo expression level does not interfere with detection of proteome stress23. Thus, transient transfection of the plasmid encoding Halo-K73T (AgHalo, 4 µg/dish) was carried out using XtremeGene™ 9 DNA transfection reagent (Roche) according to the manufacturer’s instructions in Opti-MEM® Reduced-Serum Medium. AgHalo protein was transiently expressed in the presence of 1 µM AgHalo probes for 24 h prior to stress conditions. 3. Visualization of proteome stress by fluorescence confocal microscopy. To treat cells with small molecule stressors, media containing AgHalo probes was removed by replacing it with fresh DMEM media and incubating at 37 ºC for a duration of 30 min. Subsequently, cells were treated with fresh DMEM media containing different chemical stressors: 1 M NaCl and 0.5 M sorbitol for osmotic stress, 5 min; 10 µM MG132 and 50 µM Nilotinib for drug induced proteome stress, 18 h; 50 µM Sodium (meta)arsenite for oxidative stress, 18 h. To treat cells with heat stress, excess AgHalo probes was removed by incubating cells in fresh DMEM media at 37 ºC for a duration of 30 min. An additional media change was performed to subject cells in fresh DMEM media without AgHalo probes. Subsequently, cells were transferred to a CO2 incubator that was pre-warmed to 45 ºC for 1 h incubation. The stressed live cell samples were visualized by confocal fluorescence microscope. Hoechst 33342 (0.1 µg/mL) was introduced and incubated for 30 min prior to imaging. Confocal images were obtained using Olympus FluoView™ FV1000 confocal microscope. Aggregation of the AgHalo sensor was visualized using blue argon (488 nm) laser. Nuclear counter staining was visualized using violet laser (405 nm). TMR ligand was visualized using green HeNe laser (543 nm). 4. Quantification of proteome stress by fluorescence microplate reader. Excess probe was removed by replacing it with fresh DMEM media and being subjected to a 30 min diffusion of the unbound AgHalo probes, followed by a change into fresh DMEM media. To treat cells with small molecule stressors, cells were incubated with media containing different ACS Paragon Plus Environment

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chemical stressors (10 µM MG132, 50 µM Nilotinib, 50 µM Sodium (meta)arsenite) for 18 h prior to harvesting. Harvested cells were pelleted by centrifugation to remove DMEM media, resuspended in 500 µL FluoroBrite DMEM media (without FBS), and subjected for fluorescence intensity measurement. For osmotic stress, cells were harvested and resuspended in 500 µL FluoroBrite DMEM media (without FBS) that contain 1 M NaCl or 0.5 M sorbitol for 5 min. Fluorescence intensity was measurement immediately after osmotic stress. For heat stress, cells were harvested and resuspended in 500 µL FluoroBrite DMEM media (without FBS). Subsequently, cell suspension was incubated in a 45 ºC water bath for 1 h prior to fluorescence intensity measurement. Fluorescence intensity was recorded using a Tecan M1000Pro fluorescence microplate reader using the following conditions: Ex = 440 nm/Em = 495 nm for AgHalo 495, Ex = 440 nm/Em = 550 nm for AgHalo 550, Ex = 451 nm/Em = 564 nm for AgHalo 564. The signals were then normalized against cell number measured by Biorad TC20TM automated cell counter. Experiments were also carried out using 96-well plates. HEK293T cells were transiently transfected with AgHalo and treated with 1 µM AgHalo probes during AgHalo expression for 24 h. Cells were re-seeded on a 96-well plate to a 70% confluency in fresh DMEM media with 1 µM AgHalo probes. After 12 h, excess AgHalo probes were washed away by incubating in fresh DMEM media for 30 min. Subsequently, cells were treated with fresh DMEM media with 0.5 M sorbitol for 5 min. Fluorescence intensity was recorded using a Tecan M1000Pro fluorescence microplate reader using multiple reads per well with a circular alignment: Ex = 440 nm/Em = 495 nm for AgHalo 495. To demonstrate how the fluorescence fold-of-change was derived from fluorescent intensities, we herein describe how fold-of-change for MG132-induced fluorescence increase using AgHalo 495. Non-transfected HEK 293T cells treated with 1 µM AgHalo 495 resulted in a background signal of 186.4 ± 26.9. Transfected HEK 293T cells with or without MG-132 stress yielded fluorescence signal of 3319.8 ± 913.3 and 804.5 ± 61,5, respectively. After background correction, MG-132 treatment induced a 5.07 ± 1.47-fold fluorescence increase. ACS Paragon Plus Environment

