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Screening system and application of REG Gamma-20S proteasome inhibitor

A technology of proteasome inhibitors and inhibitors, applied in the fields of biochemistry and biomaterials, to achieve the effects of high sensitivity, high specificity, and efficient detection capabilities

Active Publication Date: 2017-07-07
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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  • Screening system and application of REG Gamma-20S proteasome inhibitor
  • Screening system and application of REG Gamma-20S proteasome inhibitor
  • Screening system and application of REG Gamma-20S proteasome inhibitor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0039] The α subunit of the 20S proteasome is its regulatory subunit, which is turned on or off by the regulation of activators. REGγ can promote the degradation of substrate proteins. It is likely that REGγ may open the 20S gate by binding to the α subunit. The present invention The interaction between REGγ and 20S proteasome α1-α7 was detected by immunoprecipitation method.

[0040] Specific experimental methods:

[0041] 1. Before transfection, plant HeLa cells in a six-well plate with a concentration of 60% and culture for 12 hours.

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

[0043] 3. Vortex and shake for 10 seconds to mix well. After standing at room temperature for 15 minute...

Embodiment 2

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

[0053] Different experiments proved that REGγ specifically binds to the α7 subunit of the 20S proteasome.

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

[0055] Yeast two-hybrid results such as image 3 As shown, only when PGAD-α7 / pGBK-REGγ cotransforms, yeast can grow on the four-deficiency plate, which means that REGγ only interacts with the α7 subunit, which is consistent with the result of immunoprecipitation ( nip30 as a positive control).

[0056] Specific experimental methods of yeast two-hybrid experiment:

[0057] 1. Take out the yeast strain from the refrigerator at -80℃ and melt it at room temperature, then heat the inoculation loop to red hot on an alcohol lamp. After cooling, use the inoculation loop to dip a small amount of ...

Embodiment 3

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

[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. The present invention finally selects the efficient and convenient luciferase fragment complementation technology as the system basis for screening 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 by it can recombine well And the newly bound luciferase can regain the activity of luciferase. Such as Figure 4 As shown, the present invention selects the most...

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Abstract

The invention discloses a screening system of a REG Gamma-20S proteasome inhibitor. The screening system comprises a fusion protein lucN(416 linker1)-REG Gamma and alpha 7 lucC(linker1-394); on the basis of an internal mechanism that the REG Gamma-20S proteasome is bonded with alpha 7 by REG Gamma, the REG Gamma-20S proteasome inhibitor is selected by using a luciferase fragment complementation technology. The screening system containing two fusion proteins, lucN(416-linker1)-REG Gamma and alpha 7 lucC(linker1-394), built in the invention has a good screening effect. By optimizing the screening system, the effectiveness and the specificity of the screening system are improved, and the screening of REGGamma-20S proteasome small molecule compound inhibitor can be efficiently achieved.

Description

Technical field [0001] The present invention relates to the technical fields of biochemistry and biological materials, in particular to the molecular mechanism of REGγ binding to 20S proteasome and the application of the mechanism for REGγ inhibitor screening and REGγ-related diseases treatment. Background technique [0002] Proteasomes exist in eukaryotes, archaea and some bacteria. They regulate the degradation and renewal of most proteins in cells, and play a crucial role in maintaining the normal life activities of cells. The proteasome complex usually consists of two parts: a cylindrical 20S core particle and a proteasome activator (19S regulatory particles, 11S activator and Blm10 / PA200). [0003] As the core part of the proteasome complex, the 20s proteasome is composed of α and β subunits according to α 7 β 7 β 7 α 7 The model constitutes a 28 subunit complex. The ring composed of 7 alpha subunits is located at both ends of the 20s cylindrical structure, and the ring compo...

Claims

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Application Information

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Patent Type & Authority Applications(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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