CN102140494B

CN102140494B - Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme

Info

Publication number: CN102140494B
Application number: CN201010103035.XA
Authority: CN
Inventors: 赵英明
Original assignee: PTM Biolabs Inc
Current assignee: Hangzhou Jingjie Biotechnology Co.,Ltd.
Priority date: 2010-01-29
Filing date: 2010-01-29
Publication date: 2015-07-08
Anticipated expiration: 2030-01-29
Also published as: CN102140494A

Abstract

The invention relates to a method for screening and testing activity of a lysine propionylation removal enzyme and a butyrylation removal enzyme. The basis thought of the method is that: lysine propionylation or butyrylation modification degree of a substrate can be reflected by the sensitivity of the substrate to the catalysis removal enzyme. The method comprises the following steps of: a, lysine propionylation substrate polypeptide marked with releasable signal substances; b, the lysine propionylation removal enzyme; and c, a detection method for distinguishing the modification lysine propionylation polypeptide from corresponding non-modification polypeptide. The method can be used for screening modification removal enzymes and testing the activity of the enzymes, and also can screen substrates influencing the activity of the enzymes to be further used in quick diagnosis of several diseases.

Description

Methionin removes propionyl-enzyme and removes screening and the activity determination method of Butyrylation enzyme

Technical field

The present invention relates to a kind of Methionin that screens remove propionyl-enzyme and remove Butyrylation enzyme and measure Methionin to go propionyl-enzyme and the method for going Butyrylation enzymic activity.Removed propionyl-enzyme by Methionin especially by mensuration Boc-Lys (Prop)-AMC (Boc-Lys(Prop)-AMC) and Boc-Lys (Buty)-AMC (Boc-Lys(Buty)-AMC) and go the fluorescence intensity after the catalysis of Butyrylation enzyme, and detect the specific Methionin of confirmation in conjunction with Mass Spectrometric Identification, biochemical analysis and cytology and remove propionyl-enzyme and go the mensuration of Butyrylation enzyme and enzymic activity.

Background technology

Nucleosome is chromatinic basic repeating unit, by eight aggressiveness core histones and around the DNA of long 147 base pairs of pact form.Nucleosome can be folded into more senior structure further ⁽¹⁾.The dynamic change of chromatin Structure have adjusted the plasticity-of DNA, and then have impact on the biological procedures based on DNA profiling, as transcribed, copying and recombinating.

In the past in decades, the posttranslational modification (PTMs) of large quantity research display histone serves vital effect by regulating chromatinic structure and function in cell processes ^(2-5).Histone, especially its N-terminal afterbody, modify mode after at least there are 11 kinds of protein translations; comprise acetylize; ADP-ribosylation, citrullinated, formylation; methylate; phosphorylation, proline(Pro) isomerization, SUMOization; ubiquitination, and lysine side-chain Butyrylation and propionating (rear both are designated as K respectively ^propand K ^buty).Histone posttranslational modification is considered to have adjusted chromatinic structure, and then by two kinds of machine-processed functionatings ^(2-3,5).First, directly regulate chromatin packaging process by the electric charge or nucleosome interphase interaction changing histone, and then have adjusted the binding ability of chromatinic higher structure and DBP.Secondly, histone posttranslational modification can recruit the interaction protein of albumen and these albumen be combined with modified specificity, or stops some protein binding on chromatin.

Istone lysine acetylize is identified before 40 years.This modification is a kind of reversible posttranslational modification mode of altitude mixture control, and its decorating state is by the enzyme of two groups of opposing activity each other---histone acetyltransferase (HATs or KATs) and histon deacetylase (HDAC) (HDACs) regulate and control ^(6-7).Istone lysine acetylize by and the positive charge of lysine side-chain regulate chromatinic plasticity-, and create one and can recruit " berthing " site with bromo domain protein ^(6,8).Istone lysine is crossed acetylize and is considered to express relevant to active gene.In recent years, the association study of mass spectroscopy and genome dimension indicates forcefully between lysine acetylation with other histone posttranslational modification and there is associating of long distance.Such as, histone H 3 crosses tri-methylated (H3K4me3) significant correlation of acetylize and histone H 4 the 4th Methionin, and have adjusted active gene and transcribe ^(6,9-11).Except histone, lysine acetylation is also present in the different zones of cell, comprises nucleoprotein, cytoplasmic protein and mitochondrial protein, and these are modified with old and feeble and various diseases is closely related ^(12-14).