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5. Pulse chase experiment to monitor post-stress recovery. AgHalo protein was transiently expressed in the presence of 1 µM AgHalo 550 for 24 h prior to exposure to osmotic stress. Subsequently, excess probe was removed by incubation with fresh DMEM media for 30 min to wash off unbound AgHalo 550, followed by a change into fresh DMEM media with 0.5 M sorbitol for 5 min. Cells were restored in fresh DMEM media for 2 h at 37 ºC in a CO2 incubator. Confocal fluorescence microscopy was carried out before stress, immediately after 5 min of sorbitol stress, and after the 2 h recovery. Confocal images were obtained using Olympus FluoView™ FV1000 confocal microscope. Aggregation of the AgHalo sensor was visualized using blue argon (488 nm) laser. Nuclear counter staining was visualized using violet laser (405 nm). TMR ligand was visualized using green HeNe laser (543 nm). 6. SDS-PAGE analysis of TMR labeled-AgHalo. Cells after stress were harvested, pelleted, and lysed in RIPA buffer. Subsequently, protein concentration of lysate was measured using Bradford assay and quantified using a standard linear curve using known concentrations of BSA. Same amount of lysate was loaded on SDS-PAGE gel. Fluorescent band of TMR-labeled AgHalo protein was quantified using Typhoon 9410 scanner. 7. General synthetic and chromatography methods for AgHalo 564 are provided in Supporting Information. RESULTS Principle of AgHalo protein sensor to detect proteome stresses. A suitable proteome stress sensor should be folded in non-stressed cells but able to adopt a misfolded or aggregated state under a desired set of stress conditions. Thus, the integrity of cellular proteostasis can be assessed by monitoring aggregation status of this sensor, because its proper folding requires sufficient assistance from the proteostasis network. Under stressed conditions, the capacity of this network is largely consumed by the binding of chaperones, chaperonins and other folding enzymes to endogenous proteome denaturation and aggregation. The lack of a ACS Paragon Plus Environment

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sufficient proteostasis capacity leads to misfolding or aggregation of destabilized proteins. Thus, aggregation of a destabilized Halo variant should report on the impairment of proteostasis upon introduction of a cellular stress. We designed a screening scheme for proteins with three key features: thermodynamic instability, reliance on the proteostasis network for proper folding, and retention of an intact reactivity toward Halo ligands23. Halo K73T was identified as a suitable Halo variant to use in this context for the following reasons. Stability of Halo K73T (∆Gfolding = -2.0 kcal/mol) falls to the lower 15% of cellular proteome.25 In a previous report, we showed that AgHalo’s aggregation as measured by its fluorescence kinetics correlated with aggregation kinetics of the cellular proteome upon heat stress at 42 or 45 °C in HEK293T cells.23 Further, we showed that stress conditions inducing Halo K73T aggregation also lead to aggregation of several other cellular proteins. These proteins have previously been either identified as metastable proteins, or associated with protein misfolding diseases, or sequestered by protein aggregates in stressed cellular proteome. By contrast, the wild-type Halo is a stable protein (∆Gfolding = -5.6 kcal/mol) whose stability is close to the average of cellular proteins (∆Gfolding = -6.8 kcal/mol).25 As a representation of the stable portion of cellular proteome, we showed that Halo exhibits negligible aggregation under mild heat and chemical stressors.23 Finally, we determined that half-life of Halo K73T in mammalian cells (HEK293T) was ~ 52 hours (Figure S1), through a pulse-chase experiment. This experiment indicates that Halo K73T is a substrate of protein degradation pathway (likely the ubiquitin-proteasome pathway) and has sufficiently long lifetime to allow for fluorescence-based measurement under stress conditions. Taken together, Halo K73T serves a proper protein component of the AgHalo sensor to report on proteome aggregation induced by proteome stress conditions. Validation of fluorogenic probes that exhibit fluorescence increase upon misfolding and aggregation of the AgHalo-probe conjugates in vitro.

