Engineering Intracellularly Retained Gaussia Luciferase Reporters for

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Engineering intracellularly retained Gaussia Luciferase reporters for improved biosensing and molecular imaging applications Shuchi Gaur, Aarohi Bhargava-Shah, Sharon Hori, Rayhaneh Afjei, Thillai V. Sekar, Sanjiv S. Gambhir, Tarik F. Massoud, and Ramasamy Paulmurugan ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00454 • Publication Date (Web): 02 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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Engineering intracellularly retained Gaussia Luciferase reporters for improved biosensing and molecular imaging applications Shuchi Gaur, Aarohi Bhargava-Shah, Sharon Hori, Rayhaneh Afjei, Thillai V. Sekar, Sanjiv S. Gambhir, Tarik F. Massoud, and Ramasamy Paulmurugan

Departments of Radiology, and Bioengineering, the Bio-X Program, Molecular Imaging Program at Stanford (MIPS), Stanford University School of Medicine, 3155 Porter Drive, Palo Alto, CA 94304-1110, USA.

Key words: Gaussia luciferase, molecular imaging, biosensors, caspase, reporter gene Conflict of interest: No conflicts

Corresponding Authors: Ramasamy Paulmurugan, PhD Department of Radiology, Stanford University School of Medicine 3155 Porter Drive Palo Alto, CA 94304 Phone: 650-725-6097; Fax: 650-721-6921 Email: [email protected]

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Abstract Gaussia luciferase (GLUC) is a bioluminescent reporter protein of increasing importance. As a secretory protein it has increased sensitivity in vitro and in vivo (∼20,000-fold, and ∼1,000-fold, respectively) over its competitor, secreted alkaline phosphatase. Unfortunately, this same advantageous secretory nature of GLUC limits its usefulness for many other possible intracellular applications, e.g., imaging signaling pathways in intact cells, in vivo imaging, and in developing molecular imaging biosensors to study protein-protein interactions and protein folding. Hence, to widen the research applications of GLUC, we developed engineered variants that increase its intracellular retention by both modifying the N-terminal secretory signal peptide, and by tagging additional sequences to its C-terminal region. We found that when GLUC was expressed in mammalian cells, its N-terminal secretory signal peptide comprising amino acids 1-16 was essential for GLUC folding and functional activity in addition to its inherent secretory property. Modification of the C-terminal of GLUC by tagging a four amino acid (KDEL) endoplasmic reticulum targeting peptide in multiple repeats significantly improved its intracellular retention, with little impact on its folding and enzymatic activity. We used stable cells expressing this engineered GLUC with KDEL repeats to monitor chemically induced endoplasmic reticulum stress on cells. Additionally, we engineered an apoptotic sensor using modified variants of GLUC containing a four amino acid caspase substrate peptide (DEVD) between the GLUC protein and the KDEL repeats. Its use in cell culture resulted in increased GLUC secretion in the growth medium when cells were treated with the chemotherapeutic drugs doxorubicin, paclitaxel, and carboplatin. We thus successfully engineered a new variant GLUC protein that is retained inside cells rather than secreted extracellularly. We validated this novel reporter by incorporating it in biosensors for detection of cellular endoplasmic reticulum stress and caspase activation. This new molecularly engineered enzymatic reporter has the potential for widespread applications in biological research.

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Introduction

Pre-clinical noninvasive molecular imaging has evolved as an important tool for studying basic cellular processes in cells and living animals. Several reporter genes with different biological and physicochemical properties have been successfully adopted in studying molecular and physiological processes in cells and animal models. In particular, optical imaging reporters, such as the various luciferases and fluorescent proteins, have taken center stage in many useful lines of experimental biological investigations because they are easily available, convenient to use, and are of low-cost compared to other reporter enzymes that might require radioactive substrates or sophisticated instrumentation to image their expression. A large number of luciferase enzymes with different protein sizes, spectral properties, and activity ranges have been cloned from insects, worms and marine copepods (1-5). Based on substrate requirements, they are enzymes that use either D-Luciferin or coelenterazine. Recently, an engineered luciferase enzyme (Nanoluc) that requires a different substrate (furimazine) has been developed, but it suffers from light emission in the blue shifted wavelength (6).

Gaussia luciferase (sGLUC) is the smallest (185 amino acids) luciferase among optical reporters that use coelenterazine as a substrate. sGLUC protein is naturally secreted extracellularly when expressed in mammalian cells. In vitro, it is possible to quantify this secreted protein from analysis of cell culture medium alone, which thus avoids the need to lyse cells (7). In vivo, a similar quantification of circulating secreted reporter can be undertaken by using a small blood sample (7). Indeed, sGLUC is gaining importance as a secreted reporter, since it has been shown to have ∼20,000-fold increased sensitivity in vitro, and ∼1,000-fold increased sensitivity in vivo compared to its main competitor, secreted alkaline phosphatase (7). While the current use of sGLUC is somewhat limited to being a potential reporter on various secretory processes in mammalian cells, this very nature unfortunately also precludes its much

