Application of PHYHD1 in reducing Tau protein in cells

Abstract

The invention discloses an application of a phytanyl coenzyme A dioxygenase structural domain 1 in in-vitro reduction of an intracellular Tau protein level and a method for in-vitro reduction of an intracellular Tau protein expression level. According to the method, on the basis of early-stage research results aiming at PHYHD1 genes and proteins, effective overexpression of the PHYHD1 proteins in HEK-293 Tau cells capable of stably expressing Tau proteins is realized by preparing a transfection compound. The method can significantly reduce the Tau protein level by 33.6%, the process is simple, convenient and reliable, and the transfection operation does not have significant cytotoxicity. The method can be widely applied to the fields of AD basic research, drug screening, gene therapy strategy development and the like.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and specifically relates to an application of human phytanoyl-CoA dioxygenase domain 1 (PHYHD1) in reducing intracellular Tau protein in vitro. Background Art

[0002] The onset and progression of Alzheimer's disease (AD) can gradually lead to cognitive and memory loss, and even death. During the onset of AD, the pathological proteins involved are β-amyloid (Aβ) and Tau protein.

[0003] Among them, Tau protein is the microtubule-binding protein most closely associated with AD. Its main biological function is to stabilize the microtubule structure in neuronal axons, maintain intracellular material transport and neuronal morphology. In the brains of AD patients, Tau protein undergoes abnormal hyperphosphorylation, which leads to microtubule dissociation and aggregation, and ultimately leads to the formation of neurofibrillary tangles, which in turn promotes the development of AD. In addition, pathological changes in Tau protein are closely related to neuronal dysfunction and cognitive decline. Therefore, reducing the abnormal expression of Tau protein or promoting its clearance has become an important strategy for the treatment of AD.

[0004] The human gene PHYHD1 corresponds to the protein phytanoyl-CoA dioxygenase domain 1. As a 2-oxoglutarate-dependent dioxygenase, PHYHD1 is located in the cytoplasm and nucleus, acting as a sensor of oxygen levels and metabolic status, and participating in RNA processing and carbohydrate metabolism regulation. In recent years, numerous studies have revealed a potential link between PHYHD1 and the pathogenesis of AD through gene association, protein interaction, and expression profiling analyses, but no interactions between PHYHD1 and AD pathological marker proteins have been found. Summary of the Invention

[0005] In their research, our team discovered that PHYHD1 and Tau protein may form seven pairs of hydrogen bonds, resulting in a strong binding effect. Experiments have also shown that overexpression of PHYHD1 can reduce the level of Tau protein in cells that stably express Tau protein.

[0006] The present invention provides an application of phytanoyl-CoA dioxygenase domain 1 in reducing the level of Tau protein in cells in vitro.

[0007] In a specific embodiment, the coding sequence of the phytanoyl-CoA dioxygenase domain 1 is shown as SEQ ID NO: 1.

[0008] The present invention also provides a method for reducing the expression level of Tau protein in cells in vitro, which includes the step of overexpressing PHYHD1 gene in cells that overexpress Tau protein.

[0009] In a specific embodiment, the sequence of the PHYHD1 gene is shown in SEQ ID NO: 1.

[0010] In a specific embodiment, the cell overexpressing Tau protein is a HEK-293 cell overexpressing Tau protein.

[0011] The present invention also provides cells with reduced Tau protein expression levels prepared by the above method.

[0012] The present invention effectively overexpresses PHYHD1 in cells, significantly inhibiting Tau protein expression, providing an alternative technical approach for PHYHD1-based AD treatment strategies. Since genetic engineering methods for effectively overexpressing PHYHD1 include, but are not limited to, using Lip2000 to encapsulate plasmids for cell transfection, other reagents and methods for overexpressing PHYHD1 in cells to reduce Tau protein expression are also within the scope of this invention. The methods and cells of this invention are widely applicable to basic AD research, drug screening, and the development of gene therapy strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This figure shows a receiver operating characteristic curve (ROC) analysis of the human gene PHYHD1 for predicting AD development. The area under the curve (AUC) value in the figure is used to determine the gene's predictive ability for AD.

[0014] Figure 2 This is the strongest binding mode after docking of PHYHD1 protein and Tau protein.

[0015] Figure 3 Western blotting (WB) results of HEK-293Tau cells after PHYHD1 plasmid transfection: (a) WB results of PHYHD1 protein and β-actin; (b) protein expression analysis based on (a). .

[0016] Figure 4 Effects of PHYHD1 overexpression on Tau protein expression in HEK-293Tau cells: (a) Western blotting results of Tau protein and β-actin; (b) protein expression analysis based on (a). .

[0017] Figure 5Effects of different doses of PHYHD1 plasmid transfection on the viability of HEK-293Tau cells.

[0018] Table 1 shows the analysis results of hydrogen bonds in the strongest binding mode after docking of PHYHD1 protein and Tau protein. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0020] 1. PHYHD1 gene sequence, molecular docking analysis, and in-cell overexpression

[0021] The transcript sequence of the human gene PHYHD1 is shown in SEQ ID NO: 1. Our previous data showed that the expression of the human gene PHYHD1 is significantly upregulated with the development of AD. Therefore, we speculated that this gene has good predictive ability for AD. We then plotted a receiver operating characteristic (ROC) curve using the "qROC" package in the R language.

[0022] The results showed that the AUC value was 61.4%, exceeding 50% ( Figure 1 ). This indicates that the human gene PHYHD1 has a good predictive ability for the development of AD.