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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. In design of a fluorescence turn-on probe to sense this physical chemical change, we evaluated a set of environment-sensitive fluorophores for their ability to interact with newly exposed hydrophobic residues and emit fluorescence upon protein misfolding26. Environment-sensitive fluorophores are well known to exhibit altered fluorescence intensity and/or a shift in maximal emission wavelength upon transferring from polar (hydrophilic) solvents to non-polar (hydrophobic) solvents and from conformational flexible to rigid environment.27-32 Thus, the misfolding and aggregation of the AgHalo sensor that lead to collapse and exposure of its hydrophobic core and result in an environmental changes could be sensed by suitable environment-sensitive fluorophores via a measurable fluorescence intensity enhancement.

Figure 2: Structure of the AgHalo probes. (a) Small molecule design strategy. The AgHalo probes have three components: HaloTag warhead for bioorthogonal conjugation, extended linker to minimize background fluorescence upon conjugation, and environment-sensitive fluorophores that are sensitive to polarity or conformational rigidity of their local environment. (b) Structures of small molecule probes used in this work. Nomenclature of the AgHalo probes is based on their fluorescence emission maxima of the AgHalo-probe conjugate in forms of aggregates.

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Based on this principle, three fluorogenic probes have been thus far developed for the AgHalo sensor: AgHalo 495, 550, and 565. These probes were designed to include three structural moieties: the HaloTag warhead (Figure 2a, in black) for bioorthogonal conjugation to Halo K73T in live cells33; the extended linker (Figure 2a, in grey) that prevents interaction between Halo and probe to minimize the background fluorescence; and an environment-sensitive fluorophore to report on aggregation of Halo K73T. In addition to previously reported AgHalo 495 (Figure 2b, in blue) and AgHalo 550 (Figure 2b, in green), we introduced an optimized probe AgHalo 564 to enrich the AgHalo probe suite in this work (Figure 2b, in yellow). AgHalo 564 is a thiol-derivative of AgHalo 550 (Figure 2b). Interestingly, AgHalo 564 exhibited a higher photostability than AgHalo 550, thus it is more resistant to continuous exposure to excitation (Figure S2). We reason that this advantage primarily comes from two chemical changes: 1) the benzothiadiazole scaffold is more photostable than the benzoxadiazole scaffold as previously reported,34 and 2) the dimethyl amino group in AgHalo 564 is more resistant to oxidation-related photobleach than the methyl amino group in AgHalo 550.

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Figure 3: In vitro validation of AgHalo probes to report on heat induced misfolding and aggregation of AgHalo sensor. (a) Left: Normalized fluorescence emission spectra of aggregated AgHalo-probe conjugates. Right: Images of folded AgHalo-probe conjugate at 25 °C (top panel) and aggregated AgHaloprobe conjugate at 59 °C (bottom panel). Numbers denote the fold-of-change in fluorescence intensity increase upon aggregation. (b) AgHalo probes exhibit temperature dependent fluorescence upon heatinduced misfolding and aggregation of the AgHalo sensor. Samples (60 µM Halo K73T and 30 µM AgHalo probe in DPBS buffer) were held at indicated temperature for 15 min prior to measurement. Fluorescence intensity was recorded using a Tecan M1000Pro fluorescence microplate reader using the following conditions: Ex = 440 nm/Em = 495 nm for AgHalo 495, Ex = 440 nm/Em = 550 nm for AgHalo 550, Ex = 451 nm/Em = 564 nm for AgHalo 564.

