Facile, Fluorogenic Assay for Protein Histidine Phosphatase Activity

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A Facile, Fluorogenic Assay for Protein Histidine Phosphatase Activity Brandon S McCullough, and Amy M. Barrios Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00278 • Publication Date (Web): 09 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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A Facile, Fluorogenic Assay for Protein Histidine Phosphatase Activity Brandon S. McCullough and Amy M. Barrios* Department of Medicinal Chemistry, University of Utah Salt Lake City, UT 84412 Abstract

Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a kcat of 0.39 ± 0.02 s-1, a Km of 220 ± 30 µM, and a kcat/Km of 1800 ± 200 M-1 s-1. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 µM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition metal ions, with one apparent Cu(II) binding site with IC50 value of 500 ± 20 µM and two apparent Zn(II) binding sites with IC50 values of 25 ± 1 µM and 490 ± 20 µM.

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Introduction Protein histidine phosphorylation was first discovered in 1962,[1] nearly 20 years before protein tyrosine phosphorylation.[2] Despite this significant time advantage and the higher incidence of pHis (accounting for perhaps 6% or more of the total phosphoamino acids in the proteome)[3] as compared to pTyr (estimated at less than 1% of total phosphosites),[4] the roles of histidine phosphorylation in mammalian cells are virtually unknown compared to the roles of tyrosine phosphorylation, which are in turn much less well understood than serine and threonine phosphorylation. Nonetheless, it has become clear that histidine phosphorylation is important in mammalian cells. For example, histidine phosphatase activity is known to be important in several biological processes including regulation of T-cell receptor signaling,[5] G-protein coupled receptor signaling,[6] and potassium channel activation.[7] In addition, elevated levels of PHPT1 have been found in hepatocellular carcinoma[8] and lung cancer[9] tissue when compared to non-cancerous tissue, and high expression of PHPT1 in clear-cell renal cell carcinoma has been negatively correlated with patient survival Although interest in the biological roles of histidine phosphorylation in mammals continues to grow,[11,12] the lack of chemical tools available to study pHis and the enzymes that regulate it is a significant roadblock to the field. Highly sensitive, continuous fluorogenic assays for tyrosine phosphatase activity have been invaluable, providing substrates for enzyme assays and high throughput inhibitor screening[13–16] and tools for monitoring PTP activity in cells,[17–19] which have yielded insights into the roles of PTPs in biology.[17,20] In contrast, no commercially available inhibitors and few enzyme assays exist for studying PHP activity. The histidine phosphatase PHPT1 has been shown to hydrolyze para-nitrophenylphosphate[21] (pNPP, a colorimetric substrate commonly used to monitor general phosphatase activity,[13] see Figure 1)


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Biochemistry

and small pHis containing peptides using an HPLC-based assay[22]. However, these substrates suffer from modest turnover, low sensitivity, and discontinuous assay readout methodologies. Adding to the complexity of the problem, the assay conditions that have been used seem to have been borrowed from the literature on other protein phosphatases and have not been optimized for monitoring PHP activity. For example, some assays include DTT, which is required for protein tyrosine phosphatase activity to reduce the catalytic cysteine residue,[23] while other assays include MgCl2, which is required for serine and threonine phosphatase activity.[24] However, the PHPs are believed to be neither cysteine dependent hydrolyases nor metallohydrolases, but rather to utilize a histidine residue either as a general base to activate a water molecule to serve as the hydrolytic nucleophile[21] or to participate in phosphoryl transfer directly.[25,26] All of these issues ultimately limit the widespread use of the few existing PHP substrates and result in a high barrier of entry to studying the biochemistry and biology of the PHPs.

Figure 1. Commonly used phosphatase substrates para-nitrophenylphosphate (pNPP) and 6,8difluoromethylumbelliferyl phosphate (DiFMUP).

