Beta-hydroxybutyrylation modification marker of OXCT1 and application of beta-hydroxybutyrylation modification marker in diagnosis and treatment of ketoacidosis

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a beta-hydroxybutyrylation modification marker of OXCT1 and application of the beta-hydroxybutyrylation modification marker in diagnosis and treatment of ketoacidosis. The invention relates to a marker for ketoacidosis. The marker is modified by beta-hydroxybutyrylation on lysine at the 421st site of OXCT1 protein. The marker is applied to diagnosis of ketoacidosis, and by detecting the Kbhb modification level of OXCT1, the expression quantity of OXCT1 of each ketone body utilization organ is detected and serves as an index for auxiliary diagnosis of ketoacidosis. According to the application of the marker in treatment of ketoacidosis, Kbhb modification of lysine at the 421st site of OXCT1 protein serves as a treatment target of a ketoacidosis patient, and the marker can be applied to treatment of ketoacidosis so as to reduce the blood ketone level. The invention provides a novel diagnosis and treatment method for ketoacidosis. The marker can also be applied to preparation of a ketoacidosis diagnosis reagent or kit.

Description

Technical Field

[0001] The invention relates to a marker, belonging to the technical field of biochemistry, and in particular to a beta-hydroxybutyrylation detection of ketone body utilization rate-limiting enzyme OXCT1 and an application thereof in the diagnosis and treatment of ketoacidosis. Background Art

[0002] Ketoacidosis is often defined as an increase in blood ketones leading to a primary decrease in plasma bicarbonate concentration. According to the causes of ketoacidosis, we can divide it into diabetic ketoacidosis (DKA), euglycemic ketoacidosis and alcoholic ketoacidosis (AKA). DKA is a more common medical emergency, often caused by a decrease in circulating insulin concentration and a concomitant increase in counter-regulatory hormones (catecholamines, glucagon, growth hormone and cortisol). This type of acidosis often has an acute onset and rapid changes in the condition, posing a serious threat to the patient's life and health. Therefore, the study of the molecular mechanism of ketoacidosis and the development of targets are of great significance for the treatment of metabolic diseases such as diabetes.

[0003] At present, the preliminary diagnosis of ketoacidosis is mainly through ketone test strips and arterial blood gas analysis. Among them, ketone test strips are mainly used to detect ethyl acetate in urine, and to a lesser extent acetone. However, this detection method has a high incidence of false positives because it cannot detect β-hydroxybutyric acid. Arterial blood gas analysis cannot be widely popularized due to expensive instruments and difficulties in arterial blood sampling. In addition, the current clinical treatment methods for ketoacidosis are mainly through methods such as fluid replacement and hypoglycemic control. The research on the molecular mechanism of its onset is not very targeted, and no drugs targeting ketone body metabolism have entered clinical use. Therefore, the discovery of new specific targets for ketoacidosis is of great significance for the early diagnosis and precise treatment of ketoacidosis.

[0004] OXCT1 (3-oxoacid CoA-transferase 1) is 3-succinate CoA transferase 1, also known as SCOT (succinyl-CoA:3-ketoacid CoA transferase), which contains 521 amino acids and is located in the mitochondria of cells. The main function of OXCT1 is to transfer the coenzyme A group of succinyl-CoA to acetoacetate after catalytic isomerization of ketone bodies (mainly β-HB) in extrahepatic tissues to form acetoacetyl-CoA, and finally enter the tricarboxylic acid cycle (TCA cycle). In this metabolic process, two enzymes, 3-hydroxybutyrate dehydrogenase 1 (BDH1) and acetyl-CoA acetyltransferase 1 (ACAT1), can catalyze bidirectional reactions, while OXCT1 can only catalyze unidirectional reactions and is the rate-limiting enzyme in this reaction process. The distribution of the rate-limiting enzyme OXCT1 protein corresponds to the decomposition and utilization of ketone bodies in the body. It is rich in the heart, brain and kidneys, and can be detected in all extrahepatic tissues. The expression of OXCT1 protein in liver tissue is inhibited, which may be a mechanism to avoid the ineffective circulation of ketone body energy in liver tissue. In addition to participating in the decomposition of ketone bodies, the function of the rate-limiting enzyme OXCT1 in the occurrence and development of tumors and cardiovascular diseases has also attracted much attention.

