CN112098448B - Nuclear magnetic resonance hydrogen spectrum-based LSD1 activity detection method and application thereof - Google Patents
Nuclear magnetic resonance hydrogen spectrum-based LSD1 activity detection method and application thereof Download PDFInfo
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Abstract
The invention provides an activity detection method of LSD1 based on nuclear magnetic resonance hydrogen spectrum and application thereof, wherein the method comprises the following steps: LSD1, FAD and lysine methylated histone and H were added to the reaction buffer2O, performing demethylation reaction; detecting the reacted system by using a nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance spectrogram; and calculating the activity value of the LSD1 histone lysine demethylase according to the nuclear magnetic resonance spectrogram. The invention is based on1The detection method of H-NMR can accurately reflect the interference effect of the inhibitor on the enzyme activity of LSD1, so the method can be effectively applied to the research of inhibitor screening. The invention does not need any sample preparation step after the LSD1 catalytic reaction is finished, and the catalytic reaction and the subsequent reaction can be finished in the nuclear magnetic resonance tube1H-NMR detection, whereby a plurality of samples can be efficiently obtained using an automatic sample changer1H-NMR spectrum.
Description
Technical Field
The invention relates to the technical field of enzyme activity detection, in particular to an LSD1 activity detection method based on nuclear magnetic resonance hydrogen spectrum and application thereof.
Background
Histone lysine methylation is an important epigenetic regulation mode, and is very important for regulating gene expression. Histone lysine methylation in cells is a reversible modification, catalyzed by histone lysine methyltransferase and demethylase, respectively, for methylation and demethylation. Histone lysine residues exist in three different methylation states, monomethylation (me1), dimethylation (me2) and trimethylation (me 3).
More than 20 histone lysine demethylases have been identified. LSD1(Lysine-specific demethylase 1) is the first identified histone Lysine demethylase, belonging to the amine oxidase family, responsible for catalyzing the demethylation of H3K4me 1/2. Numerous studies have shown that LSD1 is highly expressed in lung, breast, liver, colorectal and hematological disorders. The reduction of the expression of LSD1 in lung cancer and bladder cancer cells can inhibit the proliferation of tumor cells. LSD1 interacts with NMYC in neuroblastoma to co-inhibit expression of tumor suppressor Clusterin (CLU). The expression of LSD1 in germ cell tumor such as fetal tumor, embryonic carcinoma and seminoma is also obviously improved. These studies indicate that LSD1 plays an important role in tumor development and is a potential target for tumor therapy. The screening of the LSD1 specific inhibitor has important significance for the function research of the LSD1 and the tumor treatment research.
LSD1 removes the methyl group of histone H3K4me1/2 by Flavin Adenine Dinucleotide (FAD) dependent oxidation, and the cofactor FAD is converted to a reduced form of FADH2, which is then oxidized to hydrogen peroxide, ultimately producing formaldehyde and amine. Currently, various high-throughput screening (HTS) methods are applied to the detection of LSD1 enzyme activity and screening of inhibitors thereof, including antibody-based assays (antibody-based assays), enzyme-linked assays (enzyme-based assays), and mass spectrometry (mass spectrometry-based assays). Although antibody-based detection methods have high sensitivity, their quenching characteristics can affect accuracy, additional elution steps increase the time required for detection, and require large amounts of peptide fragment substrate. Mass spectrometry can directly detect the substrate of the demethylation reaction, has higher sensitivity, but needs to optimize the sample, remove additional components, and needs to collect samples at different times when researching the time dependence of the enzyme activity. A method for indirectly detecting an enzymatic reaction catalyzed by LSD1 by utilizing an enzyme-linked reaction of a demethylation reaction byproduct, such as oxidizing the byproduct Formaldehyde into formic acid by using Formaldehyde Dehydrogenase (FDH), simultaneously reducing NAD + into NADH, and then measuring the light absorption of NADH at 340nm by using a microplate reader, wherein the method needs to consider the interference of false positive or false negative caused by the coupling enzymatic reaction. Because of the limitations of all three methods, it is necessary to establish a simpler and more accurate method for directly determining the catalytic product of LSD1 and evaluating the enzymatic activity.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for detecting the activity of LSD1 based on nuclear magnetic resonance hydrogen spectrum and application thereof, and aims to solve the problems of complex operation and low accuracy of the existing detection method.
