Application of NAT10-ac4C-mitochondrial-acetyl-CoA regulation axis in diagnosis and treatment of mitochondrial dysfunction and related metabolic diseases

Abstract

The invention discloses an application of an NAT10-ac4C-mitochondrial-acetyl-CoA regulation axis in diagnosis and treatment of mitochondrial dysfunction and related metabolic diseases, and belongs to the technical field of biology. The regulatory axis systematically regulates the expression of the nuclear coding mitochondrial gene in a physiological state, and maintains a metabolism steady state; the invention also provides a biomarker combination of the regulatory axis. The biomarker combination comprises NAT10 related indexes, ac4C modification level of nuclear coding mitochondrial gene mRNA and acetyl coenzyme A level, and can be used for diagnosis and prognosis evaluation of mitochondrial dysfunction and related metabolic diseases. Meanwhile, the invention further provides a drug screening method, a treatment method and related drug application based on the regulation axis, systematic diagnosis and universal treatment of mitochondrial dysfunction related diseases are achieved, and the regulation axis has important clinical transformation value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to the application of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis in the diagnosis and treatment of mitochondrial dysfunction and related metabolic diseases. BACKGROUND

[0002] Mitochondria, as the core hub of cellular energy generation and metabolic regulation, its functional imbalance is the common pathological basis of many diseases, including metabolic syndrome, neurodegenerative diseases, cardiovascular diseases and cancer. Mitochondria not only provides energy for cells through oxidative phosphorylation (OXPHOS), but also participates in multiple processes such as tricarboxylic acid cycle, fatty acid oxidation, amino acid metabolism and reactive oxygen species homeostasis. Although mitochondria has its own genome (mtDNA), more than 99% of the proteins required by mitochondria are encoded by the nucleus and need to be imported into mitochondria after transcription and translation, so the bidirectional signal exchange and precise coordination between the nucleus and mitochondria is the fundamental prerequisite for maintaining the metabolic homeostasis of cells and organisms. However, the molecular mechanism of nuclear-mitochondrial interaction is still lacking systematic elucidation, especially the mechanism of post-transcriptional epigenetic regulation in the coordinated expression of nuclear-encoded mitochondrial genes is still unclear.

[0003] With the rise of epitranscriptomics research, the diversification of chemical modifications on RNA molecules has been proven to play a core role in post-transcriptional gene regulation, constituting an important epigenetic regulation layer after DNA methylation and histone modification. In addition to the common N6-methyladenosine (m6A) and 5-methylcytosine (m5C), N4-acetylcytidine (N4-acetylcytidine, ac4C) as a highly conserved and biologically functional diverse RNA modification has gradually attracted widespread attention. N-acetyltransferase 10 (NAT10) is the only known "writing" enzyme in eukaryotic cells that can catalyze RNA ac4C modification, which is mainly located in the nucleus and is the core molecule for the study of the biological function of ac4C modification.

[0004] Current research on NAT10 mainly focuses on pathological states, especially in the field of metabolic reprogramming of cancer cells. For example, NAT10 stabilizes FOXP1, SEPT9 and other oncogene messenger RNAs (mRNAs) through ac4C modification, thereby enhancing glycolytic flux and promoting tumor growth; in addition, NAT10 deletion is reported to affect fatty acid metabolism and oxidative phosphorylation levels. In non-alcoholic fatty liver disease and myocardial ischemia-reperfusion injury models, sporadic studies have suggested that NAT10 regulates the expression of individual metabolism-related genes through ac4C modification.

[0005] Under normal physiological conditions, NAT10 can modify mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA), thereby regulating RNA stability and translation efficiency, and plays an important role in cell division, DNA damage response, protein synthesis, and cell aging. However, these functions are mostly focused on cell proliferation and stress response, and the role of NAT10 in maintaining mitochondrial function, metabolic homeostasis, and nuclear-mitochondrial interaction is still lacking in clear research. There is currently a lack of direct evidence on whether and how NAT10 modifies ac4C to systematically coordinate the expression of nuclear-encoded mitochondrial genes and regulate mitochondrial metabolic function.

[0006] In addition, the existing technology has obvious limitations in the metabolic regulation of NAT10. Related research mostly focuses on local metabolic changes under pathological conditions (such as cancer metabolic reprogramming and abnormal immune metabolism of T cells), lacking a systematic understanding of the global metabolic network under normal physiological conditions. Existing research only sporadically mentions "NAT10→ac4C modification" "NAT10→ACLY→acetyl-CoA" "acetyl-CoA→mitochondrial respiration" and other single links, failing to link the three into a complete regulatory network, and has not constructed a complete functional map of NAT10 at the multi-organ and multi-metabolic pathway level. There is still a lack of experimental evidence on whether NAT10 acts as a "metabolic command hub" to maintain whole-body energy and metabolic homeostasis.

[0007] In terms of mechanism research, existing literature mostly presents a "point discovery" pattern, i.e., under specific pathological conditions, NAT10 is revealed to regulate one or several specific genes, or acetyl-CoA affects a single function of mitochondria (such as the respiratory chain), but whether NAT10 can act as a core regulatory node to widely coordinate the expression and function of the entire nuclear-encoded mitochondrial genome (including oxidative phosphorylation, the tricarboxylic acid cycle, fatty acid oxidation, and all key pathways) through ac4C modification, and whether acetyl-CoA participates in this regulatory process to form a feedback loop, still lacks direct evidence and mechanistic explanation, which severely restricts the overall understanding of the molecular map of mitochondrial homeostasis regulation.

[0008] In terms of application transformation, due to the lack of understanding of the basic role of NAT10 in physiological metabolic regulation, existing technology only explores the potential of NAT10 as a cancer treatment target or the application of acetyl-CoA as a mitochondrial function detection indicator, and has not been expanded to the diagnosis and treatment of non-cancer and systemic metabolic diseases (such as primary mitochondrial disease, non-alcoholic fatty liver disease, and neurodegenerative disease). At the same time, existing research has not proposed multi-dimensional combination diagnostic markers based on NAT10, ac4C modification, and acetyl-CoA, nor has it designed treatment strategies to "enhance the function of NAT10-related regulatory pathways", resulting in a single and limited intervention means for mitochondrial systemic dysfunction.

[0009] Further, since Nat10 systemic knockout causes embryonic lethality, researchers cannot study its physiological role in metabolic homeostasis of adult individuals at the whole animal level. Existing research mainly relies on in vitro cell lines or tissue organ-specific Nat10 knockout models (such as T cell-specific knockout), lacking a system model that can reveal the integrated regulation of NAT10 in multiple tissues and multiple pathways, making it difficult to verify the regulatory effect of NAT10 on whole body metabolic homeostasis and the overall effect of related intervention strategies.