Given the current state of the field, an ideal PHP activity probe would be readily available from commercial sources and provide a highly sensitive readout. Fluorogenic substrates provide direct, continuous readouts and would greatly facilitate the study of these enzymes in vitro, the development of inhibitors, and, advance our understanding of the biological roles of histidine


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phosphatases. Based on a preliminary report that PHPT1 can hydrolyze the highly sensitive fluorogenic phosphatase substrate 6,8-difluoromethylumbelliferyl phosphate (DiFMUP),[27,28] we investigated the utility of DiFMUP as a substrate for PHPT1 activity and here provide an optimized and validated PHPT1 activity assay protocol.

Materials and Methods General Recombinant PHPT1 with an N-terminal His tag was purchased from Sino Biological (catalog number 12473-H07E, UniProt Accession ID Q9NRX4-1). All chemicals were obtained from commercial sources and used without further purification. Assays were performed using black 96 well Greiner Bio-one half area microplates with a total volume of 50 µL in each well. DMSO concentrations in each well for all assays was 10% v/v. DTT stock solutions were prepared in MilliQ water. Fluorescence data were collected using a Molecular Devices M5 plate reader using excitation and emission wavelengths of 350 nm and 455 nm. Initial velocities were determined using the linear region of the RFU vs time curves. Data points were collected in triplicate unless otherwise noted.

Assay Optimization 50 mM buffers (Tris·HCl, bicine, bis-Tris propane, and HEPES, pH 8.0) were individually prepared with a single chemical additive (10 mM, 50 mM, or 100 mM NaCl; 500 µM or 2 mM EDTA; 1 mM MgCl2, CaCl2, CuCl2, or ZnCl2; 0.01% w/v Tween 20, Triton X-100, or Brij 35; 0.5 mg/mL BSA). To a 4.93 µM aliquot of PHPT1, buffer and reducing agent (DTT, TCEP, BME) were added as appropriate to the conditions being tested to create a stock enzyme solution


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with 1.08 µM PHPT1 and optionally 5 mM DTT or TCEP or 10 mM BME. Buffer, enzyme, and DiFMUP were added to each well to result in final concentrations of 109 nM PHPT1, 100 µM DiFMUP, and 500 µM DTT/TCEP or 1 mM BME. Fluorescence values were determined every 30 s for 30 min at 37 °C unless noted.

Michaelis–Menten Kinetics To a 4.93 µM aliquot of PHPT1, buffer (unoptimized, 50 mM HEPES, pH 8.0; optimized, 50 mM HEPES, pH 8.0, 10 mM NaCl, and 0.01% Brij 35) and reducing agent (100 mM DTT) were added as appropriate to create a stock PHPT1 solution with 1.08 µM PHPT1 and 5 mM DTT. Buffer, enzyme, and variable amounts of DiFMUP were added to each well to result in final concentrations of 109 nM PHPT1, 500 µM DTT, and 20-1000 µM (unoptimized) or 20-1200 µM (optimized) DiFMUP. Fluorescence values were determined every 30 s for 30 min at 37° C. Michaelis–Menten curve and kinetic parameters were calculated using GraphPad Prism.

Inhibitor Screening A stock PHPT1 solution was prepared as described in the Michaelis–Menten kinetics section using the optimized buffer. Buffer, enzyme, and variable amounts of inhibitor were added to each well to result in 121 nM PHPT1, 556 µM DTT, and 111-889 µM CuCl2, 2.22-1110 µM ZnCl2, or 222-1110 µM Na3VO4. The enzyme and inhibitor were then allowed to incubate at room temperature for 30 min. Following incubation, DiFMUP was added resulting in final concentrations of 109 nM PHPT1, 500 µM DTT, 300 µM DiFMUP, and 100-800 µM CuCl2, 21000 µM ZnCl2 or 200-1000 µM Na3VO4. Data points for Na3VO4 were collected in duplicate.


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Fluorescence values were determined every 30 s for 30 min at 37° C. IC50 curves and values were calculated using GraphPad Prism.