The technical scheme of the invention is as follows:
a method for detecting the activity of LSD1 histone lysine demethylase, which comprises the following steps:
adding LSD1 histone lysine demethylase, FAD and lysine methylated histone and H to reaction buffer2O, performing demethylation reaction;
detecting the reacted system by using a nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance spectrogram;
and calculating the activity value of the LSD1 histone lysine demethylase according to the nuclear magnetic resonance spectrogram.
Optionally, the reaction buffer comprises: 50mM Tris-HCl, 50mM KCl,5mM MgCl, pH 8.52；
The LSD1 histone lysine demethylase is added in an amount of 0.1 mug-10 mg, the FAD is added in an amount of 0.1 mug-1 mM, and the lysine methylated histone is added in an amount of 0.1 mug-10 mg.
Alternatively, the time for the demethylation reaction is from 1 to 24 hours.
Alternatively, the temperature of the demethylation reaction is in the range of 30-42 ℃.
Optionally, detecting the reacted system by using a one-dimensional hydrogen spectrum pulse sequence of the nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance spectrogram.
Alternatively, LSD1 histone lysine demethylase has an activity value of formaldehyde concentration (nmole/mL)/reaction time (min).
Optionally, the nmr spectrometer is an Avance series 600MHz superconducting fourier transform nmr spectrometer.
The invention discloses an application of an activity detection method of LSD1 histone lysine demethylase in screening of LSD1 histone lysine demethylase inhibitors.
Alternatively, the LSD1 histone lysine demethylase inhibitor is GSK2879552, having the structural formula shown below:
the invention relates to an application of an activity detection method of LSD1 histone lysine demethylase in screening of anticancer drugs.
Has the advantages that: the invention utilizes the change of formaldehyde which is a monitoring product of the specificity of a nuclear magnetic resonance one-dimensional hydrogen spectrum to evaluate the activity of Flavin Adenine Dinucleotide (FAD) -dependent histone lysine demethylase. The specific method can be applied to screening of anti-cancer drugs.
Drawings
FIG. 1 is a flow chart of a demethylation reaction catalyzed by LSD1 and specificity of formaldehyde in an embodiment of the present invention1H-NMR spectrum.
FIG. 2 (a) shows the demethylation catalyzed by LSD1 in an embodiment of the present invention1H-NMR spectrum, (b) is the result of protein immunoblotting detection after demethylation reaction.
FIG. 3 shows the demethylation catalyzed by LSD1 at various time points in an embodiment of the present invention1H-NMR spectrum.
FIG. 4 shows (a) a standard curve obtained by plotting concentration and signal intensity in a specific example of the present invention, and (b) a calculation formula of the activity of LSD 1.
FIG. 5 is a graph of the detection of inhibitory effects in a specific embodiment of the invention; wherein A is no inhibitor, B is LSD1 specific inhibitor GSK2879552(2 μ M), C is genistein, and D is zingerone.
FIG. 6 is a graph of the detection of inhibitory effects in a specific embodiment of the invention; wherein E is ipriflavone and F is isotretinoin.
FIG. 7 is a graph of the detection of inhibitory effects in a specific embodiment of the invention; wherein G is lactulose, H is apigenin, and I is cornusine.