All three probes formed covalent conjugate with Halo K73T and remained dark when this conjugate was folded (Figure 3a, top right panel). Incubating the Halo K73T-probe conjugates at 59 ºC effectively induced aggregation of Halo K73T, resulting in fluorescence intensity increase for all probes (Figure 3a, left panel). The emission maxima and extent of intensity increase of these probes were measured and quantified as 495 nm and 14.7-fold for AgHalo 495, 550 nm and 9.2-fold for AgHalo 550, and 564 nm and 56.4-fold for AgHalo 564 (Figure 3a, lower right panel). Herein, we refer AgHalo sensor to the covalent conjugate between Halo K73T and the fluorogenic probes. Upon its aggregation, the AgHalo sensor is able to exhibit fluorescence intensity increase to different extent and different colors based on which fluorogenic probe is used. Protein aggregation is a multi-step process that includes formation of misfolded or unfolded monomer, soluble oligomers and insoluble aggregates. Our previous work established conditions wherein AgHalo sensor aggregation can be controlled when incubated under controlled temperatures for 15 min.23 While Halo K73T remains largely folded at low temperatures around 25 ºC, a series of biophysical analyses show that Halo K73T misfolds at the temperature range of 37 to 50 °C, as indicated by the loss of secondary structures from a circular dichroism (CD) spectroscopic analysis (data adapted from ref. 24, Figure S3a). The misfolded Halo K73T does not form soluble or insoluble aggregates, the lack of which is evidenced by a photo-induced cross-linking experiment (data adapted from ref. 24, Figure S3b) and a dynamic light scattering experiment (data adapted from ref. 24, Figure S3c). Halo K73T incubated at further increasing temperatures beyond 54.5 ºC effectively form significant amount of soluble oligomers and insolACS Paragon Plus Environment

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Biochemistry

uble aggregates (data adapted from ref. 24, Figures S3b and S3c).23, 24 Using this knowledge, we showed that AgHalo 495 is the most sensitive probe whose fluorescence intensity exhibited a 15fold enhancement even at the temperature where the AgHalo sensor formed misfolded or unfolded monomers (top panel in Figure 3b). By contrast, fluorescence intensity of AgHalo 550 could exhibit only moderate increase (~2-fold) with misfolded or unfolded AgHalo sensor (middle panel in Figure 3b). Formation of soluble oligomers, however, resulted in ~9-fold increase in its fluorescence intensity (middle panel in Figure 3b). The new probe, AgHalo 564, behaved similarly to AgHalo 550, primarily responded to soluble oligomers and insoluble aggregates (lower panel in Figure 3b). Validation of the AgHalo sensor to visualize and quantify proteome stress in live cells.

Figure 4: Validation of AgHalo sensor to detect proteome stress induced by MG132, a proteosome inhibitor. (a) Flow chart of experimental procedures. AgHalo sensor was transiently transfected and expressed in HEK293T for 24 h in the presence of 1 µM AgHalo probe. Media containing probes were replaced with media containing 10 µM MG132. Cells were incubated with MG132 for 18 h prior to fluorescent intensity measurement and live cell confocal fluorescence microscopy. (b) Quantification of proteome stress induced by MG132 using fluorescence plate reader. Stressed cells were harvested and transferred to 96-well plate for subsequent measurements. Fluorescence intensity was recorded using a Tecan M1000Pro fluorescence microplate reader using the following conditions: Ex = 440 nm/Em = 495 nm for AgHalo 495, Ex = 440 nm/Em = 550 nm for AgHalo 550, Ex = 451 nm/Em = 564 nm for AgHalo 564. Error bars: standard errors of biological triplicates. (c) Detection of MG132-induced proteome stress using conACS Paragon Plus Environment

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focal imaging. Upper panel: unstressed cells (treated with DMSO). Lower panel: stressed cells (treated with 10 µM MG132 for 18 h). In each image, white arrows highlight fluorescent punctate structures that correspond to insoluble aggregates, whereas diffuse fluorescence represents soluble aggregates.