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wider use as an imaging reporter to monitor intracellular processes in intact cells and living animals. To that end, Tannous et al. previously studied the removal of the N-terminal secretary signal peptide of amino acids 1-16 (producing a truncated version, tGLUC), as well as adding a four amino acid ER targeting peptide at the C-terminal end of sGLUC (8). They found a significant drop in reporter activity when they removed N-terminal signal peptide, and a moderate improvement in intracellular retention when adding an ER-targeting signal peptide to the C-terminal. Similarly, other studies have used transmembrane sequences derived from CD8 and EGFR proteins respectively attached at the C-terminus of sGLUC and vargula luciferase to image intact cells on plate and implanted in animals by displaying the proteins on the cell surface (9, 10). All these studies had set out to develop intracellular reporters either by a random approach or through a focus on a specific application, instead of systematically characterizing many possible mechanisms to produce intracellular GLUC variant(s). By contrast, a more comprehensive mechanistic analysis of induced alterations in the chemical biology of this reporter would likely result in the development of intracellular GLUC variants with much wider applications. Here, to develop intracellular GLUC variants that allow for many more biosensor reporter applications, we systematically modify both N- and C-terminals of GLUC protein using different peptides in various amino acid combinations. Moreover, we characterize the importance of the N-terminal signal peptide and its associated functional changes in GLUC protein.

Thus, we proceeded our study on two fronts: First, and as an extension of our previous observations on tGLUC, we investigated the importance of the first 16 amino acids of sGLUC and their role in sGLUC secretion and function. We thus substituted the N-terminal secretory signal sequence of sGLUC with signal peptide sequences derived from other proteins and with peptides of random amino acid sequences, and then studied consequent effects on reporter secretion and activity. Next, bearing in mind that a four amino acid (KDEL) endoplasmic

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reticulum targeting signal sequence has previously been shown to enhance intracellular retention and folding of proteins (11), we engineered a C-terminal sGLUC protein with an endoplasmic reticulum targeting KDEL peptide in multiple repeats. We comprehensively evaluated the properties of all these constructed variants in cells engineered to possess equal numbers of transgene copies. Moreover, by further molecular engineering of our new intracellular variant GLUC protein, we were subsequently able to expand its repertoire of useful biological applications beyond simple use in tracking cells and monitoring their viability. We thus validated the ability of this new GLUC reporter variant to measure chemically induced endoplasmic reticulum stress, and as an apoptotic sensor for monitoring caspase activation and the response of cancer cells to chemotherapy.

Results Comparison of the luciferase activity from secretary GLUC, tGLUC, hRLUC, FLUC1, FLUC2, TFLUC and CBLUC The HEK293T cells transfected with different luciferase genes (sGLUC, tGLUC, hRLUC, FLUC1, FLUC2, TFLUC and CBLUC) were assessed for total luciferase activity (medium and cell lysates) 48 h after transfection. For this, the pooled medium and the washed PBS, were used for measuring the secreted luciferase activity. The cell pellets lysed in 1X passive lysis buffer were used for measuring intracellular luciferase activity. The activities for GLUC and RLUC were measured from 20 µl of medium or cell lysates with 100 µl of PBS containing 100 µg of coelenterazine. The activities for FLUC1, FLUC2, TFLUC and CBLUC were measured by adding 20 µl of medium or cell lysates with 100 µl of Luciferase Assay Reagent (LARII) reagent. The total activity of each sample was calculated from the total volume of medium or the cell lysate. The total activities were normalized to protein concentration (RLU/µg protein/min). The results were compared with FLUC1 to measure the relative fold in luciferase

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activity. Overall, the total luciferase activity measured from cells transfected with sGLUC was 80.5 ± 8.39-fold higher than FLUC1, and 9.4 ± 0.87-fold higher than hRLUC. In contrast, the luciferase activity measured from cells transfected with tGLUC was 81± 5-fold lower than cells transfected with sGLUC (Figure 1a-b).

Of note, cells transfected with tGLUC showed higher amounts of intracellular protein but showed very low levels of luciferase activity compared to sGLUC.

As we had

hypothesized previously, the N-terminal secretary signal peptide (amino acids 1-16) may possibly facilitate the folding of sGLUC into a structural conformation that is important for the enzymatic activity of the protein. To test our hypothesis, we used proteins from the lysates of HEK293T cells transfected with different luciferase constructs (sGLUC, tGLUC, hRLUC, FLUC1, and FLUC2). We measured the luciferase activities of the samples over time (0, 1, 5, 24, 48 and 72 h) after incubating the lysates at 25° C. We found a significant increase in luciferase activity from tGLUC over time, while no significant changes were observed from all other constructs. At 48 h post incubation, lysates of cells transfected with tGLUC showed 70 ± 1.87% (∼105-fold more compared to activity measured immediately after lysis) of the activity of sGLUC (Figure 2a). To further explore our proposed hypothesis of slow folding of tGLUC protein, we incubated the lysates of cells transfected with tGLUC plasmid at three different temperatures (4 °C, 25 °C, and 37 °C) and measured the increased tGLUC activity over time for up to 168 h. The results showed a rapid increase in luciferase activity from samples incubated at 37 °C when compared to samples incubated at 4 °C and 25 °C. The luciferase activity of samples stored at 37 °C reached a peak signal in 24 h, while the samples incubated at 4 °C and 25 °C achieved saturation points nearly 120 h after incubation (Figure 2b). Interestingly, we observed a two-fold higher tGLUC peak signal from the samples incubated at 4 °C and 25 °C compared to samples incubated at 37 °C. We speculate that this finding may