Detailed Description
The invention provides an LSD1 activity detection method based on nuclear magnetic resonance hydrogen spectrum and application thereof, and the invention is further explained in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention aims to provide a method for evaluating the activity of the enzyme by monitoring the change of formaldehyde which is an indirect product of LSD1 in real time by utilizing nuclear magnetic resonance one-dimensional hydrogen spectrum selectivity. The method can be used for measuring enzyme activity and can be applied to the development of enzyme inhibitors. It can be seen from the structure that two hydrogen atoms in formaldehyde are covalently bonded to carbon, are in the same environment, are chemically equivalent, have the same chemical shift when measured by a nuclear magnetic resonance method, and show a single peak in a nuclear magnetic resonance one-dimensional hydrogen spectrum, and the peak appearance range is 8-9 ppm. While hydrogen covalently bonded to carbon in other organic compounds (including alkyl, benzyl, aromatic, etc.) typically peaks in the range of 0-8 ppm in the nmr spectrum; the hydrogen on oxygen/nitrogen (including alcohols, phenols, amines, etc.) typically peaks in the range of 9-13 ppm in the NMR spectrum. Therefore, the formaldehyde in the solution can be specifically detected according to the peak appearance characteristic of the nuclear magnetic resonance one-dimensional hydrogen spectrum.
Specifically, the invention provides a method for detecting the activity of LSD1 histone lysine demethylase, which comprises the following steps:
adding LSD1 histone lysine demethylase, FAD and lysine methylated histone and H to reaction buffer2O, performing demethylation reaction;
detecting the reacted system by using a nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance spectrogram;
and calculating the activity value of the LSD1 histone lysine demethylase according to the nuclear magnetic resonance spectrogram.
The invention firstly adds LSD1, FAD and histone purified from cells and D in reaction buffer solution2And O, carrying out demethylation reaction. And then, detecting by using a one-dimensional hydrogen spectrum pulse sequence of the nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance spectrogram. H3K4me2/1 demethylase LSD1 is a member of the amine oxidase family, which is known to activateA Flavin Adenine Dinucleotide (FAD) dependent oxidation reaction removes the methyl group of H3K4me2/1 of histone. One methyl group of H3K4me2/1 loses proton to generate imine intermediate product, the intermediate product generates amine group and formaldehyde after being added with water, FAD obtains proton from methylated histone lysine to generate FADH2, and then FAD and H are oxidized2O2(refer to fig. 1). Two hydrogen atoms in formaldehyde are covalently bonded to carbon in the same environment, are chemically equivalent, have the same chemical shift as measured by nuclear magnetic resonance1H-NMR is usually unimodal, with peak-off ranging from 8 to 9 ppm. And the hydrogen (including alkyl, benzyl, aromatic, etc.) on carbon in other organic compounds1The peak range in H-NMR is usually 0 to 8 ppm; on oxygen/nitrogen with hydrogen (including alcohols, phenols, amines, etc.)1The peak range in H-NMR is usually 9 to 13 ppm. Thus, according to1The peak characteristics of H-NMR can specifically detect formaldehyde in the solution, and further determine the demethylation reaction mediated by LSD 1. The method can be used for measuring enzyme activity and screening enzyme inhibitors.
In one embodiment, the reaction buffer comprises: 50mM Tris-HCl, 50mM KCl,5mM MgCl, pH 8.52；
The LSD1 histone lysine demethylase is added in an amount of 0.1 mug-10 mg, the FAD is added in an amount of 0.1 mug-1 mM, and the lysine methylated histone is added in an amount of 0.1 mug-10 mg.
In one embodiment, the time for the demethylation reaction is from 1 to 24 hours.
In one embodiment, the temperature of the demethylation reaction is in the range of 30-42 ℃.
The invention discloses an application of an activity detection method of LSD1 histone lysine demethylase in screening of LSD1 histone lysine demethylase inhibitors.
Alternatively, the LSD1 histone lysine demethylase inhibitor is GSK2879552, having the structural formula shown below:
the invention relates to an application of an activity detection method of LSD1 histone lysine demethylase in screening of anticancer drugs.
The invention is further illustrated by the following specific examples.
Examples
This example describes a method for selectively detecting formaldehyde using a nuclear magnetic resonance spectrometer.