[0005] β-Hydroxybutyrate (β-HB), also known as D-3-hydroxybutyrate, accounts for about 70% of the circulating ketone body pool and is the most abundant ketone body in the body. β-HB can not only cross the blood-brain barrier and replace glucose as a fuel for the brain, but is also used as an alternative energy source for glucose in all extrahepatic tissues. In addition to being an alternative energy source for glucose, β-HB also participates in many cellular functions as a signaling molecule, including epigenetic regulation of gene transcription, oxidative stress, and transmembrane receptor signal transduction. In 2016, β-HB was first discovered as a new type of protein post-translational modification mediated by its activated thioester form β-hydroxybutyryl-CoA, Lysine β-hydroxybutyrylation (Kbhb) modification. Subsequent studies have shown that Kbhb modification not only occurs on histones, but is also widely present in cytoplasmic proteins such as the cytoplasm and mitochondria. At the same time, mass spectrometry database retrieval showed that the key enzyme for ketone body synthesis HMGCS2 and the key enzyme for ketone body utilization OXCT1 in mice can both undergo β-HB-mediated Kbhb after translation.

[0006] In our work, we demonstrated the existence of lysine β-hydroxybutyrylation modification on OXCT1 protein through in vitro and in vivo animal and cell molecular experiments. By constructing gene mutants at specific sites, we confirmed that Kbhb modification on lysine K421 of OXCT1 can significantly affect the enzymatic activity of OXCT1, thereby regulating the accumulation of ketone bodies at the cellular or overall level.

[0007] Therefore, we believe that the detection of this site has important clinical value. On the one hand, its gene mutation or deletion may serve as a detection target for clinical ketoacidosis susceptibility genes. On the other hand, the detection of modification levels of K421 and the development of related acyltransferase activators may help in the prevention and treatment of clinical diabetic ketoacidosis. Summary of the invention

[0008] Based on the background technology, the present invention proposes a biomarker for diagnosing ketoacidosis, and based on the biomarker, the expression level of OXCT1 in each ketone body utilization organ is obtained by detecting the β-hydroxybutyrylation of OXCT1, a rate-limiting enzyme for ketone body utilization, and applied to the diagnosis of ketoacidosis;

[0009] At the same time, the present invention promotes Kbhb modification on lysine 421 of OXCT1 protein, such as β-hydroxybutyryl transferase agonist, thereby activating the enzyme activity of OXCT1 and ultimately reducing the blood ketone level, and can be used to treat ketoacidosis.

[0010] The technical solution adopted by the present invention is:

[0011] The present invention first constructed a mouse starvation ketosis model and detected the expression of OXCT1 in various ketone body utilization organs, and found that the expression of OXCT1 did not increase with the increase of ketone body levels. By detecting the Kbhb modification level of OXCT1, we found that with the increase of ketone body levels in the body, the Kbhb modification of OXCT1 was significantly upregulated;

[0012] The Kbhb modification of OXCT1 protein is significantly upregulated in ketotic mice, which makes the Kbhb modification of OXCT1 an indicator for auxiliary diagnosis of ketoacidosis. Therefore, the present invention provides a marker for ketoacidosis, which is the Kbhb modification on lysine 421 of OXCT1 protein.

[0013] At the same time, after the Kbhb modification site (lysine 421) of OXCT1 was mutated, the enzyme activity of OXCT1 was significantly reduced. The gene sequence mutation or deletion of lysine 421 of OXCT1 may be used as a detection target for clinical ketoacidosis susceptibility genes; therefore, the present invention proposes a method for diagnosing ketoacidosis by detecting β-hydroxybutyrylation of the rate-limiting enzyme OXCT1;

[0014] At the same time, the present invention has found that Kbhb modification of lysine 421 of OXCT1 protein mainly regulates the enzyme activity of OXCT1, that is, Kbhb modification of lysine 421 of OXCT1 protein can be used as a therapeutic target for patients with ketoacidosis. Therefore, the present invention proposes to promote Kbhb modification on lysine 421 of OXCT1 protein, such as β-hydroxybutyryl transferase agonist, thereby activating the enzyme activity of OXCT1, and finally reducing the blood ketone level, and applying the treatment of ketoacidosis.