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reflect the evolutionary significance and functional requirements of this protein, which was cloned from a deep-sea organism (Gaussia princeps) that lives in sea water depths below 400 m,

and

where

the

water

temperature

ranges

from

2

°C

to

14

°C

(http://eol.org/pages/340029/details).

Reporter protein complementation assays developed using tGLUC fragments can assess protein-protein interactions and protein folding but show slow folding kinetics To further study intracellular protein-protein interactions and protein folding using split-reporter protein complementation assays based on tGLUC fragments (where the split site was at amino acid position 105: N-tGLUC-16-105 and C-tGLUC-106-185, based on a truncation library approach) we used our previously optimized rapamycin mediated protein-protein interaction system (12, 13) as well as a ligand-induced ER intramolecular folding system (14, 15). We assessed HEK293T cells co-transfected with plasmids expressing fusion proteins of the rapamycin mediated protein-protein interaction system (N-tGLUC-FRB and FKBP12-CtGLUC), or transfected with the ER-intramolecular folding system (N-tGLUC-ER(LBD)-CtGLUC). We assayed for luciferase activity 24 h after treating with 40 nM rapamycin or 1µM each of different ER-ligands (estradiol (E2), diethylstilbestrol (DES), genistein (GEN), hydroxytamoxifen (OHT) and raloxifene (RAL), respectively. We further assessed the lysates from each complementation system assay for luciferase activity immediately after lysis, and at different times after incubation at 25 °C to assess the impact of slow folding of tGLUC fragments on the measured protein-protein interactions and protein folding. The results showed significant levels of luciferase activity specific to the treatment of rapamycin in the FRB-FKBP12 protein-protein interaction system immediately after lysis (19.7-fold compared to cells not treated with rapamycin), and the signal significantly increased over time when assessed after incubation of lysates at 25 °C (125-fold at 2 h, 336-fold at 5 h, 765-fold at 24 h, 715-fold at 48 h, and 753-fold at 72 h) (Figure 2c-d). Similarly, the ER-intramolecular folding

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complementation system also showed a similar specificity and trend in the obtained tGLUC activity (Figure 2e-f). Since we used an estradiol non-responsive mutant ER-LBD (G521T), as expected, we found no significant complementation from the cells treated with estradiol.

The N-terminal secretary signal peptide (amino acids 1-16) is important for sGLUC folding and the associated enzymatic activity When sGLUC was expressed in cells, we observed that more than 95% of the protein was secreted into the medium. The N-terminal 16 amino acids are considered important for secretion of this protein from cells. Our results showed that deletion of the N-terminal amino acids from sGLUC (i.e., tGLUC) significantly enhanced the retention of tGLUC inside the cells but affected its enzymatic activity. Our results also confirmed that this is likely mainly owing to slow folding of intracellular tGLUC protein (see Discussion). The above results obtained when using the complementation systems based on tGLUC fragments further confirmed the importance of the secretory signal peptide for folding of tGLUC. In an attempt to enhance intracellular retention, while maintaining reporter folding, we modified the N-terminal amino acids 1-16 using various signal peptides and different amino acid sequences. We used a nuclear localization signal peptide (TK-NLS) derived from herpes simplex virus type 1 thymidine kinase (HSV1-TK), an NLS sequence of basic amino acids (BNLS), random amino acid sequences (DGEN-Cl-7, DGEN-Cl-40) placed at the N-terminal position, as well as tGLUC and sGLUC with an additional four amino acid ER-targeting sequence at the C-terminus (tGLUC-KDEL and sGLUC-KDEL). The latter maneuver was initially preceded by running a quick prediction tool to assess for the presence of any ERtargeting sequence within sGLUC protein, and we found no positive hits for this (http://prosite.expasy.org). We tested all these constructs in transfected HEK293T cells for intracellular and secreted luciferase activities (Figure 3a). We found more than a two log order drop in total luciferase activity from all the clones where we modified the N-terminal

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secretary signal peptide sequence of sGLUC (either deleted or replaced with different amino acid sequences). In contrast, the sGLUC with KDEL at the C-terminal (sGLUC-KDEL) showed significant increase in intracellular signal (1.7-fold higher than the secreted luciferase signal, and 8-fold higher than the intracellular signal obtained from sGLUC) while maintaining higher absolute levels of signal (Figure 3b). In addition our western blot analysis correlated with the accumulation of intracellular reporter protein (Figure 3c).

