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Use of usp33 as a drug target in the preparation of drugs

A drug and protein technology, applied in the field of protein engineering, can solve problems such as reducing the survival rate of Drosophila

Active Publication Date: 2022-04-26
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After infection with L.monocytogenes in Drosophila, the function of LC3 in Drosophila lacking PARK2 will be reduced, resulting in the loss of autophagy and reducing the survival rate of Drosophila

Method used

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  • Use of usp33 as a drug target in the preparation of drugs
  • Use of usp33 as a drug target in the preparation of drugs
  • Use of usp33 as a drug target in the preparation of drugs

Examples

Experimental program
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Effect test

Embodiment 1

[0096] Example 1 Co-immunoprecipitation and mass spectrometry identification of the interaction between Parkin and the deubiquitinating enzyme USP33

[0097] 1. The present invention utilizes co-immunoprecipitation combined with mass spectrometry to study Parkin's interacting protein network. In U2OS cells, the pcDNA3-Flag-Parkin plasmid was transfected, and the cells were screened by 400ug / ml G418 to obtain a cell line stably expressing the Flag-Parkin protein SEQ ID NO.2 ( figure 1 Figure A).

[0098] Cell No. 4 with a high expression level was selected for expansion culture, the whole cell lysate was collected, and anti-FlagBeads were incubated with it, the Flag-Parkin interacting protein was enriched by co-immunoprecipitation, and SDS-PAGE gel electrophoresis was used Separation of enriched protein samples ( figure 1 Figure B), and then stained with Coomassie Brilliant Blue G250 for protein profile analysis. The control group was transfected with pcDNA3-Flag-vector plas...

Embodiment 2

[0105] Example 2 Mitochondrial localization of USP33 protein

[0106] 1. USP33 is located in the mitochondrial outer membrane

[0107] The localization of endogenous USP33 in cells was verified by immunofluorescence experiments. In U2OS cells, mitochondria were labeled with Tom20 (mitochondrial outer membrane protein) antibody, and U2OS cells were labeled with USP33 antibody, as Figure 5 As shown in Figure A, it was found that endogenous USP33 and mitochondrial protein TOM20 had obvious co-localization, indicating that USP33 protein was located on mitochondria. Next, cell fractionation was performed on HEK293 cells, as Figure 5As shown in Figure B, the contamination of nuclear LaminB and cytoplasmic Tubulin was not detected in the mitochondrial fraction, indicating that the isolated mitochondria are of good purity and can be used in the next experiment. At the same time, Western blotting experiments confirmed that USP33 exists in mitochondria, as shown in the immunofluores...

Embodiment 3

[0111] Example 3 Knockdown of USP33 Promotes GFP-Parkin Translocation to Mitochondria

[0112] When cells are treated with CCCP (Carbonyl cyanide 3-chlorophenylhydrazone, inhibitor of mitochondrial oxidative phosphorylation), the mitochondria will be damaged and depolarized, and a large amount of Parkin protein will be recruited to the mitochondria to initiate the occurrence of mitophagy. In order to study whether the knockdown of USP33 will affect the process of mitophagy initiation, this example constructed a GFP-Parkin plasmid, transfected it into U2OS cells, and established a cell line stably expressing GFP-Parkin.

[0113] Cells stably expressing GFP-Parkin were treated with different concentrations of CCCP for 2 hours, and then the translocation of GFP-Parkin was observed, as shown in Figure 7 As shown, compared with the control group, GFP-Parkin aggregated and translocated in most of the cells treated at the concentration of 20 μM and 40 μM, while GFP-Parkin aggregated...

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Abstract

The invention provides the use of USP33 as a drug target in the preparation of medicines, belonging to the field of protein engineering. The present invention utilizes co-immunoprecipitation and mass spectrometry techniques to discover the deubiquitinating enzyme USP33 that interacts with Parkin, and further discovers that USP33 is located in the mitochondrial outer membrane and can deubiquitinate and modify the E3 ligase Parkin. Knockdown of USP33 will promote the translocation of Parkin to mitochondria, thereby enhancing mitophagy to remove damaged mitochondria, confirming that USP33 regulates mitophagy by affecting the deubiquitination of Parkin, and knocking down USP33 can inhibit the expression of neurons under MPTP treatment. Apoptosis, USP33 can be used as a drug target to prepare drugs, and has good application potential and value.

Description

technical field [0001] The invention belongs to the field of protein engineering, and specifically relates to the use of USP33 as a drug target in the preparation of medicines. Background technique [0002] USP33 (VDU1) is a deubiquitinating enzyme with 942 amino acids and a molecular size of 107kD. It can remove ubiquitin molecules from the substrate, and can also rely on pVHL (mutant protein of von Hippel–Lindau (VHL) disease) way to be ubiquitinated. Structural biology revealed that it contains a ZnF UBP structure at the N-terminus, a similar structure is also found in the deubiquitinating enzyme HDAC6, and a DUSP structure at the C-terminus. The ZnF UBP structure has three zinc ions, and the 221-arginine mutation of USP33 will affect the binding to the 75-glycine ubiquitin molecule. USP33 has at least three variants. Variant 1 is full-length and has 25 exons; variant 2 lacks exon 2, and translation starts from the start codon of exon 3 lacking 31 amino acids; Body 3 i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00G01N33/573A61K38/48A61K38/53A61K45/00A61P3/00A61P9/00A61P25/28A61P31/12A61P35/00
CPCC12N9/93G01N33/573C12Y603/02019A61K38/4813A61K38/53A61K45/00A61P25/28A61P35/00A61P9/00A61P3/00A61P31/12G01N2333/948G01N2500/10
Inventor 赵永良牛凯峰方洪波魏迪
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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