KR102534180B1 - Method for producing antibodies that selectively recognize acetoacetylation of proteins - Google Patents

Abstract

The present invention relates to a method for producing an antibody that selectively recognizes acetoacetylation of a protein, and is a compound that is a mimic similar to lysine acetoacetylation of a protein, (S)-2-((((9H-fluoren-9-yl )methoxy)carbonyl)amino)-6-((5-methyloxazol-2-yl)amino)hexane((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl) An antibody that selectively recognizes acetoacetylation of proteins is prepared using amino)-6-((5-methyloxazol-2-yl)amino)hexanoic acid, and an antibody that detects lysine acetoacetylation of histones is used. By confirming that, the compound is provided as a reagent composition for producing an antibody that selectively recognizes acetoacetylation of a protein, and a method for producing an antibody that selectively recognizes acetoacetylation of a protein using the same is provided.

Description

Method for producing antibodies that selectively recognize acetoacetylation of proteins {Method for producing antibodies that selectively recognize acetoacetylation of proteins}

The present invention relates to a method for producing an antibody that selectively recognizes acetoacetylation of a protein.

Protein post-translational modifications (PTMs) of histones regulate chromatin structure and transcriptional activity. The major roles in histone protein post-translational modification are acetylation and methylation of lysine residues. Extensive studies over the past decades have shown that both types of histone marks are regulated by metabolites such as SAM, alpha ketaoglutarate, NAD and acetate, suggesting metabolically mediated epigenetic mechanisms. . Recently, a series of short-chain lysine acylations that can be stimulated by short-chain lipids and/or corresponding CoAs have been studied. Lysine acylation serves to regulate the activity of metabolic enzymes. It is associated with distinct chromatin regions and contributes to gene expression distinct from histone acetylation. These PTM pathways are associated with various pathophysiological conditions and contribute to disease progression, such as cancer.

Ketone bodies are composed of beta-hydroxylbutyrate (BHB), acetoacetate and acetone, and are stimulated in some physiological changes (eg starvation) or pathological conditions (eg diabetes). Ketone bodies are used as an energy source in tissues such as the heart and brain. Ketone body metabolism plays a central role in physiological homeostasis. BHB acts as a precursor of Kbhb to histones through the BHB CoA. These histone marks, unlike histone Kac marks, contribute to gene expression in response to starvation. The discovery of Kbhb as a dynamic and regulatory histone mark suggests the possible presence of acetoacetylation or lysine acetoacetylation (Kaa) at lysine residues in histones.

Korean Patent Registration No. 10-2083797 (2020. 03. 03. Announcement)

In order to solve the above problems, an object of the present invention is to provide a reagent composition for producing an antibody selectively recognizing acetoacetylation of a protein.

Another object of the present invention is to provide a method for producing an antibody that selectively recognizes acetoacetylation of a protein.

The present invention provides a reagent composition for producing an antibody that selectively recognizes acetoacetylation of a protein containing the compound represented by Formula 1 as an active ingredient.

In addition, the present invention produces an antibody that selectively recognizes acetoacetylation of a protein comprising the step of administering the compound represented by Formula 1 to an animal other than human to produce antisera, and separating and purifying polyclonal antibodies from the antiserum. provides a way to

According to the present invention, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((5 -Methyloxazol-2-yl)amino)hexane ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((5-methyloxazol-2-yl) amino) hexanoic acid) to prepare an antibody that selectively recognizes acetoacetylation of proteins, and confirming that histone lysine acetoacetylation is detected using the antibody, thereby making the compound selectively acetoacetylation of proteins. It is provided as a reagent composition for producing an antibody that recognizes, and a method for producing an antibody that selectively recognizes acetoacetylation of a protein using the same can be provided.

1 is a modified structure showing acetoacetylation (protein acetoacetylation, Kaa) at a lysine residue of a protein.
Figure 2 is a test schematic diagram and antigen structure for producing an antibody that specifically binds to Kaa.
3 is a schematic diagram showing a synthesis process of a Kaa analog structure to produce an antibody that specifically binds to a Kaa antibody.
Figure 4 is a result of verifying the selectivity of an antibody specifically binding to Kaa by dot-blot.
5 is a Western blot based on an antibody specifically binding to Kaa to detect three animal-derived histone proteins, detect increased Kaa after treatment with acetoacetate, and detect hymeglusin This is the result of detecting increased Kaa after treatment.
6 is a schematic diagram of acetoacetylated peptide analysis through immunoprecipitation based on an antibody specifically binding to Kaa.
7 shows Kaa sites in histones detected through immunoprecipitation and proteomic analysis based on antibodies specifically binding to Kaa.
8 shows the results of Kaa detection in proteins extracted from major organs of rats based on antibodies specifically binding to Kaa.