Engineering of sGLUC by C-terminal tagging of an ER targeting sequence (KDEL) in multiple repeats enhances its intracellular retention while improving its absolute level of luciferase activity To this point we found that any alteration of the N-terminal signal peptide significantly affected sGLUC folding and the associated luciferase activity. In contrast, the C-terminal attachment of the four amino acid ER targeting peptide (KDEL) resulted in significant improvement in intracellular GLUC retention while maintaining its absolute level of luciferase activity. Hence, we further hypothesized that placement of additional repeats of the KDEL sequence might improve its intracellular retention without significant extracellular secretion, and could thus potentially greatly expand the use of GLUC in many bio-imaging assays and research applications. Therefore, we next constructed three additional variants of sGLUC expressing repeats of KDEL (sGLUC-(KDEL)2, sGLUC-(KDEL)3, and sGLUC-(KDEL)4) in addition to sGLUC and sGLUC-KDEL, and tested all these in transfected HEK293T cells (Figure 4a and Supp-Figure 1). We found a significant rise in intracellular luciferase signal when we increased the number of KDEL repeats, while the secretory reporter fraction dropped significantly (Figure 4b). sGLUC with (KDEL)3 and (KDEL)4 showed the maximum level of intracellular luciferase signals compared to other constructs [(KDEL)3 and (KDEL)4: Intracellular: 3.2 x 1012 RLU/µg protein/min; Secretory: 5.0 x 1011 RLU/µg protein/min compared to sGLUC: Intracellular: 3.5 x 108 RLU/µg protein/min; Secretory: 3.89 x 1012

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RLU/µg protein/min, respectively] (Figure 4b). To perform an absolute comparison of these constructs in cells with similar expression levels without any perturbation from experimental transfection associated variations in reporter secretion, we constructed lentiviral systems with all four KDEL variants and GLUC constructs. We used MDA-MB231 breast cancer cells and LN229 glioma cells for this study. We isolated clones of cells with equal numbers of copies per cell by FACS sorting (Figure 4c). We further tested the cells for the presence of equal numbers of lentiviral inserts by indirectly measuring the co-expressed dTomato protein by fluorescent microscopy and for the expression of GLUC mRNA by qRT-PCR (Figure 4d-e). The results showed equal levels of dTomato fluorescence and GLUC mRNA expression, and these cells were used for further evaluation of intracellular and secreted GLUC protein levels at different time points after plating and in response to the treatment of various stressors. We also quantified the intracellular GLUC protein levels in LN229 and MDA-MB231 cells stably expressing all five different variants by immunoblot analysis using the cell lysates collected from equal numbers of cells. The results showed almost no detectable GLUC protein in both LN229 and MDA-MB231 cells stably expressing GLUC protein while other constructs with Cterminal KDEL sequences showed significant levels of GLUC protein. Importantly, the constructs with three and four repeats of KDEL sequence yielded higher levels of intracellular protein compared to the other constructs (Figure 4f). To accurately measure the level of intracellular reporter retention we plated MD-MB231 stable cells expressing different GLUC constructs in different cell numbers and measured the total activity in cells and the medium 24 h after initial plating. The relative luciferase activity in both medium and cells increased with increasing numbers of cells. As expected, the cells stably expressing sGLUC with three and four KDEL repeats showed 30-fold higher intracellular reporter, while sGLUC without any tag showed a log order higher signal in the secreted fraction (Supp-Figure 2 and 3).

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Validation of sGLUC variants in response to various stressors specifically indicates an endoplasmic reticulum signaling response Next, we evaluated the secretion of sGLUC protein from cells stably expressing different KDEL constructs in response to treatment with various stressors, including the endoplasmic reticulum stressors (tunicamycin, MITO TemPol, and TBHQ). We incubated the cells after treating them with these different stressors (in triplicates for each cell line), using DMSO as a control. The sGLUC signals in the medium and cells were assessed 24 h after treatment. The results showed significant increase in sGLUC signal in the medium of cells treated with the endoplasmic reticulum stressor, tunicamycin, compared to other stressors (Supp-Figure 3).

We further evaluated these cells in response to treatment using different doses of tunicamycin. We seeded 5,000 cells/well in 96-well plates with each stable cell line expressing sGLUC-KDEL and sGLUC-(KDEL)3 (in fact either sGLUC-(KDEL)3 or sGLUC-(KDEL)4 could have been used, since both had shown similar levels of intracellular GLUC protein previously). This was also, in part, to assess the impact of multiple repeats of KDEL on intracellular protein retention. After incubating the plates for 24 h, we washed them with PBS, and then treated 4 wells for each condition with different doses of tunicamycin starting from 10 µg/mL down to 10 ng/mL. We incubated the plates for another 24 h, after which we measured reporter activity from cells (intracellular) and medium (secretory). The results clearly showed that when treated with a stressor, the endoplasmic reticulum released more of the KDEL-tagged sGLUC protein in the medium compared to control cells. sGLUC with both single and three KDEL repeats showed a similar trend with equal efficiency (Figure 5a-c).