Example Protocol for PHPT1 Michaelis–Menten Kinetics Assay Using the Substrate DiFMUP With Optimized Buffer: Prior to assay: Lyophylized PHPT1 was dissolved in MilliQ water to a final concentration of 4.93 µM. Small (17.6 µL) aliquots were stored in a -80 °C freezer according to the supplier’s instructions. 1. Prepare the stock buffer by dissolving all components (HEPES, NaCl, and Brij 35) in MilliQ water and adjusting the pH to 8.0 to create a 50 mM HEPES, 10 mM NaCl, 0.01% Brij 35 stock buffer. 2. Prepare a 100 mM stock solution of DiFMUP by weighing the solid powder into a microcentrifuge tube and dissolving in an appropriate amount of DMSO. DiFMUP dilutions for use in the assay were prepared in DMSO as necessary from this stock. DiFMUP solutions can be reused for at least two months if stored at -20 °C. 3. Configure all computer software (plate layout, experiment duration, temperature settings, etc.) to minimize the amount of time between enzyme addition and fluorescence measurement. 4. Remove the PHPT1 aliquot(s) from a -80 °C freezer and immediately place on ice. 5. While enzyme is thawing, prepare a fresh stock solution of 100 mM DTT in a microcentrifuge tube using MilliQ water and place on ice to cool down. 6. Add 4 µL of the stock DTT and 58.4 µL buffer to an aliquot of PHPT1 to create a PHPT1 stock solution (1.08 mM PHPT1, 5 mM DTT) and mix well by pipetting up and down several times. When more than one aliquot was needed the enzyme was pooled together into a single


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Biochemistry

microcentrifuge tube and an appropriate amount of buffer and DTT were added. Immediately place the PHPT1 stock solution on ice to cool. 7. Add an appropriate volume of buffer (35-40 µL for our studies) to each well of the microplate, followed by addition of an appropriate amount of DiFMUP solution (up to 5 µL, for a total DMSO concentration of 10%). For wells with less than 5 µL DiFMUP solution, DMSO was added to each well such that the total volume of DiFMUP solution and DMSO was 5 µL. 8. Initiate the reaction by adding 5 µL of the PHPT1 stock solution to each well as quickly as possible followed by insertion of the microplate into the plate reader. 9. Shake the microplate for 5 s in the instrument prior to initial data collection to ensure proper mixing. Should the instrument not possess the ability to shake microplates it may be necessary to pipette each well up and down several times during step 8. 10. Record fluorescence values in triplicate using excitation/emission wavelengths of 350 nm / 455 nm every 30 s for 30 min at 37 °C. Use an emission cutoff filter of 455 nm to prevent lower wavelength light from interfering with data collection. 11. Determine initial enzyme rates using the linear portion of the RFU vs time curve.

Example Protocol for PHPT1 Inhibition Assay by ZnCl2 Using the Substrate DiFMUP: 1. Perform steps 1 to 6 from the example protocol for Michaelis–Menten kinetics assays. 2. Prepare a 3 mM DiFMUP dilution from the 100 mM stock. 3. Prepare a 10 mM stock solution of ZnCl2 and make 1mM and 100 µM dilutions for use in the assay.


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4. Add an appropriate volume of buffer (35-40 µL for our studies) to each well of the microplate, followed by addition of an appropriate amount of ZnCl2 (up to 5 µL) and 5 µL of the PHPT1 stock solution. 5. Incubate the microplate for 30 min at room temperature. 6. Initiate the reaction by adding 5 µL of 3 mM DiFMUP solution to each well as quickly as possible followed by insertion of the microplate into the plate reader. 7. Perform steps 9 to 11 from the example protocol for Michaelis–Menten kinetics assays to collect the data.

Results and Discussion Our initial assay conditions were based on those previously used to monitor PHPT1-mediated hydrolysis of pNPP.[21] Using 50 mM Tris·HCl pH 8.0, 500 µM DTT, 100 µM DiFMUP, and 109 nM PHPT1 at 37 °C gave a respectable amount of substrate hydrolysis (Figure 2) and was thus used as a starting point for assay optimization. A summary of the assay optimization results are shown in Table 1.