[0010] Therefore, it is urgent to break through the limitation of existing research which is only limited to pathological models or single target genes, and to establish a research system based on systematic metabolomics, nuclear-mitochondrial transcriptome integrated analysis and multi-tissue specific operation model, to reveal how NAT10 accurately regulates the expression of nuclear encoded mitochondrial genes through ac4C modification under physiological conditions, thereby maintaining energy metabolism and mitochondrial homeostasis. Based on this mechanism, it is expected to create a new diagnosis and treatment strategy for mitochondrial systemic dysfunction, and to provide new molecular targets and ideas for the intervention of metabolic diseases. SUMMARY

[0011] In view of the above shortcomings of the prior art, the present application aims to provide a new use of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis in regulating mitochondrial function and metabolic homeostasis.

[0012] The first object of the present application is to provide the use of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis in regulating mitochondrial function and metabolic homeostasis.

[0013] In a preferred embodiment, the use is to enhance the activity of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis by using a NAT10 agonist and / or a metabolic substrate that can increase the level of intracellular acetyl-CoA, thereby improving mitochondrial energy metabolism and cellular homeostasis.

[0014] In a further preferred embodiment, the metabolic substrate that can increase the level of intracellular acetyl-CoA includes acetyl-L-carnitine, citrate or pyruvate.

[0015] The second object of the present application is to provide a biomarker combination for diagnosing or prognostically evaluating mitochondrial dysfunction and related metabolic diseases, the biomarker combination comprising at least one of the following:

[0016] (a) a biomarker selected from at least one of the following: NAT10 enzyme activity, mRNA expression level of NAT10 gene, expression level of NAT10 protein; (b) a set of ac4C modification levels on nuclear-encoded mitochondrial gene mRNAs; (c) acetyl-CoA levels in cells, tissues or body fluids.

[0017] In preferred embodiments, the tissue is liver tissue, spleen tissue, intestinal tissue or heart tissue obtained by biopsy.

[0018] In preferred embodiments, the acetyl-CoA is detected by mass spectrometry.

[0019] In preferred embodiments, the mitochondrial dysfunction and related metabolic diseases include primary mitochondrial diseases and metabolic diseases characterized by secondary mitochondrial dysfunction caused by mutations in the NAT10 gene, reduced enzyme activity or down-regulated expression.

[0020] In further preferred embodiments, the metabolic diseases characterized by secondary mitochondrial dysfunction include non-alcoholic fatty liver disease, heart failure, Alzheimer's disease, Parkinson's disease, sarcopenia, type 2 diabetes, obesity or metabolic syndrome.

[0021] A third object of the present application is to provide a kit for diagnosing or prognosticating mitochondrial dysfunction and related metabolic diseases, which detects the expression levels of the combination of biomarkers in a sample.

[0022] A fourth object of the present application is to provide a method for screening candidate drugs for enhancing the function of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis, comprising the following steps: S1, constructing a reaction system: providing a reaction system comprising NAT10 full-length protein or a functional protein fragment comprising the acetyltransferase active domain of NAT10, acetyl-CoA and RNA substrate; S2, candidate compound incubation: adding the compound to be screened to the reaction system in step S1; S3, ac4C modification level detection: detecting the change in ac4C modification level in the reaction system, and the compound that significantly enhances the generation of ac4C is the potential drug.

[0023] A fifth object of the present application is to provide a use of a compound for enhancing the function of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis in the preparation of a drug for preventing or treating mitochondrial dysfunction and related metabolic diseases.

[0024] In preferred embodiments, the compound is selected from those that increase the acetyltransferase activity of NAT10, promote the formation of ac4C modification or enhance the level of acetyl-CoA.

[0025] In preferred embodiments, the dosage form of the drug comprises oral preparations, injection preparations, or targeted delivery preparations.

[0026] In further preferred embodiments, the drug is used to improve metabolic disorders, mitochondrial energy metabolism disorders, or multiple organ failure caused by decreased NAT10 activity or down-regulated ac4C modification levels.

[0027] Compared with the prior art, the present application has the following beneficial effects: 1. Scientific theory breakthrough: The present application first reveals the basic physiological function of NAT10 from the perspective of "global regulation", discovers a brand-new metabolic regulation axis, which is essential for maintaining whole-body metabolic homeostasis and survival in physiological conditions, and its disorder leads to multiple organ failure and energy depletion, breaking the local limitations of existing research and achieving global cognition of metabolic regulation.

[0028] 2. Mechanism system and depth: The present application reveals that NAT10 systemically regulates the nuclear-encoded mitochondrial gene network, including the electron transport chain (ETC), the tricarboxylic acid cycle (TCA), fatty acid oxidation (FAO), and other pathways, rather than only regulating individual genes. The regulation axis contains a unique feedback loop, i.e., its functional product acetyl-CoA is also its driving force, and the destruction of this loop is the key to causing metabolic collapse. The present application not only reveals the systemic regulation of NAT10 on the mitochondrial gene network, but also elucidates the precise feedback loop formed by NAT10 and the core metabolite acetyl-CoA, and the mechanism research is more in-depth and extensive.

[0029] 3. Application strategy universality and forward-looking: The diagnosis and treatment strategy of the present application based on the regulation axis is applicable to all diseases caused by the systematic imbalance of the regulation axis, providing a universal platform. By enhancing the function of the NAT10-ac4C-mitochondria-acetyl-CoA regulation axis, the systemic recovery of mitochondrial energy metabolism and redox homeostasis can be achieved, thereby realizing the universal intervention effect on various metabolic-related diseases. The present application does not provide a solution for a single disease, but a diagnosis and treatment platform that can be widely applied to a large class of mitochondrial dysfunction-related diseases, which has great clinical transformation potential and market value.

[0030] 4. Technical solution completeness and reliability: From in vivo models to multi-omics integration, to molecular mechanism verification, a complete evidence chain is formed, providing solid data support for the broad protection of the claims. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1Figure 1 is a Nat10 knockout mouse phenotype verification and result graph; in the figure, A is an experimental flow chart of tamoxifen-induced whole-body NAT10 gene knockout in mice; B is a NAT10 protein detection chart of intestinal tissue of Nat10-cKO and wild-type (WT) mice after tamoxifen induction; C is a chart of body weight change of Nat10-cKO female mice (n=5) compared with WT control (n=7) after induction (Note: consistent phenotype was observed in male mice); D is a survival rate chart of Nat10-cKO female mice; E is a tissue section staining (H&E) chart of Nat10-cKO female mice; F is a serum liver biochemical index chart of Nat10-cKO mice; G is a kidney biochemical index chart of Nat10-cKO mice; H is a heart biochemical index chart of Nat10-cKO mice.

[0032] Figure 2 Figure 2 is a chart of the effect of NAT10 deletion on nuclear-encoded mitochondrial gene expression; in the figure, A is a KEGG pathway enrichment analysis chart of NAT10 deletion widely inhibiting OXPHOS and thermogenic pathways in liver, spleen and rectum; B is a Venn diagram of OXPHOS and thermogenic pathway genes commonly down-regulated in three tissues; C is a digital PCR (dPCR) detection chart of key ETC mRNA levels in mouse liver; D is a flow cytometry detection chart of six representative ETC proteins in mouse liver; E is a chart of average fluorescence intensity of the proteins in Figure D.