A fusion protein of sGLUC-(KDEL)3 designed with caspase cleavable DEVD (sGLUCDEVD-(KDEL)3) peptide sequence specifically measures caspase dependent secretion of sGLUC protein in response to chemotherapeutic drugs

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We then designed a caspase sensor by constructing a vector that expresses a fusion protein with DEVD peptide flanked by sGLUC and (KDEL)3 (sGLUC-DEVD-(KDEL)3) (Figure 5d). We created MDA-MB231 breast cancer cells stably expressing this sensor by using a lentiviral transduction system, and tested the cells in response to treatment using the chemotherapeutic drugs doxorubicin (5 µM) and paclitaxel (25 nM). The secreted sGLUC signal assessed from the medium of cells treated for 24 h showed significant increases in the levels of signal compared to control cells (19.5 ± 2-fold by doxorubicin and 8.34 ± 0.3-fold by paclitaxel compared to control) (Figure 5e). Immunoblot analysis further confirmed the cleavage of sGLUC-DEVD-(KDEL)3 by caspase in cells treated with drugs (Figure 5f). In addition, we tested the signal induced by the IC50 dose of doxorubicin (5 µM), paclitaxel (25 nM), and carboplatin (25 µM) over time, as well as different doses of doxorubicin (0-1 µM) and paclitaxel (0-25 nM) over time. The results showed a dose- and time-dependent increase in secretory sGLUC signal in the medium of cells treated with chemotherapeutic drugs compared to controls (Figure 5g-i).

Discussion Reporter genes have been extensively used to study cell biology. In vitro and cell culture reporter gene techniques can also be adapted for quantitative molecular imaging in cells and in living animals. Bioluminescence reporter genes code for proteins that luminesce when exposed to their substrates, thus allowing researchers to monitor tumor growth, drug efficacy, promoter activation or inhibition, viral gene transfer, siRNA silencing, miRNA biogenesis, and proteinprotein interactions, just to name a few (7, 12-17). These reporter genes can also be used for real-time monitoring of tumor metastasis (18). The commonly used bioluminescence reporter genes are Renilla Luciferase (RLUC), Gaussia Luciferase (sGLUC), and Firefly Luciferase (FLUC). Pre-clinical non-invasive molecular imaging is generally convenient, efficient, and provides a quantitative method to evaluate molecular and cellular mechanisms in intact cells

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and within living animals. To date, intracellularly transfected or transduced reporter genes encoding proteins with different features have been extensively used to study cellular pathways and mechanisms in cells and living animals (17, 19-21). In particular, optical imaging reporters are cheap and easy systems to adopt in the laboratory; bioluminescence reporters are highly sensitive and with low background signal compared to fluorescent proteins.

In this study, we aimed to develop an intracellular version of sGLUC by engineering its N-terminal secretory signal peptide and/or tagging additional signal sequences to the Cterminal. Past researches had shown that simply removing the N-terminal secretory signal peptide of the GLUC protein was ineffective in showing intracellular GLUC activity (8) or in retaining the protein intracellularly (22). Interestingly, and somewhat contrary to that findings, we observed that truncating sGLUC (tGLUC), or replacement of the N-terminal peptide sequence with different amino acid sequences derived from other proteins or generated through random amino acid combinations, significantly improved its intracellular retention (Figure 4c). However, these modifications also markedly affected the activity of these reporter variants. We speculate that the results of that previous study (22), where significant intracellular activity was not observed after truncation of N-terminal amino acids 1-16 of sGLUC protein, may possibly reflect the particular assay system used in that investigation. Instead, in our study, the activity was measured after allowing the tGLUC protein to fold extracellularly (as it is meant to) by keeping cell lysates at different temperatures for various time periods. We also took this forward by investigating the possible motives for this observed low activity in cells, even after improving intracellular retention of reporter, by appending various additional localization sequences. We found that the low activity was most likely and mainly owing to a change in folding of the protein induced by the removal of, or a change in, the original sequence. Unfortunately, the crystal structure of sGLUC is not available, thus precluding any meaningful objective discussions about conformational changes that might follow perturbation of the protein structure. That said, our

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proposed hypothesis of slow folding of tGLUC is strongly inferred from our results addressing localization, activity, and temporal dynamics of the sGLUC variants we have investigated in detail. Taken together, these findings therefore highlights the distinct importance of the native signal peptide of sGLUC, whether or not the sGLUC molecule is subjected to any additional amino acid variations, as we have outlined above (Figure 2 and 3). Importantly, our results also confirm that tampering with the N-terminal signal peptide of sGLUC would not be a fruitful molecular engineering strategy in any future attempts to develop newer intracellular sGLUC variants.

We then explored the prospect of improving intracellular reporter retention by modifying the C-terminal of sGLUC protein instead. We chose an endoplasmic reticulum-targeting KDEL sequence as a signal peptide. We created variants of sGLUC proteins expressing different numbers of KDEL repeats at the C-terminus, and measured their efficiency by quantifying the resulting amounts of intracellular and secretory sGLUC optical imaging signals for five different constructs (sGLUC, sGLUC-(KDEL), sGLUC-(KDEL)2, sGLUC-(KDEL)3, sGLUC-(KDEL)4). We comprehensively evaluated all constructs in transiently transfected HEK293T, LN229 and MDAMB231 cells engineered to stably carry equal numbers of gene copies (Figure 4). Our results showed that sGLUC tagged with three or four repeats of KDEL sequence resulted in greater intracellular retention of sGLUC protein compared to other constructs.