Recently, we identify two kinds of structures posttranslational modification mode similar to lysine acetylation by mass spectroscopy on histone H 4--and Methionin is propionating with Methionin Butyrylation ⁽¹⁵⁾.Initial qualification is confirmed by the second mass analysis (MS/MS or tandem mass spectrum) to improvement on synthesis, external enzymatic reaction, HPLC co-elute and the western blot analysis modified ^(15-16).Our nearest research confirms that these two kinds of posttranslational modifications are not merely present in histone, some nonhistones as p53, p300 and CBP in also exist ⁽¹⁷⁾.The regulatory enzyme of research display lysine acetylation, Sirt1, p300 and CBP also can regulate that p53's is propionating ⁽¹⁷⁾.The Methionin that Escalante-Semerena and colleague thereof report the propionyl coenzyme A synthase PrpE of Salmonella enteritidis is propionating, and shows this reversibly modified effect serving regulatory enzyme activity ⁽¹⁸⁾.

, between propionating and Butyrylation, there is similarity at biochemistry level in lysine acetylation.Such as, modify for these three kinds and all use three kinds of different high energy CoA molecule, i.e. acetyl-CoA, propionyl coenzyme A and butyryl coenzyme A, as the substrate of modification reaction.Previous structural research display yeast histone acetyltransferase Hat1 has a spatially enough large acetyl-CoA binding domain, and spatially this binding domain can accept larger acetyl-CoA analogue ⁽¹⁹⁾.Therefore, butyryl coenzyme A also may utilize by some lysine acetyltransferase (KATs) with the propionating reaction of catalysis Methionin.Really, research shows that acetyl transferase p300/CBP can utilize the isotope-labeled butyryl coenzyme A of catalysis to transfer to istone lysine ⁽¹⁵⁾.In addition, some deacetylases, as Sirt1, catalysis can both go propionating reaction with external in vivo ⁽¹⁷⁾.Utilize a series of lysine acetylation similar polypeptide as substrate, Smith and Denu finds that some deacetylases have the measurable enzymic activity to external Methionin Butyrylation ⁽²⁰⁾.These limited researchs implied Methionin deacetylation and go propionating and go Butyrylation react between share the possibility of some katalaze enzymes.But, go propionating to concrete Methionin and go the screening of Butyrylation enzyme and the mensuration of enzymic activity but to know little about it, thus directly affects the biological function research of these two kinds being modified to mode.

Summary of the invention

Based on this, in conjunction with biochemical analysis, the present invention proposes a kind of method of screening Methionin and removing propionyl-enzyme and going Butyrylation enzyme and enzyme assay.

The inventive method comprises three key steps: first, Boc-Lys (Prop)-AMC and Boc-Lys (the Buty)-AMC of synthesis of high purity, secondly, Methionin to be screened is removed propionyl-enzyme or goes Butyrylation enzyme to react with Boc-Lys (Prop)-AMC or Boc-Lys (Buty)-AMC in specific reaction system, specific Methionin removes propionyl-enzyme or goes Butyrylation enzyme can excise propionyl (Prop) or the butyryl radicals (Buty) of lysine side-chain, and then AMC group is discharged under the splitting action of pancreatin, the Methionin special by the fluorescence intensity preliminary screening of the AMC group measuring release removes propionyl-enzyme or removes Butyrylation enzyme, and the activity intensity of enzyme is reflected by the fluorescence intensity measured, finally, determine that the Methionin of candidate removes propionyl-enzyme or goes Butyrylation enzyme to be that target Methionin removes propionyl-enzyme or removes Butyrylation enzyme through a series of mass spectroscopy and biochemical analysis.