[0033] Figure 3 Figure 3 is a chart of the results of NAT10 stabilizing nuclear-encoded mitochondrial mRNA through ac4C modification; in the figure, A is a KEGG pathway enrichment chart of differentially down-regulated ac4C-associated genes in WT and Nat10-cKO mouse liver ac4C-seq; B is an integrated analysis chart of OXPHOS and thermogenic pathway-enriched genes by RNA sequencing (RNA-seq) and ac4C high-throughput sequencing (ac4C-seq); C is an RNA acetylation immunoprecipitation-quantitative PCR (acRIP-qPCR) chart of mRNA ac4C modification of specific ETC genes; D is a chart of ETC mRNA stability in WRL68 human normal hepatocyte lines with NAT10 knockdown; E is a chart of polysomal analysis of ETC gene mRNA in NAT10 knockdown hepatocytes.

[0034] Figure 4Figure 8 is a result diagram of NAT10 deletion leading to mitochondrial dysfunction and energy depletion; in the figure, A is a transmission electron microscope image of Nat10-cKO mouse hepatocytes; B is a transmission electron microscope image of Nat10-cKO mouse cardiomyocytes; C is a flow cytometry histogram of reactive oxygen species (ROS) levels in Nat10-cKO mouse hepatocytes and cardiomyocytes; D is a flow cytometry histogram of mitochondrial mass in Nat10-cKO mouse hepatocytes and cardiomyocytes; E is a flow cytometry histogram of mitochondrial membrane potential in Nat10-cKO mouse hepatocytes and cardiomyocytes; and F is an adenosine triphosphate (ATP) content diagram of Nat10-cKO mouse hepatocytes and cardiomyocytes.

[0035] Figure 5 Figure 9 is a result diagram of NAT10 deletion disrupting acetyl-CoA metabolic homeostasis; in the figure, A is a cluster diagram of differentially expressed genes in the liver of NAT10 knockout mice in lipid, sugar, amino acid, and other metabolic pathways; B is a differentially expressed diagram of lipid metabolism products in the liver of WT and Nat10-cKO mice; C is a diagram of key product contents in the glycolysis pathway of liver tissues of WT and Nat10-cKO mice; D is a diagram of representative amino acid contents in liver tissues of WT and Nat10-cKO mice; E is a diagram of acetyl-CoA contents in liver tissues of WT and Nat10-cKO mice; F is a diagram of mRNA expression of key enzymes (such as Acly, Acss2, Acaca, and Fasn) for acetyl-CoA synthesis and metabolism in the cytoplasm and nucleus in the liver of WT and Nat10-cKO mice; and G is a diagram of protein expression of ACLY, ACSS2, ACACA, and FASN in the liver of Nat10-cKO mice. DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further described and illustrated by the following examples. The raw materials used in the examples can be commercially available or prepared by conventional methods.

[0037] Example 1 The animal model verifies that NAT10 deletion leads to mouse multi-organ injury, down-regulation of nuclear-encoded mitochondrial gene expression, mitochondrial dysfunction, and metabolic disorder, including the following steps: 1. Animal model construction: as shown in A-D of Figure 1 , A in Figure 1 is a flow chart of tamoxifen-induced knockout of NAT10 gene in mice: 6-week-old, littermate-paired WT and NAT10 conditional knockout mice (Nat10-cKO) Nat10 fl / fl are selected, and tamoxifen (Sigma-Aldrich, item number T5648) is intraperitoneally injected at a dose of 100 mg / kg based on body weight, continuously for 5 days, and the body weight change of the mice is continuously monitored during the treatment. Figure 1In the figure, B represents protein samples prepared from mouse intestinal tissue after tamoxifen induction. The samples were then tested with a NAT10-specific antibody (Abclonal, catalog number 19286), which confirmed that NAT10 was successfully knocked out. Figure 1 The C-value in the figure shows that after tamoxifen induction, the body weight of Nat10-cKO female mice (n=5) was significantly reduced from day 8 after induction compared with WT control (n=7) (Note: the same phenotype was observed in male mice). Figure 1 The D in the study showed that tamoxifen induction significantly reduced the survival rate of female Nat10-cKO mice (Note: a consistent phenotype was observed in male mice).

[0038] 2. Histopathological analysis: such as Figure 1 As shown in E, WT and Nat10-cKO mice induced with tamoxifen for 9 days were anesthetized with avertin at a dose of 30 μL / g, and euthanized after blood collection from the eyeballs. Major organs (including liver, spleen, heart, rectum, and kidneys) were harvested, fixed in 4% paraformaldehyde (PFA), embedded in paraffin, and histologically sectioned. After staining with hematoxylin and eosin (H&E), they were observed under a microscope. The results showed that the liver, spleen, heart, intestines, and kidneys of Nat10-cKO mice exhibited significant pathological damage. Specific phenotypes included portal hyperplasia in the liver, disordered red and white pulp structure in the spleen, disordered arrangement of myofibrils in the heart muscle, damage to intestinal villi, and glomerular hypertrophy with inflammatory cell infiltration.

[0039] 3. Blood biochemical index analysis: Figure 1 Mice induced by tamoxifen with FH on day 9 were anesthetized with afotin at a dose of 30 uL / g, and blood was collected via ocular sampling. The collected blood was centrifuged at 6000 rpm and 4℃ for 10 minutes to separate serum, which was then sent to a third-party testing institution for biochemical analysis. The results showed that multiple organ function-related indicators were significantly elevated in the serum of Nat10-cKO mice, including liver function-related aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bile acids (TBA); kidney function-related creatinine (CREA) and blood urea nitrogen (BUN); and the cardiac stress marker creatine kinase (CK) level.

[0040] 4. Mitochondrial gene expression profiling: such as Figure 2 As shown in AB, WT and Nat10-cKO mice induced by tamoxifen on day 9 were anesthetized with afodin at a dose of 30 μL / g and then sacrificed. Liver, spleen and rectal tissues were then harvested and sent to a third-party testing institution for RNA-seq. Figure 2A in FIG. 6 is a KEGG pathway enrichment analysis showing that among the differentially expressed genes in the liver, spleen and rectal tissues of the Nat10-cKO mice, the OXPHOS and thermogenesis pathways are the two most significantly down-regulated pathways. Figure 2 B in FIG. 6 is a Venn diagram showing the OXPHOS and thermogenesis pathway genes commonly down-regulated in the three tissues, which are all nucleus-encoded ETC complex-related genes.

