Use of sumo in the modulation of glycosylation for mitophagy and test methods

Abstract

The application belongs to the technical field of glycosylation and mitochondria regulation, and discloses application and test method of CARD9 protein SUMO in regulation of HOXB5 0-GlcNAc glycosylation and mitochondria autophagy, and the like. Knocking down PIAS3 inhibits CARD9 protein SUMO, promotes dissociation of CARD9 and HOXB5 and O-GlcNAc glycosylation of HOXB5, and then HOXB5 nuclear translocation promotes Parkin-induced mitochondria autophagy, and relieves myocardial I / R injury. The application adopts technologies such as immunoprecipitation, mitochondria and lysosome fluorescence tracking to study regulation of CARD9 protein SUMO on HOXB5 0-GlcNAc glycosylation and mitochondria autophagy, and further clarifies the molecular mechanism of myocardial I / R injury.

Description

Technical Field

[0001] This invention belongs to the field of glycosylation and mitochondrial regulation technology, and particularly relates to the application of a CARD9 protein SUMO in regulating the O-GlcNAc glycosylation of HOXB5 and mitophagy. Background Technology

[0002] Currently, ischemia-reperfusion (I / R) induced myocardial injury remains a challenging clinical problem, but the injury mechanism of I / R is still unclear. CARD9 is an important intracytoplasmic binding protein, and proteomic analysis shows that CARD9 binds to HOXB5, a potential transcription factor for the mitophagy factor Parkin. Analysis shows that SUMO modification of the CARD9 protein promotes the binding of CARD9 to HOXB5 but inhibits the O-GLcNAc glycosylation of HOXB5, which is an important signal for nuclear translocation of the protein. CARD9 protein SUMO is regulated by PIAS3, which is associated with I / R induced myocardial injury.

[0003] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0004] 1) Unclear injury mechanism: Although the binding of CARD9 to HOXB5 and related protein modification have been discovered, the specific mechanism of I / R-induced myocardial injury has not been fully elucidated. This limits the formulation and optimization of treatment strategies for myocardial injury.

[0005] 2) Lack of effective intervention methods: Current technologies have not yet provided effective intervention methods for the interaction between CARD9 and HOXB5, such as how to effectively regulate or affect the SUMO modification of CARD9 protein and the O-GLcNAc glycosylation of HOXB5, so as to alleviate I / R-induced myocardial injury.

[0006] 3) The regulatory mechanism of PIAS3 is unclear: Although it is known that PIAS3 regulates SUMO modification of CARD9 protein and is associated with I / R-induced myocardial injury, its specific regulatory mechanism and its association with myocardial injury are still unclear, which makes it difficult to formulate potential therapeutic strategies targeting PIAS3.

[0007] 4) Methodological limitations: Current techniques for studying I / R-induced myocardial injury may be limited by experimental methods and techniques, resulting in slow progress at the cellular and molecular levels. With the development of new technologies, more efficient and accurate research methods may emerge, helping to elucidate the relevant mechanisms of myocardial injury.

[0008] In summary, existing technologies for studying I / R-induced myocardial injury suffer from several shortcomings, including unclear injury mechanisms, a lack of effective interventions, unclear regulatory mechanisms, and methodological limitations. Addressing these issues will contribute to providing more targeted strategies for the treatment of myocardial injury, thereby improving patients' quality of life and survival rates. Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides an application of CARD9 protein SUMO in regulating HOXB5 O-GlcNAc glycosylation and mitophagy.

[0010] This invention is achieved by applying the CARD9 protein SUMO to the regulation of HOXB5 O-GlcNAc glycosylation and mitophagy.

[0011] Furthermore, it includes:

[0012] Knocking down PIAS3 inhibits CARD9 protein SUMO, promotes the dissociation of CARD9 with HOXB5 and the O-GlcNAc glycosylation of HOXB5.

[0013] HOXB5 nuclear translocation promotes Parkin-induced mitophagy and alleviates myocardial I / R injury.