The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't

Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.

Hereinafter, the present invention will be described in more detail.

The present invention provides a reagent composition for producing an antibody that selectively recognizes acetoacetylation of a protein containing the compound represented by Formula 1 as an active ingredient.

[Formula 1]

The compound represented by Formula 1 is a mimic similar to acetoacetylation (Kaa) on lysine of a protein, specifically (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl )amino)-6-((5-methyloxazol-2-yl)amino)hexane((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-( (5-methyloxazol-2-yl)amino)hexanoic acid).

Acetoacetylation of the protein occurs at lysines of histone proteins, and the antibody is a polyclonal antibody specifically binding to acetoacetylation of proteins.

The polyclonal antibody refers to an antibody produced by simultaneously activating several clones against one antigen. That is, a polyclonal antibody is a collection of antibodies that recognize various epitopes (epitopes) present in one antigen. The antibody is a term known in the art and refers to a molecule or an active fragment of a molecule that binds to a known antigen. Examples of active fragments that bind to known antigens include Fab and F(ab')2 fragments. Such active fragments can be derived from the antibodies of the present invention by a number of techniques. As a general technique for isolating the active fragment of an antibody, any method known in the art may be used. Such antibodies include bispecific and chimeric antibodies.

In addition, the present invention produces an antibody that selectively recognizes acetoacetylation of a protein comprising the step of administering the compound represented by Formula 1 to an animal other than human to produce antisera, and separating and purifying polyclonal antibodies from the antiserum. provides a way to

Animals other than humans may preferably be rabbits, mice, or goats, but are not limited thereto. In general, relatively large mammals with higher amounts of serum from which more antibodies can be isolated can be preferentially selected as the animal to be used for the production of the antibody. When the immunogen is injected into the animal, B cells of the immune system are stimulated to produce immunoglobulin (Ig) specific for the immunogen.

The antiserum (antiserum) means a serum having a specific antibody to the antigen entered from the outside. When an antigen enters the body of a human or laboratory animal, an antibody that specifically reacts to it is generated. The newly created antibody exists in the serum component of blood. called serum). Antibodies are any of IgG, IgM, IgA, IgE, and IgD immunoglobulins, but are usually a mixture of them and are mixed with many other serum proteins in serum at the same time.

Hereinafter, experimental examples and examples will be described in detail to aid understanding of the present invention. However, the following experimental examples and examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental Example> Experimental Materials and Methods

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell culture and histone extraction

- Cell culture

Human HepG2 cells, MCF7 cells and mouse MEF cells were supplemented with 10% (v/v) fetal bovine serum (FBS, Hyclone Laboratories Inc., Logan, UT, USA), 1% (v/v) GlutaMAX (Gibco, Thermo Fisher Scientific , Inc., CA, USA) and DMEM medium (Hyclone Laboratories Inc., Logan , UT, USA) at 37°C and 5% CO ₂ conditions. Drosophila S2 cells were cultured at 26°C in Schneider's medium (Gibco, Thermo Fisher Scientific, Inc., CA, USA) containing 10% (v/v) FBS. To increase the intracellular levels of Kaa, HepG2, MCF7, MEF, and S2 cells, ethyl-acetoacetate (EAA, Sigma-Aldrich, St. Louis, MO, USA) was incubated at 1, 5, and 10 mM for 24 h before harvesting. concentration was treated.

