A biomarker for β-hydroxybutyrylation modification of OXCT1 and its application in the diagnosis and treatment of ketoacidosis.

Abstract

This invention belongs to the field of biochemistry, and specifically relates to a β-hydroxybutyrylation modification biomarker of OXCT1 and its application in the diagnosis and treatment of ketoacidosis. A biomarker for ketoacidosis is a β-hydroxybutyrylation modification of lysine 421 on the OXCT1 protein. This biomarker is used in the diagnosis of ketoacidosis by detecting the Kbhb modification level of OXCT1 and the expression level of OXCT1 in various ketone body utilizing organs, serving as an auxiliary diagnostic indicator for ketoacidosis. In the treatment of ketoacidosis, the Kbhb modification of lysine 421 on the OXCT1 protein is used as a therapeutic target for patients with ketoacidosis, and can be applied to the treatment of ketoacidosis to reduce blood ketone levels. This invention proposes a new diagnostic and treatment method for ketoacidosis. The biomarker can also be used to prepare diagnostic reagents or kits for ketoacidosis.

Description

Technical Field

[0001] This invention relates to a biomarker, belonging to the field of biochemistry, and in particular to a method for detecting β-hydroxybutyrylation of ketone bodies using the rate-limiting enzyme OXCT1 and its application in the diagnosis and treatment of ketoacidosis. Background Technology

[0002] Ketoacidosis is generally defined as a primary decrease in plasma bicarbonate concentration caused by an increase in blood ketones. Based on its precipitating factors, ketoacidosis can be classified into diabetic ketoacidosis (DKA), normoglucagonous ketoacidosis, and alcoholic ketoacidosis (AKA). DKA is a relatively common medical emergency, often caused by a decrease in circulating insulin concentration and a concurrent increase in counter-regulatory hormones (catecholamines, glucagon, growth hormone, and cortisol). This type of acidosis often has a rapid onset and rapid progression, posing a serious threat to the patient's life and health. Therefore, research into the molecular mechanisms of ketoacidosis pathogenesis and the development of target therapies are of great significance for the treatment of metabolic diseases such as diabetes.

[0003] Currently, the initial diagnosis of ketoacidosis mainly relies on ketone test strips and arterial blood gas analysis. Ketone test strips are primarily used to detect ethyl acetate in urine and, to a lesser extent, acetone. However, this method has a high false-positive rate because it cannot detect β-hydroxybutyrate. Arterial blood gas analysis is also limited in widespread use due to the high cost of equipment and the difficulty of obtaining arterial blood samples. Furthermore, current clinical treatments for ketoacidosis primarily involve fluid resuscitation and glucose control, with limited research into its molecular mechanisms and no drugs targeting ketone body metabolism currently in clinical use. Therefore, discovering new specific targets for ketoacidosis is crucial for its early diagnosis and precise treatment.

[0004] OXCT1 (3-oxoacid CoA-transferase 1), also known as succinyl-CoA:3-ketoacid CoA transferase 1 or SCOT (succinyl-CoA:3-ketoacid CoA transferase), 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 acetoacetic acid, a product of catalytic isomerization of ketone bodies (mainly β-HB), in extrahepatic tissues, to form acetoacetyl-CoA, which ultimately enters 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 a unidirectional reaction, making it the rate-limiting enzyme in this process. The distribution of the rate-limiting enzyme OXCT1 protein corresponds to the breakdown and utilization of ketone bodies in the body. It is abundant in the heart, brain, and kidneys, and can be detected in all extrahepatic tissues. However, the expression of OXCT1 protein is suppressed in liver tissue, which may be a mechanism to prevent the inefficient cycling of ketone body energy in liver tissue. In addition to its involvement in ketone body catabolism, the function of the rate-limiting enzyme OXCT1 in tumorigenesis and development, as well as cardiovascular diseases, has also attracted much attention.

