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Development and validation of a fluorogenic assay to measure separase enzyme activity
Analytical Biochemistry 392 (2009) 133–138
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Development and validation of a fluorogenic assay to measure separase enzyme activity Dipanjan Basu 1, Nenggang Zhang 1, Anil K. Panigrahi, Terzah M. Horton, Debananda Pati * Department of Pediatric Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA
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Article history: Received 25 February 2009 Available online 2 June 2009 Keywords: Separase Rad21 Enzyme assay Securin Leukemia
a b s t r a c t Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples. Ó 2009 Elsevier Inc. All rights reserved.
Accurate chromosomal segregation in each cell cycle relies on a conserved protein complex called cohesin and an endopeptidase called separase. Separase is a CD clan endopeptidase (also known as Esp1 or separin in yeast) [1–3] that is bound with its inhibitory chaperone securin (also known as PTTG in human and Pds1 in yeast) prior to anaphase [1,4,5]. In metaphase, ubiquitin-mediated degradation of the securin by anaphase-promoting complex/cyclosome (APC/C)2–Cdc20 ubiquitin–ligase releases and activates separase. The activated separase proteolytically cleaves cohesin Rad21 at two sites, resulting in the removal of cohesin from chromosomes and separation of the sister chromatids [1,6–9]. Recent studies demonstrate that overexpression of separase induces aneuploidy (aberrant chromosome number) and mammary tumorigenesis in mice [10]. Small interfering RNA (siRNA)-mediated knockdown of separase and separase-deficient mouse embryonic fibroblasts also results in genomic instability [11–13]. Studies indicate that separase protein is overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma [10,14]. Furthermore, Separase overexpression
* Corresponding author. Fax: +1 832 825 4651. E-mail address: [email protected] (D. Pati). 1 These authors contributed equally to this work. 2 Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; siRNA, small interfering RNA; CSF, cytostatic factor; hCG, human chorionic gonadotropin; MMR, modified Ringer’s solution; ATP, adenosine triphosphate; IgG, immunoglobulin G; cDNA, complementary DNA; mAb, monoclonal antibody; EGTA, ethyleneglycoltetraacetic acid; MCA, methyl coumaric acid; PBS, phosphate-buffered saline; AML, acute myelogenous leukemia; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; AMC, 7-amido-4-methyl coumaric acid; RFU, relative fluorescence units; DAPI, 40 ,6-diamidino-2-phenylindole. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.046
strongly correlates with relapse, metastasis, and a lower 5-year overall survival rate in breast and prostate cancer patients [14]. Fluorogenic assay has been successfully used in enzymatic assay, such as caspases [15,16] and matrix metalloproteases [17,18], and proved to be a very reliable method. Although separase activity can be assessed by immunoblot [19,20], there is currently no other method available to quantitatively estimate separase enzymatic activity. Here, we report the development of a simple, sensitive, and robust quantitative assay for separase using a fluorogenic peptide containing the separase cleavage site of Rad21 as a substrate. This assay can be used specifically to measure separase activity from cells and tumor specimens. Materials and methods Preparation of cytostatic factor extracts from Xenopus egg Cytostatic factor (CSF) extracts were prepared as described previously [21]. Briefly, frogs were induced to lay eggs with an injection of 125 U of human chorionic gonadotropin (hCG) to the dorsal sac. Eggs were collected the next morning and dejellied with 2% cysteine in 1 modified Ringer’s solution (MMR, pH 7.8). Eggs were packed evenly by a brief spin at 500 rpm followed by a second spin at 10,000 rpm for 10 min. Clear extract with floating membranes was collected by side puncture and collected in a fresh tube. Extracts were supplemented with CSF energy mix (40 mM phosphocreatine, 4 mM adenosine triphosphate [ATP], and 0.2 mg/ml creatine phosphokinase) and 34 nM cyclin BD90 and were rotated at room temperature for 15 min. Anaphase of the CSF was initiated by adding Ca2+ up to 0.6 mM and incubating for 20 min at room
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temperature. Before being used for activating separase, the activity of CSF was confirmed using securin degradation assay.
Results and discussion Isolation and activation of separase enzyme from human cells
Overexpression, immunoprecipitation, and activation of separase We obtained active separase from human cells as described previously [19,20]. Briefly, ZZ TEV4–separase was expressed in 293T cells and immunoprecipitated from exponentially growing cells using immunoglobulin G (IgG)-conjugated Sepharose 6 (GE Healthcare, Piscataway, NJ, USA). To release active separase from its inhibitory chaperone securin, we activated separase by incubating IgG–Sepharose 6 containing ZZ TEV4–separase with anaphaseinitiated Xenopus CSF to degrade securin at room temperature for 1 h. Activated separase protein was eluted from the beads using TEV protease (Invitrogen, Carlsbad, CA, USA). Separase protease assay Separase cleavage of Rad21 was performed as described previously [19] with minor modifications. Rad21 complementary DNA (cDNA) was cloned into pFASTBac vector (Invitrogen) containing Flag tag on N terminus of Rad21 and transformed into sf9 insect cells. Flag–Rad21 was purified using Flag monoclonal antibody (mAb)conjugated agarose beads (Sigma, St. Louis, MO, USA) and eluted with 3 Flag peptide as described previously [22]. Then 5 ll of activated separase was mixed with 1 ll of recombinant Rad21 protein (expressed and purified from sf9 cells) and incubated in 20 ll of cleavage assay buffer (30 mM Hepes–KOH [pH 7.7], 50 mM NaCl, 25 mM NaF, 25 mM KCl, 5 mM MgCl2, 1.5 mM ATP, and 1 mM ethyleneglycoltetraacetic acid [EGTA]) for 1 h at 37 °C. The cleavage of Rad21 was detected by immunoblotting with Rad21 mAb. For the fluorescence assays, the same cleavage buffer was used in a 50-ll reaction volume. First, 0.5–1 ll (33.5 ng) of activated separase was combined with 2 ll of 10 mM Rad21–MCA (methyl coumaric acid) peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) (Peptide International, Louisville, KY, USA) and incubated for 3 h at 37 °C. At the end of the reaction, fluorescence was measured by spectrofluorometry (Spectrafluor Plus, TECAN, Mannedorf, Switzerland) at kex = 405 nm and kem = 465 nm.