After successful development of sGLUC variants for improved intracellular retention, we further explored the use of these reporters to establish novel biosensor imaging applications that measure specific functional properties of cells, such as endoplasmic reticulum stress, and to monitor tumor cell apoptosis in response to chemotherapeutic drugs. We first verified that our modified sGLUC proteins were localizing to the endoplasmic reticulum by treating cells with TBHQ, an oxidative stressor, MITO TemPol, a mitochondrial stressor, and tunicamycin, an

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endoplasmic reticulum stressor. We found that cells treated with tunicamycin had a stronger extracellularly secreted sGLUC signal, thus confirming the localization of KDEL-tagged sGLUC within the cell. We then evaluated the effectiveness of sGLUC variants at measuring endoplasmic reticulum stress by treating sGLUC-(KDEL)1 and sGLUC-(KDEL)3 with varying doses of tunicamycin. Our results confirm that in the presence of an endoplasmic reticulum stressor drug, more of the sGLUC protein is released from the endoplasmic reticulum and secreted extracellularly, causing a significant difference in imaging signal in treated versus untreated controls (Figure 5). Further, we developed a caspase sensor using the sGLUC(KDEL)3 variant by introducing a four amino acid caspase-3 cleavable peptide between sGLUC and the (KDEL)3 tag (Gluc-DEVD-(KDEL)3). Evaluation of MDA-MB231 cells engineered to stably express this construct showed chemotherapeutic drug dose and time dependent release of GLUC protein into the culture medium (Figure 5). Importantly, this observed highly advantageous feature of our novel engineered reporter has the potential to improve experimental noninvasive monitoring of drug therapeutic response by tumors in vivo, mostly by measuring the secreted reporter signal obtained from blood samples. We will investigate the potential utility of this application in animal models of cancer.

Conclusion We molecularly engineered variants of sGLUC with C-terminal KDEL multimers. These multimers significantly improve intracellular retention of sGLUC and further expand its repertoire and applications in designing biosensors for imaging cellular functions. In the process of developing this new reporter, we also discovered that the N-terminal secretory signal peptide (amino acids 1-16) of sGLUC is critical for proper folding of this protein into a native functional conformation, in addition to its known secretory function. We also showed that sGLUC variants possessing KDEL tag(s) at the C-terminus of the protein can be molecularly varied to act as new reporters for measuring caspase activation induced by chemotherapy, and for monitoring

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the effects of drugs that induce endoplasmic reticulum stress. Further engineering of this protein by overcoming its rapid reaction kinetics and its emission spectrum towards longer wavelengths (red shifting) in future studies could also broaden the applications of this protein into many more areas of translational cancer cell biology and therapy investigations, as well as in vivo molecular imaging research. Methods See the supporting information for details. Supporting Information Supporting information includes supplementary materials and methods section, figures, tables and references. Supplementary figure 1: Schematic map of sGLUC with different numbers of KDEL repeats used in this study; Supplementary figure 2: Intracellular and secretary GLUC proteins measured in MDA-MB231 cells stably expressing sGLUC variants with different numbers of KDEL repeats; Supplementary figure 3: Intracellular and secretary GLUC protein levels measured in MDA-MB231 cells stably expressing equal number of GLUC constructs with different numbers of KDEL repeats; Supplementary figure 4: Intracellular and secretory GLUC signal measured from MDA-MB231 cells stably expressing GLUC variants with different numbers of KDEL repeats in response to the treatment of different stressors (TBHQ: an inducer of oxidative stress, MITO-TemPol: an inducer of oxidative stress in mitochondria, and tunicamycin: an inducer of endoplasmic reticulum stress); Supplementary table 1:

List of

primers used for amplifying various GLUC variants for constructing plasmid and lentiviral vectors. This material is available free of charge via the internet at http://pubs.acs.org. Acknowledgments We thank Canary center, Department of Radiology, Stanford University, for providing facilities and support. T. F. Massoud was supported by the Ben and Catherine Ivy Foundation. This work was partially supported by the National Institutes of Health (NIH grant R01CA161091 to R.P.).

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Ray, P. (2011) Multimodality molecular imaging of disease progression in living subjects, J Biosci 36, 499-504. Yamashita, H., Nguyen, D. T., and Chung, E. (2014) Blood-based assay with secreted Gaussia luciferase to monitor tumor metastasis, Methods Mol Biol 1098, 145-151. Goetz, A. S., Liacos, J., Yingling, J., and Ignar, D. M. (1999) A combination assay for simultaneous assessment of multiple signaling pathways, J Pharmacol Toxicol Methods 42, 225-235. Kang, J. H., and Chung, J. K. (2008) Molecular-genetic imaging based on reporter gene expression, J Nucl Med 49 Suppl 2, 164S-179S. Lake, M. C., and Aboagye, E. O. (2014) Luciferase fragment complementation imaging in preclinical cancer studies, Oncoscience 1, 310-325. Luft, C., Freeman, J., Elliott, D., Al-Tamimi, N., Kriston-Vizi, J., Heintze, J., Lindenschmidt, I., Seed, B., and Ketteler, R. (2014) Application of Gaussia luciferase in bicistronic and non-conventional secretion reporter constructs, BMC Biochem 15, 14.