[0005] β-Hydroxybutyrate (β-HB), also known as D-3-hydroxybutyrate, accounts for approximately 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, replacing glucose as a fuel source in the brain, but it is also used as an alternative energy source for glucose in all extrahepatic tissues. Besides serving as 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, a novel post-translational modification of protein mediated by β-HB in its activated thioester form, β-hydroxybutyryl A—lysine β-hydroxybutyrylation (Kbhb)—was first discovered. Subsequent studies have shown that Kbhb modification occurs not only on histones but is also widely present in cytoplasmic proteins such as those in the cytoplasm and mitochondria. Meanwhile, mass spectrometry database searches revealed that both HMGCS2, a key enzyme in mouse ketone body synthesis, and OXCT1, a key enzyme in ketone body utilization, can undergo β-HB-mediated Kbhb after translation.

[0006] In our work, we demonstrated through in vivo and in vitro animal and cellular molecular experiments that the OXCT1 protein is modified with lysine β-hydroxybutyrylation. Through the construction of gene mutants at specific sites, we confirmed that the Kbhb modification at the K421 lysine of OXCT1 can significantly affect the enzymatic catalytic activity of OXCT1, thereby regulating the accumulation of ketone bodies at the cellular or whole-body 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 K421 modification level and the development of related acyltransferase activators may help prevent and treat clinical diabetic ketoacidosis. Summary of the Invention

[0008] Based on the background art, this invention proposes a biomarker for the diagnosis of ketoacidosis, and based on the biomarker, the expression level of OXCT1 in each organ utilizing ketone bodies is obtained by detecting the β-hydroxybutyrylation of the rate-limiting enzyme OXCT1 of ketone body utilization, which can be applied to the diagnosis of ketoacidosis.

[0009] Meanwhile, this invention activates the enzyme activity of OXCT1 by promoting Kbhb modification of lysine at position 421 of the OXCT1 protein, such as β-hydroxybutyryltransferase agonists, thereby ultimately reducing blood ketone levels and can be used to treat ketoacidosis.

[0010] The technical solution adopted in this invention is as follows:

[0011] This invention first established a mouse model of starvation ketosis and examined the expression levels of OXCT1 in various organs utilizing ketone bodies. It was found that OXCT1 expression did not increase with rising ketone body levels. However, by examining the Kbhb modification level of OXCT1, we discovered that Kbhb modification of OXCT1 was significantly upregulated with increasing ketone body levels.

[0012] Kbhb modification of the OXCT1 protein is significantly upregulated in ketotic mice, making it a potential indicator for the auxiliary diagnosis of ketoacidosis. Therefore, this invention provides a biomarker for ketoacidosis, namely, Kbhb modification at lysine position 421 of the OXCT1 protein.

[0013] Meanwhile, mutation of the Kbhb modification site (lysine 421) of OXCT1 significantly reduced its enzyme activity. Mutations or deletions in the lysine 421 gene sequence of OXCT1 may serve as detection targets for clinical ketoacidosis susceptibility genes; therefore, this invention proposes a method for diagnosing ketoacidosis by detecting β-hydroxybutyrylation of the rate-limiting enzyme OXCT1.

[0014] Meanwhile, this invention has discovered that Kbhb modification of lysine 421 of the OXCT1 protein primarily regulates the enzyme activity of OXCT1, meaning that Kbhb modification of lysine 421 of the OXCT1 protein can serve as a therapeutic target for patients with ketoacidosis. Therefore, this invention proposes activating the enzyme activity of OXCT1 by promoting Kbhb modification of lysine 421 of the OXCT1 protein, such as with β-hydroxybutyryltransferase agonists, thereby ultimately reducing blood ketone levels and applying this method to the treatment of ketoacidosis.

[0015] Beneficial effects of the invention:

[0016] 1. This invention proposes a biomarker for the diagnosis of ketoacidosis. A mutation or deletion in the 421st lysine residue of the OXCT1 gene sequence may serve as a detection target for clinical ketoacidosis susceptibility genes, thus providing a new direction for the diagnosis and treatment of ketoacidosis. Experimental results also show that the expression level of OXCT1 protein does not change significantly with increasing starvation time, while Kbhb modification is significantly upregulated with increasing starvation time.