[0041] 5. Mitochondrial gene dPCR detection: Figure 2C in the table is the dPCR verification result. The specific method is as follows: after the WT and Nat10-cKO mice on the 9th day of tamoxifen induction are anesthetized with avermectin at a dose of 30 μL / g and sacrificed, the liver tissues are taken out, total RNA is extracted, and a third-party testing company is invited to detect the mRNA level of key ETC complex subunit genes (including Ndufa5, Gene ID: 68202; Sdhb, Gene ID: 67680; Uqcrb, Gene ID: 67530; Cox5a, Gene ID: 12858; Atp5a1, Gene ID: 11946) by dPCR.The primer sequences used in dPCR are shown in SEQ ID NO. 1~15: Ndufa5-F: 5'-TTGAGCGGGCTTGGGAAAG-3' (SEQ ID NO. 1); Ndufa5-R: 5'-ACCCCACCAGGCCAGTTGT-3' (SEQ ID NO. 2); Ndufa5-P: 5'-FAM-TCAGCAAGCCCGCCATGACAG-BHQ1-3' (SEQ ID NO. 3); Sdhb-F: 5'-CCATGAACATCAACGGAGGC-3' (SEQ ID NO. 4); Sdhb-R: 5'-GCGTAGAAGTTACTCAAATCAGGGA-3' (SEQ ID NO. 5); Sdhb-P: 5'-VIC-ACTCTGGCGTGCACACGCAGGA-BHQ1-3' (SEQ ID NO. 6); Uqcrb-F: 5'-GATCTTGCCTAAGGATCAGTGGAC-3' (SEQ ID NO. 7); Uqcrb-R: 5'-CTTTGCCCACTCTTCTCTCTCC-3' (SEQ ID NO. 8); Uqcrb-P: 5'-CY5-ATATGAGGAGGACAAATTCTACCTTGAACC-BHQ2-3' (SEQ ID NO. 9); Cox5a-F: 5'-TGATCTGGTTCCTGAGCCCAA-3' (SEQ ID NO. 10); Cox5a-R: 5'-ATGACATAGGGATAGATTTCCTTATGAG-3' (SEQ ID NO. 11); Cox5a-P: 5'-FAM-CTGCATTGCGAGCATGTAGACG-BHQ1-3' (SEQ ID NO. 12); Atp5a1-F: 5'-GTTCCAAGACCCGCAGACGA-3' (SEQ ID NO. 13); Atp5a1-R: 5'-GGGCACCAGGCTATCCACA-3' (SEQ ID NO. 14); Atp5a1-P: 5'-VIC-CTGAAAGCCCCTGGAATTATCCC-BHQ1-3' (SEQ ID NO. 15). The results show that the mRNA levels of Ndufa5, Sdhb, Uqcrb, Cox5a and Atp5a1 in the liver of Nat10-cKO mice are significantly decreased.

[0042] 6. Mitochondrial ETC protein analysis: Figure 2D in the figure represents the flow cytometry detection of ETC protein levels. The specific steps are as follows: WT and Nat10-cKO mice induced by tamoxifen on day 9 were anesthetized with afodin at a dose of 30 μL / g and then sacrificed. Liver tissue was isolated and placed in a 5 mL EP tube. 1 mL of tissue digestion solution (PBS + 0.5 mg / mL collagenase IV [Yeasen, catalog number 40510ES60] + 0.25 mg / mL DNase I [Roche, catalog number 10104159001]) was added. The tissue was minced with scissors, and another 3 mL of tissue digestion solution was added. The mixture was incubated at 37 ℃ and 150 rpm for 30 minutes with shaking. The digested liver tissue was filtered through a 70 μm filter and washed with PBS. The filtrate was collected into a 15 mL centrifuge tube and centrifuged at 1200 rpm at room temperature for 5 minutes. The supernatant was discarded. An appropriate amount of erythrocyte lysis buffer was added for erythrocyte lysis treatment, followed by centrifugation to remove the supernatant. The tissue was then resuspended in 2 mL PBS. After cell counting, take 2 × 10⁻⁶ cells. 6 Cells were used for flow cytometry staining. After fixation and transmembrane transposition (BD Pharmingen, catalog number 554714), primary antibodies (NDUFA5 antibody, Proteintech, catalog number 16640-1-AP; NDUFS5 antibody, Proteintech, catalog number 15224-1-AP; SDHB antibody, Proteintech, catalog number 10620-1-AP; UQCRB antibody, Proteintech, catalog number 10756-1-AP; COX5A antibody, Proteintech, catalog number 11448-1-AP; ATPB antibody, Proteintech, catalog number 17247-1-AP) were added, and an IgG negative control was included. After staining the sample on ice in the dark for 1 hour, wash and centrifuge (5000 rpm, 5 minutes), discard the supernatant and gently disperse the precipitate, add secondary antibody (Goat anti-Raabit F488 antibody, Thermo Scientific, catalog number A-11008), stain on ice in the dark for 30 minutes, wash repeatedly, resuspend in PBS, and finally perform instrument detection. Figure 2 E in the figure represents the statistical average fluorescence intensity of NDUFA5, NDUFS5, SDHB, UQCRB, COX5A, and ATPB proteins.

[0043] 7. Elucidation of the ac4C modification mechanism: such as Figure 3 As shown in A–E, the NAT10-mediated ac4C modification mechanism was systematically validated. The specific steps were as follows: WT and Nat10-cKO mice induced with tamoxifen on day 9 were anesthetized with afodin at a dose of 30 μL / g and then sacrificed. Liver tissue was rapidly harvested and flash-frozen in liquid nitrogen. The samples were then sent to a third-party testing institution for ac4C-seq and data analysis.Figure 3 A in FIG. 6 is KEGG pathway enrichment analysis of differentially down-regulated ac4C-related genes in ac4C-seq results, and the results show that the down-regulated genes are mainly enriched in oxidative phosphorylation (OXPHOS) and thermogenic pathways. Figure 3 B in FIG. 6 is integrated analysis of enriched genes in OXPHOS and thermogenic pathways by RNA-seq and ac4C-seq, and the results show that the intersection genes of the two are ETC-related genes. Figure 3C is acRIP-qPCR verification of ac4C modification. The specific method is: take two 50 μg of total RNA sample, each take 1 / 50 volume as input, the remaining RNA is added to 500 μL acRIP immunoprecipitation buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH=7.4, containing RNase inhibitor) mixed for 15 minutes. Then, one group is added with 1 μg of anti-ac4C antibody (abcam, ab252215), the other group is added with the same amount of IgG antibody (abcam, ab172730) as a negative control, incubated at 4°C overnight. After incubation, 35 μL of protein G Dynabeads magnetic beads (1 μg of antibody corresponds to 300 μg of magnetic beads, the magnetic beads are pre-washed with IP buffer for 3 times) is added to each group of samples, and incubated at 4°C for 2 hours. Then, wash 6 times with IP buffer, each time thoroughly mixed to remove non-specific binding. After washing, 500 μL of Trizol reagent (Invitrogen, 15596026) is added to extract RNA, and after reverse transcription (Takara, Reverse Transcriptase M-MLV(Rnase H-), 2641B), qRT-PCR analysis of mRNA levels of key ETC complex subunit genes (including Ndufa5, Gene ID: 68202; Sdhb, Gene ID: 67680; Uqcrb, Gene ID: 67530; Cox5a, Gene ID: 12858; Atp5b, Gene ID: 11947) is performed.The reagents used for qRT-PCR (Vazyme, Catalog No. Q712) and the primer sequences used are shown in SEQ ID NO. 16~25: Ndufa5-F: 5'-ATGGCGGGCTTGCTGAAAA-3' (SEQ ID NO. 16); Ndufa5-R: 5'-GCTGCATGTTTAGGAAAGTGCTT-3' (SEQ ID NO. 17); Sdhb-F: 5'-AATTTGCCATTTACCGATGGGA-3' (SEQ ID NO. 18); Sdhb-R: 5'-AGCATCCAACACCATAGGTCC-3' (SEQ ID NO. 19); Uqcrb-F: 5'-GGCCGATCTGCTGTTTCAG-3' (SEQ ID NO. 20); Uqcrb-R: 5'-CATCTCGCATTAACCCCAGTT-3' (SEQ ID NO. 21); Cox5a-F: 5'-GCCGCTGTCTGTTCCATTC-3' (SEQ ID NO. 22); Cox5a-R: 5'-GCATCAATGTCTGGCTTGTTGAA-3' (SEQ ID NO. 23); Atp5b-F: 5'-GGTTCATCCTGCCAGAGACTA-3' (SEQ ID NO. 24); Atp5b-R: 5'-AATCCCTCATCGAACTGGACG-3' (SEQ ID NO. 25); and the qRT-PCR reaction system is as follows.