Figure 2. Representative RFU vs time curve triplicate showing the hydrolysis of DiFMUP using 50 mM Tris·HCl pH 8.0, 500 µM DTT, and 100 µM DiFMUP. ACS Paragon Plus Environment

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The first step in optimization was to identify which buffering agent to use. As pH 8.0 has been previously found to be optimal for PHPT1 activity[21] we investigated four buffers with buffering capacity at this pH: Tris·HCl, bicine, bis-Tris propane (BTP), and HEPES. PHPT1 had good activity in all four buffers, with BTP and HEPES giving slightly higher activity than Tris·HCl and bicine. All four buffers were included in our optimization experiments. To investigate the effect of temperature on PHPT1 activity, we performed the assay at both ambient temperature (~23 °C) and 37 °C. In all buffers, the enzyme was significantly more active at 37 °C. Certain phosphatases, such as the tyrosine phosphatase family of enzymes, require addition of a reducing agent for activation of the catalytic cysteine while other phosphatases, such as alkaline or acid phosphatase, do not possess a catalytic cysteine residue and are thus able to function in the absence of such compounds. While PHPT1 does not possess a catalytic cysteine, oxidation of an active site adjacent methionine by hydrogen peroxide has been reported.[29] Removal of DTT from our reaction resulted in a substantial loss of activity in both Tris·HCl and HEPES, a 40% reduction in bicine, and a 65% reduction in BTP. Neither 1 mM βmercaptoethanol (BME) nor 500 µM tris(2-carboxyethyl)phosphine (TCEP) enhanced the activity of PHPT1 as well as DTT. These data seem to conflict with a recent report that H2O2mediated oxidation of PHPT1 has little effect on the enzyme activity,[29] and merits further investigation. Many phosphatases, including SHP1[30] and PTP1[31], show a drastic change in activity upon changes in the ionic strength of the buffer, while others, such as human serum alkaline phosphatase, show little variation.[32] A high concentration of sodium chloride is often used to negate any variation in enzyme activity resulting from small, localized differences in ionic


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strength within the sample when low concentrations of salts are present. Upon addition of 100 mM NaCl to the assays, we observed a 30%-60% decrease in activity. Reducing the concentration to 50 mM showed activity reductions of 10%-25%, and a further reduction of NaCl to 10 mM nearly restored baseline enzyme activity (5-15% reduction). Metal ions play diverse roles in phosphatase activity. Some phosphatases require metal ions for proper function, such as alkaline phosphatase which contains both Zn2+ and Mg2+ ions in its active site,[33] while PP2C and PP2B respectively require Mg2+ and Ca2+ ions.[24] In other situations metal ions can act as inhibitors, as is the case with PTP1B which has been shown to be inhibited by Zn2+, Cu2+, and Cd2+.[34] Here, we examined the effect of Cu2+, Zn2+, Mg2+, and Ca2+ on PHPT1 activity. Addition of 1 mM ZnCl2 and CuCl2 to the buffer almost completely inhibited enzyme activity (0.5%-4.4% activity remaining). Addition of 1 mM MgCl2 showed a 10%-20% decrease in all buffers except HEPES, while 1 mM CaCl2 showed a 40% decrease in HEPES and a 15% decrease in Tris·HCl. Based on the observation that Zn2+ and Cu2+ inhibit PHPT1 activity, we investigated the effect of the metal chelator, EDTA. EDTA is often added to buffers to chelate any adventitious metal ion that might otherwise interfere with enzyme activity. However, we found that addition of 2 mM EDTA had a negative effect on PHPT1 activity. Aggregation and non-specific binding of proteins, substrates and inhibitors can be problematic for in vitro assays.[35] To alleviate this issue, detergents are often added to the reaction. We tested the addition of three commonly used detergents (Tween 20, Triton X-100, and Brij 35; 0.01% w/v) on PHPT1 activity and found that all three showed either increases or no change from base line activity in almost all conditions. We additionally tested bovine serum albumin (BSA) and observed minimal effects on PHPT1 activity with the exception of a 35% activity decrease in Tris·HCl.