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Figure legends Figure 1. (a). Schematic illustration of various luciferase reporter constructs with their promoter and coding sequences used in this study (CMV: cytomegalovirus promoter; LUC: coding sequences of different luciferase reporters; PA: poly A tail from SV40 virus). (b). Total luciferase activity (medium and cell lysates) measured from HEK293T cells transfected with the different luciferase constructs. The activities are represented as relative fold activity in comparison to the luciferase activity measured from FLUC1 construct transfected cells.

Figure 2. (a). Luciferase activity measured at different time points (0-72 h) from the cell lysates of HEK293T cells transfected with different luciferase constructs after incubating the lysates at 25 °C. (b). The lysate of HEK293T cells transfected with tGLUC construct measured for luciferase activity after incubating the lysate at 4 °C, 25 °C, and 37 °C for different time points (0-168 h). (c). Schematic illustration of vectors expressing fusion proteins of split-tGLUC fragments with FRB (N-tGLUC-FRB) and FKBP12 (FKBP12-C-tGLUC). (d). HEK293T cells cotransfected with constructs expressing fusion proteins in “c” measured for GLUC activity with and without exposure to 40 nM rapamycin, immediately after lysis and at different time points (0-72 h) after incubating the lysates at 25 °C. (e). Schematic illustration showing the activation of ER-intramolecular sensor fusion protein complementation in response to the binding of ERligands. (f). HEK293T cells co-transfected with construct expressing fusion protein of ER-LBD flanking N- and C- tGLUC fragments measured for GLUC activity 24 h after exposure to 1 µM of different ER-ligands, immediately after lysis and at different time points (0-18 h) after incubating the lysates at 25 °C.

Figure 3. (a). Sequence alignment of different GLUC proteins to represent N- and C-terminal amino acid modifications of different constructs developed for this study. (b). The intracellular and secretory luciferase signal measured from HEK293T cells transfected with different

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engineered GLUC constructs. (c). Immunoblot analysis of intracellular GLUC protein from HEK293T cells transfected with different engineered GLUC constructs (Note: Y-axis scales are different for each graph in ‘b’).

Figure 4. Engineering sGLUC with multiple C-terminal tags and evaluation for intracellular retention. (a) Sequences of GLUC constructs with added C-terminal KDEL repeats (Sec-GLUC: secretory GLUC). (b) Intracellular and secretory luciferase signals measured from MDA-MB231 cells transfected with these engineered GLUC constructs. (c) FACS sorting, and (d) fluorescent microscopic images of MDA-MB231 cells stably expressing these GLUC constructs and coexpressing dTomato reporter. (e) Agarose gel of qRT-PCR amplified product of GLUC mRNA (top row) from MDA-MB231 cells stably expressing these GLUC constructs (bottom row: qRTPCR product of β-Actin mRNA), with numbers 0-4 representing increasing KDEL repeats and N is negative control. (f) Immunoblot analysis of GLUC in the cell lysates of an equal number of MDA-MB231 and LN229 cells stably expressing GLUC constructs with increasing KDEL repeats.

Figure 5. Validation of C-terminal tagged sGLUC variants as biosensors for chemically induced endoplasmic reticulum stress, and for monitoring caspase activation and the response of cancer cells to chemotherapy. (a) Intracellular and secretary GLUC levels in MDA-MB231 cells stably expressing GLUC biosensor containing sGLUC-KDEL, 24 h after treatment with different concentrations of tunicamycin (0-10 µg/ml), to detect cellular endoplasmic reticulum stress. (b) Similar analysis using (sGLUC-(KDEL)3). (c) Similar analysis but using the sGLUC-KDEL biosensor and optical imaging of GLUC after adding different concentrations of tunicamycin (010 µg/ml). (d) Schematic design of sGLUC-DEVD-(KDEL)3 biosensor to measure caspase activation in cells. (e) MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 biosensor

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evaluated for secretary signal in culture medium 24 h after treatment with 5 µM doxorubicin and 25 nM paclitaxel. (f) Immunoblot analysis of cell lysates of MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 caspase biosensor after treatment with different drugs for 24 h (Dox [doxorubicin] 5 µM, Pac [paclitaxel] 25 nM, Con [control], and Car [carboplatin] 25 µM). (g) Chemotherapeutic drug-induced caspase activation measured by imaging GLUC in culture medium of MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 biosensor at different time points (0-48 h) after drug treatment (Dox: 5 µM, Pac: 25 nM, and Car: 25 µM). (h and i). Dose and time dependent activation of caspase in MDA-MB231 cells stably expressing sGLUCDEVD-(KDEL)3 biosensor in response to treatment using doxorubicin (h) and paclitaxel (i). Error bars are SEM of triplicate determinations.