[0014] Furthermore, immunoprecipitation, mitochondrial and lysosomal fluorescence tracking were used to investigate the regulation of HOXB5 O-GlcNAc glycosylation and mitophagy by CARD9 protein SUMO.

[0015] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0016] First, this invention inhibits CARD9 protein SUMO by knocking down PIAS3, promotes the dissociation of CARD9 and HOXB5 and the O-GlcNAc glycosylation of HOXB5, and then promotes HOXB5 nuclear translocation to promote Parkin-induced mitophagy and alleviate myocardial I / R injury.

[0017] Second, this invention employs techniques such as immunoprecipitation, mitochondrial and lysosomal fluorescence tracking to study the regulation of HOXB5 O-GlcNAc glycosylation and mitophagy by CARD9 protein SUMO, further elucidating the molecular mechanism of myocardial I / R injury.

[0018] Third, a deeper understanding of the mechanism: Knocking down PIAS3 to inhibit CARD9 protein SUMO, and promoting the dissociation of CARD9 and HOXB5 and the O-GlcNAc glycosylation of HOXB5, helps to understand the molecular mechanism of I / R-induced myocardial injury and provides more information for future research.

[0019] Novel treatment strategy: By modulating the interaction between CARD9 protein SUMO and HOXB5, as well as the nuclear translocation of HOXB5 and Parkin-induced mitophagy, a new potential treatment strategy for alleviating myocardial I / R injury is provided.

[0020] Optimized experimental methods: The use of methods such as immunoprecipitation, mitochondrial and lysosomal fluorescence tracking helps to more accurately study the regulatory mechanism of CARD9 protein SUMO on HOXB5 O-GlcNAc glycosylation and mitophagy, thereby improving research efficiency and accuracy.

[0021] Potential clinical applications: By regulating molecular mechanisms such as CARD9 protein SUMO, HOXB5 O-GlcNAc glycosylation and mitophagy, it is expected to alleviate I / R-induced myocardial injury, reduce patient mortality and improve quality of life in actual clinical applications.

[0022] Promoting research in related fields: This application is not only significant in the field of I / R-induced myocardial injury, but may also drive research in other disease areas related to CARD9, HOXB5, SUMO modification and mitophagy, expanding the scope and depth of research.

[0023] In summary, the application of CARD9 protein SUMO in regulating HOXB5 O-GlcNAc glycosylation and mitophagy offers positive benefits, including a deeper understanding of the mechanisms, provision of novel therapeutic strategies, optimization of experimental methods, potential clinical applications, and promotion of research in related fields. This will contribute to providing more targeted strategies for the treatment of myocardial injury, improving patients' quality of life and survival rates. Attached Figure Description

[0024] Figure 1 This is a flowchart illustrating the application of CARD9 protein SUMO in regulating HOXB5 O-GlcNAc glycosylation and mitophagy, as provided in this embodiment of the invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] The specific methods provided in the embodiments of the present invention are as follows:

[0027] 1. Gene editing technology: Using gene editing technologies such as CRISPR / Cas9, target genes (such as PIAS3) are knocked down or eliminated to study their effects on SUMO modification of CARD9 protein and O-GlcNAc glycosylation of HOXB5.

[0028] 2. Fluorescence Resonance Energy Transfer (FRET): FRET technology is used to study the interaction between CARD9 and HOXB5, and how they change under different conditions (such as PIAS3 knockdown).

[0029] 3. Protein mass spectrometry analysis: Protein mass spectrometry analysis was used to identify and quantify the O-GlcNAc glycosylation level of HOXB5 and its changes under the regulation of CARD9 protein SUMO modification.

[0030] 4. Cell and animal models: Establish cell and animal models of ischemia-reperfusion induced myocardial injury, evaluate the effects of PIAS3 knockdown and regulation of CARD9 protein SUMO modification on myocardial injury, and the roles of HOXB5 O-GlcNAc glycosylation and mitophagy in this process.