The in vitro experiments collectively suggest that these fluorogenic probes allow for detection of various conformational steps during AgHalo aggregation. Next, we validated whether all three fluorogenic probes were capable of detecting AgHalo aggregation in cells that are subjected to exogenous stress conditions. In this experiment (scheme shown in Figure 4a), HEK293T cells were transfected with the plasmid encoding Halo K73T. During transfection, cells were treated with an appropriate AgHalo probe (1 µM) to ensure that newly synthesized and folded Halo K73T was covalently labeled by the AgHalo probe to produce the AgHalo sensor. After 24 hours, unreacted AgHalo probe was removed from the growth media and exogenous stressors was added to induce proteome stress. In this experimental scheme, newly synthesized Halo K73T proteins after conjugate formation is not problematic, because unreacted probes are removed and these unlabeled proteins do not contribute to the fluorescence signal. MG132 is a small molecule proteosome inhibitor that blocks the ubiquitin-proteosome protein degradation pathway35. As a result, it is established to induce accumulation of misfolded or unfolded endogenous proteins, eventually leading to aggregation of the cellular proteome. We induced proteome stress by treating HEK293T cells harboring the AgHalo sensor with 10 µM of MG132 at 37 ºC for 18 hours and measured the fluorescence intensities of live cells using a fluorescence microplate reader. This condition, as shown in both this study and a previous work, did not induce cellular cytotoxicity as measured by a commercially available LDH assay kit (Figure S4). 23 All three AgHalo probes could permeate cell membrane, covalently label AgHalo, and quantitatively report MG132-induced AgHalo aggregation by fluorescence intensity increase (Figure 4b). Using non-stressed cells as a control (DMSO treated), AgHalo 495 yielded the highest fluorescence increase of 5.1-fold, followed by AgHalo 550 of 3.1-fold and AgHalo 564 of 2.0-fold. The value of fluorescence-fold-change was found to be consistent when we used either ACS Paragon Plus Environment

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cell number (Figure 4b) or expression level of AgHalo (Figures S5a-b) to normalize the fluorescence signal, suggesting that the AgHalo sensor produced robust fluorescence increase via two data analysis methods. Confocal fluorescence microscopy confirmed that fluorescence signal originated from aggregation of the AgHalo sensor using all three probes, demonstrated by both diffuse and punctate fluorescent structures only in cells treated with MG132 but not in nonstressed cells (Figures 4c and S6). While the fluorescent punctate structures that would most likely correspond to insoluble aggregates were similarly visible using all three probes, the diffusive fluorescence was most clearly observed by AgHalo 495, followed by AgHalo 550 and 564 (Figures 4c and S6). Because the in vitro data (Figure 3b) showed that AgHalo 495 was the most sensitive probe to misfolded and unfolded AgHalo, it would be feasible to attribute the diffuse fluorescence structures to a mixture of misfolded (or unfolded) monomers and soluble oligomers. We next subjected HEK293T cells harboring the AgHalo sensor to several commonly used stress conditions. These conditions include osmotic stress by incubating cells with 1 M NaCl or 0.5 M Sorbitol for 5 minutes,36-38 thermal stress by heating cells to 45 ºC for 1 hour,39, 40 chemical stress by incubating cells with 50 µM Nilotinib for 18 hours,23 and oxidative stress by incubating cells with 50 µM Sodium Arsenate (NaAsO2) for 18 hours.41 All these conditions have been established to induce proteome stress and drive the metastable portion of the endogenous proteins to aggregate. In particular, the AgHalo sensor was previously found to aggregate under heat and treatment of 50 µM Nilotinib. Consistent to these notions, we were able to observe fluorescence intensity increase to different extent for all these conditions using AgHalo 495 fluorescence measured by a microplate reader (Figure 5). Expression and labeling of AgHalo in HEK293T cells was confirmed by an SDS-PAGE analysis wherein the same set of experiments were carried out in the presence of the TMR-Halo ligand whose fluorescence was used as a signal (Figure S7). We found that the expression level of AgHalo was minimally perturbed by the stress conditions used herein, consistent to the 52-hour half-life of AgHalo in HEK293T cells. As a control experiment, we did not observe any fluorescence increase upon incubating either ACS Paragon Plus Environment