Because the development of an in vitro separase activity assay is limited by the availability of active enzyme, we used ZZ TEV4– separase expressed in 293T cells as a source of separase enzyme. Separase was immunoprecipitated using the IgG-conjugated Sepharose 6 and eluted from the beads by cleaving the epitope using TEV protease. Because separase is inactive when bound to its inhibitory chaperone securin, the immunoprecipitated separase was incubated in Xenopus egg extracts (pretreated with Ca2+ for APC activation) to degrade securin and activating separase. We resolved the activated separase preparation on sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and performed Coomassie staining (Fig. 1A) as well as immunostaining with separase mAb (Fig. 1B) to estimate the concentration of separase in the sample. According to the total immunoprecipitated protein loaded and the intensity ratio of separase bands to the total bands from the Coomassie-stained gel, the concentration of separase was estimated to be 33.5 ng/ll separase preparation. As an additional approach, the concentration of separase was also estimated by comparing the band intensities from a protein standard with known concentrations loaded alongside (see Fig. S1 in Supplementary material). Using both methods, we obtained the similar result that the separase concentration in the samples is approximately 30 ng/ll (Figs. 1 and S1). Activated separase was tested for activity in a Rad21 cleavage assay (Fig. 1C) and used as an enzyme source in developing the fluorogenic assay. Activated separase successfully cleaved full-length recombinant Rad21 protein, generating expected mitotic cleavage fragments in vitro that were detected on immunoblot using Rad21 antibody (Fig. 1C). Designing a synthetic fluorogenic Rad21 mitotic cleavage site peptide as separase substrate The specific mitotic separase cleavage sites of cohesin Rad21 have been mapped, and it is known that active separase cleaves Rad21 at two specific sites bearing the consensus Glu-X-X-Arg near the N-terminal and C-terminal ends of the protein, respectively
Cell culture and synchronization Mitotic cell lysates were prepared from HeLa cells according to Gaglio and coworkers with minor modifications [23]. Briefly, HeLa cells were synchronized with a double thymidine block and arrested at G2/M using nocodazole (40 ng/ml final concentration) 6 h after release from the thymidine block. After 4 h of nocodazole arrest, approximately 80% of the cells reached mitosis. Mitotic cells were collected by mitotic shake-off and released from nocodazole arrest. Typically, 105 cells were collected at 0, 60, 80, 100, 120, and 140 min after nocodazole release. Cell lysate was prepared as described by Zhang and coworkers [22]. Leukemic cell lines (HEL 92.1.7, HL60, U937, Jurkat, Molt4, RS4, and Raji, American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in RPMI 1640 supplemented with 10% bovine fetal serum (Invitrogen) to a density of 0.5–1.0 106 cells/ml. Then 10 million exponentially growing cells were pelleted and washed three times in cold phosphate-buffered saline (PBS) before making the protein lysates. Bone marrow aspirates from pediatric patients with newly diagnosed acute myelogenous leukemia (AML, n = 6) were collected prior to administration of chemotherapy using an institutional review board-approved protocol according to institutional guidelines. Mononuclear cells were isolated using Lymphoprep (Axis–Shield, Oslo, Norway) and washed twice with PBS, and then 1 107 cells were frozen as cell pellets at 80 °C. Lysates were prepared as described previously [22].
Fig. 1. Isolation and activation of separase. The separase was immunoprecipitated from 293T cells and activated with Xenopus egg extract. (A, B) Estimation of active separase protein concentration. Activated separase preparation (5, 10, and 15 ll, equivalent to 3.8, 7.6, and 11.4 lg of input protein, respectively) was resolved on 7% SDS–PAGE. The protein bands were visualized with Coomassie staining (A) and the separase bands were probed with anti-separase mAb on immunoblot. (B) Full-length and autocleaved separase bands versus total band intensities on Coomassie gel was quantified using ImageQuant 5.2 software. Activated separase protein concentration was calculated as a fraction of separase band optical density over the total band intensities from the Coomassie gel and was estimated to be 33.5 ng/ll. (C) Immunoblot of Rad21 cleavage assay using activated separase. The assay was performed at 37 °C for 1 h. The protein was resolved on 8% SDS–PAGE. Full-length Rad21 and cleavage fragments (arrows) were visualized with anti-Rad21 mAb. FL, Full length; NT, N-terminal; WB, Western Blot.