[0044] The qRT-PCR amplification procedure used is as follows:

[0045] The results show that ac4C modification of specific ETC gene mRNA is reduced in the liver of Nat10-cKO mice.

[0046] 8. mRNA stability analysis of key mitochondrial ETC genes Figure 3D is mRNA stability experiment, in human normal liver cell line WRL68, using specific small interfering RNA (siRNA) knockdown NAT10 (Gene ID: 55226). The target nucleotide sequence of NAT10 siRNA used is as follows: 5'-CUGAUAACCUCCAUACUCU-3'(SEQ ID NO. 26). After transfection of WRL68 cells with siRNA for 48 hours, actinomycin D (Actinomycin D) was added to a final concentration of 5 ug / mL to block continuous transcription, and cells were collected at different time points. Each sample was added with 500 μL Trizol reagent (Invitrogen, Cat. No. 15596026) to extract total RNA, and 1 ug of RNA was reverse transcribed into cDNA for qRT-PCR detection. The key ETC genes detected are as follows: NDUFA5 gene, Gene ID: 4698, qRT-PCR primer sequences are shown in SEQ ID NO. 27~28; SDHB gene, Gene ID: 6390, qRT-PCR primer sequences are shown in SEQ ID NO. 29~30; UQCRB gene, Gene ID: 7381, qRT-PCR primer sequences are shown in SEQ ID NO. 31~32; COX5A gene, Gene ID: 9377, qRT-PCR primer sequences are shown in SEQ ID NO. 33~34; ATP5B gene, Gene ID: 506, qRT-PCR primer sequences are shown in SEQ ID NO. 35~36.

[0047] NDUFA5-F: 5'-GCGGGTGTGCTGAAGAAGA-3' (SEQ ID NO. 27); NDUFA5-R: 5'-TTCCGCTTTAACCATAGCCAG-3' (SEQ ID NO. 28); SDHB-F: 5'-ACAGCTCCCCGTATCAAGAAA-3' (SEQ ID NO. 29); SDHB-R: 5'-GCATGATCTTCGGAAGGTCAA-3' (SEQ ID NO. 30); UQCRB-F: 5'-GGTAAGCAGGCCGTTTCAG-3' (SEQ ID NO. 31); UQCRB-R: 5'-AGGTCCAGTGCCCTCTTAATG-3' (SEQ ID NO. 32); COX5A-F: 5'-AGCTTACACCGGCTTCTCTC-3' (SEQ ID NO. 33); COX5A-R: 5'-CCCATGGGAATAGCAGCGAA-3' (SEQ ID NO. 34); ATP5B-F: 5'-GGTGAGAGGACCCGTGAAG-3' (SEQ ID NO. 35); ATP5B-R: 5'-CGCTACCTTAGAGGTGGCAT-3' (SEQ ID NO. 36); qRT-PCR reaction system and amplification procedure Figure 3 C is the same. The test results showed that after the NAT10 gene was knocked down, the mRNA degradation rate of NDUFA5, NDUFS5, SDHB, UQCRB, COX5A and ATP5B was significantly accelerated.

[0048] 9. Translation efficiency analysis of key mitochondrial ETC gene mRNAs: Figure 3 E in the figure represents polyribosome analysis. The specific steps are as follows: After transfecting WRL68 cells with NAT10 siRNA (sequence shown in SEQ ID NO. 26) for 48 hours, cyclohexylimide (CHX) was added to a final concentration of 100 μg / mL, and the cells were incubated at 37 °C for 10 min. The cells were then washed three times with pre-chilled PBS, and the cells were collected in 1.5 mL RNase-free EP tubes. The cells were centrifuged at 3000 g at 4 °C for 5 min, and the supernatant was discarded. 1 mL of ribosome lysis buffer (10 mM Tris-HCl, pH 7.4; 5 mM MgCl2; 100 mM KCl; 1% Triton X-100; 100 μg / mL CHX; 1 mM DTT; 1× protease inhibitor mixture; 40 U / mL RNasin) was added to resuspend the lysed cells and incubate on ice for 10 min. The cells were then centrifuged at 1300 g at 4 °C for 10 minutes, and the supernatant was collected, with 1 / 10 of the volume used as input. A 5%–50% sucrose gradient (5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%) was prepared, and the cells were separated into layers according to their concentration from highest to lowest (1 mL per layer). Cell lysis buffer was placed at the top of the gradient, and lysis buffer was added to the appropriate volume. The cells were then ultracentrifuged at 164000 × g at 4 °C for 4 hours. After centrifugation, the supernatant was carefully discarded. 1 mL of each sucrose gradient layer was collected, and RNA was extracted and analyzed by qRT-PCR (using the same detection method as above). Figure 3The results showed that the proportion of mRNA of NDUFA5, SDHB, UQCRB, COX5A and ATP5B genes in the polysome fraction was significantly reduced in the NAT10 knockdown cells.

[0049] 10. Mitochondrial structure detection: as shown in Figure 4 A-B, transmission electron microscopy was performed on mitochondria of WT and Nat10-cKO mice on tamoxifen induction day 9. The specific method is as follows: the WT and Nat10-cKO mice on tamoxifen induction day 9 were sacrificed after being anesthetized with Avertin at a dose of 30 μL / g, and the liver and heart tissues were cut (the sampling time was controlled within 1-3 minutes), and immediately placed in 5% glutaraldehyde fixing solution, and fixed at room temperature for 2 hours, and then transferred to 4°C for storage. The samples were sent to a third party for electron microscopy sample preparation and detection. The results of transmission electron microscopy showed that the mitochondria of hepatocytes of Nat10-cKO mice were swollen, and the mitochondrial structure of myocardial cells was broken.