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Figure 1 (a). Schematic illustration of various luciferase reporter constructs with their promoter and coding sequences used in this study (CMV: cytomegalovirus promoter; LUC: coding sequences of different luciferase reporters; PA: poly A tail from SV40 virus). (b). Total luciferase activity (medium and cell lysates) measured from HEK293T cells transfected with the different luciferase constructs. The activities are represented as relative fold activity in comparison to the luciferase activity measured from FLUC1 construct transfected cells. 168x173mm (300 x 300 DPI)



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Figure 2 (a). Luciferase activity measured at different time points (0-72 h) from the cell lysates of HEK293T cells transfected with different luciferase constructs after incubating the lysates at 25 °C. (b). The lysate of HEK293T cells transfected with tGLUC construct measured for luciferase activity after incubating the lysate at 4 °C, 25 °C, and 37 °C for different time points (0-168 h). (c). Schematic illustration of vectors expressing fusion proteins of split-tGLUC fragments with FRB (N-tGLUC-FRB) and FKBP12 (FKBP12-CtGLUC). (d). HEK293T cells co-transfected with constructs expressing fusion proteins in “c” measured for GLUC activity with and without exposure to 40 nM rapamycin, immediately after lysis and at different time points (0-72 h) after incubating the lysates at 25 °C. (e). Schematic illustration showing the activation of ER-intramolecular sensor fusion protein complementation in response to the binding of ER-ligands. (f). HEK293T cells co-transfected with construct expressing fusion protein of ER-LBD flanking N- and C- tGLUC fragments measured for GLUC activity 24 h after exposure to 1 µM of different ER-ligands, immediately after lysis and at different time points (0-18 h) after incubating the lysates at 25 °C. 362x319mm (300 x 300 DPI)


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Figure 3 (a). Sequence alignment of different GLUC proteins to represent N- and C-terminal amino acid modifications of different constructs developed for this study. (b). The intracellular and secretory luciferase signal measured from HEK293T cells transfected with different engineered GLUC constructs. (c). Immunoblot analysis of intracellular GLUC protein from HEK293T cells transfected with different engineered GLUC constructs (Note: Y-axis scales are different for each graph in ‘b’). 298x287mm (300 x 300 DPI)



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Figure 4 Engineering sGLUC with multiple C-terminal tags and evaluation for intracellular retention. (a) Sequences of GLUC constructs with added C-terminal KDEL repeats (Sec-GLUC: secretory GLUC). (b) Intracellular and secretory luciferase signals measured from MDA-MB231 cells transfected with these engineered GLUC constructs. (c) FACS sorting, and (d) fluorescent microscopic images of MDA-MB231 cells stably expressing these GLUC constructs and co-expressing dTomato reporter. (e) Agarose gel of qRT-PCR amplified product of GLUC mRNA (top row) from MDA-MB231 cells stably expressing these GLUC constructs (bottom row: qRTPCR product of β-Actin mRNA), with numbers 0-4 representing increasing KDEL repeats and N is negative control. (f) Immunoblot analysis of GLUC in the cell lysates of an equal number of MDA-MB231 and LN229 cells stably expressing GLUC constructs with increasing KDEL repeats. 426x422mm (300 x 300 DPI)


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Figure 5 Validation of C-terminal tagged sGLUC variants as biosensors for chemically induced endoplasmic reticulum stress, and for monitoring caspase activation and the response of cancer cells to chemotherapy. (a) Intracellular and secretary GLUC levels in MDA-MB231 cells stably expressing GLUC biosensor containing sGLUC-KDEL, 24 h after treatment with different concentrations of tunicamycin (0-10 µg/ml), to detect cellular endoplasmic reticulum stress. (b) Similar analysis using (sGLUC-(KDEL)3). (c) Similar analysis but using the sGLUC-KDEL biosensor and optical imaging of GLUC after adding different concentrations of tunicamycin (0-10 µg/ml). (d) Schematic design of sGLUC-DEVD-(KDEL)3 biosensor to measure caspase activation in cells. (e) MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 biosensor evaluated for secretary signal in culture medium 24 h after treatment with 5 µM doxorubicin and 25 nM paclitaxel. (f) Immunoblot analysis of cell lysates of MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 caspase biosensor after treatment with different drugs for 24 h (Dox [doxorubicin] 5 µM, Pac [paclitaxel] 25 nM, Con [control], and Car [carboplatin] 25 µM). (g) Chemotherapeutic drug-induced caspase activation measured by imaging GLUC in culture medium of MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 biosensor at different time points (0-48 h) after drug treatment (Dox: 5 µM, Pac: 25 nM, and Car: 25 µM). (h and i). Dose and time dependent activation of caspase in MDA-MB231 cells stably expressing sGLUC-DEVD-(KDEL)3 biosensor in response to treatment using doxorubicin (h) and paclitaxel (i). Error bars are SEM of triplicate determinations. 487x298mm (300 x 300 DPI)


Engineering Intracellularly Retained Gaussia Luciferase Reporters for

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