[0031] 5. Drug screening: Screening for potential small molecule compounds or biological drugs to regulate CARD9 protein SUMO modification and HOXB5 O-GlcNAc glycosylation, thereby alleviating myocardial I / R injury.

[0032] 6. Clinical Research: Following positive results from animal experiments, further clinical trials will be conducted to evaluate the safety and efficacy of regulating CARD9 protein SUMO modification and HOXB5 O-GlcNAc glycosylation in alleviating myocardial I / R injury.

[0033] These methods and experimental techniques allow for a more comprehensive study of the application of CARD9 protein SUMO in regulating HOXB5 O-GlcNAc glycosylation and mitophagy, providing new strategies and directions for the treatment of myocardial injury.

[0034] like Figure 1 As shown in the embodiments of the present invention, the application of CARD9 protein SUMO in regulating HOXB5 O-GlcNAc glycosylation and mitophagy includes the following steps:

[0035] S101: Knocking down PIAS3 inhibits CARD9 protein SUMO, promotes the dissociation of CARD9 and HOXB5 and the O-GlcNAc glycosylation of HOXB5.

[0036] S102: HOXB5 nuclear translocation promotes Parkin-induced mitophagy and alleviates myocardial I / R injury.

[0037] This invention employs techniques such as immunoprecipitation, mitochondrial and lysosomal fluorescence tracking to study the regulation of HOXB5 O-GlcNAc glycosylation and mitophagy by CARD9 protein SUMO, further elucidating the molecular mechanism of myocardial I / R injury.

[0038] As a specific optimization scheme in an embodiment of the present invention, it is as follows:

[0039] (1) Cardiac cells and cardiac cell lines: mainly used in in vitro experiments, to study the regulatory mechanism of CARD9 SUMOylation on HOXB5 glycosylation and mitochondrial autophagy through cell culture, transfection and other methods.

[0040] (2) Adult mouse myocardium or heart: PIAS3 and CARD9 were regulated in mice by feeding, intraperitoneal injection and other methods to study their effects on myocardial HOXB5 glycosylation, mitochondrial autophagy and myocardial cell damage and death.

[0041] (3) Ex vivo heart perfusion model: The effects and mechanisms of PIAS3 and CARD9 SUMOylation on myocardial I / R injury were studied in an isolated mouse heart by perfusion.

[0042] (4) Adult mouse myocardial infarction model: an infarction model was established by coronary artery ligation to study the effects of PIAS3 and CARD9 SUMOylation on the degree of infarction damage, myocardial function recovery and survival rate.

[0043] Example 1: Immunoprecipitation and Western blot were used to detect the interaction between CARD9 and HOXB5 in normal mouse cardiomyocytes, as well as the O-GlcNAc glycosylation level of HOXB5; mitochondrial autophagy was observed using mitochondrial and lysosomal specific fluorescent probes.

[0044] Example 2: PIAS3 was knocked down in mouse cardiomyocytes, and Western blot was used to detect changes in the interaction between CARD9 and HOXB5 and changes in the glycosylation level of HOXB5O-GlcNAc; changes in mitophagy were observed using mitochondrial and lysosomal specific fluorescent probes.

[0045] Example 3: Based on Example 2, the expression changes of HOXB5 in the cell nucleus were detected by immunofluorescence quantitative analysis; the expression changes of Parkin and other mitophagy-related proteins were detected by Western blot.

[0046] Example 4: In a mouse model of myocardial infarction, the effects of CARD9 SUMOylation, CARD9-HOXB5 interaction, HOXB5 glycosylation and translocation, Parkin expression, mitochondrial damage, and cardiomyocyte death were compared between the PIAS3 knockdown group and the control group.

[0047] Example 5: Immunoprecipitation, Western blot, and immunofluorescence quantitative analysis were used to detect changes in O-GlcNAc glycosylation of HOXB5, nuclear HOXB5 expression, and mitochondrial autophagy in mouse cardiomyocytes cultured in high glucose.