- Histone extraction

Core histones were extracted from HepG2, MCF7, MEF and S2 cells. Cell medium was removed when cells reached 80% confluence and washed with 1 x Dulbecco's Phosphate Buffered Saline (DPBS, Gibco, Thermo Fisher Scientific, Inc., CA, USA). It was treated with 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Inc., CA, USA) and incubated at 37°C for 3 minutes. The suspension was centrifuged at 1,000 g for 3 minutes and the supernatant was removed. Harvested cells were washed with ice cold DPBS. Cells were resuspended in lysis buffer (10 mM HEPES pH 7.0, 10 mM KCl, 1.5 mM MgCl, 0.34 M sucrose, 0.5% NP40) using pipetting and incubated for 30 minutes on ice with gentle agitation. The suspension was centrifuged at 2,000g at 4°C for 10 minutes. The supernatant was removed, the pellet was washed with wash buffer (10 mM HEPES pH 7.0, 10 mM KCl, 1.5 mM MgCl, 0.34 M sucrose) and then histone proteins were extracted with 0.4 NH ₂ SO ₄ for 4 hours at 4°C. . The suspension was centrifuged at 16,000g at 4°C for 20 minutes. The supernatant containing the histone extract was transferred to a low protein binding tube.

- Hymeglusin treatment

To increase the intracellular level of acetoacetate-CoA, HepG2 cells were treated with Hymeglusin (Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 0, 1, 5, and 10 μM for 24 hours and the cells were harvested. .

- ¹³ C ₂ Ethyl acetoacetate treatment

To confirm whether acetoacetate is involved in the formation of histone Kaa, HepG2 and MCF7 cells were treated with ethyl acetoacetate-1,3- ¹³ C ₂ (Sigma-Aldrich, St. Louis, MO, USA). 10 mM ethyl acetoacetate concentration was treated for 24 hours and the cells were harvested.

2. Animal experiments and histone extraction

To extract histones from rat tissues, 5-week-old SD male mice were purchased from Charles River, Korea (Seoul, Korea) (IRB No 2021-0025). Mice were randomly grouped, housed four per cage, and allowed to acclimatize for one week. The animals were reared under light/dark conditions with a 12-hour cycle in a space maintained at 23±3° C. and 50±10% relative humidity. All animal experiments were performed based on guidelines recommended by the American Society of Toxicology in 1989. After animals were inhaled anesthetized, liver, kidney, lung and spleen tissues were isolated. Tissue samples were homogenized using a glass Dounce homogenizer (20 strokes) in ice cold lysis buffer. The homogenate was passed through two layers of cheesecloth and centrifuged at 2,000 g at 4°C for 10 minutes. The supernatant was removed, and the pellet was briefly washed with wash buffer and extracted with 0.4 N H ₂ SO ₄ for 4 hours at 4°C. The suspension was centrifuged at 16,000 g for 20 minutes at 4°C. The supernatant containing the histone extract was transferred to a low protein binding tube. Histological analysis was performed according to YZ's Approved Animal Guide at the University of Chicago using mouse samples (ACUP # 72296).

For histone precipitation, trichloroacetic acid (TCA) was added to the histone-containing supernatant to a final concentration of 30% (v/v), followed by precipitation on ice for 12 hours. The suspension was centrifuged at 2,000g at 4°C for 10 minutes. The precipitated histone pellet was washed with ice cold acetone and dried. Histone proteins were dissolved in distilled water. Histone concentration was quantified by BCA assay (Thermo Fisher Scientific, Inc., CA, USA). The extracted histone proteins were stored in a -80°C freezer.

For the starvation experiment, 6-week-old animals were recruited and assigned to steel in two groups (control group: n = 4, starvation group: n = 4). In the case of the control group, a standard feed containing 19% protein and supplemented with water was provided. The starvation group received only water for 48 hours.

3. Co-elution of Kaa peptides with synthetic peptides

The synthetic peptides used in the experiments (H3K9aa; KaaSTGGKprAPR, H3K18aa; KaaQLATKprAAR, and H4K31aa; DNIQGITKaaPAIR) were purchased from AnaSpec, Inc. (Fremont, CA). Lysine acetoacetylated peptides from tryptic digests of human MCF7 histones, synthetic counterparts and mixtures thereof were injected into a nano-HPLC system and analyzed by Q-exactive mass spectrometry. Comparison of LC-MS/MS spectra and retention times of target proteins provided H3K9aa (m/z 523.83, KaaSTGKprAPR), H3K18aa (m/z 563.83, KaaQLATKprAAR), and H4K31aa (m/z 705.39, DNIQGITKaaPAIR).