1) HeLa cells were purchased from American Type Culture Collection (ATCC, Manassas, Va.) and grown in DMEM (Dulbecco's Modified Eagle Medium) Medium supplemented with 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS) and penicillin/streptomycin (100U/ml). HeLa cells were cultured at 37 ℃ and 5% CO2In a moist incubator.
2) Extracting histone: in lysis buffer (10mM Tris-Cl pH 8.0, 1mM KCl, 1.5mM MgCl)21mM DTT, 1% cocktail) for 30min and centrifuged to remove the supernatant. Resuspend the pellet with 0.2M sulfuric acid and disk lyse at 4 ℃ for 30min or overnight. The supernatant was collected by centrifugation and TCA was added dropwise to a final concentration of 33% (v/v) and allowed to stand on ice for 30 min. Centrifuging to remove supernatant, washing the precipitate with precooled acetone, and drying the precipitate at room temperature. Addition of H2And O, dissolving the precipitate at room temperature. In demethylation reaction buffer (50mM Tris-HCl pH 8.5,50mM KCl,5mM MgCl)2) Adding LSD1 protein 10 μ g, FAD 20 μ M and purified histone, or inhibitor. The mixture was incubated overnight at 37 ℃ before NMR measurement. Before the NMR measurement, 5% (v/v) of D was added to the sample2O, for determining the lock signal (lock signal detection).
3) The instrument used in this study was a Bruker corporation (switzerland) AVANCE series 600MHz superconducting fourier transform nmr spectrometer equipped with a cryoprobe. The sample tube is inserted into the rotor and the height of the sample tube is controlled by the depth-setting measuring cylinder. And putting the rotor into a sample injector in sequence, and setting acquisition parameters. The acquisition parameters of the one-dimensional nuclear magnetic resonance hydrogen spectrum are TD 98358, number of scan 64, Spectral width 20.48 and Receiver gain 16, and a pre-saturated noesygpr 1d pulse sequence is used for sampling. LSD1 enzymatic reaction time-dependent experiments were performed with a sweep temperature of 310K and a pre-saturation pulse sequence for 8h of sampling. After the test is finished, Fourier transform, phase correction, baseline correction and the like are carried out on the spectrogram, and a single formaldehyde peak appears in a 8.32ppm potential.
FIG. 1 shows the demethylation reaction catalyzed by LSD1 and the specificity of formaldehyde1H-NMR spectrum. H3K4me2/1 demethylase LSD1 is a member of the amine oxidase family, which removes the methyl group of H3K4me2/1 by Flavin Adenine Dinucleotide (FAD) -dependent oxidation. One methyl group of H3K4me2/1 loses proton to generate imine intermediate product, the intermediate product generates amine group and formaldehyde after being added with water, FAD obtains proton from methylated histone lysine to generate FADH2, and then FAD and H are oxidized2O2. Process for preparing formaldehyde1The H-NMR spectrum showed a single peak around 8.32 ppm.
FIG. 2 is a diagram of a LSD1 catalyzed demethylation reaction1H-NMR spectrum. FAD, histone purified from tumor cells and LSD1 are added into LSD1 reaction buffer solution, and incubation is carried out for 12h at 37 DEG C1H-NMR detection. No signal peak around 8.32ppm was detected in the control reaction without LSD1, indicating that the aldehyde proton signal was absent, and the catalytic reaction system with LSD1 showed a distinct peak around 8.32ppm, i.e., a distinct aldehyde proton signal was present, as shown in FIG. 2 (a). The results show that1The product formaldehyde of the demethylation reaction catalyzed by LSD1 was effectively detected by H-NMR.1After completion of the H-NMR detection, the methylation modification of H3K4 in the above sample was detected by a Western blot assay. As shown in fig. 2 (b), LSD1 acted to significantly reduce the levels of histones H3K4me1 and H3K4me2, and the level of protein acetylation as a negative reference was not significantly changed, compared to the control group to which LSD1 was not added, indicating that the enzymatic reaction catalyzed by LSD1 proceeded normally. The above results show that1The H-NMR detection method has high specificity to the catalytic reaction of the LSD1, a specific peak of the product formaldehyde can be detected under the action of the LSD1, and a specific peak of a formaldehyde proton cannot be generated under the condition of the loss of the action of the LSD 1.