Screening and the determination of activity of Butyrylation enzyme is gone for Methionin, by measuring the fluorescence intensity of different enzymes to be screened to the AMC group discharged after Boc-Lys (Buty)-AMC catalysis, first the present invention confirms HDAC3 and HDAC6 for candidate's Methionin and removes Butyrylation enzyme; These two kinds of enzymes have external Methionin and remove Butyrylation enzyme to go the second mass analysis after Butyrylation and external biochemical test to prove by the Methionin Butyrylation peptide section of HDAC3 and HDAC6 to synthesis subsequently; Finally, confirm that HDAC3 and HDAC6 is that Methionin removes Butyrylation enzyme by the biochemical test in body and cytologic experiment.

Except going propionyl-enzyme and screening and the enzyme assay of removing Butyrylation enzyme to Methionin, ultimate principle of the present invention and experimental technique can be used for conventional enzyme activity assay and follow-up drug screening equally.

In addition, the invention allows for a kind of test kit going propionyl-enzyme and enzyme assay for screening Methionin, this test kit comprises:

A. being marked with can the propionating substrate polypeptide of Methionin of releasor material;

B. the peptase of its digestion activity can be changed according to the propionating level of substrate polypeptide.

Another kind of the present invention is for the identification of the test kit that can suppress or strengthen the compound going propionyl-enzyme activity, and it comprises:

B. Methionin removes propionyl-enzyme;

C. the peptase of its digestion activity can be changed according to the propionating of substrate polypeptide.

Another kind of the present invention comprises for the test kit screening Methionin propionyl-enzyme and enzyme assay: a. is marked with can the substrate polypeptide of releasor material; B. the peptase of its digestion activity can be changed according to the propionating level of substrate polypeptide.

Wherein, above-mentioned arbitrary detection kit, described can releasor material be a kind of dyestuff.Described dyestuff is a kind of fluorescent substance, and the signal of release is fluorescent signal.

Described peptase comes from Lysylendopeptidase, endoproteinase Lys-C, plasmin, calpain, metalloendopeptidase and Armillaria mellea protease.

Described propionating substrate polypeptide contains a propionating lysine residue.

Described peptase is lysylendopeptidase.

The propionating substrate polypeptide of described Methionin be through 7-amino-4-methyl-coumarin (AMC) or para-nitroaniline (p-Na) fluorophore mark propionating/Butyrylation Methionin, and fluorophore is connected with Methionin peptide bond.

Described substrate polypeptide contains a lysine residue.

Accompanying drawing explanation

Fig. 1. istone lysine Butyrylation is the posttranslational modification of evolution conservative in eukaryotic cell.(A) acetyl-CoA and butyryl coenzyme A structure, and lysine acetylation and Butyrylation enzymatic reaction pictorial image.(B) the Methionin Butyrylation of histone in five kinds of eukaryotic cells.The immune-blotting method of core histones Methionin Butyrylation (on) and equivalent core histones Coomassie blue stain (under).(C) the Methionin Butyrylation Immunofluorescence test in nucleus and karyomit(e).(D) core istone lysine acetylize in five kinds of eukaryotic cells of mass spectroscopy gained, propionating and Butyrylation site schematic diagram.Ac: ethanoyl; Pr: propionyl; Bu: butyryl radicals; O: do not detect.

Fig. 2. protein lysine goes preliminary screening and the determination of activity of Butyrylation enzyme.(A) hdac inhibitor affects istone lysine Butyrylation state.HeLa cell uses I respectively, and the process of II, III, IV class hdac inhibitor is after 6 hours, immunoblotting assay core histones Methionin Butyrylation level.Hdac inhibitor is 2 μMs of trichostatin A (TSA) respectively; 50mM Sodium propanecarboxylate; 50 μMs of suberoyl anilide hydroxamic acid (SAHA); 10ng/ml depsipeptide (FK288); 30mM nicotinamide and 200 μM sirtinol.(B) synthesis of Boc-Lys (Buty)-AMC and molecular structure (synthetic method is shown in embodiment).(C) Methionin goes the screening of Butyrylation enzyme and determination of activity (screen and see embodiment with activity determination method).