[0015] Beneficial effects of the invention:

[0016] 1. The present invention proposes a biomarker for the diagnosis of ketoacidosis. The gene sequence mutation or deletion of lysine 421 of OXCT1 may be used as a detection target for clinical ketoacidosis susceptibility genes, thereby providing a new direction for the diagnosis and treatment of ketoacidosis. The experimental results also show that the expression of OXCT1 protein does not change significantly with the increase of starvation time, while Kbhb modification is significantly upregulated with the increase of starvation time.

[0017] 2. The present invention proposes a method for diagnosing ketoacidosis by detecting β-hydroxybutyrylation of the rate-limiting enzyme OXCT1. The Kbhb modification of the OXCT1 protein is significantly upregulated in ketotic mice, which makes the Kbhb modification of OXCT1 an indicator for auxiliary diagnosis of ketoacidosis.

[0018] 3. The present invention proposes that Kbhb modification of lysine 421 of OXCT1 protein can be used as a therapeutic target for patients with ketoacidosis. By activating the enzyme activity of OXCT1, the blood ketone level can be reduced, and a new treatment method for ketoacidosis is proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the changes in the weight (A), blood sugar (B), and blood ketone (C) of mice under different starvation time periods; the expression of OXCT1 protein in various ketone body utilization organs of mammals under different starvation treatments, and the changes in the overall Kbhb modification (D) of each organ; the results show that the weight and blood sugar of mice decrease with the extension of fasting time, but the blood ketone level increases with the extension of fasting time. The results also show that the expression of OXCT1 protein does not change significantly with the increase of starvation time, while Kbhb modification is significantly increased with the increase of starvation time, and the Kbhb modification level can be quickly restored after resuming diet.

[0020] Figure 2Shown are the changes in the levels of Kbhb modification of OXCT1 protein in various ketone body utilization organs of mammals under different starvation treatments; the results show that the Kbhb modification level of OXCT1 is significantly upregulated with the increase in starvation time, and is quickly restored after the diet is restored.

[0021] Figure 3 A in the middle shows the potential Kbhb modification site of OXCT1 detected by LC-MS / MS technology; Figure 3 As shown in B, the Kbhb modification sites of OXCT1 were verified by protein immunoprecipitation technology. The results showed that the 185th and 421st lysines of OXCT1 protein were Kbhb modification sites.

[0022] Figure 4 It is shown as follows: constructing a plasmid for the lysine 418 point mutation of OXCT1, and finding that the lysine 418 of OXCT1 is not a Kbhb modification site.

[0023] Figure 5 As shown: the enzyme activities of wild-type, OXCT1 proteins with point mutations at positions 421 and 418 were detected;

[0024] The results showed that when the Kbhb modification of OXCT1 was reduced, the enzymatic activity of OXCT1 was significantly inhibited. At the same time, this inhibition was not caused by the change in protein conformation caused by the point mutation. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0026] The present invention first constructed a mouse starvation ketosis model and detected the expression of OXCT1 in various ketone body utilization organs, and found that the expression of OXCT1 does not increase with the increase of ketone body levels. By detecting the Kbhb modification level of OXCT1, we found that with the increase of ketone body levels in the body, the Kbhb modification of OXCT1 was significantly upregulated; the Kbhb modification of OXCT1 protein was significantly upregulated in ketotic mice, which makes the Kbhb modification of OXCT1 an indicator for auxiliary diagnosis of ketoacidosis.

[0027] Therefore, the present invention provides a marker for ketoacidosis, which is Kbhb modification on lysine 421 of OXCT1 protein. At the same time, after we mutated the Kbhb modification site (lysine 421) of OXCT1, we found that the enzyme activity of OXCT1 was significantly reduced. The gene sequence mutation or deletion of lysine 421 of OXCT1 may be used as a detection target for clinical ketoacidosis susceptibility gene; therefore, the present invention proposes a method for diagnosing ketoacidosis by detecting β-hydroxybutyrylation of the rate-limiting enzyme OXCT1; at the same time, the present invention provides a drug target for treating ketoacidosis.