[0048] Example 6: In an in vitro simulated mouse cardiomyocyte I / R model, the changes in mitochondrial damage, cell death, HOXB5 glycosylation, HOXB5 nuclear translocation, and mitochondrial autophagy were compared between the PIAS3 knockdown group and the control group.

[0049] 1) The molecular mechanism by which CARD9 protein SUMO regulates the binding of CARD9 to HOXB5 and the O-GlcNAc glycosylation of HOXB5 protein.

[0050] In in vitro experiments, knocking down PIAS3 downregulated the SUMO molecule level of CARD9 protein in post-healing / recovery cardiomyocytes and reduced the level of HOXB5 protein on CARD9 protein, while increasing O-GlcNAc glycosylation on HOXB5 protein. This invention will further clarify the regulatory role of SUMO on the binding of CARD9 and HOXB5, and the effect of CARD9 and HOXB5 binding on O-GlcNAc glycosylation.

[0051] Example 1: Mouse and rat cardiomyocytes HL-1 and H9c2 were selected. PIAS3 was knocked down or overexpressed in HL-1 / H9c2 cells. PIAS3 was detected by PCR, and PIAS3, CARD9, and HOXB5 were detected by Western blot (WB). Immunoprecipitation was used to detect the levels of SUMO and O-GlcNAc molecules on CARD9 and HOXB5 proteins, as well as the level of HOXB5 on CARD9 protein.

[0052] Example 2: PIAS3 was overexpressed in HL-1 / H9c2 while CARD9 was knocked down. Western blotting was used to detect the expression of CARD9 and HOXB5. Immunoprecipitation was used to detect the O-GlcNAc molecule level on the HOXB5 protein.

[0053] Overexpression of PIAS3 increases the SUMO level of CARD9 protein, increases HOXB5 on CARD9 protein, and downregulates the O-GlcNAc glycosylation level of HOXB5 protein; while overexpression of PIAS3 simultaneously knocks down CARD9, the O-GlcNAc glycosylation level of HOXB5 protein increases.

[0054] This invention constructs an AAV6 serotype adenovirus, AAV6-shRNA-PIAS3, which is low in immunogenicity and suitable for specific binding to heart tissue. PIAS3 in the heart tissue of SD rats was knocked down by tail vein injection. Myocardial tissue was then collected, and PIAS3 expression was detected by PCR. The expression of PIAS3, CARD9, and HOXB5 was detected by Western blotting. Immunoprecipitation was used to detect the levels of SUMO and O-GlcNAc molecules on CARD9 and HOXB5 proteins, as well as the level of HOXB5 on CARD9 protein.

[0055] 2) The role of O-GlcNAc glycosylation and HOXB5 nuclear translocation in promoting Parkin expression and facilitating mitochondrial autophagy in cardiomyocytes after I / R

[0056] Although the overall O-GlcNAc glycosylation modification level of cardiomyocytes is increased after I / R, multiple O-GlcNAc glycosylation modification sites on HOXB5 may be occupied by HOXB5 after binding to CARD9, thereby hindering the glycosylation of HOXB5 and inhibiting the translocation of HOXB5 to the cell nucleus.

[0057] Example 1: Knockdown of CARD9 or simultaneous knockdown of CARD9 and OGT (O-GlcNAc glycosyltransferase) in HL-1 / H9c2.

[0058] Example 2: H / R treatment was performed, and CARD9 and OGT were detected by PCR; CARD9 and HOXB5 proteins were detected by Western blotting; the O-GlcNAc molecule level on HOXB5 protein was detected by immunoprecipitation; the level of HOXB5 in the cytoplasm and nucleus was detected by Western blotting; and the distribution of HOXB5 in the cytoplasm and nucleus was observed by immunofluorescence (IF).