4. Dot blot analysis

Antigen was prepared at a final concentration of 10 - 50 μg/ml in TBST buffer (Tris-buffered saline, pH 7.4, 0.5% (v/v) Tween 20). A polyvinylidene fluoride (PVDF) membrane was gridded to confirm the location of antigen spotting. Membranes were activated with methanol and washed in TBST. In the membrane, Kaa peptide, Kcr (crotonyllysine) peptide, Khib (2-hydroxyisobutyryllysine) peptide, Ksu (succinyllysine) peptide, and BSA (bovine serum albumin) were found in amounts of 1, 8, and 64 ng, respectively. Membranes were air dried and incubated with blocking buffer (5% (v/v) fat-free TBST) for 30 minutes. The membrane was washed 3 times for 10 minutes in TBST buffer. Primary antibodies were diluted to optimized concentrations in blocking buffer and incubated with membranes for 1 hour at room temperature. The membrane was then washed 3 times for 10 min in TBST buffer. Subsequently, secondary antibody was applied to the membrane and incubated for 30 minutes at room temperature. The membrane was washed 3 times for 10 minutes in TBST buffer. For Dolt detection, the membrane was treated with HRP luminol material for about 30 to 60 seconds and observed with a lumino image analyzer.

5. Sample preparation for proteomic analysis

- In-gel degradation via chemical propionylation

20 μg of each histone sample obtained from cells or animal tissue was resolved by 15% SDS-PAGE and visualized by Coomassie protein staining (Abcam, Cambridge, MA, USA). Histone bands were excised, destained with 50% ethanol and dehydrated with acetonitrile (ACN). Samples were subjected to in-gel digestion with or without chemical derivatization. For chemical derivatization, decolorized and dehydrated gel bands were hydrated in 100 mM ammonium bicarbonate (ABC) buffer. The propionylation reagent consisted of propionic anhydride with ACN in a ratio of 1:3 (v/v). 25 ul of propionylation reagent was added and ammonium hydroxide (NH ₄ OH) was immediately added to set the pH to 8.0. The reaction was held at 37° C. for 1 hour. Propionylated histones were digested with trypsin (Promega, Madison, WI, USA) at a ratio of 50:1 (w/w) at 37° C. for 12 hours. Histone peptides were extracted in 75% ACN/0.1% TFA. Peptide extracts were dried in high vacuum without heating. Histone peptides were reconstituted in 100 mM ABC buffer and propionylation.

- Kaa global immunoprecipitation

Dried histone samples were dissolved in 100 mM ABC at a protein concentration of 1 mg/mL, pH 8.0. For every 100 μL histone solution, 25 μL of propionylation reagent was added, and NH ₄ OH was immediately added to set the pH to 8.0. Propionylated histones were digested with trypsin (Promega, Madison, WI, USA) at a ratio of 50:1 (w/w) at 37°C for 12 hours. Peptides produced by digestion independently at the same time were subjected to propionylation derivatization. Propionylated histone peptides were desalted using sep-pak cartridges (Waters, Milford, MA, USA) and dried in speed-vacuum.

Lysine aceto acetylated peptides were enriched by aceto acetyl-lysine conjugated agarose beads (PTM Biolabs, Hangzhou, CN). Desalted histone peptides were dissolved in NETN buffer (2 mM EDTA, 0.04 M Tris-HCl, 0.2 M NaCl, and 1% NP ₄ O, pH 8.0). The peptide solution was centrifuged for 10 minutes at 16,000 g at 4°C to remove possible precipitates. Pan-anti-Kaa antibody conjugated beads were mixed with the peptide solution and incubated at 4°C with agitation for 12 hours. To wash unbound peptides, the peptide mixture was centrifuged at 1,000 g at 4° C. for 1 minute and the supernatant was removed. The remaining beads were washed 3 times with ice cold NETN buffer and 2 times with deionized water. The Kaa peptide was eluted 3 times with 50 μL 0.15% (v/v) trifluoroacetic acid (TFA). The eluents were combined and dried in speed vacuum without heating. Dried peptides were stored in a -80°C freezer.