[0050] 11. Mitochondrial function verification: as shown in C-E of Figure 4 , flow cytometry was used to detect mitochondrial function indicators. The specific method is as follows: the WT and Nat10-cKO mice on tamoxifen induction day 9 were sacrificed after being anesthetized with Avertin at a dose of 30 μL / g, and the mouse liver and heart cells were separated, and the liver cell separation method was the same as that in Figure 2 D, the difference in the separation method of heart cells was that collagenase II (Yeasen, item number 40508ES60) was used for tissue digestion. After cell counting, 2x10 6 cells were taken for each staining analysis. Figure 4 C of is mitochondrial ROS staining, and the specific steps are as follows: the liver and heart cells of WT and Nat10-cKO mice were centrifuged (1200 rpm, room temperature for 5 min) and the supernatant was discarded, and the cells were dispersed by tapping the bottom of the tube, and 100 μL of MitoSOX™ Red working solution (Thermo Scientific, item number M36009) with a concentration of 5 μM was added, and after resuspension, it was incubated at 37°C in a cell incubator for 30 min. 500 μL of RPMI 1640 (Gibico, item number C11875500BT) complete culture medium was added and mixed, centrifuged (1200 rpm, room temperature for 5 min) and the supernatant was discarded, and then resuspended with 200 μL of RPMI 1640 complete culture medium and detected on the machine; Figure 4D in FIG. 6 is mitochondrial mass staining, and the specific steps are as follows: after the cells are treated by the same centrifugation step, 100 μL of MitoTracker™ Deep Red dye (Thermo Scientific, item number M22426) with a concentration of 500 nM is added, and after resuspension, the cells are incubated at 37°C in a cell incubator for 30 min. Then 500 μL of RPMI 1640 complete medium is added for mixing, and after centrifugal washing, the cells are resuspended with 200 μL of RPMI 1640 complete medium for machine analysis; Figure 4 E in FIG. 6 is mitochondrial membrane potential staining, and the specific steps are as follows: 1 portion of liver and heart cells of WT and Nat10-cKO mice are taken respectively, and the supernatant is removed by centrifugation. As a negative control group, 100 μL of FCCP solution (MCE, item number HY-100410) with a concentration of 20 μM is added to the cells, and the cells are incubated at 37°C for 15 min. After incubation, 500 μL of RPMI 1640 complete medium is added for mixing and centrifugal washing, and the supernatant is discarded. Then, 100 μL of TMRE dye working solution (Thermo Scientific, item number T669) with a concentration of 2 μM is added to each portion of cells for resuspension, and the cells are incubated at 37°C for 30 min. The experimental group cells are not pretreated with FCCP, and the rest of the TMRE staining steps are the same. After incubation, 500 μL of RPMI 1640 complete medium is added again for mixing, and after centrifugal washing, the supernatant is discarded. The cells are resuspended with 200 μL of RPMI 1640 complete medium, and used for machine analysis by flow cytometry; Figure 4 F is the detection of ATP level in cells, and the specific steps are as follows: liver and myocardial cells derived from WT and Nat10-cKO mice are inoculated in a 96-well plate at a density of 5×10 4 cells / well (100 μL volume per well), and then an equal volume of CellTiter-Glo ® Reagent (Promega, item number G7570) is added, mixed by shaking for 2 min to lyse the cells, and then incubated at room temperature for 10 min to stabilize the luminescence signal. Finally, the fluorescence intensity is detected on an enzyme-labeled instrument. The comprehensive results show that the mitochondrial ROS level of Nat10-cKO mice is increased, the mitochondrial mass is increased, the membrane potential is changed, and the ATP content is significantly decreased. Figure 5 A in FIG. 6 shows that the RNA-seq differential genes of NAT10 knockout mice are enriched in lipid, sugar, and amino acid metabolism pathways. As Figure 5 B-F in FIG. 6 are the detection of metabolomics of mouse liver tissues, and the specific method is as follows: after the WT and Nat10-cKO mice induced by tamoxifen for 9 days are anesthetized by Avertin at a dose of 30 μL / g and sacrificed, the livers are quickly frozen in liquid nitrogen and transported to a third-party detection institution on dry ice for metabolomics detection.Figure 5 B in FIG. 16C shows the statistics of differential lipid metabolites in Nat10-cKO mouse liver; Figure 5 C in FIG. 16C shows that the levels of glycolysis pathway metabolites such as glucose, fructose-6-phosphate, pyruvate, etc. in Nat10-cKO mouse liver tissues are reduced; Figure 5 D in FIG. 16C shows that the amino acid content in Nat10-cKO mouse liver tissues is abnormal; Figure 5 E in FIG. 16C shows that the level of acetyl-CoA in Nat10-cKO mouse liver tissues is significantly reduced.

[0051] 13. mRNA level detection of key enzymes for acetyl-CoA synthesis and metabolism in cytoplasm and nucleus: Figure 5 F in FIG. 16F is qRT-PCR detection, and the specific method is as follows: after the WT and Nat10-cKO mice induced by tamoxifen for 9 days were anesthetized by avermectin at a dose of 30 μL / g and sacrificed, about 50-100 mg of liver tissue was taken, 1000 μL of Trizol reagent (Invitrogen, item number 15596026) was added, and the tissue grinding tube containing magnetic beads was ground thoroughly to extract total RNA. After concentration and purity detection, reverse transcription reagent (Takara, Reverse Transcriptase M-MLV (RNase H-), item number 2641B) was used to synthesize cDNA. The obtained cDNA was used as a template for qRT-PCR detection. The detected genes include Acly (Gene ID: 104112), and the qRT-PCR primer sequences are shown as SEQ ID NO. 37~38; Acss2 (Gene ID: 60525), and the qRT-PCR primer sequences are shown as SEQ ID NO. 39~40; Acaca (Gene ID: 107476), and the qRT-PCR primer sequences are shown as SEQ ID NO. 41~42; Fasn (Gene ID: 14104), and the qRT-PCR primer sequences are shown as SEQ ID NO. 43~44; Acly-F: 5'-CAGCCAAGGCAATTTCAGAGC-3' (SEQ ID NO. 37); Acly-R: 5'-CTCGACGTTTGATTAACTGGTCT-3' (SEQ ID NO. 38); Acss2-F: 5'-CACCTTCTGGCAAACAGAAAC-3' (SEQ ID NO. 39); Acss2-R: 5'-CTACACCGAAGAATGGGAAAGA-3' (SEQ ID NO. 40); Acaca-F: 5'-ACATTCCGAGCAAGGGATAAG-3' (SEQ ID NO. 41); Acaca-R: 5'-GGGATGGCAGTAAGGTCAAA-3' (SEQ ID NO. 42); Fasn-F: 5'-GGAGGTGGTGATAGCCGGTAT-3' (SEQ ID NO. 43); Fasn-R: 5'-TGGGTAATCCATAGAGCCCAG-3' (SEQ ID NO. 44); qRT-PCR amplification system, reaction conditions are consistent with Figure 3 C.

