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Method for researching phosphorylated p53 stability and intracellular localization based on phase separation

A phosphorylation and amino acid technology, applied in the fields of molecular biology and cell biology, can solve the problems of p53 original function loss, restore p53 uneven distribution, cancer activity, etc., and achieve the effect of solving complex signaling pathways

Pending Publication Date: 2022-05-24
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Despite decades of intensive research, our understanding of the p53 signaling cascade remains incomplete because of p53's complex signaling
Intracellular research is easily affected by many unknown factors, while in vitro research is difficult to restore the uneven distribution of p53 in cells
On the one hand, although p53 is usually uniformly distributed in normal nuclei, under stress p53 can be locally enriched to drive functions efficiently; on the other hand, p53 protein aggregates are often observed in cancer cells and tumor tissues, which can lead to p53 Loss of original function and even lead to carcinogenic activity

Method used

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  • Method for researching phosphorylated p53 stability and intracellular localization based on phase separation
  • Method for researching phosphorylated p53 stability and intracellular localization based on phase separation
  • Method for researching phosphorylated p53 stability and intracellular localization based on phase separation

Examples

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Embodiment 1

[0035] This example uses in vitro purified p53, MED1-IDR-IDR, Pol II and target DNA to construct simulated DNA damage compartments (also known as DNA repair compartments), and use phase separation to visualize compartment stability and p53 involvement .

[0036] 1) Amplify the corresponding DNA fragment from the template plasmid containing the desired sequence by PCR. Add homology arms or linker sequences to the amplified primers. Purified DNA fragments corresponding to the following proteins were recovered by DNA agarose gel electrophoresis and gel purification kits: EGFP, p53-wt-EGFP, p53-S392D-EGFP, MED1-IDR, Pol II, etc. The amino acid sequence of EGFP is as shown in SEQ ID NO.1 in the sequence list; the amino acid sequence of p53-wt is as SEQ ID NO.2 in the sequence list, and the amino acid sequence of p53-S392D is the same as that of p53-wt, except for the serine mutation at position 392 is negatively charged aspartic acid; the sequence of the MED1-IDR is as in SEQ ID ...

Embodiment 2

[0060] This example sees its intracellular visualization and localization, and can perform intracellular verification. p53 is an intranuclear phosphorylated protein that is phosphorylated at Ser392 upon DNA damage. Therefore, here the applicant mutated Ser392 to Ala392, which cannot be phosphorylated, as a negative control.

[0061] 1) Amplify the corresponding DNA fragment from the template plasmid containing the desired sequence by PCR. A homology arm or linker sequence is added to the amplified primers (primers are as in Example 1). Purified DNA fragments corresponding to the following proteins were recovered by DNA agarose gel electrophoresis and gel purification kit: p53-wt-EGFP, p53-S392A-EGFP, etc. The amino acid sequence of EGFP is as SEQ ID NO.1 in the sequence listing, the amino acid sequence of p53-wt is as SEQ ID NO.2 in the sequence listing, and the amino acid sequence of p53-S392D is the same as that of p53-wt, except for the serine at position 392 Mutated to ...

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Abstract

The invention discloses a method for researching phosphorylated p53 stability and intracellular localization based on phase separation. According to the invention, a system is constructed, and the system comprises MED1-IDR protein, Pol II protein, p53 protein, p53 target DNA and a phase separation inducer. By inducing p53 liquid drops through a system, monitoring the regulation effect of p53 on the repair process after DNA damage and simulating a repair compartment formed after DNA damage, the influence of post-translational modification of a certain site on the formation of the DNA repair compartment in which p53 participates can be researched, and the positioning of the p53 in a cell can be detected, so that the problems that a p53 signal channel in the cell is complicated and the result is difficult to judge are solved. The concentration of protein is locally increased by phase separation liquid drops, so that an in-vivo non-uniformly distributed environment is more truly simulated; and the process and the result are visualized through tag protein.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, in particular to a method for studying the stability and intracellular localization of phosphorylated p53 based on phase separation. Background technique [0002] The p53 gene is located on the short arm of human chromosome 17 (17p13.1), encoding an intranuclear phosphorylated protein of 393 amino acids and 53 kDa detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. p53 is an important tumor suppressor involved in many processes related to DNA repair, cell cycle arrest, senescence, apoptosis, autophagy and metabolism. In unstressed cells, p53 is usually a transient protein that is maintained at low levels mainly by its negative regulator MDM2, and upon DNA damage or other stress, p53 undergoes extensive post-translational modifications (e.g. phosphorylation ) is activated and stabilized. For example, Ser392 phosphorylation can increase p53 stability, enhance ...

Claims

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Application Information

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IPC IPC(8): G01N15/10G01N21/64G01N33/50C12N15/65C12N15/85
CPCG01N15/10G01N21/6428G01N33/5005C12N15/85C12N15/65G01N2015/1006C12N2800/107
Inventor 杨小蓉戴卓君
Owner SOUTH CHINA UNIV OF TECH
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