Fig. 3 .HDCA3 catalysed in vitro Methionin removes Butyrylation.(A-C) Methionin of HDAC3 catalysis histone H 3 polypeptide removes Butyrylation (peptide sequence: CSTGGK14 ^butyaPRK18 ^butyqLATK23 ^butyaARK).The Butyrylation histone H 3 polypeptide (A) of A-C display synthesis, or hatches (B) with HDAC3, or adds 2 μMs of TSA with HDAC3 and hatch the MALDI-TOF spectrogram after (C).The molecular weight of each main peak all marks, and the peptide spectrum peak arrow corresponding to removal one, two or three butyryl group products marks.(D-F) Methionin of HDAC3 catalysis histone H 4 polypeptide removes Butyrylation.Experimentation is identical with (A-C), except the Butyrylation histone H 4 polypeptide using synthesis is reaction substrate (peptide sequence: CSGRGK5 ^butygGK8 ^butygLGK12 ^butygGAK).(I) HDAC3 catalysed in vitro core histones removes Butyrylation.After hatching 3 hours with the restructuring HDAC3 adding or do not add TSA (10 μMs) at 37 DEG C from the core histones of HeLa cell, immunoblotting assay core histones Methionin Butyrylation level.100ng recombinates HDAC3 and 50pmol H3 polypeptide for above-mentioned analysis.

In Fig. 4 .HDAC3 body, catalysis istone lysine removes Butyrylation.(A) HeLa cell transfecting expresses Flag-HDAC3, after transfection 36 hours, immunoblotting assay core histones Methionin Butyrylation level.(B) identical with (A), except transfection expression enzyme deactivation mutant Flag-HDAC3S424A.(C) in core, HDAC3 promotes that istone lysine goes Butyrylation level.HeLa cell transfecting expresses Flag-HDAC3 and Flag-HDAC3S424A mutant, and transfection is after 36 hours, the Immunofluorescence test of core histones H4K5 Butyrylation level.

Fig. 5 .HDAC6 vivo and vitro catalysis istone lysine Butyrylation.(A) external HDAC6 catalytic core DNA methylase inhibitor.The HeLa cell core histone extracted and HDAC6 (wild-type or enzyme deactivation mutant), add or do not add TSA (2 μMs), hatch 2 hours at 37 DEG C, immunoblotting assay core histones Methionin Butyrylation level.(B) in HDAC6 body, catalytic core istone lysine removes Butyrylation.HeLa cell transfecting expresses Flag-HDAC6, after transfection 36 hours, immunoblotting assay core histones Methionin Butyrylation level.(C) HDAC6 enzyme deactivation mutant can not remove Butyrylation by catalysis istone lysine in vivo.All experimentations are identical with (B), except employing Flag-HDAC6H216/611A enzyme deletion mutant alive.(D) Leptomycin B (LMB) facilitates HDAC6 and removes Butyrylation to core histones Methionin.HeLa cell transfecting expresses Flag-HDAC6 or Flag-HDAC6H216/611A mutant.Transfection is after 36 hours, and leptomycin B (200nm) processes cell 3 hours, immunoblotting assay core histones Methionin Butyrylation level.(E-F) Leptomycin B facilitates HDAC6 and removes Butyrylation to histone H 4 K5 and K12.All experimentations are identical with (D), except the Methionin Butyrylation level using sequence-specific Methionin Butyrylation antibody immunoblotting (E) or immunofluorescence (F) to detect histone H 4 specific site.