[0052] The results show that the mRNA levels of Acly, Acss2, Acaca, Fasn, key enzymes of acetyl-CoA synthesis and metabolism in the liver of Nat10-cKO mice are down-regulated.

[0053] 14. Protein level detection of acetyl-CoA synthesis and metabolism key enzymes in cytoplasm and nucleus: Figure 5 G is a Western Blot detection. The specific method is as follows: after the WT and Nat10-cKO mice induced by tamoxifen for 9 days were anesthetized by Avertin at a dose of 30 μL / g and sacrificed, 1 small piece of liver tissue was taken, added into 1 mL RIPA lysis buffer (Bi Yun Tian, product number P0038) for grinding, and then centrifuged at 12000 rpm, 4 ℃ for 10 minutes. The supernatant was taken and added into SDS loading buffer (BioRad, product number 1610747), and then boiled at 100 ℃ for 15 minutes before protein electrophoresis separation. After electrophoresis, the membrane was transferred and blocked, and the following primary antibodies were added for incubation: ACLY antibody (Proteintech, product number 15421-1-AP), ACSS2 antibody (Proteintech, product number 16087-1-AP), ACACA antibody (Proteintech, product number 21923-1-AP), FASN antibody (Cell Signaling Technology, product number 3180), and β-actin (Cell Signaling Technology, product number 5125S), and incubated at 4 ℃ overnight. The next day, after washing with TBST for 3 times, horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (H+L) secondary antibody (Bi Yun Tian, product number A0208) was added, and incubated at room temperature for 1 hour. After washing with TBST for 3 times again, the development detection was performed. The results show that the protein levels of ACLY, ACSS2, ACACA and FASN in the liver of Nat10-cKO mice are significantly decreased.

[0054] 15. Gender universality: It is worth noting that the systemic pathological phenotype, mitochondrial dysfunction and related metabolic disorders triggered by the above-mentioned deletion of Nat10 are consistently observed in both male and female mice, indicating that the physiological function of NAT10 in maintaining metabolic homeostasis is not significantly gender-dependent, further establishing its value as a broad-spectrum therapeutic target.

[0055] Example 2 The present embodiment provides a kit for diagnosing a mitochondrial dysfunction-related disease, comprising a comprehensive biomarker detection system capable of detecting multi-level indicators of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis; the kit comprises the following components: Nucleic acid detection module: for detecting the mRNA expression level of NAT10 gene, comprising: lysis solution and purification reagent for RNA extraction, reverse transcription reagent for reverse transcription, specific primer pair for amplification of NAT10 gene, optional qRT-PCR or dPCR detection system; the Gene ID of the NAT10 gene is: 55226, and the specific primer pair of the NAT10 gene is as shown in SEQ ID NO. 45~46: NAT10-F: 5'-GGATTGCCTCAACATCACTCGG-3' (SEQ ID NO. 45); NAT10-R: 5'-CGTTGGAGGAAAACTTCAGAGGC-3' (SEQ ID NO. 46); Protein detection module: for detecting the expression level of NAT10 protein and its acetyltransferase activity state, comprising: an ELISA plate pre-coated with a NAT10 protein specific antibody, a specific primary antibody for immunoblotting or a fluorescence-labeled antibody specific for flow cytometry, and a detection reagent suitable for ELISA, immunoblotting or flow cytometry.

[0056] RNA acetylation modification detection module: for detecting the ac4C modification level of mRNA of nuclear-encoded mitochondrial ETC-related genes, comprising: ac4C specific antibody (abcam, catalog number ab252215), RNA immunoprecipitation buffer system (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH=7.4), quantitative detection primers for representative genes of ETC complexes I-V (such as NDUFA5, SDHB, UQCRB, COX5A, ATP5B) (qRT-PCR primers for mouse genes, such as SEQ ID. 16~25; or qRT-PCR primers for human genes, such as SEQ ID. 27~36); Metabolite detection module: for quantitative detection of acetyl-CoA level, including: tissue or cell extract pretreatment reagent, acetyl-CoA standard and calibration curve, solvent system suitable for liquid chromatography-mass spectrometry (LC-MS) detection; Positive and negative control system: for result comparison and data calibration, to ensure the accuracy and repeatability of the detection results.

[0057] The kit can be used to detect the NAT10 enzyme activity, mRNA and protein expression level, ac4C modification level of representative ETC gene mRNA and acetyl-CoA content in the sample of a clinical subject (including patients suspected of non-alcoholic fatty liver disease, heart failure or mitochondrial disease); through multi-level biomarker joint analysis, comprehensive diagnosis and risk assessment of mitochondrial dysfunction and related metabolic diseases are realized.

[0058] Based on the positive correlation between the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis discovered in Example 1 and mitochondrial function, this embodiment also provides a multi-level, systematic application method of the diagnostic kit for diseases related to mitochondrial dysfunction, including the following steps: S1. Sample collection: collect blood samples (peripheral blood mononuclear cells or plasma can be separated) of subjects (such as patients suspected of non-alcoholic fatty liver disease, heart failure or mitochondrial disease), or obtain tissue samples of liver, spleen, intestine, heart, etc. through biopsy; S2. RNA / protein / metabolite extraction: total RNA, total protein or metabolites are extracted from the sample using a commercial kit.

[0059] S3. Detection: the following detection schemes can be used alone or in combination: Scheme one (core regulatory factor level): detect the enzyme activity of NAT10 by enzyme activity test kit; and / or detect the mRNA or protein expression level by qRT-PCR, dPCR, Western Blot, flow cytometry or ELISA; compared with healthy controls, decreased NAT10 enzyme activity or significantly down-regulated expression level of NAT10 indicates impaired NAT10 function and increased risk of mitochondrial dysfunction; Scheme two (core epitranscriptome modification level): detect the ac4C modification level on the mRNA of a group of representative nuclear-encoded mitochondrial genes (for example, at least three genes selected from ETC complex I-V, such as NDUFA5, SDHB, UQCRB, COX5A, ATP5B) by acRIP-qPCR (using ac4C specific antibody) or ac4C-seq; compared with detecting a single gene, the combined ac4C level of this group can more reliably reflect the system state of the mitochondrial gene network, and a decreased level indicates impaired NAT10 function; Scheme 3 (Core metabolic node level - precise diagnosis scheme): Given that acetyl-CoA is the core metabolic node of the regulatory axis disclosed in the present application, its level can directly reflect the integrated functional state of the axis. By using a high-sensitivity method (such as liquid chromatography-mass spectrometry), acetyl-CoA in tissue biopsy samples (such as liver, muscle, heart) or plasma is quantified. The measured level is compared with the reference range of the healthy control population. A significant decrease in acetyl-CoA level provides the most direct metabolite level evidence for impaired mitochondrial metabolic function. This scheme is particularly suitable for precise diagnosis of difficult cases, evaluation of pharmacodynamic biomarkers in drug clinical trials; S4. Result judgment: compare the detection results of any one of the above schemes or combined schemes with the reference range of the healthy population established in advance, thereby diagnosing, typing or risk assessing the subject. Joint detection of multiple levels of markers can significantly improve the accuracy and reliability of diagnosis.