[0023] We then performed protein-protein docking of PHYHD1 and Tau, calculated the docking scores and confidence levels, and finally selected the strongest binding patterns based on the docking scores and confidence levels. In this analysis, we defined hydrophobic interactions as occurring between all hydrophobic atom pairs within a distance of 4.0 Å. Hydrogen bonds were considered to form between the acceptor and donor groups when the maximum distance between the hydrogen bond acceptor and donor was less than 4.1 Å and the minimum angle at the hydrogen bond donor was greater than 100°.

[0024] The docking results showed that the docking score of the strongest binding mode between PHYHD1 protein and Tau protein was -238.03, and the confidence score was 0.8533 ( Figure 2In this model, seven pairs of hydrogen bonds are formed between the two proteins. As shown in Table 1, PHYHD1's PRO-280 forms a hydrogen bond with Tau's LYS-145; PHYHD1's GLY-269 forms a hydrogen bond with Tau's ASP-134; Tau's GLN-33 forms a hydrogen bond with PHYHD1's GLN-288; Tau's GLN-33 forms a hydrogen bond with PHYHD1's THR-291; Tau's ARG-52 forms a hydrogen bond with PHYHD1's GLN-288; PHYHD1's ALA-282 forms a hydrogen bond with Tau's ARG-67, with a bond length of 3.5 Å, which is the weakest of the seven hydrogen bond pairs; and PHYHD1's ALA-282 forms a hydrogen bond with Tau's TYR-71, with a bond length of 1.9 Å, which is the strongest of the seven hydrogen bond pairs. The above results indicate that PHYHD1 protein may have a strong binding with Tau protein, and it is speculated that this binding may have a certain clearing effect on Tau protein.

[0025] Table 1 Statistics of seven hydrogen bond lengths between PHYHD1 and Tau protein

[0026] protein PHYHD1 Tau Bond length / Å Hydrogen bond 1 PRO-280 LYS-145 2.5 Hydrogen Bond 2 GLY-269 ASP-134 2.8 Hydrogen Bond 3 ALA-282 TYR-71 1.9 Hydrogen Bond 4 ALA-282 ARG-67 3.5 Hydrogen Bond 5 GLN-288 GLN-33 2.3 Hydrogen Bond 6 THR-291 GLN-33 2.8 Hydrogen bond 7 GLN-288 ARG-52 2.8

[0027] The full-length cDNA sequence of the human gene PHYHD1, tagged with GFP, was cloned into the pLVXpuro vector to generate the PHYHD1 plasmid and prepared into a corresponding stock solution (2142.5 ng / L). 10 μL of Lip2000 was dissolved in 250 μL of opti-MEM medium, and 5 μg of PHYHD1 plasmid was dissolved in 250 μL of opti-MEM medium. Each experiment was performed in triplicate. Both solutions were allowed to stand at room temperature for 5 minutes. The Lip2000 solution was then slowly added to the plasmid solution, gently mixed, and allowed to stand at room temperature for 15 minutes to allow for complete formation of the transfection complex. Finally, the mixture was evenly added to HEK-293 Tau cells stably expressing Tau protein that had been cultured for 12 hours and at 70% confluence. After 6 hours, the medium was replaced with complete DMEM supplemented with 10% FBS and cultured for an additional 48 hours.

[0028] Cells cultured for 48 hrs were collected and cell lysates were prepared using lysis buffer containing protease inhibitors and phosphatase inhibitors. Finally, the expression level of PHYHD1 protein was quantified by Western blotting using β-actin as an internal standard.

[0029] WB results are as follows Figure 3As shown, the cells themselves expressed little PHYHD1 protein, while PHYHD1 plasmid transfection increased its expression by 320.8% (p=0.0155). This shows that the above method can effectively achieve overexpression of PHYHD1 protein in HEK-293Tau cells.

[0030] 2. Overexpression of the PHYHD1 gene in cells reduces Tau protein expression

[0031] HEK-293Tau cells transfected with a PHYHD1 overexpression plasmid were cultured for 48 hours and then harvested for preparation of cell lysates. Tau protein expression levels were quantified by Western blotting using β-actin as an internal standard. Untransfected cells served as a control group.

[0032] Quantitative Tau protein Western blotting Figure 4 As shown in the results, the expression of Tau protein in the PHYHD1 overexpression group decreased by 33.6%, which was significantly lower than that in the Control group (p=0.00139). This indicates that overexpression of PHYHD1 in cells can reduce the excessive level of Tau protein in cells.

[0033] 3. Effect of PHYHD1 plasmid transfection on cell survival

[0034] PHYHD1 was complexed with Lip2000 at ratios of 5 μg:10 μL, 10 μg:10 μL, and 15 μg:10 μL as described above and used to transfect HEK-293Tau cells. Cell viability was assessed using the CellCounting Kit-8, with untransfected cells used as a control.

[0035] like Figure 5 As shown in the results, transfection of different concentrations of PHYHD1 plasmid did not significantly affect the viability of HEK-293Tau cells (p>0.05), which proves that the method of overexpressing PHYHD1 in cells to reduce excessive Tau protein levels in cells does not have obvious cytotoxicity.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of phytanoyl-CoA dioxygenase domain 1 in reducing intracellular Tau protein levels in vitro.

2. The use according to claim 1, characterized in that The coding sequence of the phytanoyl-CoA dioxygenase domain 1 is shown in SEQ ID NO:

1.

3. A method for reducing the expression level of Tau protein in cells in vitro, characterized in that: The method comprises the step of overexpressing the PHYHD1 gene in cells overexpressing the Tau protein.

4. The method according to claim 3, characterized in that The sequence of the PHYHD1 gene is shown in SEQ ID NO:

1.

5. The method according to claim 3, characterized in that The cells overexpressing Tau protein are HEK-293 cells overexpressing Tau protein.

6. A cell with reduced Tau protein expression level, characterized in that: It is prepared by the method according to any one of claims 3 to 5.

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