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

[0018] 3. This invention proposes that the Kbhb modification of lysine 421 of the OXCT1 protein can serve as a therapeutic target for patients with ketoacidosis. By activating the enzyme activity of OXCT1, blood ketone levels can be reduced, thus proposing a new treatment method for ketoacidosis. Attached Figure Description

[0019] Figure 1 The figures show the changes in body weight (A), blood glucose (B), and blood ketones (C) in mice under different fasting periods; the expression of OXCT1 protein in various ketone body utilization organs of mammals under different fasting treatments; and the overall changes in Kbhb modification (D) in each organ. The results show that mouse body weight and blood glucose decreased with prolonged fasting time, but blood ketone levels increased with prolonged fasting time. The results also show that the expression level of OXCT1 protein did not change significantly with the increase of fasting time, while Kbhb modification was significantly upregulated with the increase of fasting time, and the Kbhb modification level recovered rapidly after the resumption of diet.

[0020] Figure 2The figure shows the changes in Kbhb modification levels 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 increasing starvation time and is rapidly restored after the resumption of diet.

[0021] Figure 3 Figure A shows the potential Kbhb modification sites of OXCT1 detected by LC-MS / MS technology; Figure 3 Figure B shows the Kbhb modification sites of OXCT1 verified by protein immunoprecipitation. The results show that lysine residues at positions 185 and 421 of the OXCT1 protein are Kbhb modification sites.

[0022] Figure 4 The image shows the construction of a point mutant plasmid for 418 lysine in OXCT1. It was found that 418 lysine in OXCT1 is not a Kbhb modification site.

[0023] Figure 5 The image shows the enzyme activity of wild-type, 421st and 418th lysine point mutations in OXCT1 protein.

[0024] The results showed that when the Kbhb modification of OXCT1 was reduced, the enzyme activity of OXCT1 was significantly inhibited. Furthermore, this inhibition was not due to conformational changes caused by point mutations. Detailed Implementation

[0025] The invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0026] This invention first established a mouse model of starvation ketosis and examined the expression levels of OXCT1 in various organs utilizing ketone bodies. It was found that OXCT1 expression did not increase with rising ketone body levels. However, by examining the Kbhb modification level of OXCT1, we discovered that Kbhb modification of OXCT1 was significantly upregulated with increasing ketone body levels. The significant upregulation of Kbhb modification of OXCT1 protein in ketotic mice suggests that Kbhb modification of OXCT1 can serve as an auxiliary indicator for the diagnosis of diabetic ketoacidosis.

[0027] Therefore, this invention provides a biomarker for ketoacidosis, namely, the Kbhb modification at lysine 421 of the OXCT1 protein. Furthermore, mutations at the Kbhb modification site (lysine 421) of OXCT1 resulted in a significant decrease in OXCT1 enzyme activity. Mutations or deletions in the lysine 421 gene sequence of OXCT1 may serve as detection targets for clinical ketoacidosis susceptibility genes; therefore, this invention proposes a method for diagnosing ketoacidosis by detecting β-hydroxybutyrylation of the rate-limiting enzyme OXCT1; simultaneously, this invention provides a drug target for treating ketoacidosis.

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

[0029] 1. A blood ketone glucometer was used to detect changes in blood ketones and blood glucose in mice under different fasting periods.

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

[0031] Result: As Figure 1 As shown in the figure, A, B, and C represent the changes in body weight (A), blood glucose (B), and blood ketones (C) of mice under different starvation periods (expression of OXCT1 protein in various ketone body utilization organs of mammals under different starvation treatments), and the overall changes in Kbhb modification (D) in each organ, respectively. Mouse body weight and blood glucose levels decreased significantly with increasing starvation time, but rebounded significantly after resuming feeding. Blood ketones in mice increased significantly with increasing starvation time and decreased significantly after resuming feeding.

[0032] Results Analysis: During the fasting period, mice, lacking food sources, relied solely on their stored energy, resulting in rapid weight loss. Initially, they maintained blood glucose levels primarily by consuming liver glycogen. However, as fasting progressed, glycogen was gradually depleted, leading to a rapid drop in blood glucose levels. With glycogen depletion, fat became the primary energy source. In the liver, large amounts of fat were broken down and converted into ketone bodies to replace blood glucose as the main energy source, causing a rapid increase in blood ketone levels as fasting continued.