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Biochemistry

Table 1. Impact of different buffer conditions on the activity of PHPT1α Tris·HCl

Bicine

bis-Tris Propane

HEPES

Reducing Agents None

15.9% ± 4.4%

58.1% ± 3.8%

36.4% ± 2.7%

17.5% ± 5.1%

1 mM BME

23.6% ± 2.8%

87.7% ± 4.1%

55.5% ± 2.2%

37.6% ± 1.2%

500 µM TCEP

57.0% ± 6.1%

109.1% ± 3.9%

41.1% ± 7.8%

56.2% ± 1.5%

56.4% ± 1.4%

42.6% ± 2.2%

49.6% ± 2.4%

44.0% ± 0.7%

100 mM

38.8% ± 1.6%

46.8% ± 3.1%

65.5% ± 5.8%

69.6% ± 0.8%

50 mM

73.4% ± 0.8%

78.7% ± 5.3%

85.0% ± 2.2%

90.8% ± 2.5%

10 mM

93.6% ± 2.3%

85.9% ± 5.8%

95.6% ± 1.3%

93.0% ± 1.9%

Mg2+

86.0% ± 4.1%

79.3% ± 6.2%

90.4% ± 4.2%

105.9% ± 4.9%

Zn2+

0.5% ± 0.1%

0.3% ± 0.2%

2.1% ± 0.2%

4.4% ± 1.0%

Cu2+

1.5% ± 1.0%

1.4% ± 1.1%

1.1% ± 1.6%

1.1% ± 1.3%

Ca2+

85.8% ± 5.0%

99.4% ± 6.6%

106.5% ± 5.6%

61.2% ± 4.5%

2 mM

46.8% ± 1.5%

69.1% ± 9.3%

67.9% ± 5.6%

59.6% ± 0.6%

500 µM

58.1% ± 2.3%

58.6% ± 7.3%

66.8% ± 4.0%

66.1% ± 0.9%

Tween 20

103.1% ± 3.7%

132.1% ± 6.4%

112.1% ± 2.9%

118.2% ± 0.8%

Triton X-100

117.5% ± 5.4%

104.4% ± 1.7%

122.2% ± 3.7%

97.1% ± 1.1%

Brij 35

111.2% ± 0.6%

119.1% ± 2.9%

112.4% ± 6.6%

122.9% ± 6.6%

0.5 mg/mL BSA

64.9% ± 3.7%

95.7% ± 0.8%

93.3% ± 11.8%

107.5% ± 0.5%

Temperature 23 °C NaCl

Metalsβ

EDTA

Detergentsγ

α

Data are reported as a percentage of activity compared to control samples (37 °C containing 50 mM of the appropriate buffer and no additives other than 500 µM DTT). β

Metals were tested at a concentration of 1 mM.

γ

Detergents were tested at a concentration of 0.01% w/v.


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Based on the results from the optimization experiments, we identified HEPES as the optimal buffer (50 mM at pH 8.0) with 10 mM NaCl, 0.01% w/v Brij 35 and 500 µM DTT as additives. The Michaelis–Menten curve shown in Figure 3 was obtained using these conditions and gave us a kcat of 0.39 ± 0.02 s-1, a Km of 220 ± 30 µM, and a kcat/Km of 1800 ± 200 M-1 s-1. Although these values are indistinguishable from those obtained with the unoptimized buffer containing only 50 mM HEPES at pH 8.0 with 500 µM DTT (kcat of 0.44 ± 0.03 s-1, a Km of 230 ± 40 µM, and a kcat/Km of 1900 ± 400 M-1 s-1), the addition of both NaCl and detergent present distinct advantages. Specifically, the addition of NaCl provides a constant ionic strength and the addition

Figure 3. Michaelis–Menten curve of PHPT1 using the substrate DiFMUP. of a detergent such as Brij 35 will minimize aggregation and non-specific binding effects in both the enzyme assays and in inhibitor screens. These data indicate that DiFMUP is a much better substrate for PHPT1 than pNPP, with reported kinetic constants kcat = 0.35 s-1, Km = 7.4 mM, and kcat/Km = 47 M-1 s-1.[21] Notably, DiFMUP is a much more sensitive substrate for PHPT1 as well. While 2.4 µM enzyme was required for the pNPP assay, only 109 nM was required in the DiFMUP assay.