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[20]. We designed a Rad21 mitotic cleavage site peptide (Ac-AspArg-Glu-Ile-Nle-Arg-MCA) conjugated to MCA and tested this peptide as a substrate for separase activity (Fig. 2A). The standard curve of 7-amido-4-methyl coumaric acid (AMC) fluorescence for the peptide–MCA showed a detectable difference in the concentration range of 11.0–300 lM. AMC fluorescence shows a concentration-dependent linear increase in fluorescence at this range, whereas the Rad21 peptide–MCA fluorescence did not increase over background (see Fig. S2 in Supplementary material). This set of studies suggested that any product (AMC) formed due to the enzyme activity will be distinct and can be detected by fluorescence from the substrate once its concentration is greater than 11.0 lM. Thus, any fluorescence produced in the reaction was due to AMC and not to the substrate peptide under these conditions. Establishment of separase activity assay using Rad21–peptidyl MCA as substrate In a set of preliminary experiments, activated separase was used to digest the synthetic Rad21 substrate and fluorescence was measured as the concentration of free AMC liberated, indicating cleavage of the scissile bond. Unactivated securin-bound separase preparation had no appreciable fluorogenic activity in this assay (Fig. 2B). We tested a range of enzyme and substrate concentrations to determine the minimum amount of enzyme and substrate required in a single reaction to obtain appreciable fluorescence intensity over the background. These studies indicated that a minimum of 0.5 ll (16.75 ng) of active enzyme (equivalent to 58 lg of protein in the input whole cell extract) and 2 nmol of Rad21–MCA peptide substrate in a 50-ll reaction volume are
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necessary to distinguish fluorescence due to enzyme activity over the nonspecific background (data not shown). Assay optimization To optimize the amount of active enzyme in a reaction, experiments were performed to determine the correlation between the concentration of activated separase and the formation of AMC. Substrate (20 nmol) was incubated in the presence and absence of increasing concentrations of activated or inactive (securingbound) separase for 3 h at 37 °C. Fluorescence correlated linearly with activated separase concentration with a positive correlation (r2 = 0.9894) over the range of 0–134 ng of active protein. Securin-bound separase demonstrated only a background level of fluorescence with increased concentrations (Fig. 2B). The linear relationship between the amount of active separase and the amount of fluorescence was used to calculate the amount of active separase in the range of 0–35,000 relative fluorescence units (RFU) at excitation and emission wavelengths of kex = 405 nm and kem = 465 nm, respectively. To produce an appreciable difference over the unactivated negative controls, a minimum of 33.5 ng of activated separase/ll was required (Fig. 2B), and this was used as our standard experimental condition. It can be noted that a considerably less amount of activated enzyme preparation (fivefold) is required in the fluorescence assay to cleave Rad21 peptide compared with the amount necessary to cleave the full-length recombinant Rad21 in the conventional cleavage assay (Fig. 1C), indicating higher sensitivity of the fluorogenic assay compared with the conventional cleavage assay. To further optimize the assay, time and temperature dependence of AMC release after the Rad21–peptidyl MCA cleavage by
Fig. 2. Development of separase assay. (A) Outline of fluorogenic assay to measure activated separase in vitro. Activated separase was used to cleave Rad21–peptidyl MCA, and the amount of cleavage product was measured by fluorescence emission. (B) Effect of increasing concentration of activated separase enzyme on AMC release. Results were obtained as relative fluorescence units (RFU) after being corrected for nonspecific background measure in the presence of substrate alone without the enzyme. The activity was calculated in terms of nmol AMC released/h/nmol substrate (Rad21–peptidyl MCA). , activated separase; j, unactivated separase. Each data point is the mean ± standard error of three observations. (C) Time and temperature dependence of AMC release after the Rad21–peptidyl MCA cleavage by activated separase enzyme at 4, 23, 30, 37, and 55 °C. Here, 16.75 ng of active enzyme and 2 nmol of Rad21–peptidyl MCA were incubated in the presence of activated separase enzyme at different temperature conditions over a period of 9 h. Results are expressed as RFU at indicated time points after being corrected for nonspecific background measured in the absence of enzyme. Values are means ± standard errors of three observations. (D) AMC release after the Rad21–peptidyl MCA cleavage by activated separase enzyme is a function of pH. Values are means ± standard errors of three observations.
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activated separase enzyme were studied over a period of 9 h at 4, 23, 30, 37, and 55 °C (Fig. 2C). Although the optimal temperature for the assay was observed to be at a physiological temperature of 37 °C, the assay tolerated a wide range of temperatures from 30 to 55 °C. The assay reaction proceeded at a slower kinetic rate at room temperature (23 °C) and had no appreciable activity at 4 °C (Fig. 2C). Based on these observations, an incubation time of 3 h at 37 °C was used as the standard experimental condition for subsequent studies. Optimal cleavage activity in terms of released AMC was studied over a pH range of 6.0–9.0. Maximum AMC release was obtained at pH 7.7 (Fig. 2D). Enzyme kinetics Activated separase enzyme was incubated with increasing concentrations of Rad21–peptidyl MCA (100–3200 lM) for a period of 3 h at 37 °C. AMC release was corrected for nonspecific background measured in the absence of the enzyme. The ability of separase to cleave the synthetic peptide in vitro follows distinct Michaelis– Menten kinetics and was saturated at a substrate concentration of more than 1000 lM Rad21–MCA (Fig. 3A). We calculated a maximum velocity (Vmax) of 1.25 nmol of AMC released/h and a KM of 2.93 104 M based on 33.5 ng (0.145 pmol) of activated separase (Fig. 3B). Thus, the Vmax translates into 8620.6 mol of AMC released/mol activated separase/h, indicating robustness of separase enzyme activity. The KM value (a measure of the affinity of the enzyme for its substrate) of separase was comparable to that observed for caspase 3, a distant member of this family [24]. Enzyme stability We tested the stability of the activated Separase at several storage conditions, including multiple freeze–thaw cycles, to assess enzyme stability. Allowing the activated enzyme to stay for 48 h at room temperature (23 °C) reduced its activity by approximately 17%, whereas activity was reduced only 12% when stored at 4 °C. However, storage below 20 °C for 1 week had no significant effect on separase activity. The activated enzyme could tolerate up to three freeze–thaw cycles without significant loss in activity (see Fig. S3A in Supplementary material). Assay variability To assess inter- and intraassay variability, significant difference between the positive and negative controls was calculated using Z0 values (Fig. S3B). Z0 compares the mean value of the maximum signal control with the mean value of the minimum control and will have a higher value when (i) there is a wide separation band between maximum and minimum controls and (ii) the standard deviations are low. Both the substrate and enzyme alone as well as the buffer yielded Z0 values greater than 0.5 (Z0 substrate only = 0.6513,