Embodiment

Plasmid, plasmid and corresponding enzyme deactivation mutant plasmid HDAC3S424A and HDAC6H216/611A thereof of antibody and other reagent coding HDAC3, HDAC6cDNA are so kind as to give by the Edward Seto of H.Lee Moffitt Cancer center.Plasmid information is reported before seeing ^(21-22).The antibody of fight against press-ganging acyl Methionin antibody and anti-histone locus specificity butyryl Methionin has the present inventor oneself to prepare.Other antibody used has: anti-acetylated lysine antibody (ImmunoChemPharmaceuticals, Burnaby, British Columbia, Canada); Anti-histone H3 antibody, anti-histone H4 antibody (Abcam, Cambridge, UK); Anti-HA antibody (Roche Diagnostics, Indianapolis, IN); Anti-Flag antibody (Sigma-Aldrich, St.Louis, MO).Restructuring HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10 and HDAC11 purchase to BPSBioscience Inc. (BPS Bioscience Inc., San Diego, CA).Butyryl coenzyme A, acetyl-CoA, Leptomycin B and acetic acid are purchased from Sigma-Aldrich Co. (St.Louis, MO).HPLC level acetonitrile, water and ethanol are purchased from EMD Chemicals Inc. (Gibbstown, NJ).Polypeptide with one or several Butyrylation Methionin synthesizes (GL Biochem Ltd., Shanghai, P.R.China) by gill biochemistry.

The synthesis of Boc-Lys (Buty)-AMC is at 23 DEG C, to containing Boc-Lys-AMC (50mg, 0.124mmol, 1.0 equivalents) and triethylamine (35 μ L, 0.248mmol, 2.0 equivalents) methylene dichloride (1mL) solution in add butyryl chloride (16 μ L, 0.149mmol, 1.2 equivalents).Stirring is spent the night.Reaction solution methylene dichloride (1mL) dilution, then uses saturated sodium bicarbonate solution (2mL × 2) and water (2mL) washing.Organic phase with sodium sulfate is dry, filters, and obtains Boc-Lys (Buty)-AMC after concentrated.

Histone measures the method for fluorescence excitation after going the confirmation histone of the screening of Butyrylation enzyme and Enzyme assay and enzyme to go the screening of Butyrylation enzyme and Enzyme assay to adopt modification group catalyzed removal.First, contriver has been developed a kind of Methionin based on fluorescent strength determining and has been gone the screening of Butyrylation enzyme and the method for enzyme assay.The method is similar with the method for measuring Methionin deacetylase ^(23-24).Boc-Lys (Buty)-AMC and Boc-Lys (Prop)-AMC the present inventor synthesized is used for fluorimetric substrate.For screening and mensuration Methionin go Butyrylation enzyme and enzymic activity, subsequently by restructuring deacetylase (2ng or 200ng) to be screened and 85 μ l ddH ₂o, 10 μ l 10X HDAC reaction buffer (25mM Tris-Cl (pH 8.0), 137mM NaCl, 2.7mM KCl, 1mM MgCl ₂) and 5 μ l 4mM Boc-Lys (Buty)-AMC mix.Mixture was 37 DEG C of reactions 30 minutes.In reaction system, add 10 μ l contriver homemade Lysine Developer termination reaction, and hatch 30 minutes at 37 DEG C.The fluorescence intensity produced after pancreatin cracking is measured at Ex=350-380nm and Em=440-460nm place by SPECTRAFluor Plus fluorescence microplate reading apparatus (MTX Lab Systems Inc., Vienna, VA).Only have and the enzyme of catalytic elimination butyryl radicals or propionyl can could produce fluorescent signal, and fluorescence intensity represents this enzyme and goes Methionin Butyrylation or propionating enzymic activity.

The Methionin that istone lysine goes the mass spectroscopy of Butyrylation enzyme preliminary screening to be gone out go the Methionin Butyrylation polypeptide of Butyrylation enzyme and synthesis in 10 μ l HDAC reaction buffers 37 DEG C reaction 2 hours.In one typically experiment, Butyrylation enzyme and 50pmol peptide substrate need be removed with 100ng candidate Methionin.For confirming that candidate's Methionin goes the catalysed in vitro ability of Butyrylation enzyme further, adding 2 μMs of TSA and going Butyrylation enzymic activity to suppress Methionin.Reacted substrate polypeptide eventually passes μ C18Zip Tips purifying, carries out MALDI-TOF mass spectroscopy.