6. LC-MS/MS Analysis

Prior to MS analysis, dried peptide samples were desalted using Zip-Tip (5 μg) (Millipore, Milford, MA, USA). The desalted peptide was dissolved in 20 μL solvent A (solvent A: 2% ACN and 0.1% formic acid, solvent B: 0.1% formic acid in ACN). A 2 μL peptide sample was injected into a nano-LC (Eksigent, Dublin, CA, USA) integrated with an autosampler. Peptides were separated using 60 min gradient nano-LC, and the flow rate was set at 300 nl/min. The LC gradient was set to start at 3% solvent B and end at 28% solvent B. For peptide analysis, the nano-LC was integrated with the LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA) at the Mass Spectrometry Convergence Research Center. For data-dependent scans, the precursor ion scans were from 300 m/z to 1,500 m/z with a resolution of 60,000 and were scanned at 445.12 m/z with the lock mass activated. Automatic gain control (AGC) target value is 1.0 x 10 ⁶ for MS scan. Tandem MS was obtained using collision induced dissociation mode. For the MS/MS scans, 10 of the most abundant ions with intensities greater than 1.0e5 in the full scan were separated in the high-energy collision dissociation mode with a resolution of 7,500 and a normalized collision energy of 28%, with an isolation width of 2.0 m/z. appeared as MS was run in positive mode.

7. Peak Alignment

MS-generated raw data were used with MASCOT software (version 2.3.0, Matrix Science Ltd, London, UK) against human, rat, or mouse databases for protein and peptide identification. Lysine acetoacetylation (Kaa), lysine acetylation (Kac), lysine propionylation (Kpr), methionine oxidation and protein N-terminal acetylation were designated variable modifications. Mass tolerances were set at 10 ppm for precursor ions and 0.05 Da for fragment ions. To reduce the number of low-quality PTM identifications, Mascot score cutoff <20, significance threshold p<0.05, and all peptides with C-terminal lysine modifications were further discounted (except when the modified peptide was a protein C-terminal peptide). box). All results indicating detected Kaa-modified peptides were manually inspected. Mass spectrometry proteomics data were stored in the ProteomeXchange Consortium through the PRIDE partner repository with the dataset identifier PXD025392.

8. Quantification of Kaa sites by PRM

For Kaa sequence relative quantification, peptides were analyzed using Parallel Reaction Monitoring (PRM) based on a high-resolution mass spectrometry analyzer (HR/MS). PRM was performed with total mass and PRM tandem mass integration methods. The target m/z of the Kaa sequence was isolated from that identified by the DDA scan. The target m/z of the ¹³ C ₂ labeled Kaa sequence was calculated by replacing ¹² C ₂ with ¹³ C ₂ for the Kaa sequence identified in the DDA scan. For the full scan, the precursor ion scan was from 300 m/z to 1,500 m/z with a resolution of 60,000 and was scanned at 445.12 m/z with the lock mass activated. Automatic gain control (AGC) target value is 1.0 X 10 ⁶ . For ms/ms, the resolution was set to 7,500 with 35% normalized collision energy, and the isolation width was 1.0 m/z. MS was run in positive mode. The fold change of Kaa sequences between the two samples was quantified as a ratio of peaks.

9. Western Blot

10 μg of histone protein was mixed with 4x SDS buffer (200 mM Tris-Cl, pH 6.8, 400 mM dithiothreitol (DTT), 8% SDS, 0.4% bromophenol blue, 40% glycerol) to a final concentration of 1x SDS. The protein mixture was denatured by heating at 95° C. for 3 minutes. The denatured protein was cooled to room temperature (RT) and loaded onto a 15% SDS-PAGE gel. The SDS-PAGE gel was run at 120 v for 90 minutes. The protein was then electrotransferred to a PVDF membrane (Roche, Basel, CH) by wet transfer. Transfer was carried out on ice for 120 min at a voltage of 90 V. After transfer, the membrane was coated with Tween-20 (TBST, 137 mM sodium chloride). , 20 mM Tris, and 0.1% Tween-20) in 1x Tris-buffered saline for 2 hours at room temperature with 5% (v/v) BSA (bovine serum albumin, GenDEPOT, Barker, TX, USA) did

Membranes were incubated with primary antibodies: anti-Kaa, anti-Kac, anti-Kpr, anti-Kcr, anti-Kbhb, anti-Kbu (PTM Biolabs, Hangzhou, CN), anti-histone H3 (Cell Signaling Technology, Beverly, MA, USA). Antibodies were diluted 1:100 (v/v) in 5% (v/v) BSA and membranes were incubated with primary antibodies overnight at 4°C. The membrane was washed gently in TBST buffer for 10 minutes. The washed membrane was incubated for 90 minutes at room temperature with a secondary antibody, which is a corresponding horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA). The membrane was washed 3 times in TBST buffer. For visualization of the membrane, the membrane was dampened using 1 ml ECL reagent (GE Healthcare, Chicago, IL, USA) and stained with mage Quant LAS 4000 mini (GE Healthcare, Chicago, IL, USA) and iBright 1500 (Thermo Fisher Scientific, Inc., CA, USA) was used to perform exposure.

