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Application of deubiquitinating enzymes USP42 as drug target in preparation of drugs

A deubiquitinating enzyme and a technology for preparing a drug, which is applied in the application field of treating lung cancer, can solve the problems of unclear correlation phase separation and the like, and achieve the effect of promoting tumor growth

Pending Publication Date: 2021-06-15
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although ubiquitination is strongly associated with phase separation, the relevance of human deubiquitinases to phase separation and the ubiquitination-mediated phase separation of deubiquitinase substrates are unclear

Method used

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  • Application of deubiquitinating enzymes USP42 as drug target in preparation of drugs
  • Application of deubiquitinating enzymes USP42 as drug target in preparation of drugs
  • Application of deubiquitinating enzymes USP42 as drug target in preparation of drugs

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Example 1 Identification of USP42 as a deubiquitinating enzyme with phase separation properties

[0036] USP42 is distributed in a dot-like aggregation in the nucleus. In this example, the Hela cells transiently transfected with USP42 were first treated with 1,6-hexanediol (1,6-HD) to detect changes in the intracellular localization of USP42 , it was found that 1,6-Hexanediol treatment could induce USP42 to change from a punctate aggregation state to a diffuse distribution in the nucleus【 figure 1 A]. Then, using laser confocal microscopy, fusion experiments and FRAP experiments were used to prove that the punctate aggregation of USP42 was fluid and recovered after fluorescence bleaching.【 figure 1 B and C]. The above results indicated that USP42 was distributed in liquid phase in the nucleus.

[0037] Then the phase separation properties of USP42 were further verified in vitro. The GFP-USP42 fusion protein was purified in vitro, and the ability of USP42 to aggregat...

Embodiment 2

[0038] Example 2 USP42 localizes in nuclear speckles in an enzyme activity-dependent manner

[0039] After the phase separation of USP42 was confirmed, its physiological function was further studied. First, GFP-USP42 was overexpressed in cells, and the co-localization of USP42 and nuclear spot marker SC35 was detected by laser confocal microscopy. The results showed that exogenously overexpressed USP42 could co-localize with SC35【 figure 2 A]. Then, using USP42 antibody, the localization of endogenous USP42 protein and nuclear spot markers was detected, and the results showed that endogenous USP42 could co-localize with SC35【 figure 2 B]. This shows that USP42 is localized in nuclear speckles.

[0040]Cysteine ​​at position 120 is the enzymatic active site of USP42. In order to prove the effect of USP42 enzymatic activity on its localization, mutants with inactive mutations in the enzymatic active site were constructed, including point mutation (USP42-C120A) and USP seque...

Embodiment 3

[0041] Example 3 USP42 regulates the phase separation of PLRG1

[0042] In order to further study the mechanism of USP42 regulating tumor growth through phase separation, the downstream regulatory protein PLRG1 of USP42 was found through database analysis. PLRG1 is an important component of the splicing complex, regulating mRNA alternative splicing. First, GFP-tagged USP42 full-length protein, USP42-C with retained C-terminal sequence, and USP42-C120A with active site mutation were overexpressed in cells, and GFP-tagged USP42 and its mutants were detected by immunofluorescence experiments. The co-localization of overexpressed PLRG1 and endogenously expressed PLRG1 in cells showed that USP42 could co-localize with both exogenous and endogenous PLRG1【 image 3 A and B]. In order to further clarify the combination of USP42 and PLRG1, HEK293 cells were co-transfected with GFP-tagged USP42 (GFP-USP42, GFP-USP42-C) and Flag-tagged PLRG1 (Flag-PLRG1), and USP42 was detected by co-i...

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Abstract

The invention relates to application of deubiquitinating enzymes USP42 as a drug target in preparation of drugs, in particular to a mechanism of the deubiquitinating enzymes USP42 for promoting lung cancer growth by regulating and controlling mRNA variable splicing through phase separation and application of the deubiquitinating enzymes USP42 in treatment of lung cancer. It is found through research that USP42 is positioned in a cell nucleus and is in a liquid phase separation state, a carbon-end disordered region with positive charges of USP42 drives phase separation to occur and enter a nuclear spot, and the activity of the deubiquitinating enzymes USP42 plays an important role in the process. The deficiency of USP42 can inhibit growth of tumor cells. USP42 regulates and controls the phase separation state of a splicing body component PLRG1 and mRNA splicing, and therefore gene expression participating in cell function regulation is affected. In conclusion, inhibition of USP42 can serve as a potential mode for tumor treatment.

Description

technical field [0001] This application relates to the field of medical technology, specifically to the use of deubiquitinating enzyme USP42 as a drug target in the preparation of medicines, and more specifically to the mechanism by which deubiquitinating enzyme USP42 promotes the growth of lung cancer by regulating mRNA alternative splicing through phase separation and its role in Application in the treatment of lung cancer. Background technique [0002] Lung cancer is a malignant tumor with high morbidity and mortality worldwide, which seriously threatens human health. Intracellular partitioning is an important means for eukaryotic cells to coordinate and regulate the normal operation of various physiological functions in time and space. These cellular compartments contain both membrane-encased and membraneless organelles. Recent studies have shown that liquid-liquid phase separation represents a general way of forming membraneless organelles. This structure is an aggre...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61P35/00
CPCA61K45/00A61P35/00
Inventor 刘晗刘书言王太术石玉林张迎秋
Owner DALIAN MEDICAL UNIVERSITY
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