[0059] In this invention, knocking down CARD9 exposes the O-GlcNAc glycosylation site of HOXB5, and O-GlcNAc glycosylation promotes the translocation of HOXB5 to the cell nucleus. However, knocking down both CARD9 and OGT simultaneously blocks the O-GlcNAc glycosylation of HOXB5 and inhibits the translocation of HOXB5 to the cell nucleus.

[0060] This invention aims to clarify that HOXB5 nuclear translocation can promote mitophagy in cardiomyocytes after hemophagocytosis (H / R) by increasing Parkin expression. The method involved knocking down CARD9 in HL-1 / H9c2 cells, overexpressing HOXB5, simultaneously knocking down both CARD9 and HOXB5, or overexpressing HOXB5 and simultaneously knocking down Parkin, followed by H / R treatment. PCR was used to detect the expression of CARD9, HOXB5, and Parkin; Western blotting was used to detect the expression of CARD9, HOXB5, Parkin, PINK1, LC3Ⅱ / Ⅰ, and p62; chromatin immunoprecipitation was used to detect the binding of HOXB5 to the Parkin gene promoter; dual-luciferase reporter gene assays were used to detect HOXB5 transcriptional regulation of Parkin; electron microscopy was used to observe mitophagy; and the dynamic process of mitophagy was tracked using the mitochondrial-specific fluorescent probe Mito Tracker and the lysosome-specific fluorescent probe Lyso Tracker. In this invention, knocking down CARD9 and overexpressing HOXB5 both promote Parkin transcription and post-H / R mitophagy, but knocking down CARD9 while simultaneously knocking down HOXB5 or overexpressing HOXB5 while simultaneously knocking down Parkin inhibits post-H / R mitophagy.

[0061] This invention constructs AAV6 serotype adenoviruses AAV6-shRNA-CARD9, AAV6-shRNA-OGT, and AAV6-shRNA-HOXB5. A rat I / R model is established by tail vein injection into SD rats. Myocardial tissue is collected, and CARD9, OGT, and HOXB5 are detected by PCR. Western blotting detects CARD9, OGT, HOXB5, Parkin, PINK1, LC3Ⅱ / Ⅰ, and p62 proteins. Immunoprecipitation is used to detect the O-GlcNAc molecule level on the HOXB5 protein. Western blotting is used to detect the HOXB5 level in the cytoplasm and nucleus. Chromatin immunoprecipitation is used to detect the binding of HOXB5 to the Parkin gene promoter. Mitochondrial autophagy is observed under electron microscopy.

[0062] 3) Verify that PIAS3 downregulates CARD9 protein SUMO levels by promoting HOXB5-induced mitophagy, thereby alleviating I / R-induced myocardial injury.

[0063] Analysis showed that CARD9 binds to HOXB5, preventing the latter from entering the nucleus to initiate Parkin expression and mitophagy after I / R (injury / reperfusion injury). However, CARD9 has cardioprotective effects, and it is not advisable to activate HOXB5 function by knocking down CARD9. This invention promotes HOXB5-induced mitophagy and alleviates I / R-induced myocardial injury by knocking down PIAS3 to downregulate CARD9 protein SUMO levels.

[0064] Example 1: HL-1 / H9c2 transfection with PIAS3 knockdown vector, simultaneous transfection with PIAS3 and HOXB5 knockdown vector, or simultaneous transfection with PIAS3 and Parkin knockdown vector.

[0065] Example 2: H / R treatment was performed, and PCR was used to detect the expression of PIAS3, HOXB5, and Parkin; Western blotting was used to detect the expression of CARD9, HOXB5, Parkin, PINK1, LC3Ⅱ / Ⅰ, and p62; mitochondrial membrane potential was detected; and mitophagy was observed under an electron microscope.

[0066] Simultaneously, the dynamic process of mitochondrial autophagy was tracked using the mitochondrial-specific fluorescent probe Mito Tracker and the lysosome-specific fluorescent probe LysoTracker; cardiomyocyte viability and apoptosis rate were detected.