Preparation Example: Kaa-specific antibody development

Kaa structural analogues were synthesized by predicting the molecular structure of the acetoacetyl group. PTM Biolabs Inc. (Hangzhou, Zhejiang, China), a peptide library containing synthesized analogs was prepared and used as an antigen. The Kaa structural analogue is a compound represented by Formula 1, specifically (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((5-methyl Oxazol-2-yl)amino)hexane((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-((5-methyloxazol-2-yl)amino) hexanoic acid). For immunization, the antigen solution was mixed with complete Freund's adjuvant (Sigma-Aldrich, St. Louis, MO, USA) in a 1:1 (v/v) ratio and injected into 5 New Zealand rabbits. Blood was drawn from the carotid artery after 9 weeks of vaccination. Blood samples were stored at 37°C for 2 hours and at 4°C overnight. Blood samples were centrifuged at 6,000g at 4°C for 20 minutes, and the antiserum-containing supernatant was collected. The resulting antibody was purified using an affinity column. The column was activated with 20 mL of phosphate-buffered saline (PBS). Anti-serum was loaded onto the affinity column and the passaged serum was collected and reloaded. The column resin was washed with PBS until OD ₂₈₀ < 0.05. The purified antibody was eluted with 0.2 M glycine (pH 2.8) and neutralized with 1 M Tris buffered saline (pH 8.0).

Example 1. Hypothesis of lysine acetoacetylation in core histones

The identification and epigenetic function of Kbhb on histones supports the possibility of acetoacetate-mediated Kaa on histones (Fig. 1). The mass shift determined from the chemical structure of Kaa is calculated to give rise to +84.0211 Da (monoisotopic mass, C ₄ H ₄ O ₂ ). As shown in FIG. 1 , histones were extracted from HepG2 cells and rat livers treated with 10 mM ethyl acetoacetate (EAA) for initial Kaa identification. Core histones were subjected to in-gel degradation with or without chemical propionylation. Peptide samples were analyzed using a data-dependent acquisition mode with a high-resolution mass spectrometer. Mascot software (version 2.3.0, Matrix Science Ltd, London, UK) was used to retrieve MS/MS data containing Kaa (+ 84.0211) as a variable correction.

Example 2. Identification of lysine acetoacetylated residues in core histones

To further confirm histone Kaa, a pan antibody against Kaa was prepared. A peptide library containing acetoacetylated lysine was chemically synthesized and used as an antigen, but no pan anti-Kaa antibody was generated. The chemical structure of an acetoacetyl analog under physiological conditions was predicted, and ^ε N-lysine was introduced into methylisoxazole as a structural analog (FIG. 2). Pan anti-Kaa antibodies were generated by chemically synthesizing lysine containing methylisoxazole analogs as replacement antigens (FIG. 3). The specificity of the resulting anti-Kaa antibody was confirmed by dot blot analysis using a peptide library containing acetoacetyl analogues (FIG. 4). The pan anti-Kaa antibody successfully recognized peptides containing Kaa but not three other peptide libraries in which the immobilized lysine residue was unmodified, crotonylated, β-hydroxybutylated, or succinylated. As shown in Figure 5, the two most selective anti-Kaa antibody batches were selected according to the dot-blot results. Antibodies were used for western blotting and immunoprecipitation (IP). Using a pan anti-Kaa antibody, histone Kaa was detected in core histones isolated from Homo sapiens, Rattus norvegicus, and Danio rerio. This provides an additional boost to histone modifications that are evolutionarily conserved among diverse eukaryotic species. After attaching agarose beads to the anti-Kaa antibody, Kaa peptides were concentrated from tryptic peptides of core histones isolated from HepG2 and rat livers (FIG. 6).