[0028] Example 1: Kbhb modification of OXCT1 can be used as a diagnostic target for ketoacidosis

[0029] 1. The blood ketone and blood glucose meter was used to detect the changes in blood ketone and blood glucose of mice at different fasting times.

[0030] Methods: Tail blood samples were collected from mice between 8:00 and 10:00 on the day of testing. The collected whole blood samples (5 μl) were immediately dropped onto a test strip and tested using a blood glucose and ketone body monitoring system (FreeStyle Optium Neo).

[0031] Results: Figure 1 As shown in the figure, A, B, and C are the changes in the weight (A), blood sugar (B), and blood ketones (C) of mice under different starvation time periods (the expression of OXCT1 protein in various ketone body utilization organs of mammals under different starvation treatments), as well as the changes in the overall Kbhb modification (D) of each organ. The weight and blood sugar levels of mice decreased significantly with the increase of starvation time, and after resuming the diet, the weight and blood sugar of mice showed a significant correction. The blood ketones of mice increased significantly with the increase of starvation time, and decreased significantly after resuming the diet.

[0032] Analysis of results: During the fasting period, mice had to rely on their own stored energy due to lack of food sources, so their weight dropped rapidly. In the early stage of fasting, the blood sugar level was maintained mainly by consuming liver glycogen. However, as the fasting time prolonged, glycogen was gradually depleted, causing blood sugar levels to drop rapidly. With the depletion of glycogen, fat became the main source of energy. In the liver, fat was broken down and converted into ketone bodies in large quantities to replace blood sugar as the main source of energy, which caused the level of blood ketones in the body to rise rapidly as the fasting time prolonged.

[0033] 2. Western blot was used to detect the expression of OXCT1 protein in different tissues of mice at different fasting times, as well as the overall changes in Kbhb modification.

[0034] Methods: BABL-c mice were dissected at different fasting time periods to obtain brain, heart, liver, kidney, lung and skeletal muscle tissue samples. Each tissue was broken into tissue homogenate using a tissue homogenizer, and then the cells of each component were broken using the cell lysis buffer RIPA. The protein of the cell lysate was quantified under the same conditions, and finally the equal amount of protein samples of each component were detected and analyzed by Western bolt. The specific steps are as follows: After protein electrophoresis separation by SDS-PAGE, the protein was transferred to a PVDF membrane, and the protein was blocked with 5% skim milk; two specific antibodies, OXCT1 and Kbhb, were used to fully bind to it, and then the secondary antibody corresponding to the species of the primary antibody was used to bind it, and then it was exposed and developed.

[0035] Results: The study found that Figure 1 As shown, the expression of OXCT1 in various ketone body utilizing organs does not change with the change of fasting time, but the level of Kbhb modification in various organs increases with the increase of fasting time.

[0036] Analysis of results: Fasting promotes ketone body metabolism in the body, thereby increasing the level of β-HB in the body. As β-HB is the raw material for Kbhb modification, the increase in β-HB content will naturally lead to an increase in the level of Kbhb modification. OXCT1 is a key enzyme in the ketone body utilization pathway. In theory, when the ketone body level in the body increases rapidly, the expression or activity of OXCT1 should increase accordingly. However, we observed that the expression level of OXCT1 does not change with the change of ketone body level, so we speculate that OXCT1 regulates the ketone body level in the body by changing its own activity.

[0037] 3. Protein immunoprecipitation (IP) was used to detect the changes in Kbhb modification of OXCT1 in different tissues of mice at different fasting times. The process is as follows:

[0038] (1) When the cells treated with the experiment have grown to 90%-100%, remove the culture medium, add phosphate buffered saline (PBS), wash three times, and then remove all the solution.

[0039] (2) Freshly prepare NP-40 + PMSF lysis buffer (1:100) and add it to the culture dish containing cells after washing. Use a cell scraper to scrape the cells into the lysis buffer. After blowing the cell-containing lysis buffer evenly, aspirate it into a 5 ml pre-cooled collection tube and perform sonication.