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

[0034] Methods: BABL-c mice were dissected at various fasting periods to obtain brain, heart, liver, kidney, lung, and skeletal muscle tissue samples. Each tissue was homogenized using a tissue homogenizer, and then the cells in each component were lysed using RIPA cell lysis buffer. Protein quantification of the cell lysis buffer was performed under the same conditions. Finally, equal volumes of protein samples from each component were analyzed using Western blot. Specific steps were as follows: After protein separation by SDS-PAGE, the proteins were transferred to a PVDF membrane and blocked with 5% skim milk. The proteins were then fully bound to the membrane with two specific antibodies, OXCT1 and Kbhb, followed by binding with a secondary antibody corresponding to the primary antibody species, and then exposed for development.

[0035] Results: The study found that, Figure 1 As shown, OXCT1 levels in various organs utilizing ketone bodies do not change with fasting time, but the level of Kbhb modification in each organ increases with increasing fasting time.

[0036] Results Analysis: Fasting promotes ketone body metabolism, thereby increasing the level of β-HB in the body. Since β-HB is a raw material for Kbhb modification, an increase in β-HB content naturally leads to an increase in Kbhb modification levels. OXCT1 is a key enzyme in the ketone body utilization pathway. Theoretically, when ketone body levels rise rapidly, the expression level or activity of OXCT1 should increase accordingly. However, we observed that the expression level of OXCT1 did not change with the change in ketone body levels. Therefore, we hypothesize that OXCT1 regulates ketone body levels in the body by altering its own activity.

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

[0038] (1) When the cells treated in the experiment grow to 90%-100%, remove the culture medium, add phosphate buffer solution (PBS), wash 3 times, and remove the solution.

[0039] (2) Prepare fresh NP-40+PMSF lysis buffer (1:100) and add it to a culture dish containing cells after cleaning. Use a cell scraper to scrape the cells into the lysis buffer, blow the lysis buffer containing cells evenly, and then aspirate it into a 5ml pre-cooled collection tube for sonication.

[0040] (3) Before sonication, clean the probe of the cell sonicator with double-distilled water, wipe it clean with filter paper, and put the probe into the cell lysis buffer (the probe should be suspended and should not touch the centrifuge tube wall and bottom). Sonicate each sample for 5 seconds at 30% for one minute, and stop for 5 seconds.

[0041] (4) Add the sonicated liquid to a 1.5 ml microcentrifuge tube and centrifuge at 12000 rpm for 20 min.

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

[0043] (6) Take the supernatant of the centrifuged sample, separate 100 μl of protein solution and put it into a new 1.5 ml ep tube (microcentrifuge tube) as input. Add the required antibody to the remaining protein solution as IP sample (the content is calculated according to the concentration in the instruction manual). Put all samples into a rotating shaker at 4℃ and rotate overnight.

[0044] (7) Wash the beads: Take 500 μl of beads, centrifuge at 3000 rpm for 3 min, discard the supernatant, add 1 ml of NP-40 solution, invert and mix well, place in a centrifuge, and centrifuge at 3000 rpm for 3 min. Repeat the washing process 4 times. For the last wash, place the beads on an ice box, shake horizontally for 10 min, centrifuge again to discard the supernatant, add an equal volume of NP-40 solution to resuspend the beads, and store in a 4℃ refrigerator.

[0045] (8) Add 50 μl of Beads to the overnight IP sample and put it back into the rotary shaker for 5 h; add 25 μl of 5×loading Buffer to the Input sample and mix well.

[0046] (9) Pre-cool the high-speed centrifuge, put the IP sample into the pre-cooled centrifuge, centrifuge at 3000 rpm for 3 min, discard the supernatant, add 1 ml of NP-40 solution, invert and mix well, put it into the centrifuge again, and centrifuge at 3000 rpm for 3 min.

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

[0048] (11) Use a syringe to remove the supernatant (as clean as possible), add 2×Loading 60μl, and mix well; put the IP sample and Input sample into a metal bath at 100℃ for 10min.

[0049] (12) After boiling, the IP sample is placed in a pre-cooled centrifuge and centrifuged at 12,000 rpm for 5 min. The supernatant can be directly loaded or stored at -20℃.

[0050] (13) The sample was validated by Western Blot (the experimental steps are the same as above).

[0051] Results analysis: The Kbhb modification level of OXCT1 in various ketone body utilizing organs increased with increasing fasting time, and the Kbhb modification of OXCT1 rapidly recovered upon resumption of diet. This suggests that Kbhb modification of OXCT1 may be involved in the regulation of ketone body metabolism and could serve as a diagnostic target for ketoacidosis.