Istone lysine goes the biological chemistry of Butyrylation enzyme and cytology to confirm as and analyzes the external activity that candidate set protein lysine removes Butyrylation enzyme; the 6 μ g core histones that restructuring Methionin preliminary screening gone out goes Butyrylation enzyme to be separated with from eukaryotic cell in 15 μ l HDAC reaction buffers 37 DEG C react 2 hours, reaction product after SDS-PAGE with the change of the immune-blotting method core histones Methionin Butyrylation of the antibody of anti-Methionin Butyrylation.For confirming that candidate set protein lysine removes the activity in vivo of Butyrylation enzyme, go plasmid (wild-type and the enzyme deactivation mutant) transfection of Butyrylation enzyme cDNA in eukaryotic cell by containing encodes candidate istone lysine.Transfection is after 36 hours; cell through cracking and after SDS-PAGE after with the change of the immune-blotting method core histones Methionin Butyrylation of the antibody of anti-Methionin Butyrylation, or antibody cell being fixed rear histone locus specificity Methionin Butyrylation carries out immunofluorescence analysis.

Result

HDAC3 and HDAC6 is that candidate set protein lysine removes propionyl-enzyme and removes Butyrylation enzyme

Istone lysine acetylize is a kind of reversible posttranslational modification, being closely connected of the dynamic adjustments effect of its decorating state and Transacetylase (HATs) and deacetylase (HDACs).Consider lysine acetylation and Methionin is propionating and the similarity of Butyrylation, it is propionating and go the effect of Methionin butyryl enzyme that we suppose that some HDACs may exercise Methionin equally.

For checking this to suppose, we first analyze the propionating and Butyrylation of istone lysine whether can in the cell adding hdac inhibitor Selective long-range DEPT.Be changed to example with histone Butyrylation, histone Butyrylation level significantly increases (TSA, NaBu, SAHA and FK288) after adding I, II and VI class HDACs inhibitor; But the process of III class hdac inhibitor has an impact (Nicotinamide and sirtinol) (Fig. 2 A) to histone Butyrylation level hardly.This result has implied that I, II and VI class HDACs may be that main istone lysine removes Butyrylation enzyme.

Going Butyrylation to react to screen concrete which kind of I, II and VI class HDACs preferential catalysis istone lysine, inventors have developed a kind of screening of Methionin deacetylase and the fluorescence intensity detection method (see embodiment) of enzyme assay.In this detection; HDAC fluorogenic substrate Boc-Lys (the Buty)-AMC of all possible I, II and VI class HDACs and synthesis is carried out hatching (Fig. 2 B); only possessing Methionin goes the HDACs of Butyrylation enzymic activity the butyryl radicals of lysine side-chain can be removed; with after the AMC group that produces through pancreatin cracking under fluorescent reagent effect, inspire detectable fluorescent signal, simultaneously the power of fluorescent signal has reacted the height of enzymic activity.This test class is similar to the method detecting lysine acetylation with Boc-Lys (Ac)-AMC ^(23-24).The removal of HDCAs (2ng) on the butyryl radicals of Boc-Lys (Buty)-AMC lysine side-chain that this detection method shows all low dosages does not almost affect; then, when the dosage of raising HDACs is to 200ng, HDAC3 and HDAC6 demonstrates the activity more much higher than other HDACs and removes in butyryl group Boc-Lys (Bu)-AMC (Fig. 2 C).The above results has implied that HDAC3 and HDAC6 potential removes Butyrylation enzyme, and embodies high enzymic activity.

HDAC3 catalysed in vitro istone lysine removes Butyrylation

For confirming that the external Methionin of HDAC3 goes Butyrylation enzymic activity, external Methionin being carried out to the histone H 3 synthesized and H4 Methionin Butyrylation polypeptide and has gone Butyrylation to react (H3 peptide sequence: CSTGGK14 ^butyaPRK18 ^butyqLATK23 ^butyaARK; H4 peptide sequence: CSGRGK5 ^butygGK8 ^butygLGK12 ^butygGAK), reaction product is through mass spectroscopy determination mass shift.Result shows, under the katalysis not having HDAC3, and improvement on synthesis H3K _14,18,23 ^butyand H4K _5,8,12 ^butymolecular weight be 2182Da and 1728Da (Fig. 3 A and 3D) respectively; When after interpolation HDAC3, the peptide of improvement on synthesis spectrum peak have gradually lose ~ 70Da is of poor quality, this is of poor quality loses 1,2 or 3 butyryl groups consistent (Fig. 3 B and 3E) respectively with polypeptide.But after HDAC3 activity is suppressed by TSA, the mass shift that improvement on synthesis is lost is resumed again (Fig. 3 C and 3F).