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AgHalo ligand 495 (Figure S8a) or the AgHalo-495 conjugate (Figure S8b) with these stressors in vitro, indicating that observed fluorescence increase was largely due to aggregation of the AgHalo sensor within a stressed proteome. One of the major uses of the AgHalo sensor could be its application in a screening platform. To this end, we showed that the osmotic stress induced by 0.5 M sorbitol was able to produce a 1.85-fold fluorescence increase (Figure S9), comparable to the 1.74-fold change from a bulk measurement (Figure 5). It is important to point out that this data is insufficient to state that the AgHalo sensor system is suited for a high-throughput application at its present stage. Optimization of AgHalo probes for specific proteome stress conditions may be necessary to produce sensitive and robust signal towards a desired high-throughput screening platform. However, data demonstrated herein speaks for the potential of the AgHalo sensor to be developed into screening assays.

Figure 5: Quantification of proteome stress using fluorescence intensity of AgHalo 495. AgHalo sensor was transiently transfected and expressed in HEK293T for 24 h in the presence of 1 µM AgHalo 495. Experimental details are described the Experimental Procedure section. Fluorescence intensities were taken by Tecan M1000 Pro fluorescence plate reader (Ex = 440 nm/Em = 495 nm). Error bars: standard errors of biological triplicates.

The fluorogenic AgHalo sensor visualizes proteome stress that could be invisible to alwaysfluorescent methods in live cells. To further demonstrate advantage of the AgHalo sensor, we carried out a two-color live cell imaging experiment by combining a commercial HaloTag TMR ligand (1 µM) with the ACS Paragon Plus Environment

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Biochemistry

AgHalo 550 probe (1 µM) during the in-situ labeling step (experimental scheme shown in Figure 6a). This experimental scheme would allow the newly synthesized and folded Halo K73T proteins to be equally labeled with either the HaloTag TMR ligand or the AgHalo 550 probe. Different from the AgHalo 550 probe, the HaloTag TMR ligand is fluorescent both before and after aggregation of the AgHalo sensor. Thus, signal from the TMR ligand represents alwaysfluorescent methods using fluorescent protein fusions or fluorescent probes to monitor cellular location of the AgHalo sensor (Figure 1a). By contrast, signal of the AgHalo 550 probe exclusively arises from AgHalo aggregates in stressed proteome (Figure 1b).

Figure 6: Fluorescence confocal imaging reveals that AgHalo sensor forms soluble oligomers and/or insoluble aggregates in HEK293T cells subjected to osmotic, chemical, thermal, and oxidative stress conditions. (a) Experimental scheme. AgHalo sensor was transiently transfected and expressed in HEK293T for 24 h in presence of 1 µM AgHalo 550 and 1 µM TMR ligand. Media containing probes was replaced by media containing different stressors at indicated concentrations. (b) Detection of proteome stress using confocal imaging. White arrows highlight fluorescent punctate structures that correspond to insoluble aggregates, whereas diffuse fluorescence represents soluble aggregates. Red: TMR fluorescence. Green: AgHalo 550 conjugate. Blue: Hoechst 33342, nucleus staining.

