A screening system and application of regγ-20s proteasome inhibitors

A proteasome inhibitor, inhibitor technology, applied in the field of biochemistry and biomaterials, to achieve the effect of high sensitivity, high specificity, and efficient detection ability

Active Publication Date: 2021-02-02
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since the proteasome plays an extremely important role in the body, these proteasome inhibitors as drugs can completely inhibit the activity of the proteasome, so the use of this proteasome inhibitor will also have relatively strong side effects on patients. For example, patients may have severe insomnia, headache, nausea, diarrhea and other adverse reactions

Method used

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  • A screening system and application of regγ-20s proteasome inhibitors
  • A screening system and application of regγ-20s proteasome inhibitors
  • A screening system and application of regγ-20s proteasome inhibitors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Co-immunoprecipitation experiment proves that REGγ specifically binds to α7 subunit of 20S proteasome

[0039] The α subunit of the 20S proteasome is its regulatory subunit, which is opened or closed under the control of activating factors. REGγ can promote the degradation of substrate proteins. It is likely that REGγ may open the gate of 20S by combining with the α subunit. The present invention The interaction between REGγ and 20S proteasome α1-α7 was detected by co-immunoprecipitation.

[0040] Specific experimental methods:

[0041] 1. Before transfection, HeLa cells were planted in six-well plates at a concentration of 60%, and cultured for 12 hours.

[0042] 2. Add 300 μL of serum-free DMEM medium to a 1.5 mL centrifuge tube, and then add the target plasmid and Young’s transfection reagent. The ratio of the two is 1 μg: 2 μL. Each well of a six-well plate can transfer 1 μg of plasmid.

[0043] 3. Vortex and vibrate for 10 seconds to mix well. After sta...

Embodiment 2

[0052] Example 2 Yeast two-hybrid experiment proves that REGγ specifically binds to α7 subunit of 20S proteasome

[0053] Using different experiments, it was again demonstrated that REGγ specifically binds to the α7 subunit of the 20S proteasome.

[0054] PSMA1-PSMA7 (α 1 -α 7 ) was constructed on the pGADT7 vector, and the yeast two-hybrid experiment was used to further verify the interaction between REGγ and α7.

[0055] The results of yeast two-hybrid image 3 As shown, only when PGAD-α7 / pGBK-REGγ was co-transfected, yeast could grow on the four-deficient plate, that is to say, REGγ only interacted with the α7 subunit, which was consistent with the results of co-immunoprecipitation ( nip30 as a positive control).

[0056] The specific experimental method of yeast two-hybrid experiment:

[0057] 1. Take out the yeast strain from the -80°C refrigerator and melt it at room temperature, then burn the inoculation loop on the alcohol lamp until it is red hot, and after cooli...

Embodiment 3

[0068] Example 3 Using luciferase fragment complementation technology to construct a screening system for inhibitors of the interaction between REGγ and α7

[0069] By comparing several commonly used molecular imaging techniques for studying protein-protein interactions, such as energy resonance transfer technology, green fluorescent protein fragment complementation technology and luciferase fragment complementation technology, etc. The present invention finally selects the highly efficient and convenient luciferase fragment complementation technology as the system basis for screening the inhibitors of the interaction between REGγ and α7. In the design experiment, the present invention considers three key issues: the first key issue is (1) the cleavage site of luciferase, how to cut the luciferase so that the two fragments produced can recombine well , and the newly bound luciferase can regain luciferase activity. Such as Figure 4 As shown, the present invention selects the...

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Abstract

The invention discloses a screening system for REGγ-20S proteasome inhibitors, the screening system includes fusion proteins lucN(416-linker1)-REGγ and α7-lucC (linker1-394); based on REGγ-20S proteasome through REGγ Intrinsic Mechanism of Binding to α7, Using Luciferase Fragment Complementation, Screening for REGγ‑20S Proteasome Inhibitors. The screening system constructed by the present invention comprising two fusion proteins of LucN(416-linker1)-REGγ and α7-lucC(linker1-394) has a good screening effect. The invention improves the effectiveness and specificity of the screening system by optimizing the screening system, and can efficiently screen REGγ-20S proteasome small molecule compound inhibitors.

Description

technical field [0001] The invention relates to the technical fields of biochemistry and biomaterials, in particular to the molecular mechanism of REGγ binding to 20S proteasome and the application of the mechanism to screen REGγ inhibitors and treat REGγ-related diseases. Background technique [0002] Proteasome exists in eukaryotes, archaea and some bacteria, regulates the degradation and renewal of most proteins in cells, and plays a key role in maintaining normal life activities of cells. The proteasome complex usually consists of two parts: a cylindrical 20S core particle and a proteasome activator (19S regulatory particle, 11S activator, and Blm10 / PA200). [0003] The 20s proteasome, as the core part of the proteasome complex, is composed of α and β subunits according to the α 7 beta 7 beta 7 alpha 7 The model constitutes a 28-subunit complex. The ring composed of 7 α subunits is located at both ends of the 20s cylindrical structure, and the ring composed of 7 β s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K14/47G01N33/68
CPCC07K14/47C07K2319/61C07K2319/70G01N33/68
Inventor 李晓涛李磊张变红张坤薛燕燕翟万里
Owner EAST CHINA NORMAL UNIV
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