Example 3. Evaluation of histone lysine acetoacetylation kinetics by isotope labeling

It was hypothesized that the Kaa reaction would proceed in a similar manner to other lysine short-chain acylations, such as succinylation and malonylation leading to succinyl-CoA and malonyl-CoA, respectively. To examine the kinetics of histone Kaa responses to intracellular acetoacetate levels, HepG2 cells were treated with 1 - 10 mM EAA for 24 hours. As shown in FIG. 5, it was confirmed by Western blot that histone Kaa was significantly increased after EAA treatment. Meanwhile, Western blotting was also performed for five known histone acylations, including Kac, Kpr, Kcr, and Kbhb, and a slight increase was observed only in Kpr, while no change was observed in other modifications. In addition, to investigate the effect of acetone acetyl-CoA levels on Kaa dynamics, cells were transfected with acetoacetyl-CoA to HMG-CoA, a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Exposure to Hymeglusin , which inhibits conversion. As shown in Figure 5, the expression of Kaa increased after Hymeglusin treatment due to acetoacetyl-CoA accumulated in the cells. HMGCS inhibitor treatment decreased Kpr and increased Kcr in histones of HepG2 cells. The above results indicate that histone Kaa is produced by an intermediate such as acetoacetyl-CoA, and the expression of Kaa is dependent on the level of acetoacetate in cells.

To further validate that acetoacetate is used as a whole of acetoacetyl-CoA to generate histone Kaa, HepG2 cells were metabolically labeled with ¹³ C ₂ -EAA (10 mM) for 24 hours. After ¹³ C ₂ -EAA treatment, the expression level of histone Kaa increased similarly to that of 10 mM EAA treatment. Histone peptides were analyzed by parallel reaction monitoring (PRM) scanning, which was performed by theoretically calculating the replacement value of ¹² C-acetoacetyl group by ¹³ C ₂ -acetoacetyl group in the measured peptide sequence. As shown in Figure 7, a total of 30 ¹² C-Kaa sites were identified in HepG2 cells, of which 6 sites had detectable isotopically labeled acetoacetylation in response to treatment with ¹³ C ₂ -ethyl acetoacetate. and four sites in the core histones. Six isotope-labeled sites were identified in MCF-7 cells under the same conditions. A high-resolution MS/MS spectrum of KaaQLATKprAAR (including Kaa at H3K18) labeled with representative ¹³ C ₂ -acetoacetate is shown, and isotopically labeled Kaa peptides are readily identifiable by an additional 2 Da-mass shift relative to Kaa. there is. The above results demonstrate that acetoacetate can be converted to acetoacetyl-CoA as a high-energy molecule and used in histone Kaa in a manner similar to Kac, Kbhb, Ksu and Kmal.

Example 4. Broad distribution and stoichiometry of histone lysine acetoacetylation in rat tissues

To confirm the distribution of histone Kaa in vivo, histones were extracted from liver, kidney, lung and spleen, and Kaa was analyzed in the same manner as described above. As shown in FIG. 8 , 11, 13, 10, and 14 Kaa quartiles were detected in core histones isolated from liver, kidney, lung and spleen, respectively. Kaa of H2BK46, H3K27, and H4K20 were observed in all tissues, and 10 Kaa sites were detected in two or more tissues. Kaa sites, like other histone acylations, are prevalent in the globular core, N-terminus, which includes a central histone-fold domain and a C-terminal domain. Kaa was found to coexist at 13, 15, and 13 sites with Kac, Kcr, and Ksu, respectively. As a result, Kaa is known to play a role similar to histone acylation, which differs from Kac, Ksu and Kcr in chromatin regulation.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (5)

A reagent composition for producing an antibody that selectively recognizes acetoacetylation of a protein comprising the compound represented by Formula 1 as an active ingredient.
[Formula 1]

The reagent composition according to claim 1, wherein the acetoacetylation of the protein occurs at the lysine of the histone protein. The reagent composition according to claim 1, wherein the antibody is a polyclonal antibody specifically binding to acetoacetylation of a protein. A method for producing an antibody that selectively recognizes acetoacetylation of a protein comprising the steps of administering a compound represented by Formula 1 to an animal other than human to produce antisera, and separating and purifying polyclonal antibodies from the antiserum.
[Formula 1]

5. The method of claim 4, wherein the acetoacetylation of the protein occurs at the lysine of the histone protein.

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