Z0 separase only = 0.7141, Z0 buffer = 0.7314) when compared with positive control, indicating acceptable variability. Validation of separase activity assay Specificity of the assay To validate whether separase activity assay is substrate specific, we compared Rad21–peptidyl MCA and LEHD–CHO–MCA. The latter is a caspase 9/6-specific substrate. Incubating under similar conditions in the presence and absence of activated separase, the caspase substrate LEHA–AMC did not produce any detectable fluorescence over the negative control, indicating that separase could not cleave LEHA–AMC (see Fig. S4 in Supplementary material). It is possible that, besides separase, other proteases in crude cell extract might be able to cleave Rad21–peptidyl MCA. Unlike the activated separase, the unactivated separase failed to cleave Rad21– peptidyl MCA (Fig. 2B), implying that separase cleavage of Rad21–peptidyl MCA is specific. To further validate the specificity of separase activity in cleaving Rad21–peptidyl MCA, we knocked down separase expression in 293T cells by siRNA as described previously [22]. Whole cell extract was made and used for separase fluorogenic assay. Compared with the cells transfected with control siRNA, the cleavages of Rad21–peptidyl MCA were reduced in separase siRNA-transfected cells; this was directly proportional to the reduction in separase protein level (Fig. S4) and further confirmed the specificity of separase-mediated cleavage of Rad21– peptidyl MCA. Separase activity at the metaphase-to-anaphase transition To measure the separase enzymatic activity during mitotic progression of the cell cycle, we synchronized HeLa cells with double thymidine block followed by nocodazole arrest. Approximately 80% of the cells were found to be in mitosis as determined by microscopic analysis of the 40 ,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Synchronized mitotic cells were collected by mitotic shake-off at various time points after release from the nocodazole arrest, whole cell extracts were prepared, and separase activity was measured. Separase activity significantly increased after 60 min of nocodazole release and then declined in a graded fashion over the next 40 min and remained stable thereafter (Fig. 4A). To corroborate separase activation at the metaphase– anaphase transit with other cell cycle events, we examined the securin and cyclin B levels in these cells at the same time points after nocodazole release (Fig. 4B). As expected, Western blot analysis showed a time-dependent degradation of securin and cyclin B starting 60 min after release and was nearly undetectable by 100 min. Therefore, the fluorogenic assay can successfully detect separase in these synchronized cells when most of the cells are at the metaphase-to-anaphase transition.
Fig. 3. Characterization of separase enzyme kinetics. (A) Cleavage reaction was carried out in the presence of increasing concentrations of the Rad21–peptidyl MCA (50– 1600 lM) and the activated separase enzyme (33.5 ng) at 37 °C for 3 h. Results are expressed as nmol AMC released/h after being corrected for nonspecific background measured in the absence of the enzyme. Saturation kinetics is plotted as a Michaelis–Menten curve. (B) Vmax and KM values were calculated using a Lineweaver–Burk plot.
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Fig. 4. Determination of separase activity in cells and leukemia samples. (A) Separase activity during mitotic progression. HeLa cells were synchronized with double thymidine and arrested at G2/M with nocodazole. Metaphase arrested cells were harvested for lysis at 0, 60, 80, 100, 120, and 140 min after release from the nocodazole block. Separase activity assay was performed with 20 lg of protein from each time point. Each time point is the mean ± standard error of three observations. (B) Immunoblotting of cyclin B and securin levels in HeLa cells after release from nocodazole arrest. (C) Separase activity assay in a panel of leukemic cell lines. Here, 20 lg of total protein from each line was analyzed. (D) Separase activity in adult and pediatric AML samples. P1 was a 16-year-old Caucasian male with inversion 16 [inv(16)], M4AML; P2 was a 5-year-old African American male with M3-AML in relapse; P3 was an 11-year-old Hispanic male with M1-AML; P4 was a 2-year-old Hispanic female with inv(16) AML; P5 was an 11-year-old African American male with fulminant AML; and P6 was a 2-day-old Hispanic with trisomey 21 and fulminant AML/MDS. Peripheral blood cells from a normal individual were used as control. For each sample, 25 lg of total protein was analyzed.
Separase activity in leukemic samples Separase has been identified as an important regulator of metaphase–anaphase transition in vertebrate cells. We showed recently that overexpression of separase leads to aneuploidy and tumor development in murine mammary glands [10]. We also reported that separase is overexpressed in a number of human cancers, including breast, prostate, and osteosarcoma [14]. Therefore, we wanted to investigate whether cancer cells exhibit higher separase enzyme activity. Using the fluorogenic assay, here we measured the activity of separase enzyme in a number of leukemia cell lines and pediatric leukemia patient samples. In leukemic cell lines (Fig. 4C), a basal level of separase activity was expected because these are rapidly dividing cells. However, significantly higher separase activity could be detected in two (HEL and HL60) of the seven cell lines tested, indicating increased separase activity in AML cells. Next, we extended our assay to quantify separase activity in six pediatric AML patient samples. Compared with normal individuals, the AML samples studied showed significantly greater separase activity than did the control (Fig. 4D).
Summary Although attempts have been made to quantify separase from cells and tissues, those approaches relied mostly on modifications of separase protein (e.g., phosphorylation) and were unable to quantitatively measure its enzymatic activity [25]. Our fluores-
cence-based assay is a simple, sensitive, specific, inexpensive, and user-friendly method. Importantly, this is a robust assay that tolerates a wide range of physical parameters and can effectively detect a wide range of separase activity even in crude cell extract. Given the tight regulatory mechanisms that lead to a very finetuned protease activity of separase for a brief window during the cell cycle, it would be worth looking at the activity profile of this protease in different tumors. Separase activity profile in tumors may provide insights into the process of chromosomal instability, tumorigenesis, and tumor progression. In addition to its potential clinical diagnostic and prognostic tool to assess tumor status, separase assay can also be used to develop a high-throughput screen for inhibitors of separase. Separase assay could be an important tool for researchers as well as clinicians studying sister chromatid cohesion, aneuploidy, and/or cancer. Acknowledgments This study was supported by award 1RO1 CA109330 from the National Cancer Institute, a pilot project grant from Dan L. Duncan Cancer Center at Baylor College of Medicine, and 2008 Virginia and L.E. Simmons Family Foundation Collaborative research award to D. Pati. ZZ TEV4–separase construct was a kind gift from Hui Zhou (University of Texas Southwestern Medical Center at Dallas). We thank the research administration at the Texas Children’s Hospital, particularly Denise Treece, for generous support in establishing the frog facility that played a key role in the completion of this work.