[0040] (3) Before sonication, clean the probe of the cell ultrasonic disruptor with double distilled water, wipe it clean with filter paper, and place the probe in the cell lysis solution (the probe should be suspended in the air and should not touch the wall and bottom of the centrifuge tube). Sonicate each sample at 30% for 5 seconds within one minute and then stop for 5 seconds.

[0041] (4) Add the sonicated liquid into a 1.5 ml microcentrifuge tube and centrifuge at 12,000 rpm for 20 min.

[0042] (5) Take the supernatant after centrifugation and continue centrifugation at 12000 rpm for 5 min.

[0043] (6) After centrifugation, take the supernatant of the sample, separate 100 μl of the protein solution and put it into a new 1.5 ml ep tube (microcentrifuge tube) as input. The remaining protein solution is used as IP sample and the required antibody is added (the content is converted by volume according to the concentration in the instruction manual). All samples are placed on a rotary shaker in a 4°C refrigerator and rotated overnight.

[0044] (7) Wash the beads. Take 500 μl of beads, centrifuge at 3000 rpm for 3 min, remove the supernatant, add 1 ml of NP-40 solution, mix by inversion, put into centrifuge, and centrifuge at 3000 rpm for 3 min. Repeat the washing 4 times. For the last time, put the beads on an ice box, shake horizontally for 10 min, centrifuge again to remove the supernatant, add an equal volume of NP-40 solution to resuspend the beads, and store in a 4°C refrigerator.

[0045] (8) Add 50 μl of Beads to the overnight IP sample, place it in the rotary shaker again, and rotate for 5 h; add 25 μl of 5× loading buffer to the input sample and mix well.

[0046] (9) Precool the high-speed centrifuge, place the IP sample in the precooled centrifuge, centrifuge at 3000 rpm for 3 min, discard the supernatant, add 1 ml of NP-40 solution, invert to mix, place the sample in the centrifuge again, and centrifuge at 3000 rpm for 3 min.

[0047] (10) Repeat step 9 5 times.

[0048] (11) Use a syringe to remove the supernatant (try to remove it completely), add 60 μl of 2× Loading, and mix well; place the IP sample and Input sample in a metal bath at 100°C for 10 min.

[0049] (12) Place the boiled IP sample in a precooled centrifuge and centrifuge at 12,000 rpm for 5 min. Take the supernatant and load it directly or store it at -20°C.

[0050] (13) The samples were verified by Western Blot (same experimental steps as above).

[0051] Results: The Kbhb modification level of OXCT1 in each ketone body utilizing organ increased with the increase of fasting time, and the Kbhb modification of OXCT1 was rapidly restored after the diet was restored. This indicates that the Kbhb modification of OXCT1 may be involved in the regulation of ketone body metabolism and can be used as a diagnostic target for ketoacidosis.

[0052] Figure 2 The results show the changes in the level of Kbhb modification of OXCT1 protein in various ketone body utilization organs of mammals under different starvation treatments; the results show that the body weight and blood glucose of mice decrease with the extension of fasting time, but the blood ketone level increases with the extension of fasting time. The results also show that the expression of OXCT1 protein does not change significantly with the increase of starvation time, and the Kbhb modification level of OXCT1 is significantly increased with the increase of starvation time, and it is quickly restored after the diet is restored.

[0053] Example 2: Markers of ketoacidosis: Kbhb modification sites of OXCT1 are lysine 185 and 421

[0054] 1. Construction of OXCT1 point mutation plasmid

[0055] According to the CDS sequence of OXCT1 found on NCBI, the corresponding PCR primers were designed, and the gene fragment of OXCT1 was obtained from HEK-293T cells using the primers. The fragment was then integrated into the PCDH plasmid vector by molecular cloning technology to obtain the overexpression plasmid of wild-type OXCT1. In the wild-type OXCT1 overexpression plasmid, we mutated adenine A at position 553 to guanine G according to the base sequence of OXCT1, and designed PCR primers at the mutation position to amplify the full-length plasmid, and then the wild-type plasmid was enzymatically digested with Dpn1 enzyme, thereby obtaining the plasmid in which lysine at position 185 of OXCT1 was mutated to arginine. The OXCT1 K421R plasmid was also obtained by the above steps.