[0060] Example 3 Based on the verified NAT10 enzyme activity function and its key role in mitochondrial health in Example 1, this embodiment provides a method for screening candidate drugs for enhancing the function of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis, comprising the following steps: 1. Expression and purification: express and purify the tagged recombinant human NAT10 full-length protein or functional protein fragment containing the acetyltransferase active domain of NAT10 in E. coli or insect cell system; the amino acid sequence of the NAT10 full-length protein is shown in SEQ ID NO. 47; the amino acid sequence of the functional protein fragment containing the acetyltransferase active domain of NAT10 is shown in SEQ ID NO. 48: Amino acid sequence of human NAT10 acetyltransferase active domain (SEQ ID NO. 48): RLMALYVASHYKNSPNDLQMLSDAPAHHLFCLLPPVPPTQNALPEVLAVIQVCLEGEISRQSILNSLSRGKKASGDLIPWTVSEQFQDPDFGGLSGGRVVRIAVHPDYQGMGYGSRALQLLQMYYEGRFPCLEEKVLETPQEIHTVSSEAVSLLEEVITPRKDLPPLLLKLNERPAERLDYLGVSYGLTPRLLKFWKRAGFVPVYLRQTPNDLTGEHSCIMLKTLT; 2. Establishing in vitro acetylation reaction system: containing acetyl-CoA, purified NAT10 full-length protein or functional protein fragment containing NAT10 acetyltransferase active domain, and RNA substrate; The RNA substrate can be: (1) an in vitro transcribed or synthesized RNA fragment containing the NAT10 preferred recognition motif "CCGGGCAGAG", or (2) a mixture of RNAs containing multiple ac4C modification sites of different mitochondrial genes, including but not limited to: NDUFA5 (Gene ID: 4698), SDHB (Gene ID: 6390), UQCRB (Gene ID: 7381), COX5A (Gene ID: 9377), ATP5B (Gene ID: 506); by reacting in the above system, the acetylation modification activity of NAT10 on the mitochondrial nuclear encoded gene network can be simulated; 3. Adding candidate compounds: adding the small molecule compound library to be screened into the reaction system; 4. Detecting enzyme activity: after the reaction is completed, extract the RNA, and detect the amount of ac4C generated by Dot Blot or LC-MS / MS; compared with the control group without adding compounds, the compounds with significantly increased ac4C production are the NAT10 agonist candidates.

[0061] Example 4 Based on the disease phenotype caused by the deletion of NAT10 confirmed in Example 1, this embodiment provides the application of the NAT10-ac4C-mitochondria-acetyl-CoA regulatory axis in preventing or treating mitochondrial dysfunction and related metabolic diseases, including the following steps: 1. Drug preparation: preparing a pharmaceutical composition containing a NAT10 agonist (obtained by screening according to the method described in Example 3) or an acetyl-CoA precursor (such as acetyl-L-carnitine); 2. A method of treatment: administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition to improve mitochondrial function, treat diseases such as nonalcoholic fatty liver disease, heart failure, and the like.

[0062] It should be understood that the above examples are only used to illustrate the content of the present application but not to limit the scope of protection of the present application. Furthermore, it should be understood that after reading the content of the present application, those skilled in the art can make various modifications or changes to the present application, and these equivalent forms also fall within the scope defined by the claims of the present application.

Claims

1. Application of the NAT10–ac4C–mitochondrial–acetyl-CoA regulatory axis in the diagnosis or prognostic assessment of mitochondrial dysfunction and related metabolic diseases.

2. The application according to claim 1, characterized in that, The combination of biomarkers for the diagnosis or prognostic assessment of mitochondrial dysfunction and related metabolic diseases includes at least one of the following: (a) A biomarker selected from at least one of the following: NAT10 enzyme activity, mRNA expression level of the NAT10 gene, and expression level of the NAT10 protein; (b) The level of ac4C modification on a set of nuclear-encoding mitochondrial gene mRNAs; (c) Acetyl-CoA levels in cells, tissues or body fluids.

3. The application according to claim 2, characterized in that, The tissues referred to are liver tissue, spleen tissue, intestinal tissue, or heart tissue obtained through biopsy.

4. The application according to claim 2, characterized in that, The detection of acetyl-CoA was performed by mass spectrometry.

5. The application according to claim 2, characterized in that, The aforementioned mitochondrial dysfunction and related metabolic diseases include primary mitochondrial diseases or secondary mitochondrial dysfunctions characterized by mutations in the NAT10 gene, decreased enzyme activity, or downregulation of its expression.

6. A kit for diagnosing or prognostically assessing the aforementioned mitochondrial dysfunction and related metabolic diseases, characterized in that, The kit detects the expression level of the combination of biomarkers as described in claim 2 in a sample.

7. A method for screening candidate drugs to enhance the function of the NAT10–ac4C–mitochondrial–acetyl-CoA regulatory axis as described in claim 1, comprising the following steps: S1. Constructing the reaction system: Provide a reaction system containing the full-length NAT10 protein or a functional protein fragment containing the NAT10 acetyltransferase active domain, Acetyl-CoA, and RNA substrate; S2, Candidate Compound Incubation: Add the compound to be screened to the reaction system in step S1; S3, ac4C modification level detection: Detecting changes in RNA ac4C modification levels in the reaction system; compounds that significantly enhance ac4C generation are candidate drugs.

8. A drug that enhances the function of the NAT10–ac4C–mitochondrial–acetyl-CoA regulatory axis as described in claim 1, characterized in that, The drug is selected from compounds or compositions that enhance NAT10 acetyltransferase activity, promote ac4C modification formation, or increase acetyl-CoA levels.

9. The medicament according to claim 8, characterized in that, The dosage forms of the drug include oral formulations, injectable formulations, or targeted delivery formulations.

10. The medicament according to any one of claims 8-9, characterized in that, The drug is used to improve metabolic disorders, mitochondrial energy metabolism disorders, or multiple organ failure caused by decreased NAT10 activity or downregulated ac4C modification levels.

11. The application of the NAT10–ac4C–mitochondrial–acetyl-CoA regulatory axis as described in claim 1 in the prevention or treatment of mitochondrial dysfunction and related metabolic diseases.

12. The application according to claim 11, characterized in that, By using NAT10 agonists and / or metabolic substrates that can increase intracellular acetyl-CoA levels, the activity of the NAT10–ac4C–mitochondrial–acetyl-CoA regulatory axis can be enhanced, thereby improving mitochondrial energy metabolism and cellular homeostasis.

13. The application according to claim 12, characterized in that, The metabolic substrates that can increase intracellular acetyl-CoA levels include acetyl-L-carnitine, citrate, or pyruvate.

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