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Development and validation of a fluorogenic assay to measure separase enzyme activity Dipanjan Basu 1, Nenggang Zhang 1, Anil K. Panigrahi, Terzah M. Horton, Debananda Pati * Department of Pediatric Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA
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Article history: Received 25 February 2009 Available online 2 June 2009 Keywords: Separase Rad21 Enzyme assay Securin Leukemia
a b s t r a c t Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples. Ó 2009 Elsevier Inc. All rights reserved.
Accurate chromosomal segregation in each cell cycle relies on a conserved protein complex called cohesin and an endopeptidase called separase. Separase is a CD clan endopeptidase (also known as Esp1 or separin in yeast) [1–3] that is bound with its inhibitory chaperone securin (also known as PTTG in human and Pds1 in yeast) prior to anaphase [1,4,5]. In metaphase, ubiquitin-mediated degradation of the securin by anaphase-promoting complex/cyclosome (APC/C)2–Cdc20 ubiquitin–ligase releases and activates separase. The activated separase proteolytically cleaves cohesin Rad21 at two sites, resulting in the removal of cohesin from chromosomes and separation of the sister chromatids [1,6–9]. Recent studies demonstrate that overexpression of separase induces aneuploidy (aberrant chromosome number) and mammary tumorigenesis in mice [10]. Small interfering RNA (siRNA)-mediated knockdown of separase and separase-deficient mouse embryonic fibroblasts also results in genomic instability [11–13]. Studies indicate that separase protein is overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma [10,14]. Furthermore, Separase overexpression
* Corresponding author. Fax: +1 832 825 4651. E-mail address: [email protected] (D. Pati). 1 These authors contributed equally to this work. 2 Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; siRNA, small interfering RNA; CSF, cytostatic factor; hCG, human chorionic gonadotropin; MMR, modified Ringer’s solution; ATP, adenosine triphosphate; IgG, immunoglobulin G; cDNA, complementary DNA; mAb, monoclonal antibody; EGTA, ethyleneglycoltetraacetic acid; MCA, methyl coumaric acid; PBS, phosphate-buffered saline; AML, acute myelogenous leukemia; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; AMC, 7-amido-4-methyl coumaric acid; RFU, relative fluorescence units; DAPI, 40 ,6-diamidino-2-phenylindole. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.046
strongly correlates with relapse, metastasis, and a lower 5-year overall survival rate in breast and prostate cancer patients [14]. Fluorogenic assay has been successfully used in enzymatic assay, such as caspases [15,16] and matrix metalloproteases [17,18], and proved to be a very reliable method. Although separase activity can be assessed by immunoblot [19,20], there is currently no other method available to quantitatively estimate separase enzymatic activity. Here, we report the development of a simple, sensitive, and robust quantitative assay for separase using a fluorogenic peptide containing the separase cleavage site of Rad21 as a substrate. This assay can be used specifically to measure separase activity from cells and tumor specimens. Materials and methods Preparation of cytostatic factor extracts from Xenopus egg Cytostatic factor (CSF) extracts were prepared as described previously [21]. Briefly, frogs were induced to lay eggs with an injection of 125 U of human chorionic gonadotropin (hCG) to the dorsal sac. Eggs were collected the next morning and dejellied with 2% cysteine in 1 modified Ringer’s solution (MMR, pH 7.8). Eggs were packed evenly by a brief spin at 500 rpm followed by a second spin at 10,000 rpm for 10 min. Clear extract with floating membranes was collected by side puncture and collected in a fresh tube. Extracts were supplemented with CSF energy mix (40 mM phosphocreatine, 4 mM adenosine triphosphate [ATP], and 0.2 mg/ml creatine phosphokinase) and 34 nM cyclin BD90 and were rotated at room temperature for 15 min. Anaphase of the CSF was initiated by adding Ca2+ up to 0.6 mM and incubating for 20 min at room
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temperature. Before being used for activating separase, the activity of CSF was confirmed using securin degradation assay.
Results and discussion Isolation and activation of separase enzyme from human cells
Overexpression, immunoprecipitation, and activation of separase We obtained active separase from human cells as described previously [19,20]. Briefly, ZZ TEV4–separase was expressed in 293T cells and immunoprecipitated from exponentially growing cells using immunoglobulin G (IgG)-conjugated Sepharose 6 (GE Healthcare, Piscataway, NJ, USA). To release active separase from its inhibitory chaperone securin, we activated separase by incubating IgG–Sepharose 6 containing ZZ TEV4–separase with anaphaseinitiated Xenopus CSF to degrade securin at room temperature for 1 h. Activated separase protein was eluted from the beads using TEV protease (Invitrogen, Carlsbad, CA, USA). Separase protease assay Separase cleavage of Rad21 was performed as described previously [19] with minor modifications. Rad21 complementary DNA (cDNA) was cloned into pFASTBac vector (Invitrogen) containing Flag tag on N terminus of Rad21 and transformed into sf9 insect cells. Flag–Rad21 was purified using Flag monoclonal antibody (mAb)conjugated agarose beads (Sigma, St. Louis, MO, USA) and eluted with 3 Flag peptide as described previously [22]. Then 5 ll of activated separase was mixed with 1 ll of recombinant Rad21 protein (expressed and purified from sf9 cells) and incubated in 20 ll of cleavage assay buffer (30 mM Hepes–KOH [pH 7.7], 50 mM NaCl, 25 mM NaF, 25 mM KCl, 5 mM MgCl2, 1.5 mM ATP, and 1 mM ethyleneglycoltetraacetic acid [EGTA]) for 1 h at 37 °C. The cleavage of Rad21 was detected by immunoblotting with Rad21 mAb. For the fluorescence assays, the same cleavage buffer was used in a 50-ll reaction volume. First, 0.5–1 ll (33.5 ng) of activated separase was combined with 2 ll of 10 mM Rad21–MCA (methyl coumaric acid) peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) (Peptide International, Louisville, KY, USA) and incubated for 3 h at 37 °C. At the end of the reaction, fluorescence was measured by spectrofluorometry (Spectrafluor Plus, TECAN, Mannedorf, Switzerland) at kex = 405 nm and kem = 465 nm.