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Biochemistry

Having observed a drastic decrease in enzyme activity in the presence of Zn2+ and Cu2+, we decided to further investigate these results using our newly validated assay (Figure 4). Interestingly, for ZnCl2 we observed a biphasic curve with IC50 values of 25 ± 1 µM and 490 ± 20 µM, indicating that there may be two zinc binding sites in the enzyme. In contrast, CuCl2 showed a more traditional sigmoidal curve, with an IC50 of 500 ± 20 µM. To our knowledge, no metal binding sites have been identified on PHPT1, so this new finding warrants further future investigation. We additionally tested the commonly used phosphatase inhibitor vanadate, and found that vanadate does not inhibit PHPT1 activity up to a concentration of 1 mM, consistent with previous literature.[25] (A)

(B)

Figure 4. IC50 curves of (A) ZnCl2 and (B) CuCl2 against PHPT1. IC50 values were found to be 25 ± 1 µM and 490 ± 20 µM for ZnCl2 and 500 ± 20 µM for CuCl2.

A lack of facile chemical probes to measure and manipulate PHPT1 activity has hampered progress in the field. Here, we have demonstrated that DiFMUP is an excellent substrate for monitoring PHPT1 activity, providing superior kinetic parameters and sensitivity when compared with the previously established pNPP assay. To demonstrate the utility of our new assay, we performed inhibitor testing on two promising compounds, ZnCl2 and CuCl2, and determined IC50 values for both. In the process of optimizing the DiFMUP assay, we discovered


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that PHPT1 shows higher activity in a reducing environment and may potentially bind up to two zinc ions, which inhibit enzyme activity. We expect that this new PHPT1 activity assay will decrease the barrier of entry to studying PHPT1 and will be an invaluable tool in discovering high potency inhibitors needed for biochemical studies. Author Information Corresponding Author: A. M. Barrios, 30 South 2000 East, Skaggs Research Building, University

of

Utah

College

of

Pharmacy,

Salt

Lake

City,

UT,

84112.

Email:

[email protected] ORCID: Brandon S. McCullough: 0000-0002-4496-0914 Funding: This work was supported by a Teva Pharmaceuticals Mark A. Goshko Memorial Grant award (56426-TEV) and an NSF award (CHE 1308766) to AMB. Notes: The authors declare no competing financial interest. Abbreviations PHP, protein histidine phosphatase; PHPT1, protein histidine phosphatase 1; DiFMUP, 6,8difluoromethylumbelliferyl phosphate; pHis, phosphorylated histidine; pTyr, phosphorylated tyrosine; PTP, protein tyrosine phosphatase; pNPP, para-nitrophenylphosphate; HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; DTT, dithiothreitol; RFU, relative fluorescence unit; BTP, bis-Tris propane; BME, β-mercaptoethanol; TCEP, tris(2-carboxyethyl)phosphine; SHP1, tyrosine-protein phosphatase non-receptor type 6; PTP1, Protein tyrosine phosphatase PTP1; PP2C, protein phosphatase 2C; PP2B, protein phosphatase 2B; PTP1B, Tyrosine-protein phosphatase non-receptor type 1; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum


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Biochemistry

albumin; DMSO, dimethyl sulfoxide; IC50, concentration of compound at which enzyme activity is reduced by 50%.

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For Table of Contents Use Only Title: A Facile, Fluorogenic Assay for Protein Histidine Phosphatase Activity Authors: Brandon S. McCullough and Amy M. Barrios


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Facile, Fluorogenic Assay for Protein Histidine Phosphatase Activity

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