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Acetylation of human parkin protein and application of parkin protein in medicine preparation

An acetylation and deacetylase technology, applied in the field of physiology and molecular biology, can solve undisclosed, enhanced mitophagy and anti-tumor functions, undisclosed parkin and other problems

Active Publication Date: 2020-12-04
ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, neither of these two patent applications discloses whether parkin is acetylated, nor does it disclose whether acetylation modification can enhance parkin-mediated mitophagy and anti-tumor functions

Method used

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  • Acetylation of human parkin protein and application of parkin protein in medicine preparation
  • Acetylation of human parkin protein and application of parkin protein in medicine preparation
  • Acetylation of human parkin protein and application of parkin protein in medicine preparation

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Experimental program
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Effect test

Embodiment 1

[0025] Since the acetylation of parkin protein has not been clearly reported, there is no commercial acetylation antibody for parkin. HEK293 cells were used to overexpress Flag-parkin, treated with histone deacetylase inhibitor (HDACI) SAHA drug at 2.5 μM for 12 hours, and protein samples were precipitated by FLAG or acetyl-lysine (Ac-lysine) affinity purification gel, Western blot (abbreviated as WB) detected acetyl-lysine or parkin protein levels, and found that the acetylation level of parkin in cells treated with SAHA drug was significantly increased, as shown in figure 1 As shown in A.

[0026] In order to reveal the molecular mechanism that regulates the acetylation of parkin protein, protein spectrum analysis was performed on the protein complex after GFP affinity purification gel precipitation, and the histone acetylase HAT (ACAT1, EP300) and Histone deacetylase HDAC (HDAC1, HDAC2, HDAC3), the obtained HAT and HDAC protein name and protein score (protein score), such ...

Embodiment 2

[0031] Parkin protein is mainly regulated by phosphorylation modification, and acetylation modification has not been reported. In order to further clarify the molecular mechanism regulating parkin acetylation, HEK293 cells overexpressing Flag-parkin were treated with 2.5 μM SAHA drug for 12 hours, protein samples were analyzed by SDS-PAGE gel after FLAG affinity purification gel precipitation, and parkin strips were cut out After digestion of strips and strips, the acetylation sites of parkin were found by protein spectrum LC-MS analysis. It was found that SAHA treatment caused acetylation of parkin protein sites K76, K129, K220, K349, and K408, such as Figure 2B1 , Figure 2B2 , Figure 2B3 with Figure 2B4 shown. At the same time, multiple acetylation sites screened by protein spectrum were verified and analyzed, and KR (lysine to arginine) point mutation was carried out to construct KR mutant plasmid. After HEK293 cells were transfected with parkin wild-type (parkin-WT...

Embodiment 3

[0033] Recent studies suggest that the level of mitochondrial protein acetylation is closely related to mitophagy and tumorigenesis. In order to clarify the effect of parkin protein acetylation on the level of parkin-mediated mitophagy, HeLa tumor cells were first transfected with parkin-WT plasmid and parkin-KR plasmid, and after 24 hours, the transfected HeLa tumor cells were treated with SAHA drug 2.5 μM was treated for 12 hours, and Western blot was used to detect the changes in the expression of mitochondrial membrane proteins before and after SAHA treatment, and the results were as follows image 3 As shown in A and 3B, before SAHA treatment, overexpression of parkin-WT reduces the levels of mitochondrial membrane proteins such as VDAC, MFN2, TIM23 and HSP60, and increases the levels of autophagy proteins such as LC3 and P62 in mitochondria, while the parkin acetylation site mutation After that, it weakened the degradation effect of parkin-WT on mitochondrial membrane pr...

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Abstract

The present invention discloses acetylation of parkin protein and an application of the parkin protein in preparation of medicines for regulation and control of mitophagy, a histone deacetylase inhibitor is used for inducing the acetylation of the parkin protein, so that a mitophagy level of cells is enhanced and growth of tumor cells is inhibited. A plurality of acetylation sites of the parkin protein are screened by using a protein spectrum technology, the acetylation sites are confirmed to be K129, K220, K349 and K408 through a KR point mutation verification analysis, and parkin-KR mutant plasmids are constructed after multi-site mutation. Functional analysis results show that after parkin mutation, the mitophagy level of the cells is weakened and an anti-tumor effect of a cancer suppressor gene parkin is inhibited, indicating acetylation modification is an important regulation and control mode influencing the parkin function.

Description

technical field [0001] The invention relates to the fields of physiology and molecular biology, in particular to the application of the acetylation of parkin protein and the mutation of its acetylation site in the preparation of mitophagy regulation medicines. Background technique [0002] Mitochondrial autophagy refers to the fact that under the stimulation of reactive oxygen species, nutrient deficiency, cell aging and tumors, the mitochondria in the cells undergo depolarization damage, the mitochondrial potential decreases, and the damaged mitochondria are specifically wrapped into autophagosomes. , forming mitophagosomes (early stage), which fuse with lysosomes to degrade damaged mitochondria (late stage), and then play an important role in maintaining the homeostasis of cells. [0003] The pathways that regulate cellular mitophagy mainly include parkin-dependent pathways, that is, the PINK1-parkin signaling pathway, and parkin-independent pathways, such as the mitochond...

Claims

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Application Information

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IPC IPC(8): C12N9/00A61K38/53A61K31/167A61P35/00
CPCC12N9/93A61K31/167A61P35/00C12Y603/02019A61K38/00
Inventor 张建宾黄东胜孙馨王继刚高瑞兰舒雨涵郑国湾徐梦婷
Owner ZHEJIANG PROVINCIAL PEOPLES HOSPITAL
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