Method for identifying functional kinase for regulating cell autophagy

A kinase and cell technology, applied in the field of bioinformatics, can solve the problem of low accuracy of protein kinases, achieve the effect of narrowing the screening range, reducing workload, and accurately identifying

Active Publication Date: 2020-04-07
HUAZHONG UNIV OF SCI & TECH
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  • Application Information

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Problems solved by technology

[0004] The present invention solves the technical problems that the identification of protein kinases regulating autophagy in the prio

Method used

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  • Method for identifying functional kinase for regulating cell autophagy
  • Method for identifying functional kinase for regulating cell autophagy
  • Method for identifying functional kinase for regulating cell autophagy

Examples

Experimental program
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Effect test

Embodiment 1

[0043] This example provides a method for predicting regulatory autophagy kinases based on multi-omics integration, such as figure 1 and figure 2 shown, including the following steps:

[0044] Using silica nanomaterials with a size of 16nm as an autophagy inducer at a final concentration of 60ug / mL, normal rat kidney cells were treated for 0 hours, 8 hours, 16 hours, 20 hours, and 24 hours to treat 0 The normal rat kidney cells for 4 hours were used as the control group, and cell samples at 4 time points after treatment were collected.

[0045] Using the second-generation gene sequencer HiSeq 4000system, RNA sequencing was performed on cell samples before and after treatment with 16nm silica nanomaterials to obtain transcriptome data.

[0046] The Bowtie-Tophat-Cufflinks series software was used to analyze and process the transcriptome data to obtain quantitative information of genes. Among them, a total of 6059 genes were significantly changed after nanomaterial treatment...

Embodiment 2

[0068] Subsequently, small molecule interfering RNA was designed for the above kinase genes, and the expression changes of the autophagy marker molecule LC3-II were detected by Western blotting to explore the effect of protein kinases on autophagy. At the same time, the GFP-LC3 cell line was used to detect the effect of protein kinase on the formation of autophagosomes. Such as image 3 , Figure 4 As shown, knocking down Cdk4 reduced the expression level of LC3-II protein induced by nanomaterials, and inhibited the accumulation of GFP-LC3 polysites induced by nanomaterials. Such as Figure 5 and Figure 6 As shown, knocking down Cdk7 reduces the expression of LC3-II protein induced by nanomaterials, and reduces the number of GFP-LC3 aggregation points induced by nanomaterials. It was confirmed that two protein kinases, Cdk4 and Cdk7, played important regulatory functions in nanomaterial-induced autophagy.

[0069] Based on the above results, it is proved that this method...

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Abstract

The invention discloses a method for identifying functional kinase for regulating cell autophagy, and belongs to the technical field of biological information. The method comprises the following steps: carrying out transcriptome, proteome and phosphorylated proteome analysis on a cell sample before and after autophagy inducer treatment to obtain gene expression data, protein expression data and phosphorylation site data of the cell sample before and after autophagy inducer treatment; normalizing the obtained phosphorylation site data to obtain kinase intensity information; according to the obtained kinase intensity information, calculating kinase which is remarkably changed in the cell sample before and after treatment of the autophagy inducer; according to the obtained kinase with significant change, carrying out the significant change analysis on transcriptional expression, protein expression and phosphorylation modification levels of the kinase, and acquiring the functional kinase for regulating and controlling cell autophagy by combining the autophagy regulation and control function of the known kinase. According to the method, the kinase screening range can be effectively reduced, the workload of experimental verification is reduced, and the functional kinase for regulating and controlling cell autophagy is accurately identified.

Description

technical field [0001] The invention belongs to the field of biological information technology, and more specifically, relates to a method for identifying functional kinases that regulate autophagy, Background technique [0002] Protein phosphorylation is one of the most important post-translational modifications, which play key regulatory roles in different biological processes and signaling pathways. From a biochemical point of view, the principle of phosphorylation is that under the catalysis of protein kinase, the γ-phosphate group on adenosine triphosphate is transferred to specific receptor residues on the substrate protein, usually serine, threonine and tyrosine acid. The discovery of functional phosphorylation events is an important basis for understanding the biological effects of phosphorylation. Traditional experimental methods need to consume huge manpower and financial resources, and the time period is long. With the rapid development of liquid chromatography...

Claims

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Application Information

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IPC IPC(8): G16B25/00G16B30/00G16B50/00C12Q1/6869G01N30/72G01N30/88G01N33/68
CPCC12Q1/6869G01N30/72G01N30/88G01N33/6848G01N2030/8831G16B25/00G16B30/00G16B50/00C12Q2535/122C12Q2537/165
Inventor 薛宇彭迪阮晨王晨玮
Owner HUAZHONG UNIV OF SCI & TECH
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