Fig. 3 is a graph of LSD1 reaction product detection over time. The detection method based on nuclear magnetic resonance can not change the characteristics and the state of the sample, and can ensure the integrity of the sample, thereby being used for real-time monitoring of chemical reaction. While other analytical methods involve a certain destructive sample processing step prior to detection, if the time dependence of the enzymatic activity is to be detected, samples are collected which are subjected to different times of reaction. To verify the feasibility of the method established in this study for real-time monitoring of the enzymatic reaction, the same LSD1 catalytic reaction was monitored continuously for 8h in real-time, FIG. 3 shows the reaction at 6 different time points1The H-NMR spectrum shows that the height of the aldehyde proton signal peak is gradually increased along with the time, which shows that the generation of formaldehyde is continuously increased along with the prolonging of the reaction time. The results show that the detection method established in the research can be used for detecting the demethylation reaction catalyzed by LSD1 in real time. The enzyme activity is usually reflected in the enzyme kinetics by the conversion rate of the enzyme-catalyzed reaction under certain conditions (expressed as "nmole/min/ml" ═ milliunit/ml). To reflect the kinetic characteristics of the LSD 1-mediated enzymatic reaction, absolute quantification of the product was first required. First, 5 kinds of formaldehyde solutions with different concentrations are prepared and measured1An H-NMR spectrum, and then a standard curve (shown in FIG. 4 (a)) was plotted based on the concentration and the signal intensity, and the relative amount of the product was calculated from the results of the nuclear magnetic resonance measurement of the reaction product. FIG. 4 (b) shows the calculation method of LSD1 activity, and the LSD1 activity in this experiment was 0.52 milliunit/ml.
FIGS. 5-7 are inhibitory effect detection graphs. A is no inhibitor added; b is LSD1 specific inhibitor GSK2879552(2 μ M); c to I are 7 different cell membrane permeable natural compound treatments (10 μ M), respectively: genistein (C), zingerone (D), ipriflavone (E), isotretinoin (F), lactulose (G), apigenin (H), and cornusine (I). The enzyme activity detection is a key link for screening the inhibitor. Then is based on1H-NMR LSD1 enzyme activity assays the feasibility of application in LSD1 inhibitor screening was examined. First use1The H-NMR method examined the effect of known LSD1 inhibitors on the enzymatic reaction. As shown in the figure, the reaction product was enzymatically reacted with LSD1When the LSD1 specific inhibitor GSK2879552 (B in figure 5) is added into the system, the 1H-NMR signal intensity of aldehyde protons is obviously weakened compared with that of a control group (A in figure 5), which shows that the generation of formaldehyde is reduced and the enzymatic reaction is inhibited. The detection method established in the research can effectively reflect the inhibition effect of the inhibitor on the enzyme activity of the LSD 1. The present study also examined the effect of 7 natural compounds with cell membrane permeability on the LSD1 enzymatic reaction, genistein (C in fig. 5), zingerone (D in fig. 5), ipriflavone (E in fig. 6), isotretinoin (F in fig. 6), lactulose (G in fig. 7), apigenin (H in fig. 7) and cornisine (I in fig. 7). As shown, the detected signal peak intensities after 7 compounds treatment were less altered compared to the control group, indicating that these compounds act with less interference with LSD1 enzyme activity. The results of these experiments are based on1The detection method of H-NMR can accurately reflect the interference effect of the inhibitor on the enzyme activity of LSD1, so the method can be applied to the research of inhibitor screening. Moreover, the research does not need any sample preparation step after the LSD1 catalytic reaction is finished, and the catalytic reaction and the subsequent reaction can be finished in the nuclear magnetic resonance tube1H-NMR detection, whereby a plurality of samples can be efficiently obtained using an automatic sample changer1H-NMR spectrum.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (8)
1. A method for detecting the activity of LSD1 histone lysine demethylase, which is characterized by comprising the following steps:
adding LSD1 histone lysine demethylase, FAD and lysine methylated histone and H to reaction buffer2O, performing demethylation reaction;
detecting the reacted system by using a nuclear magnetic resonance spectrometer to obtain a nuclear magnetic resonance spectrogram;
calculating the activity value of LSD1 histone lysine demethylase according to the nuclear magnetic resonance spectrogram;
according to1And (3) specifically detecting formaldehyde in the reacted system by using the peak characteristics of H-NMR (hydrogen-nuclear magnetic resonance), and further determining the demethylation reaction mediated by LSD1, so as to obtain the activity value of LSD1 histone lysine demethylase, namely formaldehyde concentration/reaction time.