Subsequently, contriver have detected restructuring HDAC3 further and goes Butyrylation ability to the Methionin of core histones in eukaryotic cell in vitro.As expection, HDAC3 significantly reduces the Methionin Butyrylation level of histone H 3 and H4 in vitro, and after HDAC3 activity is suppressed by TSA, the Methionin Butyrylation level of histone H 3 and H4 returns to normal level (Fig. 3 G).It is that istone lysine removes Butyrylation enzyme that these experiment in vitro confirm HDAC3.

HDAC3 is that the istone lysine in body removes Butyrylation enzyme

Whether be that istone lysine in body removes Butyrylation enzyme for detecting HDAC3, we introduce the dose-dependent ectopic expression of HDAC3 in HeLa cell, then by immune-blotting method istone lysine Butyrylation level.Result shows; istone lysine Butyrylation reduces along with the process LAN of wild-type HDAC3 (Fig. 3 A), but the process LAN of HDAC3 enzyme deactivation mutant HDAC3 S424A can not cause the decline (Fig. 3 B) of istone lysine Butyrylation level.On cell level; compare with the cell of untransfected; in body, the HDAC3 of process LAN significantly reduces the Methionin Butyrylation of histone H 4 K5, and the expression of its enzyme deactivation mutant HDAC3 S424A can not make H4K5 remove Butyrylation (Fig. 3 C, shown in arrow).Confirming HDAC3 is thus that istone lysine in body removes Butyrylation enzyme.

Istone lysine removes Butyrylation to HDAC6 with catalysed in vitro in vivo

With the fluorescence intensity detection display that Boc-Lys (Buty)-AMC is substrate there is significant Methionin and go Butyrylation enzymic activity (Fig. 2 B).For confirming the effect of HDAC6 further, contriver have detected the ability that HDAC6 catalysed in vitro istone lysine removes Butyrylation subsequently.Result shows, the HDAC6 of restructuring significantly reduces the Methionin Butyrylation level of histone H 3 and H4.But, by TSA inhibit active HDAC6 then istone lysine Butyrylation level without impact.In addition, the HDAC6 H216/611A of enzyme deactivation sudden change cannot go Butyrylation to react (Fig. 4 A) by catalysis istone lysine, has implied that istone lysine goes Butyrylation to need the enzymic activity of HDAC6.

For katalysis in the body of detection HDAC6, contriver introduces the wild-type of process LAN and the HDAC6 H216/611A of enzyme deactivation mutant in cell.What result showed that the process LAN of wild-type HDAC6 facilitates istone lysine removes Butyrylation (Fig. 4 B), and HDAC6 H216/611A is then without this effect (Fig. 4 C).

HDAC6 is considered to the deacetylase of kytoplasm usually ⁽²⁵⁾but this albumen can dynamically shuttle back and forth in kytoplasm and nucleus ⁽²⁶⁾.Nearest research shows that HDAC6 can combine with chromatin, marks different DNA fragmentations ⁽²⁷⁾, imply the function of HDAC6 in nucleus of knowing clearly.When utilizing core output inhibitor leptomycine B process cell, contriver observed HDAC6 and is trapped in core.All these results enhance HDAC6 dynamic positioning in nucleus, exercise the possibility that istone lysine removes Butyrylation.