[0052] Figure 2 The results show the changes in Kbhb modification levels of OXCT1 protein in various ketone body utilization organs of mammals under different starvation treatments. The results indicate that mouse body weight and blood glucose decreased with prolonged fasting time, but blood ketone levels increased with prolonged fasting time. The results also show that the expression level of OXCT1 protein did not change significantly with increasing starvation time; the Kbhb modification level of OXCT1 was significantly upregulated with increasing starvation time and rapidly recovered after the resumption of feeding.

[0053] Example 2: Biomarkers of ketoacidosis: The Kbhb modification sites of OXCT1 are lysine residues at positions 185 and 421.

[0054] 1. Constructing point mutant plasmids for OXCT1

[0055] Based on the CDS sequence of OXCT1 obtained from NCBI, corresponding PCR primers were designed. Using these primers, the OXCT1 gene fragment was obtained from HEK-293T cells. This fragment was then integrated into a PCDH plasmid vector using molecular cloning techniques to obtain an overexpression plasmid of wild-type OXCT1. In the wild-type OXCT1 overexpression plasmid, adenine A was mutated to guanine G at position 553 according to the OXCT1 base sequence. PCR primers were designed at the mutation site to amplify the full-length plasmid. Subsequently, the wild-type plasmid was digested with Dpn1 enzyme, resulting in a plasmid with a lysine-arginine mutation at position 185 of OXCT1. The OXCT1 K421R plasmid was also obtained using the same steps.

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

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

[0058] Example 3: Kbhb modification of OXCT1 can enhance the enzyme activity of OXCT1, which can serve as a therapeutic target for patients with ketoacidosis. This can be applied to the treatment of ketoacidosis.

[0059] 1. Enzyme activity detection of OXCT1

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

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

[0062] (3) Place the EP tube on ice and ultrasonically break it for 15 to 20 seconds, then place it in a pre-cooled centrifuge at 4°C and centrifuge at 15,000 rpm for 10 minutes.

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

[0064]

[0065] (5) Add 100 μL of cell lysis buffer 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 313 nm wavelength every 2 minutes for 50 minutes.

[0066] Results analysis: Mutation of the Kbhb modification site of OXCT1 significantly inhibited its enzyme activity, indicating that Kbhb modification of lysine 421 of OXCT1 can enhance its enzyme activity. Therefore, Kbhb modification of lysine 421 of OXCT1 may serve as a potential target for the treatment of ketoacidosis.

[0067] like Figure 5The results show that the enzyme activity of wild-type OXCT1 protein with point mutations at positions 421 and 418 was detected. The results indicate that the enzyme activity of OXCT1 was significantly inhibited when the Kbhb modification of OXCT1 was reduced. Furthermore, this inhibition was not due to conformational changes caused by the point mutation.

[0068] The results showed that Kbhb modification of lysine 421 of the OXCT1 protein mainly regulates the enzyme activity of OXCT1. By promoting Kbhb modification of lysine 421 of the OXCT1 protein, such as β-hydroxybutyryltransferase agonists, the enzyme activity of OXCT1 is activated, ultimately reducing blood ketone levels. Therefore, Kbhb modification of lysine 421 of the OXCT1 protein can serve as a therapeutic target for patients with ketoacidosis and can be applied to the treatment of ketoacidosis.

Claims

1. The application of a reagent for detecting biomarkers in the preparation of diagnostic reagents or kits for ketoacidosis, characterized in that: The marker is the OXCT1 protein modified with Kbhb at lysine position 421.

2. The application according to claim 1, characterized in that: The OXCT1 protein modified with lysine Kbhb at position 421 serves as a detection target for clinical ketoacidosis susceptibility genes. By detecting the expression level of OXCT1 in ketone body utilizing organs, an indicator for the auxiliary diagnosis of ketoacidosis can be obtained.

3. The application as described in claim 1, in the diagnosis of ketoacidosis.

4. The application according to claim 3, characterized in that: By detecting the level of OXCT1 protein modified by Kbhb at lysine 421, the expression level of OXCT1 in various ketone body utilizing organs was determined, serving as an indicator for the auxiliary diagnosis of ketoacidosis.

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