2. The method for detecting the activity of LSD1 histone lysine demethylase of claim 1, wherein said reaction buffer comprises: 50mM Tris-HCl, 50mM KCl,5mM MgCl, pH 8.52；
The LSD1 histone lysine demethylase is added in an amount of 0.1 mug-10 mg, the FAD is added in an amount of 0.1 mug-1 mM, and the lysine methylated histone is added in an amount of 0.1 mug-10 mg.
3. The method for detecting the activity of LSD1 histone lysine demethylase of claim 1, wherein the time for the demethylation reaction is 1-24 h.
4. The method for detecting the activity of LSD1 histone lysine demethylase of claim 1, wherein the temperature of the demethylation reaction is 30-42 ℃.
5. The method for detecting the activity of the LSD1 histone lysine demethylase of claim 1, wherein the nmr spectrometer is an Avance series 600MHz superconducting fourier transform nmr spectrometer.
6. The use of the method of detecting the activity of LSD1 histone lysine demethylases of any of claims 1-5 in the screening of LSD1 histone lysine demethylase inhibitors.
7. The use of claim 6, wherein said LSD1 histone lysine demethylase inhibitor is GSK2879552, having the formula:
8. use of the LSD1 histone lysine demethylase activity assay method of any one of claims 1-5 in anticancer drug screening.
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| WO2011096196A1 (en) * | 2010-02-02 | 2011-08-11 | Oncotherapy Science, Inc. | Lsd1 for target genes of cancer therapy and diagnosis |
| CN109825551A (en) * | 2019-02-21 | 2019-05-31 | 深圳大学 | A method of evaluation istone lysine demethyl transferase active |
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| CA2591717A1 (en) * | 2004-12-16 | 2006-07-06 | President And Fellows Of Harvard College | Histone demethylation mediated by the nuclear amine oxidase homolog lsd1 |
| US8367366B2 (en) * | 2010-12-04 | 2013-02-05 | The Board Of Trustees Of The University Of Arkansas | Methods and kits for quantitative methyltransferase and demethylase measurements |
| US9273343B2 (en) * | 2011-09-02 | 2016-03-01 | Promega Corporation | Compounds and methods for assaying redox state of metabolically active cells and methods for measuring NAD(P)/NAD(P)H |
| US9556170B2 (en) * | 2013-08-30 | 2017-01-31 | University Of Utah Research Foundation | Substituted-1H-benzo[d]imidazole series compounds as lysine-specific demethylase 1 (LSD1) inhibitors |
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| WO2011096196A1 (en) * | 2010-02-02 | 2011-08-11 | Oncotherapy Science, Inc. | Lsd1 for target genes of cancer therapy and diagnosis |
| CN109825551A (en) * | 2019-02-21 | 2019-05-31 | 深圳大学 | A method of evaluation istone lysine demethyl transferase active |
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| Title |
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| Synthesis, LSD1 Inhibitory Activity, and LSD1 Binding Model of Optically Pure Lysine-PCPA Conjugates;Yukihiro Itoh et al.;《Computational and Structural Biotechnology Journal》;20140215;第9卷(第4期);1-9 * |
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