For confirming that HDAC6 goes Butyrylation effect to istone lysine in core, leptomycin B process or have detected process LAN under not processing HDAC6 and HDAC6H216/611A on the impact of istone lysine Butyrylation.Add separately leptomycin B, or independent process LAN HDAC6 produces obvious histone H 3 and H4 Methionin removes Butyrylation.But the synergy of leptomycin B process and HDAC6 process LAN significantly can reduce the Methionin Butyrylation level of histone.Otherwise HDAC6H216/611A and leptomycin B does not have synergistic effect (Fig. 4 D) substantially.By detecting the Methionin Butyrylation level of histone H 4 specific position, observe under leptomycine B effect, process LAN HDAC6 significantly can reduce H4K5 and H4K12 Butyrylation level (Fig. 4 E).This result with act on process LAN HDAC6 and HDAC6 H216/611A cell with leptomycin B after, the immunofluorescence dyeing result consistent (Fig. 4 F) marked with the sequence specific antibody of anti-histone H4K5 and H4K12 Butyrylation.In sum, the HDAC6 that contriver confirms in nucleus can remove Butyrylation by catalysis istone lysine in vivo.

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Claims

1. Methionin removes propionyl-enzyme and removes qualification and an activity determination method for Butyrylation enzyme, it is characterized in that, comprises three steps:

First, Boc-Lys (Prop)-AMC and Boc-Lys (the Buty)-AMC of synthesis of high purity;

Secondly, Methionin to be identified is removed propionyl-enzyme or goes Butyrylation enzyme to react with Boc-Lys (Prop)-AMC or Boc-Lys (Buty)-AMC in specific reaction system, specific Methionin removes propionyl-enzyme or goes Butyrylation enzyme can excise propionyl or the butyryl radicals of lysine side-chain, and then AMC group is discharged under the splitting action of pancreatin, the Methionin special by the fluorescence intensity preliminary evaluation of the AMC group measuring release removes propionyl-enzyme or removes Butyrylation enzyme, and reflects the activity intensity of enzyme by the fluorescence intensity measured;

Finally, determine that the Methionin of candidate removes propionyl-enzyme or goes Butyrylation enzyme to be that target Methionin removes propionyl-enzyme or removes Butyrylation enzyme through a series of mass spectroscopy and biochemical analysis;

In above-mentioned three steps, described Methionin to be identified removes propionyl-enzyme or goes Butyrylation enzyme to comprise HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10 and HDAC11 of restructuring;

Described specific reaction system refers to that the Methionin to be identified of 2ng or 200ng removes Butyrylation enzyme and 85 μ l ddH ₂o, 10 μ l 10X HDAC reaction buffers and 5 μ l 4mM Boc-Lys (Buty)-AMC mix, and mixture was 37 DEG C of reactions 30 minutes; In reaction system, add the Lysine Developer termination reaction of 10 μ l, and hatch 30 minutes at 37 DEG C; Wherein HDAC reaction buffer is 25mM Tris-Cl, the 137mM NaCl of pH 8.0, the mixed solution of 2.7mM KCl and 1mM MgCl2; Described a series of mass spectroscopy refer to the Methionin that preliminary evaluation is gone out go the Methionin Butyrylation polypeptide of Butyrylation enzyme and synthesis in 10 μ l HDAC reaction buffers 37 DEG C reaction 2 hours, add 2 μMs of TSA and go Butyrylation enzymic activity to suppress Methionin; Reacted substrate polypeptide eventually passes μ C18Zip Tips purifying, carries out MALDI-TOF mass spectroscopy;

Described biochemical analysis comprises, the 6 μ g core histones that restructuring Methionin preliminary evaluation gone out goes Butyrylation enzyme to be separated with from eukaryotic cell in 15 μ l HDAC reaction buffers 37 DEG C react 2 hours, reaction product after SDS-PAGE with the change of the immune-blotting method core histones Methionin Butyrylation of the antibody of anti-Methionin Butyrylation; For confirming that candidate set protein lysine removes the activity in vivo of Butyrylation enzyme, go the plasmid transfection of Butyrylation enzyme cDNA in eukaryotic cell by containing encodes candidate istone lysine; Transfection is after 36 hours, cell is through cracking and with the change of the immune-blotting method core histones Methionin Butyrylation of the antibody of anti-Methionin Butyrylation after SDS-PAGE, or antibody cell being fixed rear histone locus specificity Methionin Butyrylation carries out immunofluorescence analysis;

The Methionin of described candidate removes propionyl-enzyme or goes Butyrylation enzyme to be HDAC3 and HDAC6.