[0067] This invention constructs a rat I / R model by injecting AAV6 serotype adenovirus AAV6-shRNA-PIAS3, AAV6-shRNA-HOXB5, and AAV6-shRNA-Parkin into SD rats via tail vein. Echocardiography was used to detect LVFS, LVEDD, and LVEF. Serum CK, CK-MB, cTnI, cTnT, and LDH levels were detected using a kit. HE staining was used to observe pathological changes in myocardial tissue of each group of rats. TUNEL staining was used to assess cell apoptosis. Myocardial tissue was harvested, and PCR was used to detect the expression of PIAS3, HOXB5, and Parkin. Western blotting was used to detect the expression of PIAS3, HOXB5, Parkin, PINK1, LC3Ⅱ / Ⅰ, and p62. Mitochondrial autophagy was observed under electron microscopy.

[0068] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for testing the regulatory effect of CARD9 protein SUMO modification on HOXB5 O-GlcNAc glycosylation and mitophagy, characterized in that, Includes: PIAS3 knockdown inhibits CARD9 protein SUMO modification, promotes the dissociation of CARD9 and HOXB5 and O-GlcNAc glycosylation of HOXB5; HOXB5 nuclear translocation promotes Parkin-induced mitophagy; The testing method includes the following steps: (1) Clarify the molecular mechanism by which CARD9 protein regulates the binding of CARD9 to HOXB5 and the O-GlcNAc glycosylation of HOXB5 protein through SUMO; Mouse and rat cardiomyocytes HL-1 and H9c2 were selected. PIAS3 was knocked down or overexpressed in HL-1 / H9c2. PIAS3 was detected by PCR, and PIAS3, CARD9 and HOXB5 were detected by Western blot. Immunoprecipitation was used to detect the SUMO molecule level on CARD9 protein, the O-GlcNAc molecule level on HOXB5 protein, and the HOXB5 level on CARD9 protein. PIAS3 was overexpressed in HL-1 / H9c2 while CARD9 was knocked down. Western blot was used to detect the expression of CARD9 and HOXB5. Immunoprecipitation was used to detect the O-GlcNAc molecule level on HOXB5 protein. (2) O-GlcNAc glycosylation and HOXB5 nuclear translocation enhance Parkin expression and promote mitochondrial autophagy in cardiomyocytes after ischemia-reperfusion I / R; Knock down CARD9 in HL-1 / H9c2 or knock down both CARD9 and O-GlcNAc glycosyltransferase; H / R treatment was performed, and PCR was used to detect CARD9 and O-GlcNAc glycosyltransferases. Western blot was used to detect CARD9 and HOXB5 proteins. Immunoprecipitation was used to detect the O-GlcNAc molecule level on HOXB5 protein. Western blot was used to detect the HOXB5 level in the cytoplasm and nucleus, respectively. Immunofluorescence was used to observe the distribution of HOXB5 in the cytoplasm and nucleus. (3) Verify the effect of PIAS3 downregulating CARD9 protein SUMO levels to alleviate I / R-induced myocardial injury by promoting HOXB5-induced mitophagy. HL-1 / H9c2 transfection with PIAS3 knockdown vector, simultaneous transfection with PIAS3 and HOXB5 knockdown vector, or simultaneous transfection with PIAS3 and Parkin knockdown vector. H / R treatment was performed, and PCR was used to detect the expression of PIAS3, HOXB5, and Parkin; Western blot was used to detect the expression of CARD9, HOXB5, Parkin, PINK1, LC3Ⅱ / Ⅰ, and p62; mitochondrial membrane potential was detected; and mitophagy was observed under electron microscopy. Simultaneously, the dynamic process of mitophagy was tracked using the mitochondrial-specific fluorescent probe Mito Tracker and the lysosome-specific fluorescent probe LysoTracker; cardiomyocyte viability and apoptosis rate were detected.

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