Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of promoting ATG5-ATG12 conjugation to enhance cell autophagy by removing wild type p53 protein

A ATG5-ATG12, wild-type technology, applied in the field of biomedicine, can solve the problem of unclear inhibition mechanism and achieve the effect of enhancing the level of cell autophagy

Active Publication Date: 2022-04-22
ACADEMY OF MILITARY MEDICAL SCI
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under normal physiological stress conditions, p53 inhibits autophagosome formation, but its specific inhibitory mechanism is unclear

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of promoting ATG5-ATG12 conjugation to enhance cell autophagy by removing wild type p53 protein
  • Application of promoting ATG5-ATG12 conjugation to enhance cell autophagy by removing wild type p53 protein
  • Application of promoting ATG5-ATG12 conjugation to enhance cell autophagy by removing wild type p53 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Interaction between p53 and key molecules in the autophagy pathway

[0028] In order to confirm the interaction between p53 and key molecules in the autophagy pathway, HEK293T overexpression wild-type Flag-p53 plasmid vector and key molecule expression plasmids in the autophagy pathway (Myc tag and HA tag expression vector plasmids) purchased from ATCC were selected. Co-immunoprecipitation technique was used to detect the interaction.

[0029] Wherein the implementation steps of the protein co-immunoprecipitation method are:

[0030] 1. Collect the cells that have been co-transfected with the plasmid for 36 hrs (T25 culture flask);

[0031] 2. Wash twice with pre-cooled PBS, centrifuge at 3000 rpm for 3 min;

[0032] 3. Add 600 μl lysate (add protease inhibitors and phosphatase inhibitors to the lysate, choose HEPES lysate or RIPA lysate according to specific experimental needs), lyse on ice for 10 minutes, and then ultrasonically disrupt the cells (power 30...

Embodiment 2

[0050] Example 2 p53 inhibits ATG5-ATG12 conjugation

[0051] After obtaining the interaction between p53 and ATG12, the wild-type p53 was firstly overexpressed in the cells by transfection, and the level of the ATG5-ATG12 conjugate was detected. It was found that overexpressing the wild-type p53 protein in the cells inhibited the ATG5-ATG12 The formation of the yoke ( Figure 4 ). The specific implementation steps are:

[0052] 1. After 24 hours of cell transfection, blot dry the cell culture medium in the culture plate, shake gently with PBS and wash twice;

[0053] 2. Blot up the PBS washing solution, add cell lysate, add an equal volume of 2×loading buffer, mix well, completely lyse the cells, transfer to EP tube, boil in boiling water for 15 minutes, and analyze by SDS-PAGE electrophoresis;

[0054] Further, the ATG5-ATG12 conjugate generation system was constructed in the extracellular environment, and the purified p53 protein was added. After detection, it was found ...

Embodiment 3

[0063] Example 3 p53 inhibits autophagy in mice

[0064] Since the occurrence of autophagy under physiological conditions needs to be induced by starvation, tissue samples from postnatally starved mice were used for detection. In order to verify the effect of p53 on autophagy in vivo, the tissues of newborn mice starved for 6 hrs with high expression of p53 were taken, the tissue cell protein was extracted, and the conjugated level of ATG5-ATG12 was detected. The level of ATG5-ATG12 conjugation was significantly reduced ( Figure 7 ); while the ATG5-ATG12 conjugate level in p53 knockout mouse tissue was significantly increased ( Figure 8 , Figure 9 ). Figure 9 Scale bar is 50 μm. The specific implementation steps are as follows:

[0065] 1. Tissue lysate sample preparation and analysis

[0066] 1. Isolate the organs and tissues of neonatal starved mice;

[0067] 2. Cut out 50 mg of tissue and add 500 μl of RIPA lysate (add protease inhibitors and phosphatase inhibito...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an application of promoting ATG5-ATG12 conjugation to enhance cell autophagy by removing p53 protein, firstly discloses a mechanism of inhibiting cell autophagy by p53 protein, and finds that the conjugation effect of key regulatory proteins ATG5 and ATG12 of cell autophagy is obviously weakened after the p53 protein is increased; by removing the p53 protein, the inhibition of the p53 protein on ATG5-ATG12 conjugation is reduced, so that the cell autophagy process is recovered; the invention proves that the cell autophagy inhibition mechanism of p53 is that p53 inhibits the conjugation effect of ATG12-ATG5 by combining with ATG12, and can promote the cell autophagy process by clearing p53 protein, so that inhibition of p53 expression or clearing of p53 protein becomes a potential drug target for relieving cell autophagy inhibition and promoting cell autophagy.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an application of promoting ATG5-ATG12 conjugation to enhance cell autophagy by clearing p53 protein. Background technique [0002] Autophagy is an important process in cells, which is highly conserved in the evolution of eukaryotes, and can carry out material turnover by degrading the components of cells themselves. In addition to normal nutrient metabolism, autophagy can also engulf some damaged organelles or proteins in the cell into autophagic vesicles with a double-membrane structure, and then fuse with lysosomes (animals) to form autolysosomes. Remove damaged organelles or proteins from cells. At present, it is found that autophagy plays an important role in the body's adaptation to starvation, immune response to infection, prevention and treatment of tumors and neurodegenerative diseases, and the deletion or mutation of key autophagy genes will cause newborn mice to be unable t...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/12A01K67/027
CPCC07K14/4746A01K67/0276A01K2227/105A01K2267/03
Inventor 张令强李洪昌李超男崔春萍翟文静
Owner ACADEMY OF MILITARY MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com