[0056] Result analysis: Figure 3 A in the middle shows the potential Kbhb modification site of OXCT1 detected by LC-MS / MS technology; Figure 3Figure B shows the Kbhb modification site of OXCT1 verified by protein immunoprecipitation. Based on mass spectrometry data, we mutated lysine 185 and lysine 421 of OXCT, and further detected the Kbhb modification changes of OXCT1 after point mutation by immunoprecipitation. The results showed that lysine 185 and lysine 421 of OXCT1 protein are potential Kbhb modification sites of OXCT1.

[0057] like Figure 4 As shown, by constructing a lysine 418 point mutation plasmid of OXCT1, it was found that lysine 418 of OXCT1 was not a Kbhb modification site.

[0058] Example 3: Kbhb modification of OXCT1 can enhance the enzymatic activity of OXCT1 and can be used as a therapeutic target for patients with ketoacidosis.

[0059] 1. OXCT1 enzyme activity detection

[0060] (1) Wash the adherent cells 2-3 times with PBS and then dry them;

[0061] (2) Add an appropriate amount of TBST to the culture dish, scrape the cells with a clean cell scraper and transfer them to an EP tube;

[0062] (3) Place the EP tube on ice for 15 to 20 seconds for ultrasonic disruption, then place in a 4°C precooled centrifuge and centrifuge at 15,000 rpm for 10 minutes;

[0063] (4) The enzyme reaction system was prepared according to the following table:

[0064]

[0065] (5) Add 100 μL of cell lysate to 800 μL of enzyme reaction buffer. Set up a group without cells and a control group. Then add acetoacetate (final concentration of 10 mM) to each well to initiate the reaction. Measure the absorbance (OD value) at a wavelength of 313 nm every 2 minutes for 50 minutes.

[0066] Results: When the Kbhb modification site of OXCT1 was mutated, the enzyme activity of OXCT1 was significantly inhibited, indicating that Kbhb modification of lysine 421 of OXCT1 can enhance its enzyme activity. Therefore, Kbhb modification of lysine 421 of OXCT1 can be used as a potential target for the treatment of ketoacidosis.

[0067] like Figure 5As shown: The enzyme activity of wild-type OXCT1 protein and lysine point mutations at positions 421 and 418 were detected; the results showed that when the Kbhb modification of OXCT1 was reduced, the enzyme activity of OXCT1 was significantly inhibited. At the same time, this inhibition was not caused by the change in protein conformation caused by the point mutation.

[0068] The results showed that Kbhb modification of lysine 421 of OXCT1 protein mainly regulated the enzymatic activity of OXCT1. By promoting Kbhb modification on lysine 421 of OXCT1 protein, such as β-hydroxybutyryltransferase agonist, the enzymatic activity of OXCT1 was activated, and finally the blood ketone level was reduced. That is, Kbhb modification of lysine 421 of OXCT1 protein can be used as a therapeutic target for patients with ketoacidosis and applied to the treatment of ketoacidosis.

Claims

1. A marker for ketoacidosis, characterized in that: The marker is Kbhb modification on lysine 421 of OXCT1 protein.

2. Use of the marker according to claim 1 in the diagnosis of ketoacidosis.

3. The use according to claim 2, characterized in that: By detecting the Kbhb modification level of OXCT1, the expression level of OXCT1 in each ketone body utilizing organ was detected as an indicator for auxiliary diagnosis of ketoacidosis.

4. Use of the marker according to claim 1 in the treatment of ketoacidosis.

5. The use according to claim 4, characterized in that: Kbhb modification of lysine 421 of OXCT1 protein is used as a therapeutic target for patients with ketoacidosis and is applied to the treatment of ketoacidosis to reduce blood ketone levels.

6. The marker according to claim 1 is used in the preparation of a diagnostic reagent or kit for ketoacidosis.

7. The diagnostic reagent or kit according to claim 6, characterized in that: Kbhb modification of OXCT1 is used as a detection target for clinical ketoacidosis susceptibility genes. By detecting the expression level of OXCT1 in ketone body-utilizing organs, indicators for auxiliary diagnosis of ketoacidosis are obtained.

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