Because the development of an in vitro separase activity assay is limited by the availability of active enzyme, we used ZZ TEV4– separase expressed in 293T cells as a source of separase enzyme. Separase was immunoprecipitated using the IgG-conjugated Sepharose 6 and eluted from the beads by cleaving the epitope using TEV protease. Because separase is inactive when bound to its inhibitory chaperone securin, the immunoprecipitated separase was incubated in Xenopus egg extracts (pretreated with Ca2+ for APC activation) to degrade securin and activating separase. We resolved the activated separase preparation on sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and performed Coomassie staining (Fig. 1A) as well as immunostaining with separase mAb (Fig. 1B) to estimate the concentration of separase in the sample. According to the total immunoprecipitated protein loaded and the intensity ratio of separase bands to the total bands from the Coomassie-stained gel, the concentration of separase was estimated to be 33.5 ng/ll separase preparation. As an additional approach, the concentration of separase was also estimated by comparing the band intensities from a protein standard with known concentrations loaded alongside (see Fig. S1 in Supplementary material). Using both methods, we obtained the similar result that the separase concentration in the samples is approximately 30 ng/ll (Figs. 1 and S1). Activated separase was tested for activity in a Rad21 cleavage assay (Fig. 1C) and used as an enzyme source in developing the fluorogenic assay. Activated separase successfully cleaved full-length recombinant Rad21 protein, generating expected mitotic cleavage fragments in vitro that were detected on immunoblot using Rad21 antibody (Fig. 1C). Designing a synthetic fluorogenic Rad21 mitotic cleavage site peptide as separase substrate The specific mitotic separase cleavage sites of cohesin Rad21 have been mapped, and it is known that active separase cleaves Rad21 at two specific sites bearing the consensus Glu-X-X-Arg near the N-terminal and C-terminal ends of the protein, respectively
Cell culture and synchronization Mitotic cell lysates were prepared from HeLa cells according to Gaglio and coworkers with minor modifications [23]. Briefly, HeLa cells were synchronized with a double thymidine block and arrested at G2/M using nocodazole (40 ng/ml final concentration) 6 h after release from the thymidine block. After 4 h of nocodazole arrest, approximately 80% of the cells reached mitosis. Mitotic cells were collected by mitotic shake-off and released from nocodazole arrest. Typically, 105 cells were collected at 0, 60, 80, 100, 120, and 140 min after nocodazole release. Cell lysate was prepared as described by Zhang and coworkers [22]. Leukemic cell lines (HEL 92.1.7, HL60, U937, Jurkat, Molt4, RS4, and Raji, American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in RPMI 1640 supplemented with 10% bovine fetal serum (Invitrogen) to a density of 0.5–1.0 106 cells/ml. Then 10 million exponentially growing cells were pelleted and washed three times in cold phosphate-buffered saline (PBS) before making the protein lysates. Bone marrow aspirates from pediatric patients with newly diagnosed acute myelogenous leukemia (AML, n = 6) were collected prior to administration of chemotherapy using an institutional review board-approved protocol according to institutional guidelines. Mononuclear cells were isolated using Lymphoprep (Axis–Shield, Oslo, Norway) and washed twice with PBS, and then 1 107 cells were frozen as cell pellets at 80 °C. Lysates were prepared as described previously [22].
Fig. 1. Isolation and activation of separase. The separase was immunoprecipitated from 293T cells and activated with Xenopus egg extract. (A, B) Estimation of active separase protein concentration. Activated separase preparation (5, 10, and 15 ll, equivalent to 3.8, 7.6, and 11.4 lg of input protein, respectively) was resolved on 7% SDS–PAGE. The protein bands were visualized with Coomassie staining (A) and the separase bands were probed with anti-separase mAb on immunoblot. (B) Full-length and autocleaved separase bands versus total band intensities on Coomassie gel was quantified using ImageQuant 5.2 software. Activated separase protein concentration was calculated as a fraction of separase band optical density over the total band intensities from the Coomassie gel and was estimated to be 33.5 ng/ll. (C) Immunoblot of Rad21 cleavage assay using activated separase. The assay was performed at 37 °C for 1 h. The protein was resolved on 8% SDS–PAGE. Full-length Rad21 and cleavage fragments (arrows) were visualized with anti-Rad21 mAb. FL, Full length; NT, N-terminal; WB, Western Blot.