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We first induced osmotic stress by incubating cells with high concentration of NaCl (1 M) or sorbitol (0.5 M) for 5 min36, 38. Signals from the red channel that collects TMR fluorescence did not exhibit obvious differences between stressed and non-stressed cells (compare images in column 1 of rows 1-3, Figure 6b). By contrast, a rapid fluorescence increase was found from the green channel that monitors fluorescence of AgHalo 550 (compare images in column 2 of rows 1-3, Figure 6b). It is noted that we observed largely diffused fluorescent structures, instead of punctate structures, indicating that the AgHalo sensor primarily formed soluble oligomers but not insoluble aggregates in these stress conditions. Taken together, these interesting observations demonstrate that the fluorogenic AgHalo sensor is capable of detecting proteome stress conditions that are nearly invisible to either fluorescent protein fusion or probe incorporation method. Similar differences between the always-fluorescent and fluorogenic methods were found when we tested other stress conditions. In cells stressed with MG132 (10 µM), Nilotinib (50 µM), and heat (45 ºC), it was difficult to identify obvious changes, e.g. formation of punctate fluorescent structures, from images of the TMR channel (red channel of rows 4-6, Figure 6b). By contrast, both diffuse and punctate green fluorescence signals were easily obtained using the AgHalo 550 channel (green channel of rows 4-6, Figure 6b), suggesting that the AgHalo sensor could form of both soluble oligomers and insoluble aggregates in these cells. Different from these stress conditions, cells treated with 50 µM NaAsO2 displayed quite clear fluorescent puncta in images from the TMR channel (red channel of row 7, Figure 6b). While these punctate structures were also visible in images from the AgHalo 550 channel, we could only observe very low diffusive fluorescence (green channel of row 7, Figure 6b). Under these stress conditions, expression and labeling of AgHalo in HEK293T cells were minimally altered in relative to the non-stressed cells, as measured by an SDS-PAGE analysis wherein the fluorescence of the AgHalo-TMR conjugate was used as a signal (Figure S10). Collectively, these data suggest that the AgHalo sensor

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Biochemistry

could unambiguously visualize its aggregation via a fluorogenic signal under all above stress conditions, rendering a clear advantage over the non-fluorogenic methods. It is worth noting that fluorescence resonance energy transfer (FRET) may occur in twocolor imaging. In particular, the AgHalo 550 and TMR fluorescence profiles bear potential to be a FRET pair, wherein the AgHalo 550 probe is the donor and TMR is the acceptor. If they transfer energy with a high efficiency, the AgHalo 550 fluorescence would significantly decrease and the TMR fluorescence would increase. This scenario will undermine the ability of AgHalo 550 to report on AgHalo aggregation. However, we did not observe an obvious reduction of AgHalo 550 (donor) fluorescence using fluorescent confocal imaging (Figure 6). Consistent to this observation, an in vitro co-aggregation experiment of Halo K73T that was equally labeled by AgHalo 550 and TMR exhibited no FRET effect, suggesting that these two fluorophores are not spatially close enough to conduct energy transfer (Figure S11). A successful dual-probe experiment relies on similar rates of labeling between the TMR ligand and the AgHalo ligand. To demonstrate this notion, we carried out an in vitro kinetic competition experiment. In this experiment, we first incubated 5 µM of purified AgHalo with either 5 µM of AgHalo 550 (lane 1 in Figure S12a) or 5 µM of TMR Halo-Tag ligand (lane 2 in Figure S12a). TMR fluorescence was used as a signal to evaluate extent of AgHalo being labeled by the TMR Halo-Tag ligand. After confirming the absence of TMR labeling in lane 1 and presence of TMR labeling in lane 2 (Figure S12a), we then incubated 5 µM of AgHalo with a mixture of 5 µM of AgHalo 550 and 5 µM of TMR Halo-Tag ligand (lane 3 in Figure S12a). If these two ligands were able to label AgHalo with equal or similar kinetics (kTMR ~ kAgHalo-550), we should expect ~50% of the AgHalo to be labeled with TMR. Otherwise, we would expect either most of the AgHalo (kTMR >> kAgHalo-550) or very little portion of AgHalo (kTMR