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[20]. We designed a Rad21 mitotic cleavage site peptide (Ac-AspArg-Glu-Ile-Nle-Arg-MCA) conjugated to MCA and tested this peptide as a substrate for separase activity (Fig. 2A). The standard curve of 7-amido-4-methyl coumaric acid (AMC) fluorescence for the peptide–MCA showed a detectable difference in the concentration range of 11.0–300 lM. AMC fluorescence shows a concentration-dependent linear increase in fluorescence at this range, whereas the Rad21 peptide–MCA fluorescence did not increase over background (see Fig. S2 in Supplementary material). This set of studies suggested that any product (AMC) formed due to the enzyme activity will be distinct and can be detected by fluorescence from the substrate once its concentration is greater than 11.0 lM. Thus, any fluorescence produced in the reaction was due to AMC and not to the substrate peptide under these conditions. Establishment of separase activity assay using Rad21–peptidyl MCA as substrate In a set of preliminary experiments, activated separase was used to digest the synthetic Rad21 substrate and fluorescence was measured as the concentration of free AMC liberated, indicating cleavage of the scissile bond. Unactivated securin-bound separase preparation had no appreciable fluorogenic activity in this assay (Fig. 2B). We tested a range of enzyme and substrate concentrations to determine the minimum amount of enzyme and substrate required in a single reaction to obtain appreciable fluorescence intensity over the background. These studies indicated that a minimum of 0.5 ll (16.75 ng) of active enzyme (equivalent to 58 lg of protein in the input whole cell extract) and 2 nmol of Rad21–MCA peptide substrate in a 50-ll reaction volume are
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necessary to distinguish fluorescence due to enzyme activity over the nonspecific background (data not shown). Assay optimization To optimize the amount of active enzyme in a reaction, experiments were performed to determine the correlation between the concentration of activated separase and the formation of AMC. Substrate (20 nmol) was incubated in the presence and absence of increasing concentrations of activated or inactive (securingbound) separase for 3 h at 37 °C. Fluorescence correlated linearly with activated separase concentration with a positive correlation (r2 = 0.9894) over the range of 0–134 ng of active protein. Securin-bound separase demonstrated only a background level of fluorescence with increased concentrations (Fig. 2B). The linear relationship between the amount of active separase and the amount of fluorescence was used to calculate the amount of active separase in the range of 0–35,000 relative fluorescence units (RFU) at excitation and emission wavelengths of kex = 405 nm and kem = 465 nm, respectively. To produce an appreciable difference over the unactivated negative controls, a minimum of 33.5 ng of activated separase/ll was required (Fig. 2B), and this was used as our standard experimental condition. It can be noted that a considerably less amount of activated enzyme preparation (fivefold) is required in the fluorescence assay to cleave Rad21 peptide compared with the amount necessary to cleave the full-length recombinant Rad21 in the conventional cleavage assay (Fig. 1C), indicating higher sensitivity of the fluorogenic assay compared with the conventional cleavage assay. To further optimize the assay, time and temperature dependence of AMC release after the Rad21–peptidyl MCA cleavage by
Fig. 2. Development of separase assay. (A) Outline of fluorogenic assay to measure activated separase in vitro. Activated separase was used to cleave Rad21–peptidyl MCA, and the amount of cleavage product was measured by fluorescence emission. (B) Effect of increasing concentration of activated separase enzyme on AMC release. Results were obtained as relative fluorescence units (RFU) after being corrected for nonspecific background measure in the presence of substrate alone without the enzyme. The activity was calculated in terms of nmol AMC released/h/nmol substrate (Rad21–peptidyl MCA). , activated separase; j, unactivated separase. Each data point is the mean ± standard error of three observations. (C) Time and temperature dependence of AMC release after the Rad21–peptidyl MCA cleavage by activated separase enzyme at 4, 23, 30, 37, and 55 °C. Here, 16.75 ng of active enzyme and 2 nmol of Rad21–peptidyl MCA were incubated in the presence of activated separase enzyme at different temperature conditions over a period of 9 h. Results are expressed as RFU at indicated time points after being corrected for nonspecific background measured in the absence of enzyme. Values are means ± standard errors of three observations. (D) AMC release after the Rad21–peptidyl MCA cleavage by activated separase enzyme is a function of pH. Values are means ± standard errors of three observations.
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activated separase enzyme were studied over a period of 9 h at 4, 23, 30, 37, and 55 °C (Fig. 2C). Although the optimal temperature for the assay was observed to be at a physiological temperature of 37 °C, the assay tolerated a wide range of temperatures from 30 to 55 °C. The assay reaction proceeded at a slower kinetic rate at room temperature (23 °C) and had no appreciable activity at 4 °C (Fig. 2C). Based on these observations, an incubation time of 3 h at 37 °C was used as the standard experimental condition for subsequent studies. Optimal cleavage activity in terms of released AMC was studied over a pH range of 6.0–9.0. Maximum AMC release was obtained at pH 7.7 (Fig. 2D). Enzyme kinetics Activated separase enzyme was incubated with increasing concentrations of Rad21–peptidyl MCA (100–3200 lM) for a period of 3 h at 37 °C. AMC release was corrected for nonspecific background measured in the absence of the enzyme. The ability of separase to cleave the synthetic peptide in vitro follows distinct Michaelis– Menten kinetics and was saturated at a substrate concentration of more than 1000 lM Rad21–MCA (Fig. 3A). We calculated a maximum velocity (Vmax) of 1.25 nmol of AMC released/h and a KM of 2.93 104 M based on 33.5 ng (0.145 pmol) of activated separase (Fig. 3B). Thus, the Vmax translates into 8620.6 mol of AMC released/mol activated separase/h, indicating robustness of separase enzyme activity. The KM value (a measure of the affinity of the enzyme for its substrate) of separase was comparable to that observed for caspase 3, a distant member of this family [24]. Enzyme stability We tested the stability of the activated Separase at several storage conditions, including multiple freeze–thaw cycles, to assess enzyme stability. Allowing the activated enzyme to stay for 48 h at room temperature (23 °C) reduced its activity by approximately 17%, whereas activity was reduced only 12% when stored at 4 °C. However, storage below 20 °C for 1 week had no significant effect on separase activity. The activated enzyme could tolerate up to three freeze–thaw cycles without significant loss in activity (see Fig. S3A in Supplementary material). Assay variability To assess inter- and intraassay variability, significant difference between the positive and negative controls was calculated using Z0 values (Fig. S3B). Z0 compares the mean value of the maximum signal control with the mean value of the minimum control and will have a higher value when (i) there is a wide separation band between maximum and minimum controls and (ii) the standard deviations are low. Both the substrate and enzyme alone as well as the buffer yielded Z0 values greater than 0.5 (Z0 substrate only = 0.6513,
Z0 separase only = 0.7141, Z0 buffer = 0.7314) when compared with positive control, indicating acceptable variability. Validation of separase activity assay Specificity of the assay To validate whether separase activity assay is substrate specific, we compared Rad21–peptidyl MCA and LEHD–CHO–MCA. The latter is a caspase 9/6-specific substrate. Incubating under similar conditions in the presence and absence of activated separase, the caspase substrate LEHA–AMC did not produce any detectable fluorescence over the negative control, indicating that separase could not cleave LEHA–AMC (see Fig. S4 in Supplementary material). It is possible that, besides separase, other proteases in crude cell extract might be able to cleave Rad21–peptidyl MCA. Unlike the activated separase, the unactivated separase failed to cleave Rad21– peptidyl MCA (Fig. 2B), implying that separase cleavage of Rad21–peptidyl MCA is specific. To further validate the specificity of separase activity in cleaving Rad21–peptidyl MCA, we knocked down separase expression in 293T cells by siRNA as described previously [22]. Whole cell extract was made and used for separase fluorogenic assay. Compared with the cells transfected with control siRNA, the cleavages of Rad21–peptidyl MCA were reduced in separase siRNA-transfected cells; this was directly proportional to the reduction in separase protein level (Fig. S4) and further confirmed the specificity of separase-mediated cleavage of Rad21– peptidyl MCA. Separase activity at the metaphase-to-anaphase transition To measure the separase enzymatic activity during mitotic progression of the cell cycle, we synchronized HeLa cells with double thymidine block followed by nocodazole arrest. Approximately 80% of the cells were found to be in mitosis as determined by microscopic analysis of the 40 ,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Synchronized mitotic cells were collected by mitotic shake-off at various time points after release from the nocodazole arrest, whole cell extracts were prepared, and separase activity was measured. Separase activity significantly increased after 60 min of nocodazole release and then declined in a graded fashion over the next 40 min and remained stable thereafter (Fig. 4A). To corroborate separase activation at the metaphase– anaphase transit with other cell cycle events, we examined the securin and cyclin B levels in these cells at the same time points after nocodazole release (Fig. 4B). As expected, Western blot analysis showed a time-dependent degradation of securin and cyclin B starting 60 min after release and was nearly undetectable by 100 min. Therefore, the fluorogenic assay can successfully detect separase in these synchronized cells when most of the cells are at the metaphase-to-anaphase transition.
Fig. 3. Characterization of separase enzyme kinetics. (A) Cleavage reaction was carried out in the presence of increasing concentrations of the Rad21–peptidyl MCA (50– 1600 lM) and the activated separase enzyme (33.5 ng) at 37 °C for 3 h. Results are expressed as nmol AMC released/h after being corrected for nonspecific background measured in the absence of the enzyme. Saturation kinetics is plotted as a Michaelis–Menten curve. (B) Vmax and KM values were calculated using a Lineweaver–Burk plot.
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Fig. 4. Determination of separase activity in cells and leukemia samples. (A) Separase activity during mitotic progression. HeLa cells were synchronized with double thymidine and arrested at G2/M with nocodazole. Metaphase arrested cells were harvested for lysis at 0, 60, 80, 100, 120, and 140 min after release from the nocodazole block. Separase activity assay was performed with 20 lg of protein from each time point. Each time point is the mean ± standard error of three observations. (B) Immunoblotting of cyclin B and securin levels in HeLa cells after release from nocodazole arrest. (C) Separase activity assay in a panel of leukemic cell lines. Here, 20 lg of total protein from each line was analyzed. (D) Separase activity in adult and pediatric AML samples. P1 was a 16-year-old Caucasian male with inversion 16 [inv(16)], M4AML; P2 was a 5-year-old African American male with M3-AML in relapse; P3 was an 11-year-old Hispanic male with M1-AML; P4 was a 2-year-old Hispanic female with inv(16) AML; P5 was an 11-year-old African American male with fulminant AML; and P6 was a 2-day-old Hispanic with trisomey 21 and fulminant AML/MDS. Peripheral blood cells from a normal individual were used as control. For each sample, 25 lg of total protein was analyzed.
Separase activity in leukemic samples Separase has been identified as an important regulator of metaphase–anaphase transition in vertebrate cells. We showed recently that overexpression of separase leads to aneuploidy and tumor development in murine mammary glands [10]. We also reported that separase is overexpressed in a number of human cancers, including breast, prostate, and osteosarcoma [14]. Therefore, we wanted to investigate whether cancer cells exhibit higher separase enzyme activity. Using the fluorogenic assay, here we measured the activity of separase enzyme in a number of leukemia cell lines and pediatric leukemia patient samples. In leukemic cell lines (Fig. 4C), a basal level of separase activity was expected because these are rapidly dividing cells. However, significantly higher separase activity could be detected in two (HEL and HL60) of the seven cell lines tested, indicating increased separase activity in AML cells. Next, we extended our assay to quantify separase activity in six pediatric AML patient samples. Compared with normal individuals, the AML samples studied showed significantly greater separase activity than did the control (Fig. 4D).
Summary Although attempts have been made to quantify separase from cells and tissues, those approaches relied mostly on modifications of separase protein (e.g., phosphorylation) and were unable to quantitatively measure its enzymatic activity [25]. Our fluores-
cence-based assay is a simple, sensitive, specific, inexpensive, and user-friendly method. Importantly, this is a robust assay that tolerates a wide range of physical parameters and can effectively detect a wide range of separase activity even in crude cell extract. Given the tight regulatory mechanisms that lead to a very finetuned protease activity of separase for a brief window during the cell cycle, it would be worth looking at the activity profile of this protease in different tumors. Separase activity profile in tumors may provide insights into the process of chromosomal instability, tumorigenesis, and tumor progression. In addition to its potential clinical diagnostic and prognostic tool to assess tumor status, separase assay can also be used to develop a high-throughput screen for inhibitors of separase. Separase assay could be an important tool for researchers as well as clinicians studying sister chromatid cohesion, aneuploidy, and/or cancer. Acknowledgments This study was supported by award 1RO1 CA109330 from the National Cancer Institute, a pilot project grant from Dan L. Duncan Cancer Center at Baylor College of Medicine, and 2008 Virginia and L.E. Simmons Family Foundation Collaborative research award to D. Pati. ZZ TEV4–separase construct was a kind gift from Hui Zhou (University of Texas Southwestern Medical Center at Dallas). We thank the research administration at the Texas Children’s Hospital, particularly Denise Treece, for generous support in establishing the frog facility that played a key role in the completion of this work.
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